Abstract Book - Eurobiotech-IsSN 0001 527X

Contents
Plenary Lectures:....................................................................................................................... 1
White Biotechnology
Session 1:

Nanotechnologies in industrial processes.............................................................................. 3

Chair: Zygmunt Kowalski
Session 2:

Development of renewable energy in biotechnology........................................................... 13

Chair: Henryk Kooczek
Session 3:

Bioeconomy................................................................................................................................ 22
Chair: Stanisaw Bielecki
Session 4:

Bioplastics and biobased polymers......................................................................................... 26

Chair: Andrzej Kononowicz, Krzysztof Pielichowski
Session 5:

Pharmaceutical biotechnology................................................................................................ 33
Chair: Alicja Jzkowicz, Katarzyna Kie-Kononowicz
Green Biotechnology
Session 6:

Plant molecular breeding......................................................................................................... 60

Chair: Dariusz Grzebelus
Session 7:

Environmental biotechnology................................................................................................. 75
Chair: Katarzyna Turnau
Session 8:

Plant genetic engineering......................................................................................................... 125

Chair: Rafa Baraski
Session 9:

Animal biotechnology in biomedicine................................................................................... 134

Chair: Zdzisaw Smorg, Ryszard Somski
Session 10:

Animal biotechnology in agriculture...................................................................................... 148

Chair: Krystyna Koziec, Arieh Gertler
Session 11:

Legislation for biotechnologists............................................................................................... 165

Chair: Tomasz Twardowski
Satellite Panels
Session 1:

Induced pluripotent stem cells: a future of biomedicine...................................................... 168

Chair: Alicja Jzkowicz
Session 2 :

Business Session Life Science Open Space

Chair : Kazimierz Murzyn
Preface by the Organizing Committee

Dear Madame/Sir,
It gives me great pleasure to welcome you, on behalf of the Organizing Committee, to the V Central European
Congress of Life Sciences 2013 Eurobiotech in the Royal City of Crakow. The Congress is held under
the honorary patronage of the Voivode of the Malopolska Voivodeship, the Marshal of the Malopolska
Voivodeship and the Mayor of the Royal Capital City of Crakow.
The Congress has been organized by Crakow University of Technology (member of the Organizing
Committee for the first time this year), the University of Agriculture, the Jagiellonian University,
Biotechnology Committee of Polish Academy of Sciences, Polish Federation of Biotechnology, Malopolska
Center of Biotechnology, Life Science Center in Crakow, Jagiellonian Center of Innovation Ltd. and Targi
w Krakowie Ltd.
Satellite sessions devoted to stem cells and bio-business discussions will precede the opening ceremony of
the Congress.
The scientific programme of the Eurobiotech 2013 will combine white and green biotechnology topics as
well as legislation problems in the biotechnology sector.
Outstanding scientists from Europe, USA and other countries have been invited to deliver lectures on
such important issues as nanotechnologies, renewable energy, environmental biotechnology or animal
biotechnology in biomedicine and agriculture and many others.
We hope that the V Eurobiotech Congress and the accompanying exhibitions will enable you to exchange
your scientific opinions and to present the latest biotechnological achievements and future trends in the area
of white and green biotechnologies.
We are convinced that the Congress being of great value to the Polish science will encourage the invited
scientists and businessmen to start long-term scientific and business cooperation.
We warmly welcome you to the Congress hoping that its main goal, providing you with a scientific
programme of the highest quality, will be achieved and that you will spend wonderful time in our vibrant
city with great cultural and historical dignity.
On behalf of the Congress Organizing Committee

Henryk Koloczek
Congress Chair
Crakow University of Technology
Crakow, October 2013
Plenary lectures
PL1
PL2
Synthetic Plant Biotechnology Tools

for Precise Transgene Expression C
Oxidative Stress Signal Transduction

in plants
Neal Stewart, Jr.
Frank Van Breusegem
Department of Plant Sciences, University of Tennessee,

Knoxville, TN, USA
VIB Department of Plant Systems Biology, UGent Technologie

Park 927
B-9052 Gent, Belgium
Synthetic biology tools have the ability to greatly increase

the precision and amount of gene expression in transgenic plants. The presentation will discuss synthetic promoters for inducible gene expression and how they are being
utilized in our phytosensors research program. Here, we
are producing phytosensor tobacco plants with an orange
fluorescent protein reporter gene that is induced upon
pathogen infection. This research is designed to fit into
precision agriculture systems to allow farmers to treat for
plant pathogens where needed and prior to when disease
symptoms are observed. We are also studying how synthetic transcription activator-like effectors (TALEs) can
be used as transcription factors to increase gene expression. We are using designer TALE-TFs that bind to the
flanking regions of the TATA-box motif on the CaMV 35S
promoter for the purpose understanding the engineerable hot-spots for increasing transgene expression. We
demonstrated that the de novo-engineered TALEs could
increase reporter gene expression by up to 3 fold in stable
transgenic tobacco harboring an orange fluorescent protein reporter gene under the control of synthetic inducible, minimal, or full-length 35S promoters. These results
provide novel insights into the potential applications of
synthetic promoters and TALE-TFs for targeted gene activation of transgenes in plants.
Different biotic and abiotic stresses adversely affect plant

growth and development. A common theme within these
environmental factors is the perturbation of reactive oxygen species (ROS) homeostasis. The signal transduction
mechanisms of the oxidative stress response in plants are
poorly understood. We conducted genetic and chemical
screens, which combined with proteomic approaches
will lead to a more comprehensive overview on the components of the network that govern the oxidative stress
response. These efforts resulted in the identification
of a number of genes as new members of the oxidative
stress gene network in plants. In our recent studies, we
have identified a novel transcriptional regulator of the
mitochondrial retrograde regulation and are assessing
the modulation of cellular NAD(P) homeostasis as anew
strategy to improve plant performance in stress conditions.
Eurobiotech 2013
PL3
PL4
Teaching from the Plant Kingdome;

Energy, Economics and the Environment
Luuk A.M. van der Wielen
Stanisaw Karpiski
Warsaw University of Life Sciences,
Department of Genetics, Breeding and Plant Biotechnology,
Nowoursynowska 159, 02-776 Warsaw, Poland
Almost all of the modern infrastructure of our civilization

relies on fossil fuels for energy. Despite clear evidence
that the combustion of fossil fuels over the 20th and the
beginning of 21st century has led to significant increase
in atmospheric carbon dioxide that is altering the global
environment, there has been little collective political
will to alter the energy sources of the largest industrial
societies.
This economic positive feedback is further exacerbated by
the abundance of large quantities of fossil fuels, especially
coal and natural gas, remaining in known reservoirs.
To alter this trajectory in the 21st century, concerted
investment in alternative, clean energy technologies must
be made both by governments and private industries.
In this lecture, I will discuss potential of plant and
photosynthesis biotechnologies, and I will present lesson
of economy from the Plant Kingdome for encouraging
investment by government and private industries.
Delft University of Technology, The Netherlands, Industrial

Biotechnology (tentative title)
Nanotechnologies in industrial processes
Lectures
L1.2
L1.1
Use of titanium dioxide for purification,

self-cleaning and disinfection in urban
infrastructures
The use of biofiltration in the rendering plants

L. Tymczyna1, A. Chmielowiec-Korzeniowska1, Z. Paluszak2,
M. Banach3, J. Pulit3
Uniwersytet Przyrodniczy w Lublinie;
Uniwersytet Technologiczno-Przyrodniczy w Bydgoszczy;
3
Politechnika Krakowska
1
2
The study evaluated the effectiveness of air biofiltration of

rendering plants. The biofilter material comprised compost soil (40%) and peat (40%) mixed up with coconut
fiber (medium A) and oak bark (medium B). During biofiltration average reduction of VOCs was 88.4% for medium A and 89.7% for medium B. There was a positive
relationship of the reduction of aldehydes from material
humidity (r = 0.502, < 0.05). Other parameters of the
biomaterial does not have a significant impact on the efficiency of the treatment.
Agata Markowska-Szczupak1, Beata Tryba1,

Antoni W. Morawski1
Institute of Chemical and Environment Engineering,
West Pomeranian University of Technology, Szczecin
1
Titanium dioxide is commonly used as a white pigment

for paints and lacquers (about 70% of its total production). Recently it has emerged as an excellent photocatalytic material for environment purification, self-cleaning
materials and disinfection. In this review, current progress in this area of TiO2 applications is presented.
Titanium dioxide, particularly in the anatase form, can be
use as a photocatalyst when it is excitated with the UV
light. The overall photocatalytic activity of titanium dioxide particles depends on number of parameters such as:
method of preparation, phase composition, ratio between
the anatase and rutile crystal phases, anatase crystallite
size, excitation light wavelength and many others. The
strong oxidative potential of hydroxyl radicals which are
generated on the surface of TiO2 particles after its excitation is responsible for oxidation a huge number of organic and inorganic compounds such as: nitrogen oxides,
aromatic hydrocarbons, aldehydes, ketones, VOCs, drugs,
pesticides etc. [1].
The development of new photocatalysts for the optimization of photocatalytic performance is promoted in various
directions. To achieve improved photocatalytic activity of
TiO2, particularly under visible light excitation, different
modifications are performed such as: doping of both,
metal or non-metal ions, creating of nonstructured materials (e.g. nanotubes, nanowires, nanoshells etc.), and
formation of co-catalysts.
New generation photocatalysts based on titanium dioxide are added to some building materials (e.g. paints, cements, gypsum, glasses, tiles etc.) in order to form their
self-cleaning properties. The most effective use of photocatalytic materials can be seen on large-scale construction
projects, in which the large surfaces are exposed to the
light promoting self-cleaning and anti-pollutant performance. For this reason facades, roadways and pavements
in the traffic urban areas became the target for the first examples of utilisation these photocatalytic materials. Such
applications are already in progress in Japan and Europe
and are recently initiated in the United States.
Under solar or UV light irradiation TiO2 exhibits the superhydrophilic properties by reducing the water contact
Eurobiotech 2013
angle, thus gives increased self-cleaning capacity and

cooling effect. It was found the application in production
of anti-fogging windows, car mirrors, glasses. The cooling
properties let to reduce the amount of energy consumption used for operation of air-conditioners and also artificial heat emission in glass buildings [2].
Titanium dioxide is internationally recognized as one of
the new sterilization materials, which can kill wild spectrum of organisms including viruses, bacteria, fungi and
algae. It has been widely used in places with high sterilization demanding such as hospitals, institutions, schools
etc. Self-cleaning fabrics could revolutionize the sport
and army apparel industry [3].
The titania nano-biotechnology is considered as alternative method of cancer therapy, which targets only cancer
cells and does not affect normal living tissue [4].
Titanium dioxide is used in numerous applications because of its compatibility with modern technology. New
applications involving TiO2 can improve our lives in areas
such as water treatment, indoor and outdoors purification and energy saving.
Acknowledgements
This work was supported by National Centre for Science and Ministry
of Science and Higher Education of Poland under project No. MNiSW/
DPN/4878/TD/2010.
References
[1] Carp O., Huisman C.L., Reller A. (2004) Photoinduced reactivity of
titanium dioxide, Prog. Solid State Chem. 32: 33177.
[2] Nakata K., Fujishima A., (2012) TiO2 photocatalysis: design and
applications. J. Photochem. Photobiology C: Photochem. Rev. 13:
169189.
[3] Markowska-Szczupak A., Ulfig K., Morawski A.W. (2011) The
application of titanium dioxide for deactivation of bioparticulates: an
overview, Catal. Today, 169: 249257.
[4] Rozhkova E., Ulasov I., Lai B., Dimitrijevic N.M., Lesniak M.S., Rajh
T. (2009) Nanobio photocatalyst for targeted brain cancer therapy, Nano
Lett. 9: 33373342.
L1.3
Nanosilver a new product for disinfecting
treatment
Marcin Banach1, Jolanta Pulit1, Leszek Tymczyna2,
Anna Chmielowiec-Korzeniowska2
Institute of Inorganic Chemistry and Technology, Crakow
University of Technology; 2University of Life Sciences in Lublin
1
The human body is exposed to microorganisms such as

bacteria, viruses or fungi. Micro-organisms and their
remnants, may cause imbalance of enzymatic homeostasis in ones organism and may exert immunotoxic effects
on the immune system [1].
The amount of chemicals used for disinfecting is limited due to their negative impact on the body and because
of the difficulties with their solubility and with the possibility of direct application. In addition, the ecological
consumer lifestyle forces to use only those chemicals that
occur naturally in nature, or whose use does not pose
athreat to the environment. Organic acids and their salts,
1-Hexadecylpyridinium chloride, sodium orthophosphate, hydrogen peroxide and sodium bicarbonate are
a large group of compounds among preferred additives
destroying bacterial flora. However, not all of these compounds appear to be fully effective, so new methods of
microorganisms disposing still have been looked for. Silver nanoparticles, which, according to the literature exhibit good biocide effect, may be an alternative for those
disinfectants [24].
Nanostructured silver is an effective destructing
agent against a broad spectrum of Gram-negative and
Gram-positive bacteria, not excluding antibiotic-resistant strains [5]. It is characterized by a specific surface
and a high content of surface atoms which induces a rare
physicochemical properties. The results suggest that metal which reacts with a thiol group (SH) binds with a protein of the microorganism, which leads to its inactivation
[4, 6].
Therefore, nanotechnology takes a considerable challenge, which aims to reduce pathogenic bacterial activity.
However, nanometals toxicity against eukaryotic cells, including the cells of vertebrates is an important issue. Results of in vitro studies presented in the literature confirm
nanometals cytotoxicity, particularly nanostructured silver
which is destroying to the rat liver cells, neurons, human
lung epithelial cells and murine stem cells. Silver nanoparticles may also act genotoxic. Some in vivo studies have
also shown nanometals effect on the cardiovascular system, respiratory system, central nervous system and liver.
However, these data are incomplete and their confirmation
requires further research in this direction [6].
The work presents results of research related to obtaining
nanostructured silver, its characteristics, biocidal
properties and uses for disinfection purposes.
Acknowledgements
The scientic work is nanced for the years 20102013 as research
project 4111/B/H03/2010/39.
Eurobiotech 2013
References
[1] Burrell R. (1995) Ann. Agric. Environ. Med. 2, 11.
[2] Kowalski Z., Banach M., Powaka E. (2009) Przem. Chem. 88, 478.
[3] Konopka M., Kowalski Z., Wzorek Z. (2009) Arch. Environ. Prot.
35, 107.
[4] Banach M., Kowalski Z., Wzorek Z. (2007) Chemik 9, 435.
[5] Wright J.B., Lam K., Hansen D., Burrell R.E. (1999) Am. J. Infect.
Control 27, 344.
[6] Banach M., Pulit J. (2013) Przem. Chem. 92, 6, 1056.
L1.4
Pig manure treatment by filtration
Zygmunt Kowalski1, Agnieszka Makara1, Dalibor Matsek2,
Jzef Hoffmann3, Krystyna Hoffmann3
Institute of Chemistry and Inorganic Technology,
Crakow University of Technology, Warszawska 24,
31-155 Crakow, Poland; 2Institute of Geological Engineering,
VB-Technical University of Ostrava, 17. listopadu 15,
708 33 Ostrava-Poruba, Czech Republic; 3Institute of Inorganic
Technology and Mineral Fertilizers, Wroclaw University
of Technology, Wybrzeze Wyspianskiego 27, 50-370 Wroclaw,
Poland
1
Numerous livestock farms in Europe import fodders including the nitrogen (N) and phosphorus (P)-bearing
ingredients that these fodders contain. This results in
local nutrient surpluses because home-grown crops take
up less N and P than available in the manure. We have
developed in our study new pig manure treatment and
filtration process. The advantage of worked out technology is the method of incorporation in organic phase of the
manure of 4059% of crystalline phase. This resulted in
achievement of high filtration rate over 1000 L/m2/h with
the used pressure filters and good quality of filtrate. The
method is able to maintain an overall average pollutant
removal performance ~90% for the chemical oxide demand COD, > 99% for the suspended solids SS, to 47%
for the total nitrogen content determined with Kjeldahls
method TKN. Preliminary investigation indicated that
the mineralization process eliminates over 75% of the
odor intensity coming from the filtrate obtained in comparison to odor intensity coming from the pig manure.
Acknowledgements
This study has been carried out in the framework of the Development
Project No. 14-0003-10/2010 granted by the National Centre for
Research and Development.
Eurobiotech 2013
L1.5
L1.6
Phosphorus cycle possibilies of its

rebulidng
Nanoemulsion as a base to controlled

release of forskolin
Katarzyna Gorazda, Zbigniew Wzorek, Anna K. Nowak,

Barbara Tarko
Magorzata Jaworska1, Elbieta Sikora1,

Maria Jose Garcia-Celma3, Elvira Escribano3, Conxita Solans4,
Jan Ogonowski1
Crakow University of Technology, Warszawska 24 St.,

31-155 Crakow, Poland
Institute of Inorganic Chemistry and Technology
The recovery of phosphorus from waste can take place

in many ways: from the waste water streams, sewage
sludge, animal waste, industrial waste and other industrial emissions. It is proposed, that thermal processing of
sewage sludge, where phosphorus is removed by chemical precipitation, will be a preferable method for their
utilisation. Obtain product in the form of ash could be
potentially used as a substitute of natural ore. Developed
by Cracow University of Technology and patented method of phosphorus recovery by acid extraction methods
allows for the phosphorus recovery efficiency between
8096%. The average phosphorus content in Polish ashes is around 9.7%. 3000 to 4000 tons of phosphorus per
year can be recycled and introduced to the environment.
That amount is equivalent to 30 770 t of phosphorus ore.
The proposed technology allowed finding out alternative
sources of phosphorus for individual industries.
Faculty of Chemical Engineering and Technology,

Institute of Organic Chemistry and Technology,
Crakow University of Technology; 2Dept. of Pharmacy and
Pharmaceutical Technology, Faculty of Pharmacy, University
of Barcelona, Spain; 3Institute for Advanced Chemistry
of Catalonia, Consejo Superior de Investigaciones Cientficas
(IQAC-CSIC), Centro de Investigacin Biomdica en Red
en Bioingeniera, Biomateriales y Nanomedicina (CIBER-BBN),
Barcelona, Spain
1
Forskolin is one of labdane diterpenoids. It is isolated

from the roots of Coleus Forskohlii, an aromatic herb
belongs to the family of mints and lavenders originated
from India. It is characterized by many biological effect
e.g. stimulates adenylate cyclase, resulting in increase of
cAMP levels which is related to biochemical mechanism
of maintaining or increasing lean body mass [1]. Moreover, its applied in the treatment of atopic dermatisis
and used as an antiallergic agent [2]. Nanoemulsions are
anew form of cosmetic and pharmaceutic products. They
show some advantages comparing to classic emulsions:
relatively easily penetrate into the skin, exhibit a high
degree of hydration and soften the skin and have a very
good user properties. They are one of the most promising
formulations used to enhance percutaneous penetration
of active substances as well as their bioavailability [3, 4].
The aim of this work was to determine the release of forskolin from nanoemulsion. The nano-emulsion based on
caprylic/capric triglycerides and stabilized by Polisorbate
80 was obtained. The formulation was prepared by stepwise addition of water to a mixture of polisorbate/oil/
forskolin, at room temperature. Drug release study of forskolin was carried out using the Spectra/Por Standard
Regenerated Cellulose (RC). The concentration of the
active in the receptor solution (i.e. Ethanol/PBS mixture)
was analyzed by means spectrophotometer UV-VIS and
HPLC. The results have shown that nanoemulsions can
be used as carriers for controlled release of forskolin
Acknowledgements
The research (work) was supported by the European Union through the
research scholarship Doctus Maopolski fundusz stypendialny dla
doktorantw.
References
[1] Bagchi D., Preuss H.G. (2012) Obesity: Epidemiology, Pathophysiology and Prevention, Taylor and Francis Group.
[2] Ciotonea C., Cernatesul C. (2001) Biological active effects of
Forskolin extract, Buletinul InstitutulviPolitehnic DIN IASI, 4, 95106.
[3] Gugliemini G. (2006) Evaluating droplet size in nanoemulsions from
a novel emulsifier system, Cosmetics and Toiletries. 121 (12), 6772.
[4] Tharwat Tadros, Izquierdo P., Esquena J., Solans C. (2004) Formation
and stability of nano-emulsions, Advances in Colloid and Interface
Science.108-109, 303318.
Eurobiotech 2013
Posters
P1.2
P1.1
Studies on the formation of O/W nanoemulsions, by low-energy emulsification

method, suitable for cosmeceutical
applications
Preparation of Ag nanoparticles using

plant extracts
Zygmunt Sadowski, Hanna Hy, Szcepan Gorzynik
Wroclaw University of Technology, Chemical Engineering
Department
Present study reports a green chemistry approach for

the synthesis of silver nanoparticles. A simple procedure using Aloe vera and Pelargonium graveolens leaf
extracts was used to produce of silver nanoparticles. The
extract from dried powder of Punica granatum was also
used as reducining agent. The silver nanoparticles synthesized using the plant extracts were characterized by
UV-vis spectroscopy. The results from UV-vis show that
the reduction mechanism of silver ions was affected by
the extract concentration and precursor solution (silver
nitrate). The temperature of extract preparation was responsible for the synthesis of silver nanoparticles. The
bioreduction of silver ions occurred at room temperature
in a few hours. The kinetics of reduction reaction was
investigated. Biosynthesis of silver nanoparticles using
plant extract is considered to be a novel, effective and
eco-friendly method.
Acknowledgements
Zlecenie Nr S20072/Z0307.
Magorzata Jaworska1, Elbieta Sikora1, Micha Zielina2,

Jan Ogonowski1
Faculty of Chemical Engineering and Technology, Institute
of Organic Chemistry and Technology, Crakow University
of Technology; 2Faculty of Environmental Engineering,
Institute of Water Supply and Environmental Protection,
1
Nanoemulsions offer several advantages for cosmeceutical use. They are easy to prepared, they characterized
by long term stability, high solubilization capacity for
hydrophilic and lipophilic actives and additionally improved transdermal active delivery [1, 2]. The formation
of O/W nano-emulsions suitable for cosmeceutical application was studied. Nano-emulsions were prepared by
using phase inversion composition (PIC) method, one of
low-energy emulsification methods. The process consist
of stepwise water addition to oil/surfactant mixture, at
25C. Caprylic/capric triglycerides, propylene glycol dicaprylate/dicaprate and oleic acid were applied as an oil
phase. Polisorbate 80 was used as the surfactant. Kinetic
stability of the nano-emulsions was analyzed by measuring droplet diameter as a function of time for different
oil/surfactant ratio. The particles size distribution was
analyzed by means DLS measurement technique (Dynamic Light Scattering), using Zetasizer Nano ZS (Malvern Instruments). One of triterpenoic acid, practically
non-water soluble substance was selected as an active and
incorporated into the stable formulation. The obtained
results proved that the nanoemulsion based on caprylic/
capric triglycerides was the most stable one and additionally showed the highest solubilisation for the triterpene.
The maximum concentration of the active achieved in
oil/surfactant mixture (20/80) was 2,5 mg/ml.
Acknowledgements
The research (work) was supported by the European Union through the
research scholarship Doctus Maopolski fundusz stypendialny dla
doktorantw.
References
[1] Maghraby G.M., Transdermal delivery of hydrocortisone from
eucalyptus oil microemulsion: effects of cosurfactants. International
Journal of Pharmaceutics, 2008, 355(12), 285292.
[2] Peltola S., Saarinen-Savolainen P., Kiesvaara J., Suhonen T.M., Urtii A.,
Microemulsions for topical delivery of estradiol, International Journal
of Pharmaceutics, 2003, 254, 99.
Eurobiotech 2013
P1.3
P1.4
Influence of process parameters on

properties of Nanostructured Lipid Carriers
(NLC) formulation
Nanometals as tool in fight against plant

pathogens
Elwira Laso, Elbieta Sikora, Jan Ogonowski

Institute of Organic Chemistry and Technology,
Crakow University of Technology, Poland
Nanostructured lipid carriers (NLC) are stabile colloidal

formulations with notable advantages for drug delivery
systems, i.e. physicochemical stability, biocompatibility,
biodegradability and controlled drug release. NLC are derived from o/w emulsions by simply replacing the liquid
lipids (oils) by a solid lipids. Due to controlled nanostructure, the particles offers enough space to accommodate
the active ingredient [1, 2]. The particles are produced
through a mixing of solid and liquid lipid with water
phase in the presence of surfactants. The most preferred
NLC production methods are high pressure homogenization and ultrasound technology.
The aim of the study was preparation and characterization of nanostructured lipid carriers (NLC). Both, effect
of process parameters and effect of preemulsion composition on the NLC properties were investigated. In the
work, different type of surfactants (i.e. decyl glucoside,
Poloxamer188, Tween 80, sodium cholate) and their combinations were used to stabilize NLC dispersions. Moreover, several kinds of solid lipids (modified beeswax, gliceryl behenate, cetyl palmitate and berry wax) and liquid
lipids (caprilic/capric triglyceride and decyl oleate) were
applied. An ultrasonication method using a probe type
sonicator (Sonics Vibra-Cell, Sonics & Materials, INC.),
were used to obtained NLC, the time and energy of the
process were modifying. The physicochemical properties
of the formulations, such as particle size, size distribution, polidispersity index and zeta potential were studied
using dynamic light scattering (DLS) method (Malvern
Zetasizer instrument).
The obtained results showed that the process parameters
and the composition of preemulsion significant influence on the nanoparticles structure. Stable formulation,
of particles size approximately 80 nm, were prepared by
ultrasonified the mixture for 10 min at 35W, maintaining
the temperature at least 5C above the lipid melting point.
As a stabilizing agent alkylpolyglucoside and its combination with some other surfactants give the best effect.
Acknowledgements
The research (work) was supported by the European Union through
the European Social Fund within Cracow University of Technology
development program top quality teaching for the prospective
Polish engineers; University of the 21st century project (contract No.
UDA-POKL.04.01.01-00-029/10-00).
References
[1] Mller R.H., Petersen R.D., Hommoss A., Pardeike J. (2007)
Nanostructured lipid carriers (NLC) in cosmetic dermal products, Adv.
Drug Deliv. Rev. 59: 522530.
[2] Radtke M., Souto E., Mller R.H. (2005) Nanostructured lipid
carriers: a novel generation of solid lipid drug carriers, Pharm. Technol.
Eur. 17: 4550.
Agnieszka Sobczak-Kupiec1, Dagmara Malina1,

Boena Tyliszczak2, Zbigniew J. Burgie3
Institute of Inorganic Chemistry and Technology, Crakow
University of Technology, 24 Warszawska Str., 31-155 Crakow,
Poland; 2Department of Chemistry and Technology of Polymers,
Crakow University of Technology, 24 Warszawska St.,
31-155 Crakow, Poland; 3Department of Plant Protection,
University of Agriculture in Crakow, al. 29 Listopada 54,
1
In recent years, a great interest in nanotechnology has

been observed. Nanostructured metallic particles because of their unusual properties, have been the subject
of numerous intensive research due to their unusual
properties, which are dependent on the nanometric sizes. Products based on nanoparticles are applied in the
electronic, optic, chemical, textile industries, as well as in
pharmacy, cosmetology, and medicine. Moreover, certain
types of nanometals (e.g. silver and gold nanoparticles)
also demonstrate strong antimicrobial activity against
bacteria, fungi and viruses.
The main aim of this study was to investigate antifungal activity of selected metallic nanoparticels towards
common pathogenic fungi of crop species. For this purpose suspensions of nanosized Ag and Au, proceeded by
chemical reduction using a suitable stabilizer, were added
to the medium for fungi growth. In the next step, media
with appropriate amount of nanoparticles were inoculated with mycelial fragments of selected fungi strains.
Nanometals applied in research exhibit high fungicidal
activity against all fungi tested independently of concentration. Based on the obtained results it is assumed
that in the future, pesticides harmful for plants may be
replaced by nanoparticles free from negative effects on
plant growth.
Acknowledgements
The research was funded from the budget for science for years
20132014, No. IP2012 062872.
Eurobiotech 2013
P1.5
Carbon aerogels based on natural starches
synthesis and characterization
Monika Bakierska, Marcin Molenda, Magorzata Maria Zaitz,
Roman Dziembaj
Faculty of Chemistry, Jagiellonian University
Introduction. Carbon aerogels are a novel class of nanostructured and porous carbon materials which exhibit
unusual properties and can be used in a wide variety of
applications (eg. energy storage, catalysis, biomedicine
etc.) [13]. These materials can be obtained by the pyrolysis of organic aerogels at elevated temperatures under
inert atmosphere. The most widely used organics for the
fabrications of the aerogels via the sol-gel polycondensation are resorcinol and formaldehyde [4]. However, the
use of natural polysaccharides and their derivatives is
considered to be more attractive because of their stability,
availability, renewability, low cost and non toxicity [57].
The purpose of this study was to prepare and characterize carbon aerogels based on natural starches of different
botanical origin (potato, maize and rice). Moreover, to
decrease the process cost and shorten processing time, an
ambient pressure drying instead of supercritical drying
was applied.
Experimental. Carbon aerogels were prepared by the
carbonization of organic aerogels. Four main steps can
be distinguished in the preparation of these materials,
and the first was the gelation process in which different
types of starches (potato, maize and rice) were dissolved
in water and stirred while maintaining the appropriate
gelatinization temperature for each type of starch. The
second step was the solvent exchanging carried out either by soaking the gel directly in the new solvent ethanol. The third step was the wet gel drying performed at
50C for 1 day under ambient pressure and the fourth
was the carbonization of dried gel under constant flow
of argon at 600C and after that carbon aerogels were
obtained.
Optimal conditions for carbonization process of organic
aerogels were determined by thermal analysis methods
(TGA/DTG/SDTA/EGA-QMS). The structure and the
morphology of the prepared carbon aerogels were investigated using X-ray powder diffraction (XRD) and low
temperature N2 adsorption measurements (N2-BET).
Electrical properties of the obtained aerogels were examined by electrical conductivity studies (EC) using
4-probe method within temperature range of 20C to
+40C.
Conclusions. To conclude, starch-based carbon aerogels were synthesized by the pyrolysis of organic aerogels, which were prepared in gelatinization process of
natural starches (potato, maize and rice). This method
made it possible to obtain nanostructured materials
with well-developed porosity and high surface area. The
electrical conductivity measurements proved that pre-
pared materials provide good electrical properties. The

obtained carbon aerogels seem to be perspective materials which can be widely used in many fields of science
and industry.
Keywords: carbon aerogels, starches, gelatinization process, textural
properties, conductivity
Acknowledgements
This work is supported by the European Institute of Innovation and
Technology under the KIC InnoEnergy NewMat project.
References
[1] Mirzaeian M., Hall P.J. (2009) Electrochimica Acta 54: 7444.
[2] Moreno-Castilla C., Maldonado-Hdar F.J. (2005) Carbon 43: 455.
[3] Garca-Gonzlez C.A., Alnaief M., Smirnova I. (2011) Carbohydrate
Polymers 86: 1425.
[4] Pekala R.W. (1989) Journal of Materials Science 24: 3221.
[5] Aaltonen O., Jauhiainen O. (2009) Carbohydrate Polymers 75: 125.
[6] Tsioptsias C., Michailof C., Stauropoulos G., Panayiotou C. (2009)
Carbohydrate Polymers 76: 535.
[7] Chang X., Chen D., Jiao X. (2010) Polymer 51: 3801.
10
Eurobiotech 2013
P1.6
P1.7
Characterization and properties of silver

nanoparticles
for agro-engineering applications
Silver nanoparticles as an effective

biocidal factor
Agnieszka Sobczak-Kupiec1, Katarzyna Bialik-Ws2,

Boena Tyliszczak2, Dagmara Malina1, Zbigniew Burgie3
Institute of Inorganic Chemistry and Technology,
31-155 Crakow, Poland; 2Department of Chemistry and
Technology of Polymers, Crakow University of Technology,
Warszawska 24 St., 31-155 Crakow, Poland;
3
Department of Plant Protection, Agricultural University
1
Nowadays nanomaterials and nanotechnology are very

important in medicine, pharmacy, tissue engineering as
well as in the world of agriculture. Today protect plants
against pests is carried out mainly by chemical methods
that use synthetic pesticides. Its advantage is the effective
action and thus possibility of preventing the occurrence
epidemic of plant diseases. However this method is very
detrimental to the human health and environment because of several active substances with high toxicity [1].
It turns out that formulations containing nanometals particles can be particular useful, because they characterize
higher biochemical activity than the macro-structures
and more effective influence on the bacteria or fungi.
According to the literature, silver and gold nanoparticles
have high fungistatic activity and biocidal properties. The
biocidal activity of the silver nanoparticles is dependent
on the size and shape of the particles [24].
In carried out investigations were developed stable suspensions of silver nanoparticles containing degradable
polymer dispersant. The use of polymers for the preparation of dispersions improve the homogeneity, rheological
properties and stability of nanoparticles. The agrochemical formulations containing metallic nanoparticles of
silver, were developed. The physicochemical characterization of the obtained materials was carried out: particle
size distribution, stability and sedimentation, analysis of
the morphology of the particles.
Acknowledgements
The research was funded from the budget for science for years
20132014 No. IP2012 062872.
References
[1] Weber Z., Kryczyski S. (red.) (2010) Fitopatologia, PWRiL, Pozna.
[2] Nair R., Varghese S.H., Nair B.G., Maekawa T., Yoshida Y.,
Kumar D.S. (2010) Plant Science, 179: 154163.
[3] Rai M., Yadav A., Gade A. (2009) Biotechnology Advances, 27:
7683.
[4] Zielinska A., Skwarek E., Zaleska A., Gazda M., Hupka J. (2009)
Procedia Chemistry 1: 15601566.
Jolanta Pulit1, Marcin Banach1, Renata Szczygowska2,

Mirosaw Bryk3
Crakow University of Technology, Institute of Inorganic
Chemistry and Technology, Warszawska 24 St.,
31-155 Crakow, Poland; 2Silesian Environmental Ph.D. Studies
Centre, Central Mining Institute, Gwarkow Sq., 1,
40-166 Katowice, Poland; 3Implementing Company
1
Nanosilver is one of the most thoroughly investigated

nanomaterials, and owes its popularity to its biocidal properties [1]. Its antimicrobial activity is associated
with the characteristic structure of nanoparticles. They
are characterized by a high fraction of surface atoms so
that nanoparticles have a greater affinity for interactions
with thiol groups, after which the destruction of the bacteria follows [2]. Biological activity is therefore a very important factor, so that nanosilver is used where asepsis
is highly desirable. Mostly, this applies in biomedical engineering, cosmetology, food packaging industry, textile
industry, crops, animal husbandry etc. [3].
Recently, using nanosilver as biological agent has become
increasingly common. Such an idea aims at the synthesis
of nanometric silver in accordance with the principles of
green chemistry. The development of nanosilver preparation processes while respecting green chemistry principles was the intention of the authors. It was decided to
find a solution that would allow the use of one substance
acting as both a stabilizing and a reducing agent. This
assumption was carried out using a raspberry extract,
which largely reflects the ideas of environmental technologies. The use of raspberry extract as a source of ellagic
acid offers the possibility of direct silver ion reduction,
as it has strong antioxidant effects, similar to many other
polyphenols. Furthermore, minor amounts of other compounds with antioxidant properties, such as gallic acid,
ascorbic acid, and quercetin that also contribute to efficiently carrying out the chemical reduction are also present in raspberry extract. The presence of other organic
compounds from the group of polyphenols, tannins and
flavonols allows to stabilize emerging suspensions with
no need to add other factors that inhibit the growth of
metallic silver agglomerates [4].
Authors also raise the problem of asepsis. Microbial
colonization, for instance, in painted buildings causes
aesthetic problems and may lead to degradation of the
coating and splintering [5]. Much more dangerous effects may be caused by the influence of mould on human
health. Spores and mycelial fragments escaping from the
ground, together with bacteria and dust, form bioaerosols
that are a source of air pollution. They may contain toxic compounds called mycotoxins, which cause allergies.
Penicillium spp., Aspergillus spp. and Alternaria spp. are
significantly responsible for the formation of many of the
allergic symptoms and respiratory diseases in people exposed to high levels of mould spores and allergens.
Eurobiotech 2013
Thus, according to the varying degrees of effectiveness

of the antimicrobial properties of nanosilver obtained
via green technology and the need to recognize the application in the coating field, researches were undertaken
whose aim was to evaluate the antifungal activity of silicon paint, plaster and ground plaster containing silver
nanoparticles as well as perform a mycological efficacy
resistance analysis of tested preparations in relation to
different concentrations of nanostructured silver. The
use of nanoparticles with antimicrobial properties in
construction materials can bring numerous potential advantages, such as reinforcing their hygienic properties,
preventing microbial growth and maintaining their mechanical properties.
The work presents the way of obtaining an aqueous raspberry extract, its physicochemical and analytical characteristic as well as the method of preparation of nanosilver
suspensions based on raspberry extract. Moreover, results of microbiological studies on biocidal effect of obtained nanosilver against Cladosporium cladosporoides,
Aspergillus Niger, Rhodotorula mucilaginosa, Aspergillus
niger and Geotrichium candidum are described.
Acknowledgements
The work is part of a project Synthesis and application of innovative
nanomaterials with antimicrobial properties supported by
National Centre for Research and Development under the contract
No. LIDER/03/146/L-3/11/NCBR/2012 for the period of 20122015.
References
[1] Vaidyanathan R., Kalishwaralal K., Gopalram S., Gurunathan
S., Nanosilver the burgeoning therapeutic molecule and its green
synthesis. Biotechnol. Adv. 2009, 27, 924.
[2] Sadeghi B., Jamali M., Kia S., Amininia A., Ghafari S., Synthesis and
characterization of silver nanoparticles for antibacterial activity. Int. J.
Nano. Dim. 2010, 1, 119.
[3] Pulit J., Banach M., Kowalski Z., Nanosilver making difficult
decisions. Ecol. Chem. Eng. S. 2011, 18, 185.
[4] Hakkinen S. H., Karenlampi S.O., Mykkanen H.M., Heinonen I.M.,
Torronen A.R., Ellagic acid content in berries: Influence of domestic
processing and storage. Eur. Food Res. Technol. 2000, 212, 75.
[5] Bellotti N., Salvatore L., Dey C., Del Panno M.T., del Amo B.,
Romagnoli R., The application of bioactive compounds from the
food industry to control mold growth in indoor waterborne coatings.
Colloids Surf. B, 2013, 104, 140.
11
P1.8
The impact of industrial nanoaerosols
on the pulmonary surfactant
Dorota Kondej1, Tomasz R. Sosnowski2
Central Institute for Labour Protection National Research
Institute/ Department of Chemical, Aerosol and Biological
Hazards/ Czerniakowska 16/ 00-701 Warsaw/ Poland;
2
Warsaw University of Technology/ Faculty of Chemical and
Process Engineering/ Warynskiego 1/ 00-645 Warsaw/ Poland
1
The development of nanotechnologies and the related

increasing use of nanomaterials create a potential risk of
workers exposure to inhalation of nanoparticles realised
during industrial processes. The aim of this study was
to evaluate the impact of selected mineral nanoparticles
(bentonite PGV, halloysite HN, surface-modified montmorillonite I.28E) used as nanofillers in the production
of polymer nanocomposites on the surface activity of the
pulmonary surfactant (PS). PS being the first barrier separating the inhaled air present in pulmonary alveoli from
the lung tissue plays an important role in the physiology
of the respiratory system. PS is responsible for a significant reduction of the surface tension of the liquid covering the lung tissue which has important implications for
the mechanics of the respiratory cycle and mass transfer
processes in the lung. The research was done using two
complimentary techniques (the Langmuir-Wilhelmy
film balance and the pulsating bubble method) allowing to study different aspects of the influence of mineral
nanoparticles on the interfacial activity of model pulmonary surfactants (DPPC and Survanta). It was found the
mineral nanoparticles alter the surface activity of model
pulmonary surfactants under dynamic conditions related to lung behaviour during breathing. The presence of
PGV and HN nanoparticles causes a reduction in the
activity of model pulmonary surfactants which may be
attributed to the adsorption of PS molecules on the surface of the nanoparticles investigated. Surface-modified
montmorillonite I.28E nanoparticles induce increase of
the surface pressure in the system which can be explained
by a release of surface-active compounds from I.28E particles. Inhalable nanoparticles used in the production of
polymer nanocomposites may cause the disturbance of
surface activity of PS. By this mechanism industrial nanoaerosols may contribute to adverse health effects in the
respiratory system of workers involved in the production
process.
Acknowledgements
This paper has been prepared on the basis of the results of research
project No. I.B.10 carried out within the National Programme
Improvement of safety and working conditions partly supported
in 20112013 within the scope of research and development by the
Ministry of Science and Higher Education. CIOP-PIB has been the
Programme main coordinator.
12
Eurobiotech 2013
P1.9
The impact of nanosilver addition
on element ions release form light-cured
dental composite and compomer into
0,9% NaCl
Krzysztof Sokoowski1, Magorzata Iwona Szynkowska2,
Aleksandra Pawlaczyk2, Jerzy Sokoowski3
Department of Conservative Dentistry, Medical University
of Lodz, Pomorska 251, 92-213 Lodz, Poland; 2Institute
of General and Ecological Chemistry, Technical University
of Lodz, Zeromskiego 116, 90-924 Lodz, Poland; 3Department
of General Dentistry, Medical University of Lodz, Pomorska 251,
92-213 Lodz, Poland
1
Nowadays, a wide spectrum of dental materials is available, material properties depend on the basic material
type e.g. metals, ceramics or polymers. In fact, a detailed
understanding of the key concept for each of them gives
an insight into how each class of material can behave
as a restorative dental material, as well as an idea of the
potential of these materials if some of their limitations
can be overcome. There is no surprise that only certain
specific material properties determine their selection
for application in restorative dentistry. Basing on that
knowledge, it is possible to make a proper selection of
the material being the best compromise of desired properties versus inherent limitations. Due to the fact that
any class of basic type of presented materials possesses
all the desired properties, in many cases they are used in
combination limiting their usefulness [1]. The main objective of this study was to assess the release of metal ions
from dental fillings into 0.9% NaCl solution in different
time intervals. The investigated dental materials were
samples of flowable composite material with nanosilver
addition and flowable compomer material with nanosilver addition, both being a light-cured dental restorative
material with flow characteristics, which makes it ideal
material for filling small cavities in anterior and posterior teeth. Composite restorations seem to represent excellent aesthetics properties, but due to the fact that they
undergo polymerization shrinkage on setting, they are
associated with marginal leakage, which causes bacterial
penetration and, in consequence, the potential damage
to the tooth. Notwithstanding, fluoride releasing materials in combination with silver ions can influence the
surrounding micro environment, involving bacteria.
Each of the samples was first washed with ethanol and
then placed in a test-tube in 10 mL 0.9% NaCl solution.
A number of counts of metal ions released into NaCl
solution from the dental materials were determined using Optimass 8000 ICP-TOF MS spectrometer (GBC
Scientific Equipment, Australia) after a week, a month
and three months time of storage. The results confirmed
the significant increase in the number of counts of metal
ions originating form the main components of studied
dental material with the time of storage. The gathered
spectra were also compared with some previous out-
comes obtained for other restorative materials not containing silver in their composition.
Acknowledgements
The financial support of this work by the Polish Scientific Research
Council (grant N N209 343237 ) is gratefully acknowledged.
Development of renewable energy in biotechnology
Lectures
L2.1
Metabolic Engineering of Non-Conventional
Yeasts for Construction of the Advanced
Producers of Lignocellulosic Ethanol
Andriy A. Sibirny1, 2
Department of Molecular Genetics and Biotechnology,
Institute of Cell Biology, NAS of Ukraine, Drahomanov Street,
14/16, Lviv 79005 Ukraine; 2Department of Biotechnology and
Microbiology, University of Rzeszow, Zelwerowicza 4, Rzeszow
35-601, Poland
1
Non-conventional yeasts are attractive ethanol producers from lignocellulose. One of the most promising
ethanol producer from lignocellulose is Hansenula polymorpha which belongs to the best studied non-conventional yeasts. Its genome has been sequenced and publicly available (http://genomeportal.jgipsf.org/Hanpo2/
Hanpo2.info.html) and methods of molecular genetics
and cell biology are well developed. It is a popular host
system for expression of heterologous proteins and some
metabolites as well as favorite model organism for studying peroxisome biogenesis and autophagic degradation, methanol catabolism and stress response. Besides,
H. polymorpha is apparently the most thermotolerant
yeast known with maximal growth temperature at 50C.
Several years ago, we have found that H. polymorpha is
capable of high-temperature xylose alcoholic fermentation though ethanol production is quite low. At the
same time is effectively ferments glucose and cellobiose
(Ryabova et al., 2003, Ishchuk et al., 2009). More recently, it was shown that H. polymorpha can effectively produce ethanol from glycerol, the byproduct of biodiesel
production (Suvannarangsee et al., 2010). To construct
effective ethanol producers from xylose, overexpression
of the modified gene XYL1m (encoding xylose reductase)
and the native genes XYL2 (xylitol dehydrogenase), XYL3
(xylulokinase) and PDC1 (pyruvate decarboxylase) and
additionally, the isolation of 3-bromopyruvate-resistant
mutants from metabolically engineered strains have been
conducted (Dmytruk et al., 2008; Ishchuk et al., 2008).
Using mentioned approaches, the strains of H. polymorpha have been constructed which accumulate elevated
amounts of ethanol from xylose, up to 1012 g/L or near
22 g/L after correction for ethanol evaporation. However,
to be economically feasible, available level of ethanol synthesis from xylose has to be increased for several times,
up to 3035 g/L. We have found that increase in ethanol
production from xylose can be achieved by either disrup-
tion or knock out of ATG13 gene involved in autophagy

and pexophagy. Increase in xylose conversion to ethanol
could be also achieved by overexpression of genes coding
peroxisomal enzymes dihydroxyacetone synthase (DAS1)
and transaldolase (TAL2). DAS1 overexpression resulted
in increase in ethanol production from xylose both on
the backgrounds of the wild-type strain and previously
isolated metabolically engineered strain with enhanced
ethanol synthesis. Other promising organism for xylose
alcoholic fermentation is Pichia stipitis. Ethanol accumulation in the last organism could be also increased using
methods of classic selection and metabolic engineering.
References
[1] Dmytruk O.V. et al. (2008) Microb Cell Fact. 7, 21.
[2] Ishchuk O.P. et al. (2008) FEMS Yeast Res. 8, 11641174.
[3] Ishchuk O.P. et al. (2009) Biotechnol. Bioeng. 104, 911919.
[4] Ryabova O.B. et al. (2003) FEMS Yeast Res. 4, 157164.
[5] Suvannarangasse S. (2010) Appl. Microbiol. Biotechnol. 88, 497507.
14
Eurobiotech 2013
Posters
P2.1
The influence of phytase addition during
the enzymatic starch hydrolysis process
with the use of basic amylolytic enzymes
on yield and the course of alcoholic
fermentation process
Dawid Mikulski, Grzegorz Kosowski
Department of Biotechnology, Institute of Experimental Biology,
Kazimierz Wielki University, 85-667 Bydgoszcz,
Chodkiewicza 51 St., Poland
Phytic acid (myo-inositol hexa-phosphoric acid, IP6) is

the major phosphorus storage compound of most cereal grains. Phosphorylated form of inositol due to ionized
phosphates groups has strong chelating properties. Phytic acid creates complexes, the so-called phytin complexes
with divalent cations (Mg2+, Ca2+, Fe2+, Zn2+), with proteins
and polysaccharides, i.e. starch [1]. These complexes limit
the availability of the divalent cations, proteins and starch
for organisms which do not produce the phosphohydrolases enzymes. The applications of phytase, the enzyme
hydrolysing phytin complexes during the preparation of
the fermentation medium, should result in the increase
of availability of biogenic compounds [2]. The aim of the
study was to determine the influence of phytin hydrolysis before and after starch hydrolysis process present in
high gravity maize mashes (HG mashes) on yield and the
course of alcoholic fermentation process. The raw material used in preparation of HG mashes according to pressureless liberation of starch technology (PLS technology)
was milled maize grain (starch concentration 53.34%,
phytic acid concentration 2.76 mg/g, total phosphorus
concentration 2.93 mg/g). The process of enzymatic
starch hydrolysis was conducted with heat-stable -amylase (Termamyl SC DS preparation) and glucoamylase
(Saczyme preparation) produced by Novozymes company. Phytin complexes hydrolysis was conducted with
the use of Phytase 10000L preparation produced by Erbsloh company. During the alcoholic fermentation the
basic technological indicators (alcohol concentration,
productivity, yield and fermentation energy) and indicators of the physiological condition of yeast (number,
viability, phosphorus concentration in yeast cells) were
determined. Alcoholic fermentation volatile by-products
were determined with capillary gas chromatography (GC
method). The analysis of variance (significant level 3/100
kg of starch was observed in the variant with phytin hydrolysis before starch hydrolysis process. In the initial
fermentation phase irrespectively of the research variants
the viability of yeast was ca. 98%. In subsequent phases
the increase of ethanol concentration caused the decrease
of viability and number of yeast cells in variants with addition of phytase in relations to the control variant. The
highest concentration of acetaldehyde (524.125.6 mg/L)
and higher alcohols (4144.2154.0 mg/L) was observed

in the distillate obtained in the fermentation medium
with phytin hydrolysis before hydrolysis of starch.
Acknowledgements
The study financed from the science resources in the years 20132014, as
an experimental study of the National Science Centre (No. 2012/05/N/
NZ9/02436).
References
[1] Garcia-Estepa R.M., Guerra-Hernndez E., Garcia-Villanova B.
(1999) Phytic acid content in milled cereal products and breads. Food
Research International 32, 217221.
[2] Lei X.G., Porres M. (2003) Phytase enzymology, applications, and
biotechnology. Biotechnology Letters, 25, 17871794.
Eurobiotech 2013
P2.2
Determination of the impact of lactose
suplementation of synthetic cellulolytic
media on the activity of cellulase complex
Dorota Macko, Beata Miklaszewska, Dawid Mikulski,
Grzegorz Kosowski
Cellulose is linear -D-glucopyranose polymer, in which

individual units (8-12 thousand) are connected with
-1-4-glycosidic bonds, no other chemical compound
of the polysaccharides group is not produced in the natural environment, in such large amounts. Cellulose is
arenewable, and potential source of glucose for the production of ethanol. Effective enzymatic hydrolysis of this
polymer depends on proper activation of lignocellulotic
biomass (pre treatment method) [Mosier et al. 2005],
and also on the composition, concentration, and activity
of enzymes comprising the cellulase complex: endo-1,4-D-glucanase, exo-1,4--D-glucanase and 1,4--D-glucosidase. Great importance on the polymer degradation
has a process of induction of cellulase secretion in fungal
hyphae, it is followed by natural activators such as oligosaccharides: cellobiose, sophorose, gentobiose [Suto and
Tamita, 2001]. The aim of the study was to evaluate the
influence of lactose supplementation of culture medium
on enzymatic activity of endoglucanase (CMC-ases), celobiase, and total celulase activity (FPaza) produced by
isolate of Penicillium sp. selected from cellulolytic industry materials. The strain was cultivated by liquid fermentation on medium contained different substrates of
cellulose: microcrystalline (AVICEL 2%), carboxymethyl
cellulose high viscosity (CMChv 1%), and carboxymethyl cellulose low viscosity (CMClv 2%) in Vogels medium
(according to Ahmed et. al 2009). The experiment was
carried out in two different variants: on pure Vogels medium with and without addition of 10g lactose per one
liter of medium. All analyses of the enzymatic activity
and concentrations of reducing sugars were conducted according to the IUPAC procedure (International
Union of Pure and Applyed Chemistry). The analysis of
variance (significant level deviation was carried out by
using STATISTICA software, ver. 10). Statistical significant influence on FPase and CMCase activity by supplementation medium with lactose was detected. During the
fermentation reaction in medium contained with AVICEL, total FPase activity grew from 0,690 (without lactose addition) to 4,893 mg of glucose/ min/ mg of protein
(with lactose addition). Similar the activity of CMCase
increased from 11,910 to 43,796 mg of glucose/ min/ mg
of protein, reaching a maximum value.
References
[1] Suto M, Tomita F. (2001) Induction and catabolite repression
mechanisms of cellulase in fungi, Journal of Bioscience and
Bioengineering, Vol. 92, No. 4, 305311.
15
[2] Mosier N., Wyman Ch., Dale B., Elander R., Lee Y.Y., Holtzapple M.,
Ladisch M. (2005) Features of promising technologies for pretreatment of
lignocellulosic biomass, Bioresource Technology 96: 673686.
[3] Ahmed S., Bashir A., Saleem H., Saadia M., Jamil A. (2009)
Production and purification of cellulose-degrading enzymes from
a filamentous fungus, Trichoderma harzianum Pakistan Journal of
Botany 41(3): 14111419.
16
Eurobiotech 2013
P2.3
P2.4
Perspectives of biofuel production

from cellulosic crop waste
Biogas generation from sewage sludge

in the anaerobic digestion process
Dariusz Dziga1, Dominika Jagieo-Flasiska2
Mariusz Makowski, Pawe Wolski, Lidia Wolny
Department of Plant Physiology and Development, Faculty

of Biochemistry, Biophysics and Biotechnology, Jagiellonian
University, Gronostajowa 7, Crakow, Poland; 2Department of
Plant Biotechnology, Faculty of Biochemistry, Biophysics and
Biotechnology, Jagiellonian University, Gronostajowa 7, Crakow,
Poland
Institute of Environmental Engineering, Czestochowa University

of Technology
Cellulose is a major component of plant biomass and

could be applied in the production of biofuels, especially
bioethanol. An alternative approach is to produce hydrogen from cellulosic biomass. In this paper an innovatory
model of cellulosic waste utilization has been proposed
to verify the possibility of cellulose degradation product
utilization by purple non-sulfur bacteria. The concept is
based on a two-step process of microbial cellulose conversion in order to obtain an organic acid mixture. In the
next stage such products are consumed by Rhodobacter
sphaeroides which is capable of hydrogen production
in a photofermentation process using different organic
compounds. It has been documented that R. sphaeroides employed in this process can effectively consume
organic acids released from straw by Cellulomonas uda
and Lactobacillus rhamnosus and that it is able to grow
rapidly in the presence of these substrates. Additionally,
an enhanced nitrogenase activity of R. sphaeroides has
been shown when bacteria were cultivated in the presence of cellulose derivatives which suggest that hydrogen
production occurs. These promising results indicate the
necessity to carry out further research using the proposed
model of cellulose transformation into biohydrogen. The
key aim of further experiments is to confirm whether
R. sphaeroides is able to produce hydrogen effectively using a mixture of organic acids originating from cellulose.
Anaerobic digestion is a process used in the most of sewage treatment plants. Volume reduction in relation to cost
is a big opportunity in the sludge management, both in
relation to less volume of digesters and less final product.
Volume reduction can be achieved by converting more of
the sludge to biogas and also by better mechanical dewatering of sludge.
Those aims are achieved with advanced digestion. Ultrasonic field as physical agent is very effective pretreatment
method to enhance the biodegradability of sludge in the
anaerobic digestion. Decomposition of organic (nutrient)
substances is limited mainly by the velocity of the first
step of methane fermentation process named as hydrolysis. During this hydrolysis the organic compounds can
be decomposed into liquid and finally becomes methane
and carbon dioxide. Intensification of the methane fermentation hydrolytic phase and the increase of biogas
generation depends on dispersion of sewage sludge solid
phase.
The aim of investigations was the preliminary treatment
process evaluation before the anaerobic digestion of excess sludge influencing on the amount of biogas generation and the energetic balance. The ratio of energy costs
during the sludge ultrasonication process to the amount
of biogas was evaluated. Excess sludge from the wastewater treatment plant of paper industry was the substrate
of investigations. For sewage sludge ultrasonication the
ultrasonic disintegrator VCX-1500 (Sonics Company)
of frequency 20 kHz was used. Ultrasonication of tested
sludge was made at four vibration amplitudes 16, 24, 32
and 39 m in the time of 300 seconds. The anaerobic digestion of preliminary modified sewage sludge with ultrasonic field was conducted in the digestion chamber at
temperature 37C during 25 days.
In the paper the effective preliminary method of sludge
pretreatment was presented, which improve degree of
sludge disintegration and biogas output. In the anaerobic digestion of tested excess sludge after this preliminary
treatment two time increase of biogas generation was
observed.
Keywords: anaerobic digestion, sewage sludge, ultrasonic field, biogas
Eurobiotech 2013
P2.5
P2.6
Zeolite nano-adsorbents for fuel ethanol

dehydratio
Production of butane-2,3-diol by Bacillus

licheniformis NCIMB 8059 in batch
and fed-batch cultures and its up-scaling
Tomasz Tokarz
Wydzia Inynierii I Technologii Chemicznej Politechniki
Krakowskiej, Instytut Chemii i Technologii Organicznej, Katedra
Technologii Organicznej i Procesw Rafineryjnych
Anhydrous ethanol from fermentation process of biomaterials is widely used in chemical, pharmaceutical and
cosmetic industries. This is also used as a fuel additive
in the internal combustion engines. Technologically ethanol production of a high concentration is a major problem, due to the presence in the system of ethanol/water
azeotropic point (p = 1,013 bar, T = 78,15C, 4,43% m/m
H2O). High energy consumption in the traditional azeotropic distillation is a main disadvantage of this process.
One of the most widely used alternative method is Pressure Swing Adsorption (PSA) in which zeolite nano-adsorbent is applied. The aim of this study is to find relations between surface area and pore structure for mass
production zeolite nano-adsorbents with their efficiency
in dehydration of raw ethanol.
Keywords: PSA, adsorption, separation, zeolite, ethanol
17
Joanna Krysiak1, Ewa Gromek1, Halina Kalinowska1,

Marianna Turkiewicz1, Aneta Biakowska1, Siegmund Lang2
Institute of Technical Biochemistry Lodz University
of Technology;2Institute for Biochemistry, Biotechnology and
Bioinformatics Braunschweig University of Technology
1
2,3-butanediol (2,3-BD) is a colorless and odorless polyhydroxyalcohol with a chemical formula C4H10O2. This
liquid is characterized by a high boiling temperature
and a low melting temperature. Because of attractive
physical properties and feasibility of diverse chemical
conversions this alcohol has found multiple applications
in cosmetic, pharmaceutical and fuel industries etc.
2,3-BD has been hitherto produced from crude oil by
chemical technologies, but due to the depletion of fossil
fuels and their growing prices its biosynthesis by microorganisms has recently become an interesting alternative. It is to note that the majority of microorganisms
synthesizing appreciable amounts of this compound are
pathogens, which cannot be used in industry. Therefore,
investigations on improvement of 2,3-BD biosynthesis
by non-pathogenic bacteria like Bacillus strains are so
important.
This study aimed at maximizing 2,3-BD biosynthesis by
a non-pathogenic Bacillus licheniformis strain through
fed-batch cultures and process up-scaling using bioreactors with volumes of 0.75 and 30 L.
Tested carbon sources used in 2,3-BD production
by this strain were by-products from food processing such as molasses and an enzymatic hydrolysate
of apple pomace. Fermentations conducted in 0.75 L
bioreactors enabled selection of the most appropriate
carbon source and optimum agitation intensity (tested rotation rates of a stirrer: 150, 250 or 450 rpm).
These results enabled efficient 2,3-BD production in
fed-batch cultures conducted in 30L bioreactors. The
most suitable carbon source both in batch and fedbatch cultures (irrespective of the scale) was found
to be molasses. Concentration of 2,3-BD synthesized
by the B. licheniformis strain in 30 L bioreactor (fedbatch cultures, fed 4-times with 50% glucose solution)
reached 80 g/L after 72 h. When carbon sources were
mixtures of the apple pomace hydrolysate and pure
glucose, the maximum 2,3-BD concentration was only
slightly lower (73 g/L) but it was achieved after 96 h of
fermentation.
These results are comparable to 2,3-BD yields presented
in literature for its most efficient pathogenic producer
Klebsiella pneumoniae strains. We found that among
tested by-products from food processing the most suitable for 2,3-BD production in industrial scale is molasses
from sugar beets. Advantages of this raw material are its
low price and good availability as well as, in contrast to
18
Eurobiotech 2013
the apple pomace, the lack of necessity of its earlier hydrolysis before fermentation.
Acknowledgements
Presented study was accomplished with the scope of the international
project Production and upgrading of 2,3-butanediol from biomass.
P2.7
Biogas from physical modified excess
sludge of pulp industry
Mariusz Baraski, Lidia Wolny, Iwona Zawieja
of Technology
Physical conditioning methods of sludge as for example

ultrasonic field operate mainly to the disintegration of
sludge, which can intensify the hydrolysis process as the
first stage of anaerobic digestion. Ultrasonication before
anaerobic digestion causes that hydrolytic phase is shorter as well as the degree of organic compounds decomposition is better in comparison to not conditioned sludge.
The aim of investigations was the evaluation of excess
sludge disintegration and biogas generation in the anaerobic stabilization after ultrasonic generator VCX-750
(Sonics Company) application. Tested substrate was
excess sludge from paper industry. Results obtained after anaerobic stabilization of ultrasonically conditioned
sludge were compared with the results of unconditioned
sludge.
The feed/inoculum ratio in the anerobic digestion was
9/1. Two series of investigations were conducted.: for
unconditioned excess sludge (mixture A) and for conditioned excess sludge (mixture B) at vibration amplitude
of ultrasonic field 35% and 60% (mixture C). In both cases the sonication time was 360 seconds. The most advantageous results of ultrasonic field action were observed
at amplitude of 60% and sonication time 360 seconds.
For this parameters the 70% degree of fermentation was
obtained and 50% increase of unitary biogas production
(3.54dm/g s.m.org.) in comparison to unconditioned
sludge.
Eurobiotech 2013
19
P2.8
P2.9
Polymer-enzyme catalyst systems

for the hydrolysis of cellulose
Effect of cellulase immobilization upon

digestion of cellulose
Joanna wider1, Katarzyna Gaweek1, Ewa Witek1,

Edyta Podstawka- Proniewicz1, Anna Konieczna-Molenda2
Katarzyna Gaweek1, Agnieszka Tta1, Ewa Witek1,

Edyta Podstawka-Proniewicz1, Anna Konieczna-Molenda2
Faculty of Chemistry, Jagiellonian University, Ingardena 3 St.,

30-060 Crakow, Poland; 2Department of Chemistry and Physics,
University of Agriculture, Balicka 122 St., 30-149 Crakow,
Poland
Immobilization of enzymes used in the food, pharmaceutical and biofuels synthesis on polymeric supports significantly reduces the cost and improves the hydrolysis of
polysaccharides [1, 2].
The study involved polymer carriers based on crosslinked
N-vinyl formamide (NVF) synthesized by the reaction
of free radical suspension polymerization of N,N-methylenebisacrylamide (NMBA), optionally with ethylene
glycol dimethacrylate (EGDMA) [3].
Developed optimum parameters for copolymerization
NVF/NMBA and NVF/EGDMA in inverse suspension
giving spherical polymer grains having a low coefficient
of swelling in water. It has been shown that the most effective medium in this process is a silicone oil.
Selected six most preferred polymeric carriers characterized by physicochemical parameters and used it
to immobilize the enzyme mixture from the group of
carbohydrases under the trade name Viskozyme L
(Sigma-Aldrich). Received systems used in the test reactions of hydrolysis microcrystalline cellulose and hydrolysis of wood pulp as application process. Immobilization
of enzyme was carried out by means of glutaraldehyde,
as well as without its participation. The morphology of
carriers determined based on microscopic images. Physico-chemical methods for the characterization of obtained
catalytic polymer-carrier systems included CHN analysis,
FTIR spectroscopy and Raman spectroscopy.
It has been shown that reactive formamide groups do
not provide a permanent binding medium glutaraldehyde-enzyme, hence the catalytic tests showed less than
the immobilized enzyme activity of native enzyme.
Cellulases play an important role in specialized commercial applications such as fabric modifications, paper
and pulp industry, food industry [1] etc. Enzymatic hydrolysis of cellulose has been extensively studied in the
past decades since the utilization of cellulosic biomass
as a renewable resource has great potential for reducing
emissions of carbon dioxide and thereby prevents global
warming [2]. Cellulase can be used as the biocatalyst for
cellulose hydrolysis, but its activity is rather lower than
that of other hydrolases.
Cellulase was immobilized on N-vinylformamidecrosslinked with divinylbenzene as a result of copolymerization in reverse suspension [3]. The morphology of carriers determined based on microscopic images. Physico-chemical methods for the characterization of obtained
catalytic polymer-carrier systems included CHN analysis,
FTIR spectroscopy and Raman spectroscopy.
The enzyme Novozym 476 (Novozym) was covalently
bound directly to the supports or using glutaraldehyde
as a spacer [4]. The immobilization procedure was optimized involving such factors as temperature, pH, time,
sequence of reactions, and kind of carrier employed [5].
Received systems used in the test reactions of hydrolysis
microcrystalline cellulose and hydrolysis of paper as application process. The results of the immobilization were
evaluated in base on analyses of the enzyme activity and
stability prior and after immobilization, as well as on the
immobilization yield and stability.
Highly active biocomposites were designed that provided
their multiple application for cellulose hydrolysis.
Acknowledgements
The authors wish to thank the Ministry of Science and Higher Education
for the funding of the research. The grant N N204 354840.
References
[1] Konieczna-Molenda A., Kochanowski A., Walaszek A., Bortel E.,
Tomasik P. (2009) Chemical Engineering Journal, 146: 515519
[2] Farrell A.E., Plevin R.J., Turner B.T., Jones A.D., OHare M., Kammen
D.M. (2006) Science, 311 (5760), 506508
[3] Witek E. (2008) Polimery, 53: 477480.
Faculty of Chemistry, Jagiellonian University, Ingardena 3 St.,

30-060 Crakow, Poland; 2Department of Chemistry and Physics,
University of Agriculture, Balicka 122 St., 30-149 Crakow,
Poland
Acknowledgements
The authors wish to thank the Ministry of Science and Higher Education
for the funding of the research. The grant N N204 354840.
References
[1] Li C., Yoshimoto M., Fukunaga K., Nakao K. (2007) Bioresearch
Technology, 98: 13661372.
[2] Li C., Yoshimoto M., Fukunaga K., Nakao K. (2004) J. Chem. Eng.
Jpn., 37: 680684.
[3] Witek E. (2008) Polimery, 53: 477480.
[4] Bryjak J., Bachmann K., Paww B., Maliszewska I., Trochimczuk A.,
Kolarz B.N. (1997) Chem. Eng. J.,65: 249.
[5] Konieczna-Molenda A., Kochanowski A., Grabiec, A., Bortel E.,
Tomasik P. (2008) PTTZ-Malopolska Branch, Krakow, Ch. 8: 103120.
20
Eurobiotech 2013
P2.10
P2.11
Application of wastes from alcohol

distillation in two-step process
of biohydrogen production
Electrochemical studies of chemically

modified and native Sinorhizobium meliloti
laccase
Roman Zagrodnik
Anna Pawlik, Grzegorz Janusz, Jerzy Rogalski
Faculty of Chemistry, Adam Mickiewicz University,

Umultowska 89b, 61-614 Poznan, Poland
Maria Curie-Skodowska University, Faculty of Biology

and Biotechnology, Department of Biochemistry
Recent years showed that the cost of obtaining energy

from traditional sources is increasing in a manner difficult to predict. The solution to these problems is utilization of renewable energy sources such as biomass. The
necessity of recycling and use of organic compounds
contained in waste materials and wastewater is inevitable.
However, regardless of the source, the energy has to reach
the user through an efficient carrier, that can be used in
a wide range of applications. Hydrogen is considered to
be one of the most promising fuels of the future, and is
widely recognized as a potential substitute for fossil fuels.
Biological processes of hydrogen production are found
to be the most environmentally friendly processes of hydrogen production, and dark and photo fermentations of
organic wastes are considered as the most promising processes for biological hydrogen production. These processes are operated at ambient temperature and atmospheric
pressures, thus are less energy intensive and more environmentally friendly as compared to thermochemical
and electrochemical processes. Moreover, the biological
hydrogen generation can be carried out as a continuous
process, which is appealing to large scale systems.
In this work the hybrid system applying both dark fermentation and photofermentation was tested. At first, the fermentation of wheat distillation effluent containing certain
amounts of simple sugars was conducted in bioreactor.
Active sludge from municipal wastes was a source of microorganisms in dark fermentation. Here, starch and simple
sugars were transformed into hydrogen and liquid metabolites (lactic acid, acetic acid, simple alcohols effective
substrates for photo fermentative bacteria). This step was
followed by photofermentation process with Rhodobacter
sphaeroides bacteria. Here, flat plate photobioreactor of our
own construction, working under continuous conditions
was applied. Continuous condition were provided by two
peristaltic pumps. The COD of waste water from wheat distillation was 45.7 g O2/l and after dark fermentation 27.8 g
O2/l. The amount of the lactic and acetic acid during photofermentation process was reduced by 98 and 88%, respectively. Average hydrogen production rate in the case of photofermentation was 10,8 ml H2/l medium/h, and was slightly lower than in the case of dark fermentation 97,2 ml H2/l
medium/h. Experiments with effluent from dark fermentation as the source of organic carbon proved that wastes
originating from food industry can be efficiently applied for
hydrogen production in photofermentation process.
Laccases (EC 1.10.3.2) are propably the most numerous

members of the multicopper protein family, being distributed in all domains of life. Because of their broad substrate specifity and interesting chemical properties (e.g.:
high thermostability, alkaline pH-stable activity), laccases harbor great potential to biotechnological processes.
A significant number of reports have been published in
the past decade that have focused on the biochemical
properties of these proteins and/or on their applications
in the paper and pulp, food, cosmetic, textile and petrochemical industry, nanobiotechnology, medical diagnostics and for bioremediation and clinical purposes. The use
of laccase in electrochemical biosensors and biofuel cells
has been the subject of basic as well as applied research
[1]. Wild laccases are not always suitable for industrial
purposes. For example, they suffer from several limits: in
some cases, their activity needs the presence of a redox
mediator, their pH activity is acidic and does not correspond to environmental conditions. Chemical modification may provide recombinant enzymes with wider substrate specificity, higher oxidation potential and neutral
pH of activity.
The present work mainly focused on electrochemical
measurements of chemically modified and native bacterial laccases.
The crude enzyme extract of Sinorhiozbium meliloti laccase was purified by means of chromatographic techniques. Laccase modification was performed as described
elsewhere [2]. AUTOLAB instrument and GPES software was used for electrochemical measurements and
analysis of modified and native enzymes.
The use of modified enzyme allowed to determine the
effect of chemical modifications on laccase activity and
stability. The basic bioelectrochemical studies of modified
and native S. meliloti proteins enable further implementation research and application of laccases in bioenergetics.
Acknowledgements
The project was financed by the National Science Centre granted on the
basis of decision DEC-2012/05/N/NZ9/01577.
Acknowledgements
The research was supported by the the Polish Scientific Project
BS/ZBioch/Maria Curie-Skodowska University.
References
[1] Kunamneni A., Plou F.J., Ballesteros A., Alcalde M. (2008) Laccases:
a patent review. Recent Pat Biotechnol. 2: 1024.
[2] Kucharzyk K.H., Janusz G., Karczmarczyk I., Rogalski J. (2012)
Chemical modifications of laccase from white-rot basidiomycete
Cerrena unicolor. Appl Biochem Biotechnol. 168: 19892003.
Eurobiotech 2013
21
P2.12
P2.13
Micelle-mediated extraction of elderberry

blossom by whey protein and
naturally-derived surfactants
Biosynthesis of 2,3-butanediol from waste

biomass by RaoultellaPlanticola CECT 843
Karolina liwa, Anna Tomaszkiewicz-Potpa, Elbieta Sikora,

Jan Ogonowski
Department of Biotechnology and Food Microbiology,

Poznan University of Life Sciences, Poland
Faculty of Chemical Engineering and Technology,

Classical methods for the extraction of active ingredients from the plant material are expensive, complicated
and often environmentally unfriendly. Popular methods
of extraction require the use of an expensive equipment,
a large quantity of electricity, heat, and large volumes of
flammable and toxic organic solvents such as methanol,
ethyl acetate or n-propanol [1, 2].
The micelle-mediated extraction method (MME) seems
to be a good alternative. The extraction temperatures are
relatively low and there is no need to apply organic solvents. In addition, this method enables to receive a higher
yield of polyphenols than the standard methods. The use
of surfactants that are suitable for human consumption
could also reduce costs of the process. The additional advantages of micelle-mediated extraction are no need for
separation of used surfactants from the polyphenols containing extract and protection of the polyphenols against
oxidation [2].
The aim of this work was the study of the possibility of
whey protein application as extraction agent. In this work,
extractions of elderberry blossoms (Flos Sambuci) were
performed. The micelle-mediated method using several
popular surfactants and whey protein was applied. The
results were compared with those obtained for extraction
with methanol. Antioxidative properties of the extract
and collected, after separation on the chromatographic
column, fractions were analyzed. Two different methods
were applied: reaction with DPPH reagent, Follins method. Futhermore, the flavonoid content was determined.
The results confirmed that the MME method with using
whey protein might be an alternative and a convenient
method for the preparation of rich in natural antioxidants
plant extracts.
Reference
[1] Katsoyannos E., Chatzilazarou A. (2006) Fresenius Environmental
Bulletin, 15: 1122.
[2] Chatzilazarou A., Katsoyannos E. (2010) Journal of The Air & Waste
Management Association, 60: 454.
Mariusz Lesiecki, Adrian Czerniak
Introduction. 2,3-Butanediol (2,3-BD) is a chiral bivalent alcohol which exhibits a wide range of potential utilizations as a solvent, liquid fuel or precursor for 1,3-butadiene an intermediate for synthetic rubber production.
Several microorganisms are able to synthesis 2,3-BD.
So far the highest yields were reached using risk class 2
strains e.g. Klebsiella species, however an industrial process with risk class 1 microorganisms are cheaper and less
recalcitrant. Based on this fact, the present studies deal
with the production of 2,3-BD from sugar beet pulp hydrolysates by risk class 1 bacteria, namely Raoultella planticola CECT 843 strain.
AIM. The main study objective was to statistically optimise the biosynthesis process of 2,3-BD from sugar beet
pulp hydrolysates by Raoultella planticola CECT 843
strain.
Materials and methods. Response surface methodology
(RSM) was used in this work to optimise the biosynthesis of 2,3-Butanediol by Raoultella planticola CECT 843
strain. Sugar beet pulp hydrolysates were used as a medium culture. The optimisation experiment was planned
according to D-optimal design and consisted of 25 runs.
The effect of yeast extract (YE) and concentration of
CH3COO, Fe2+ and Mg2+ ions was evaluated on the final level of 2,3-butanodiol. Sugars and other carbon compounds
concentration were determined preferably by HPLC
or GC.
Results. Obtained results showed statistically significant
influence (p < 0,05) of the yeast extract, CH3COO and
Mg2+ ions concentration on the studied process. On the
basis of statistic analysis, optimal values of analysed variables were determined as follows (gdm3): yeast extract
4; CH3COONH4 4; FeSO47H2O 0,1; MgSO4H2O
0,3; that corresponded to 19,7 gdm3 final 2,3-Butanediol
concentration.
Conclusions. Under optimised conditions Raoultella
planticola CECT 843 can effectively convert fermentable
sugars from sugar beet pulp hydrolysates to 2,3-BD. Production of diol achieved nearly 20 gdm3 and efficiency
of production reached 0,42 gg1glucose in 24h of cultivation.
It shows that risk class 1 bacteria strains are promising
direction in research of microbial production of 2,3-Butanediol.
Acknowledgements
This work was supported by ERA-IB EU-project called Production and
Upgrading of 2,3-Butanediol from Biomass (ERA-NET-IB/03/2009).
Bioeconomy
Lectures
L3.2
L3.1
Biomass as a source of raw materials for

the preparation of environmentally friendly
polymer materials
Agri-food industry a key element

of Polish bioeconomy
Lech Michalczuk
Research Institute of Horticulture, Skierniewice, Poland
Agri-food industry is one of the most important sectors of

Polish economy. In 2011 total value of its production sold
exceeded 37 bln (app. 22% of total industrial production
sold) and gross value added (GVA) amounted to 10 bln ,
which accounted for 12,7% of total industrial gross value
added and was 1.2 times higher than GVA in mining industry, 2.2 times higher than GVA in car manufacturing,
4.1 times higher than GVA in fuel and petrochemical industry and 6.5 times higher than GVA in electronics and
computer manufacturing. Polish agri-food industry has
also stronger position in the European Union than the
whole Polish economy; its GVA accounts for 6.5% of total
industrial gross value in EU, compared with 4.1% share of
Polish GDP in Gross European Product.
The main challenges facing Polish agri-food industry are
low labour productivity (83% of mean labour productivity in Polish economy and 50% of mean labour productivity in European Union) and relatively low innovativeness. Serious problems are also with soil degradation and
shrinking water resources for irrigation, which are affecting yield and crop quality in primary agricultural production. Results of foresight project Food and nutrition in 21
century, completed during 20092011 show that the best
strategy for further development of Polish agri-food industry is investment in innovative technologies increasing
food quality and safety, especially those based on biotechnology, nanotechnology, nutigenomics and nutrigenetics.
Much efforts shall be also done to ameliorate prognosed
effects of global climate changes affecting agricultural
production. Especially important is prevention of further
soil degradation, which shall involve both basic research
on soil biology and developing technologies aimed at increasing abundance and diversity of soil microflora and
organic matter content.
Andrzej Okruszek
Lodz University of Technology, Institute of Technical
Biochemistry
The aim of the project (acronymBIOMASA) is utilization of various kinds of plant biomass and textile waste
materials by their transformation with biotechnological
methods, involving either enzymatic or microbial processes, into fibrous polymer materials. The intermediate
products in those transformations are: cellulose nanofibres, tactic polylactide and aliphatic-aromatic co-polyesters, which all are known to be important raw-materials for the production of biodegradable fibrous materials
as well as other kinds of biodegradable polymer composites.
For the preparation of cellulose nanofibres, a cellulose-rich plant biomass is being utilized, including grass
and straw of various cereals as well as waste fibres from
textile industry (cotton, linen). The biomass is first pretreated with physical and/or chemical methods including
boiling, steam-explosion or treatment with certain chemicals. Multienzyme complex obtained from Aspergillus
niger mould is utilized as the main enzymatic tool. The
fibrous materials and composites prepared within this
project on the basis of abovementioned intermediates will
be further utilized for obtaining new functional textiles
and nonwovens with potential sanitary or technical applications, such as sweat-absorbing textile inserts, sanitary
textiles, filtration materials, geotextiles and agrotextiles.
Within this project, the processes of ageing and controlled
biodegradation of prepared materials will be studied, as
well as the conditions of their recycling and possible use
of degradation products in agriculture.
The synthesis of tactic polylactide is being performed by
chemical polymerization of L,L-lactide, prepared from
L-lactic acid. The latter is obtained by stereoselective fermentation of plant biomass, after its saccharization by
ppropriate enzymes (Aspergillus niger preparations). The
microorganisms (bacteria), used for the fermentation,
were selected by classical microbiology methods from the
environment. In this case patatoes, cereal grains or beet
pulp are employed as starting biomass. The tactic polylactide will be further utilized for fiber formation and hermoforming.
The third path involves utilization of various oil-plant biomass, which on sequential treatment with lipase prepara-
Eurobiotech 2013
tions obtained from Mucor circinelloides and Mucor racemosus moulds (transesterification with 2-methylbutanol)
and dimerization of obtained esters (cycloaddition) are
transformed into dimeric esters containing fatty acid
residues. These will be co-polymerized with appropriate
reagents in order to produce new biodegradable aliphatic-aromatic co-polyesters. The polyesters will be utilized
as components in the preparation of various fibrous polymers and composites.
The project is being realized by nine research groups
from Poland belonging to four different research establishments, with the Lodz University of Technology being the leader. The elaboration within this project new
methods of preparation of polymer fibrous materials and
composites will positively influence development of science-based economy and will increase the innovativeness
of connected areas of research and production. The main
recipients of elaborated methods will be producers of fibers and nonwovens from thermoplastic materials, sanitary textiles, filtration materials, geotextiles, agrotextiles
and packing materials.
Acknowledgements
This research is realized within the project BIOMASA (POIG 01.01.0210-123/09) and partially financed by the European Union from the
European Regional Development.
23
L3.3
Biotransfromations as a tool
for Biorafineries
Pawel Kafarski
Department of Bioorganic Chemistry, Faculty of Chemistry,
Wroclaw University of Technology, Wybrzeze Wyspianskiego 27,
50-370 Wroclaw, Poland
The biorefinery concept is analogous to petrolium refinery, which produce multiple fuels and products from petroleum. At the beginning this term was used to describe
a facility that integrates biomass conversion processes and
equipment to produce fuels, power, heat, and value-added chemicals. Athough still being related to biofuel production in a broader sense biorefining is the sustainable
processing of biomass into a spectrum of marketable
products, especially high-value fine chemicals. Ideally it
is focused on integrated production for food, feed and
non-food applications, while aiming for zero waste and
getting maximum added value from co-products. It is believed that in 2030 many key chemicals using biological
and/or chemical processes.
Biotransformations are chemical reactions that are catalyzed by microorganisms in terms of growing or resting
cells or that are catalyzed by isolated enzymes. Because
of the high stereo- or regioselectivity combined with
high product purity and high enantiomeric excesses, biotransformations are considered as technically superior to
traditional chemical synthesis. They appeare to be a superiour tool in production of high-value chemicals upon
biorefining processes. For example, the Department of
Energy, USA has identified 30 platform chemicals composed of one to six carbon atoms as potential candidates
for biobased production with biotransformations being
leading processes in this respect.
Acknowledgements
This work was supportedby the project Biotransformations for
pharmaceutical and cosmetics industry No. POIG.01.03.01-00-158/09,
which is partly-financed by the European Union within the European
Regional Development Fund for the Innovative Economy.
24
Eurobiotech 2013
L3.4
L3.5
Bionidustry in Poland current status,

major obstacles, driving force and open
opportunities
Biobased production an answer

to societal challenges? The importance
of sustainability and communication
Tomasz Kapela
Patricia Osseweijer
Biotechnika
Delft University of Technology, Department of Biotechnology
The main goal of the presentation is to outline in general the current shape of bioindustries in Poland, briefly define what are the foundations of this new industrial branch and try to outline what pushes it forward and
what holds it back. An author will try, basing on his own
experience, to draw a picture of current biobusinesses in
Poland and characterise major fields of activities. On one
hand Poland is a country of great opportunities and expectations in terms of its bioresources. But on the other
it is still struggling numerous problems of various nature
(legal, financial, public awareness related, etc.). Hopefully the presentation will flash some light on positives as
well as those issues that still need to be tackled in order to
ensure, that Poland takes its chance to built strong, biobased economy. Several case studies will help the guests
to have a complete picture of discussed ideas
There are many reasons why we should replace our present fossil-based products such as plastics, chemicals and
carpets to products made from biorenewable sources.
One of them is that we have to be much more sustainable
if we wish to mitigate climate change and feed a growing world population. In that sense biobased production
does contribute to societal challenges. However, the way
we do this will matter, so far integrated approaches for
production of materials, chemicals and energy do seem
to provide the best economical, environmentally sustainable and social benefits.
However to introduce such practices successfully, we
not only require innovation in industrial biotechnology
to deliver the best routes to develop new materials from
waste products but also a better management of our agricultural production and novel collaboration between
agricultural, chemical and energy sectors.
In order to achieve this and the adequate policy measures
to support this we need to have the support of citizens
and consumers as well. That is not straightforward as for
example the debate on food versus (bio)fuel shows. The
presentation will address the different views on sustainability and the importance of (scientists to be involved
in) communication to achieve a supported and well governed transition to a biobased society.
Eurobiotech 2013
Posters
P3.1
Isolation of bacterial strains capable
of converting glycerol into succinic acid
Marcin Podleny, Piotr Jarocki, Jakub Wyrostek,
Tomasz Czernecki, Zdzisaw Targoski
Department of Biotechnology, Human Nutrition and Food
Commodities, University of Life Sciences in Lublin
Succinic acid is currently used mainly in industries producing food, pharmaceutical products, detergents, surfactants and ingredients stimulating plant and animal
growth [1]. Enormous versatility of succinic acid and
its derivatives applications leads to treatment of this dicarboxylic acid as one of the top twelve building block
chemicals of great interest according to the United States
Department of Energy [2]. Development of economically efficient biotechnology process for succinic acid
production could extend application market for this
chemical to biodegradable plastics and green solvents.
Present production scheme of succinic acid is based on
petroleum as the starting source. Attempts for petroleum substitution by renewable low-cost raw material
are intensively made [3, 4]. These promising efforts are
inseparable connected with the usage of high-yield producing microorganisms. Therefore searching for microorganisms capable of efficient utilization of renewable
low-cost substrates during succinic acid production is
of the great interest [5]. Among considered inexpensive
compounds for succinic acid production glycerol gained
significant attention. This byproduct generated during
biodiesel production process is desirable substrate because having the same level of reduction as succinic
acid. Bioconversion of glycerol to succinic acid seems
therefore a redox balanced pathway [6].
Present study describes isolation and screening of microorganisms for succinic acid production on glycerol. Isolation of bacteria from different eight eco-niches
was carried out. A total number of 187 bacterial isolates
were obtained using microbiological techniques. After
96 hours of anaerobic cultivation on glycerol containing
medium isolates were screened for succinic acid production by HPLC. Succinic acid was not detected in nearly
half of the examined bacterial strains. About eight percent of bacterial isolates screened showed succinic acid
production rates exceeding 2 g/l. Succinic acid was not
the only product of glycerol utilization in anaerobic condition. Ethanol and acetic acid were the most common
byproducts followed by formic acid. Isolate KRBGAL,
afacultative anaerobe isolated from Holstein Freisian cow
rumen fluid, showed maximum yield of 4,6 g/l of succinic
acid from 20 g/l of glycerol. The glycerol utilization rate
during growth of KRBGAL isolate was nearly 68%. This
isolate was identified as Escherichia coli by 16S rDNA sequence analysis followed by biochemical identification
using API 20E. This strain may become a candidate for
25
further genetic improvement of succinic acid production

because of relatively high succinic acid yields and glycerol
utilization rate.
Acknowledgements
This study was co-funded by The European Union project No. PO
IG 01.01.02-00-074/09 from The European Regional Development
Fund within the framework of the Innovative Economy Operational
Programme 20072013.
References
[1] McKinlay J.B., Vieille C., Zeikus J.G. (2007) Prospects for a biobased succinate industry. Appl Microbiol Biotechnol 76: 72740.
[2] Werpy T., Petersen G. Eds. (2004) Top Value Added Chemicals from
Biomass. US DoE.
[3] Du C., Lin S.K.C., Koutinas A., Wang R., Webb C. (2007) Succinic
acid production from wheat using biorefining strategy. Appl Microbiol
Biotechnol 76: 126370.
[4] Liu Y.P., Zheng P., Sun Z.H., Ni Y., Dong J.J., Zhu L.L. (2008)
Economical succinic acid production from cane molasses by
Actinobacillus succinogenes. Biores Technol 99: 173642.
[5] Scholten D., Daegele D. (2008) Succinic acid production by a newly
isolated bacterium. Biotechnol Lett 30: 214346.
[6] Yazdani S.S., Gonzalez R. (2007) Anaerobic fermentation of glicerol:
a path to economic viability for the biofuels industry. Current Opinion
in Biotechnology 18: 213-19.
Bioplastics and biobased polymers
Lectures
L4.1
Novel bioplastic fibres for tissue
engineering and biodegradable packaging
materials
Magdalena Wrbel-Kwiatkowska1, Jan Szopa1, 2, 3
Linum Foundation, Wroclaw, Poland; 2Faculty of Biotechnology,
University of Wroclaw, Wroclaw, Poland; 3WCB EIT+, Wroclaw,
Poland
1
Flax (Linum usitatissimum L.) is best known as a plant cultivated for industrial purposes. Flax seeds are the source
of oil used as the basal component or an additive for various paints or polymers, flax stems are the source of bres
that can be used in the textile and paper industries. A new
generation of novel bioplastic bres contain polyhydroxybutyrate (PHB) were obtained from transgenic ax plants.
Transgenic plants were generated by introduction of three
bacterial genes necessary for PHB synthesis [1]. Biochemical analysis of bioplastic bres revealed presence of several antioxidative compounds of hydrophilic (phenolics)
and hydrophobic (cannabidiol CBD, lutein) nature, indicating their high antioxidant potential.
IR spectra of transgenic ax bres revealed structural
changes in comparison to unmodified fibres, i.e. observed
higher ordering of cellulose in transgenic bres resulted
from the intra- and/or intermolecular hydrogen bonds of
glucopyranosyl residues and from the formation of the
PHB-cellulose composite [2]. The presence of PHB naturally incorporated into the bres increases their compatibility with other polymers of biodegradable (polyhydroxyalconate) and non-biodegradable (polypropylene,
polystyrene) nature.
Natural bioplastic ax fibres with stronger physical and
higher bioactive properties may serve as the source of
material for industry (automotive industry, packaging
materials) or for biomedical application (tissues scaffold,
implant).
Thus biodegradable and bioactive composites with polylactic acid (PLA) or poly-e-caprolactone (PCL) as the
matrix and bioplastic ax bres as reinforcement were
prepared and analyzed [3]. FTIR analysis of composites
showed intermolecular hydrogen bonds between the
constituents in composite PLA-ax bers which were
not detected in PCL-based composite. Mechanical analysis of prepared composites revealed improved stiffness
and a decrease in tensile strength. The viability of human
dermal broblasts on the surface of composites made of
PLA and transgenic ax bres was the same as for cells
cultured without composites and only slightly lower (up

to 9%) for PCL-based composites. The amount of platelets and Escherichia coli cells aggregated on the surface of
the PLA based composites was signicantly lower than for
pure polymer. Thus, composites made of PLA and transgenic ax bres seemed to have bacteriostatic, platelet anti-aggregated, and non-cytotoxic effect.
The composites made of PLA and transgenic bioplastic
flax fibres implanted into rat musculus latissimus dorsi [4]
did not have any negative effect on muscle function and
genes expression (i.e. vascular endothelial growth factor,
insulin like growth factor 1, insulin like growth factor 2,
collagen-1, collagen-2 and myostatin). The composites
also did not cause any inflammation response after insertion to the muscles and exhibited good biocompatibility
to the muscle.
The promising effect of bioplastic fibres which enhanced
polypropylene resin was also detected. The preliminary
data showed that composite of polypropylene and bioplastic fibres may serve as novel packaging material with
increased rate of degradation in soil.
References
[1] Wrbel M., ebrowski J., Szopa J. (2004) Polyhydroxybutyrate
synthesis in transgenic ax. J. Biotech. 107: 4154.
[2] Wrbel-Kwiatkowska M., Zuk M., Szopa J. et al. (2009) Poly3-hydroxy butyric acid interaction with the transgenic flax fibers:
FT-IR and Raman spectra of the composite extracted from a GM flax.
Spectrochim Acta A Mol Biomol Spectrosc 73: 286294.
[3] Wrbel-Kwiatkowska M., Czemplik M., Kulma A., uk M., Kaczmar
J., Dymiska L., Hanuza J., Ptak M., Szopa J. (2012) New biocomposites
based on bioplastic flax fibres and biodegradable polymers. Biotech.
Prog. 13361316.
[4] Gredes T., Kunert-Keil C., Dominiak M., Gedrange T., Wrbel-Kwiatkowska M., Szopa J. (2010) The influence of biocomposites
containing genetically modified flax fibers on gene expression in rat
skeletal muscle. Biomedizinische Technik. Biomedical engineering
55(6): 3239.
Eurobiotech 2013
27
L4.2
L4.3
Bacterial nanocellulose scaffolds

for regenerative medicine
Biomedical applications of dendrimers
Karolina Ludwicka, Marzena Jedrzejczak-Krzepkowska,

Katarzyna Kubiak, Marek Kolodziejczyk, Stanislaw Bielecki
University of Lodz, Department of General Biophysics
Institute of Technical Biochemistry, Lodz University

of Technology, Poland
Dendrimers are a family of synthetic macromolecules

of considerable interest as a platform for biomedical research because they can be designed and synthesized having specific desirable properties. Dendrimers are characterized by a globular shape which is a result of their internal structure. All bonds in dendrimers emerge radially
from a central core to which monomers are attached. This
unique molecular architecture means that dendrimers
have a number of distinctive properties that distinguish
them from other polymers.
Dendrimers are monodisperse macromolecules with
plenty of surface groups and internal cavities. The specific
structure makes dendrimers suitable for many biomedical applications.
Due to a large number of terminal groups to which drug
molecules can be conjugated, one molecule of dendrimer
is capable of carrying drugs at a high density. Encapsulating drugs inside dendrimers offers potential benefits such
as prolonging the circulation time in the blood, protecting unstable drugs from the environment, enhancing solubility of poorly soluble drugs, and achieving controlled
release.
Dendrimers, in addition to being excellent carriers for
active compounds, have biological activity in their own
right. They can be therapeutic agents in the prevention
of neurodegenerative disorders and viral or bacterial infections.
However, there is still a substantial gap of knowledge in
understanding how dendrimers interact with biological
structures, which has considerable consequences for the
translation of research from bench to bedside.
Bacterial nanocellulose (BNC), synthesized by Gluconacetobacter strain, is the natural biopolymer of exceptional
purity and outstanding biocompatibility. Bionanocellulose is the material of known properties being already investigated in a wide range of industrial and medical applications, including clinical trials for wet wound dressing
and as a potential implant e.g. in vascular surgery (small
vascular grafts), bone, dental fillings, peripheral nerves
regeneration and many others. Many publications confirm that as a nondegradable biomaterial BNC implants
fulfil their function by appropriate high hydrophilicity,
inertness, lack of cytotoxicity, outstanding biocompatibility and stability in a wide range of temperatures and
pH. It was recently investigated that the neurotubes made
of BNC are of very good biocompatibility and allow the
accumulation of neurotrophic factors inside, thus, facilitating the process of peripheral nerves regeneration. The
successful application of BNC fragments in trachea reconstruction was also described. Following the obtained
results the direction of the current research is set to the
improvement of BNC scaffolds in vivo functionality.
A new method of production of novel composites made
of BNC and polymeric or titanium meshes has been developed and investigated. The potential application of
such constructs include cranioplasty and herniorraphy.
Also a cartilage-like BNC scaffolds mechanical properties
were evaluated together with in vitro biocompatibility
confirmation. The results suggest the possible application
of such a material in cartilage reconstruction. Although
still, the stable connection with the growing cells is crucial for the effective tissue regeneration. Since the properly adjusted structure of BNC membrane determines the
success of such constructs adaptation, it is necessary to
improve the manufacturing process to produce a number
of both suitably dense and porous BNC scaffolds, in order
to create the efficient drug reservoir system, as well as the
proper conditions for the proliferation of host and in vitro
introduced tissue-engineered cells.
References
Kowalska-Ludwicka K. et al. (2013) Arch Med Sci 9(3), 527534.
Bielecki S. et al. (2012) In Bacterial Nanocellulose: A Sophisticated
Multifunctional Material; Eds. Boca Raton, Florida: CRC Press,
157174.
Kolodziejczyk M. et al. (2010) Military Pharmacy and Medicine 3(1),
6265.
Barbara Klajnert-Maculewicz
28
Eurobiotech 2013
L4.4
L4.5
Bionanocellulose production control from

molecular point of view
Functionalized microfibrillar cellulose

for bio-based polyamide composites
Katarzyna Kubiak, Marzena Jdrzejczak-Krzepkowska,

Marta Kurzawa, Karolina Ludwicka, Marek Koodziejczyk,
Stanisaw Bielecki
Krzysztof Pielichowski, Agnieszka Leszczyska

of Technology, Stefanowskiego 4/10 St., 90-924 Lodz, Poland
Bionanocellulose (BNC) is an innovative bio-material

tested in numerous applications, among which medical
ones are on main focus of research conducted at IBT
TUL. Development of new products is limited by diversity in Ga. xylinus strains productivity and time needed
for whole process. Most challenging aspect of BNC production is planning the structural composition of fibrils
building up the BNC membrane. The product parameters
may be modulated by technological modifications done
during or after bacteria culture only to some extent. The
mentioned above aspects limiting technology development (productivity of a strain, time needed for biosynthesis and BNC structure control) are goals which may
be reached after understanding of molecular processes
responsible for control over polysaccharides synthesis
and secretion, cell motility and rate of bacteria growth.
In order to find molecular targets for productive strain
modifications we performed genome sequencing and
comparative transcriptomic analysis. Conclusions driven
from results of both projects have opened a number of
promising lines of molecular research. At the moment we
are investigating the most intensively one of them, namely cellular stress response mechanisms. Initial results of
BNC production with genetically engineered strains evidence that molecular control over production of BNC is
realistic in a near future.
Department of Chemistry and Technology of Polymers,

Crystalline cellulose nanoparticles, such as microfibrillated cellulose (MFC), have been recently considered for
manufacturing of short fiber reinforced polymeric composite materials as an alternative for artificial fibers, especially glass fibers. Due to submicronic dimensions and
crystalline structure cellulose nanofibers present higher
mechanical strength and better thermal stability as compared to natural fibers. However, initial degradation temperature of MFC is still lower than processing temperatures of numerous engineering polymers and restrain
wide implementation of green composites in structural
applications. With respect to that problem a heterogeneous surface modification of never dried MFC was
carried out by acetylation with acetic acid as well as esterification with hexanoyl and decanoyl chlorides. The
effect of molar ratio of total cellulose hydroxyl groups
to modifying agent (OH:MA) and time of reaction on
the thermal stability, structure, morphology and surface
properties of cellulose fibrils were discussed. As a result of
modification an increase in thermal stability of MFC was
achieved while crystalline structure and fibrous morphology of the cellulose filler was preserved. Moreover, modified MFC displayed significantly improved compatibility
with a new bio-based engineering polymer polyamide
4,10. Thus blending of modified MFC with polymer melt
was possible at temperatures as high as 285C. PA 4,10/
acetylated MFC composites showed significantly better
structural and morphological properties than polyamide
4,10/neat MFC system. The composite structure clearly
depended on the processing conditions.
Acknowledgements
Authors are grateful to the PolishNational Science Center for financial
support under contract No. DEC-2011/01/M/ST8/06834. J. Rettenmeier
& Shne GmbH +CO KG is kindly acknowledged for sending free
samples of microfibrillated cellulose.
Eurobiotech 2013
Posters
P4.2
P4.1
Biocatalytic synthesis of gluconolactone

and -caprolactone copolymers
Oxidative polymerization of lignins by

laccase in water-organic solvent system
Ionita Firuta Fitigau1, 4, Francisc Peter1,
Carmen Gabriela Boeriu2, 3
University Politehnica of Timioara, Faculty of Industrial
Chemistry and Environmental Engineering, C. Telbisz 6,
300001 Timioara, Romania; 2Wageningen UR Food
& Biobased Research, Bornse Weilanden 9, 6708 WG
Wageningen, Netherlands; 3University Aurel Vlaicu
of Arad, Department of Life Sciences, str. E. Dragoi nr 2, Arad,
Romania; 4National Institute of Research-Development for
Electrochemistry and Condensed Matter, A. P. Podeanu 144,
300569 Timioara, Romania
1
The enzymatic oxidative polymerization of five technical lignins with different molecular properties, i.e. Soda
Grass/Wheat straw Lignin, Organosolv Hardwood Lignin, Soda Wheat straw Lignin, Alkali pretreated Wheat
straw Lignin, and Kraft Softwood was studied. All lignins were previously fractionated by acetone/water 50:50
(v/v) and the polymerisation of the low molecular weight
fractions (Mw < 4000 g/mol) was carried out in the same
solvent system. Reactivity of lignin substrates in laccase-catalysed reactions was determined by monitoring
the oxygen consumption. The oxidation reactions in 50%
acetone in water mixture proceed with high rate for all
tested lignins but the enzyme lost its catalytic activity at
acetone concentration above 55%. The syringyl type Organosolv Hardwood Lignin had the highest reactivity and
the reaction proceeded with 19,66 (M/min) initial rate.
Polymerisation products were analysed by Size Exclusion
Chromatography, Fourier Transform Infrared Spectroscopy, 31P-NMR. The results provide evidence of important
lignin modifications after incubation with laccase. Structural oxidation and a notably molecular weight increase
(Mw up to 17500 g/mol) were attained. 31P-NMR spectral
analysis revealed a decrease of the total phenolic hydroxyl
groups and a substantial increase of the condensed phenolic OH content for all the polymerisation products. The
obtained polymers have potential for applications in bioplastics, adhesives and polymeric dispersants production.
Acknowledgements
The work of I.F. Fiigu was partially supported by the strategic grant
POSDRU 107/1.5/S/77265, inside POSDRU Romania 20072013
co-financed by the European Social Fund Investing in People.
The work at WUR FBR was carried out in the frame of the ERA-IB
project Products from Lignocellulose (EIB 10.013).
29
Anamaria Todea1, Lajos Nagy2, Valentin Badea1, Sandor Keki2,

Francisc Peter1
University Politehnica of Timioara, Faculty of Industrial
Chemistry and Environmental Engineering, C. Telbisz 6,
300001 Timisoara, Romania; 2Department of Applied Chemistry,
University of Debrecen, H-4010 Debrecen, Hungary
1
Synthesis of copolymers based on bio-derived monomers

by in vitro enzymatic catalysis has attracted many research
interests in the past years. The biodegradability and biocompatibility properties of -caprolactone homopolymers place it as a valuable raw material, particularly for
controlled drug delivery and tissue engineering applications. However, the usefulness of such materials is limited by their low hydrophylicity and slow biodegradation
rate. In order to improve polycaprolactone properties and
functionalities, copolymerization of -caprolactone with
gluconolactone was investigated. Since enzymatic reactions involving sugars are usually hindered by the low solubility of these compounds in common organic solvents,
finding the best reaction medium was a major objective of
this research.The optimal copolymerization conditions of
the reactions were carried out by using different organic
solvents (solvents mixtures), ionic liquids or solvent free
systems that are able to dissolve (completely or partially) sugars and are nontoxic for enzymes. Native and immobilized lipases by different immobilization techniques
from Candida antarctica B and Mucor miehei have been
used as biocatalyst and the reaction temperatures were set
up to 80C. Although the main copolymer amount was
synthesized in DMSO:t-BuOH=20:80 medium, the highest polymerization degrees, up to 16 for the copolymer
product, were achieved in solventless conditions. The
reaction product analysis revealed the formation of the
cyclic products that could be probably the major impediment of further increase of the chain length. The products, cyclic and linear polyesters, have been characterized
by FT-IR, MALDI-TOF-MS and NMR analysis. Carbohydrate derivatives represent an important monomer
source and insertion into non-polysaccharide backbones
can lead to functional polymers with specific properties
and application.
30
Eurobiotech 2013
P4.3
P4.4
New methods of poly(itaconic acid)

synthesis
Medium engineering for 2-methylbutyl-1ol

esters synthesis
Szczepan Bednarz, Diana Baejewska, Alicja Baszczyk,

Aleksandra Nowak, Piotr Krzywda
Mirosawa Szczsna-Antczak, Emilia Grabarczyk,

Agnieszka Borowska, Jakub Szelg, Katarzyna Chudzik,
Magdalena akowicz-Siuda, Tadeusz Antczak
Crakow University of Technology, Faculty of Chemical

Engineering and Technology
Itaconic acid (IA) is a one of twelve most important platfrom chemicals that can be produced from sugars via
biological conversions. Large scale fermentation processes using molasses as carbohydrate source and fungus Aspergillus Terreus have been developed. The acid
and its derivatives are frequently used in production of
various polymers, which are viable alternatives to acrylic and methacrylic acid based polymers. Recently, due
to non-toxic nature of the acid and biocompatibility,
IA-based copolymers have been synthesized in form of
hydrogels for biomedical applications. Itaconic acid is
a well-know, unsaturated monomer which polymerizes
via radical mechanism with difficulties. Persulfates and
water are used as a standard initiators and the most common solvent, respectively. However, the process takes
quiet a long time and for that reason, persulfate activators
such as amino alcohols (N,N-dimethylenethanoloamine) or sodium hypophosphite have been developed to
increase the polymerization rate or decrease the process
temperature. As stated above, polymerization IA is usually carried out in aqueous phase and to the best of our
knowledge application of ionic liquids as the polymerization medium or the polymerization catalysts has not
been previously reported. In this study, a preliminary results of application choline salts or choline-based ionic
liquids as polymerization catalyst or solvents, respectively, were shown. The catalytic effect of choline cation on
decomposition rate of the polymerization initiator was
demonstrated. Additionally, possibility of polymerization
of ionic liquid choline hydrogen-itaconate to obtain
poly(itaconic acid) was presented.
Acknowledgements
This work was supported by the European Union Funds under Human
Capital Programme within Bioinynier chemiczny (BINC) project
(contract number POKL.04.01.02-00-217/11-00).

of Technology, Stefanowskiego 4/10 St., 90-924 Lodz, Poland
This study aims at the development of chemo-enzymatic

methods of conversion of oleaginous biomass into diols
components of alkyl-aryl co-polyesters. It is one of objectives of the Biomass project realized by Ludz University
of Technology. The main concept of the proposed (within
the scope of task 3.2) procedure is based on chemo-enzymatic conversions of triacylglycerols (TAGs), contained
in various oils (mainly rapeseed, sunflower and waste
oils), into dimers of fatty acid esters, followed by their
conversion into diols and polymerization of the latter.
In the first step plant oils are subjected to enzymatic alcoholysis by a branched alcohol (e.g. 2-methylbutan-1-ol)
and resulting esters are physico-chemically converted
into dimers (cycloaddition reaction), which are then
converted into diols. Enzymatic step of this procedure is
mainly based on application of Mucor circinelloides membrane-bound lipase, as whole-cell preparations, which are
predestined to catalysis in non-aqueous media [14].
Presented study focused on medium engineering (molar ratio of substrates, type of organic medium and water
content in reaction medium, as well as addition of some
polar substances modifying the lipase micro-environment such as diethylamine, triethylamine and ectoine [4]
in concentration of 0.560mM) of enzymatic alcoholysis
of rape and sunflower oils in batch and semi-continuous
processes.
Water content was controlled by water quantification by
Karl Fischer method and water activity measurement
(Rotronik probe). Reaction progress was assayed by GC
method (yield of esters) as well as by TLC plates analysis using JustTLC program (Sweday) that enables estimation of percentage content of esters, mono-, di- and
triacylglycerols and free fatty acids in reaction mixtures.
Free fatty acids were additionally assayed by copper soaps
method [5]. The reaction mixtures were also analyzed by
HPLC (RP-C18 column, NQAD detector) method. Also
the stability of lipases (by residual lipolytic activity measurement) was estimated.
Acknowledgements
The study was realized within the scope of the project POIG 01.01.0210-123/09 Application of biomass in production of environmentally
friendly polymer materials tasks 3.2., co-financed from the funds
of European Fund of Regional Development within the frames of
Operation Program Innovative Economy 20072013.
References
[1] Szczesna-Antczak M. et al. (2009) Renew. Energy 34, 11851194.
[2] Szczesna-Antczak M. et al. (2006) Enzyme Microb. Technol. 39,
12141222.
[3] Szczesna-Antczak M. et al. (2004) J. Mol. Cat. B, Enzymatic, 29, 163171.
[4] Antczak T. et al. (2002) J. Mol. Cat. B Enzymatic, 19-20, 287294.
[5] Lowry R.R., Tinsley I.J. (1976) J Am. Oil Chem. Soc, 53, 470472.
Eurobiotech 2013
P4.5
P4.6
An influence of water content on enzymatic

transesterification
Synthesis of fluorescent polyesters from

citric acid
Jakub Szelg, Mirosawa Szczsna-Antczak, ukasz Staczyk,

Magorzata Piotrowicz-Wasiak, Tadeusz Antczak
Wiktor Kasprzyk, Szczepan Bednarz, Dariusz Bogda
Institute of Technical Biochemistry, Technical University of Lodz,

Stefanowskiego 4/10 St., 90-924 Lodz, Poland
Water strongly affects reaction efficiency in non-aqueous

systems. An impact of water content was studied for sunflower oil and 2-methylbutan-1-ol transesterification reaction catalyzed by immobilized lipases: Mucor circinelloides (mycelial preparation from ITB) and Thermomyces
lanuginosa (from Novozymes).
The influence of water content in reaction mixture on
esters yield and stability of lipases preparations (residual lipolytic activity measurement) was determined. The
transesterification processes were carried out using either
only a mixture of substrates (without any additional solvent) or with the addition of petroleum ether. Water content in reactions mixtures, which was controlled by Karl
Fischer method, varied from 0 to 3% in the first variant
and from 0 to 5% in the variant with petroleum ether.
Products of transesterification reaction (esters, mono-,
di- and triacylglycerols and free fatty acids) were quantified using JustTLC analysis program (Swedey) and the
yield of esters was determined also by GC method. Free
fatty acids were additionally assayed by a colorimetric
method [1]. Water content was found to be a key factor deciding of transesterification efficiency. The highest
yield of 2-methylbutan-1-ol esters was achieved at ~0.5%
water concentration for both M. circinelloides (67.5%,
without petroleum ether) and T. lanuginosa (80%, also in
the absence of petroleum ether) lipases. The decrease in
lipolytic activity of both immobilized lipase preparations
was the lowest at 1.5% water concentration.
Acknowledgements
The study was realized within the scope of the project POIG 01.01.0210-123/09 Application of biomass in production of environmentally
friendly polymer materials task PZ 3.2., co-financed from the funds
of European Fund of Regional Development within the frames of
Operation Program Innovative Economy 20072013.
References
[1] Lowry R.R., Tinsley I.J. (1976) J Am. Oil Chem. Soc, 53, 470472.
31
Chair of Biotechnology and Physical Chemistry, Faculty

of Chemical Engineering and Technology, Crakow University
of Technology, Warszawska 24 St., 31-155 Crakow, Poland
Novel and carefully prepared biomaterials can facilitate

growing demands of medicine. Excellent examples of
these include fluorescent proteins, fluorescent organic
dyes, lanthanide chelates, and quantum dots as well as
recently developed carbon nanodots (CD). Carbon nanodots can be easily synthesized from low-temperature pyrolysis of mixtures of citric acid with amines or diamines.
Furthermore, relatively high quantum yields (up to 62%)
have also been achieved for biodegradable photoluminescent polyesters (cys-BPLP). This was accomplished
through introduction of different L-amino acids into the
polyester structure made of biocompatible monomers of
citric acid and aliphatic diols. Thus produced polyester
solutions, films, scaffolds and nanoparticles emit strong
and tunable fluorescence (up to 725 nm). Moreover,
BPLPs demonstrate in vitro cytocompatibility, minimal
chronic inflammatory responses in vivo, controlled degradability, good photostability and desired mechanical
properties. Initial studies on the fluorescence mechanism
of BPLPs have indicated morpholine-2,5-dione as the
structure responsible for the fluorescent properties. The
fluorescence mechanism of this ring was explained based
on its planarity and hyperconjugation theory, though it is
not fully understood yet.
Here we present results of our investigations of chemical
structure of the moiety responsible for cys-BPLP luminescence. We found that as a result of polycondensation
reaction of citric acid and amino acids, among many others, luminescent derivatives of 2-piridone are formed.
In turn, the luminescent compound (5-oxo-2,3-dihydro-5H-[1,3]thiazolo[3,2-a]pyridine-3,7-dicarboxylic
acid TPA) was synthesized directly from citric acid and
L-cysteine as well as being separated from cys-BPLP hydrolyzate. Furthermore, attempts of introducing TPA to
poly(diol citrate) films revealed that it can be incorporated into the polyester chain through formation of ester bonds and even minor quantities led to luminescent
materials.
Acknowledgements
The authors gratefully acknowledge the support for this work from
the National Centre of Science of Poland (grant number: 2011/01/N/
ST5/05591). We are also grateful to Marek ylewski and Pawe mudzki
for performing NMR and ESI-MS analyses, respectively.
32
Eurobiotech 2013
P4.7
P4.8
Application of selectively-hydrogenated
rapeseed oil for modification of flexible
foams
Microwave-enhanced chemical
modification of chitosan
Sylwia Dworakowska1, Dariusz Bogdal1, Federica Zaccheria2,

Nicoletta Ravasio2
Chair of Biotechnology and Physical Chemistry,

Crakow University of Technology, Crakow, Poland
Department of Biotechnology and Physical Chemistry,

Crakow University of Technology, Warszawska 24, Crakow,
Poland; 2CNR-Institute of Molecular Science and Technologies,
Via Golgi 19, Milan, Italy
Marek Sawiski, Marek Pitkowski
Recently, renewable resources are getting more and more

interesting because of decreasing fossil feedstocks and
their higher prices. Natural oils have been shown to be
a potential bio-renewable feedstock since they can be
derivatized towards different monomers for polymer
chemistry e.g. for various polyurethane materials.
Selective hydrogenation represents a valuable tool for
standardization of vegetable oils composition. The use
of selectively hydrogenated oil (with increased monoenic
chains in the triglycerides) constitutes promising way of
normalisation of vegetable oils [13].
This work demonstrates synthesis of flexible polyurethane
foams modified with rapeseed oil-based polyols being
30wt.% replacement of petroleum polyols [4]. The aim
of this study was to apply rapeseed oil stabilized through
a selective hydrogenation process for synthesis of polyols
and afterwards obtained flexible foams. Two-stage method for preparation of selectively hydrogenated oil-derived
polyols was presented. The bio-based polyols with different hydroxyl values were used for modifications of flexible foams which allowed obtaining tailored properties of
the final products for bedding and furniture applications
i.e. increased compressive strength as well as elongation
at break and tensile strength in comparison to reference
ones made from 100% petrochemical polyol [5].
Acknowledgements
The authors thank the UBIOCHEM COST Action CM0903 for Short
Term Scientific Missions of S. Dworakowska and F. Zaccheria. Authors
also gratefully acknowledge Regione Lombardia for financial support
through the VeLiCA Project and the European Union through the
European Social Found within Cracow University of Technology
development program top quality teaching for the prospective Polish
engineers; University of the 21st century project (contract No. UDAPOKL.04.01.01-00-029/10-00).
References
[1] Zaccheria F., Psaro R., Ravasio N., Bondioli P. (2012) Eur. J. Lipid Sci.
Technol. 114: 2430.
[2] Zaccheria F., Psaro R., Ravasio N. (2009) Green Chem. 11: 462465.
[3] Trasarti A.F., Segobia D.J., Apesteguia C.R., Santoro F., Zaccheria F.,
Ravasio N. (2012) J. Am. Oil Chem. Soc. 89: 22452252.
[4] Dworakowska S., Bogdal D., Prociak A. (2012) Polymers 4:
14621477.
[5] Dworakowska S., Bogdal D., Zaccheria F., Ravasio N., Catal. Today
(in press).
Recently in many research centres intense efforts towards

obtaining new biobased polymers of high purity applicable in biomaterial engineering, are made. Such macromolecular compounds have to fulfil specific requirements,
such as non-toxicity, biocompatibility and biodegradability. Both polymers and products of their decomposition inside organism must be non-toxic, cause no inflammation states or other immune responses, as well as
any changes in body fluids. Currently, derivatives of lactic
acid and poly(ethylene glycol)s are used for this purpose.
In addition, due to specific properties, various kinds of
chitosan derivatives are more often applied in biomedical
engineering [13].
An innovative and original method of the synthesis of
chitosan derivative in form of copolymer with aspartic
acid and various glycols under microwave radiation, was
developed. The use of microwave technology allows the
complete elimination of toxic catalyst, significantly reduces reaction time and improves the yield. Moreover,
in aqueous solution chitosan-based copolymer forms
hydrogel selectively sorbing heavy metal ions, especially
from very diluted solutions. The swelling properties are
dependent on synthesis parameters as well as type of diol
used in reaction.
Presented research comprises synthesis route and also
determination of properties of novel biobased materials with use of Fourier Transform Infrared Spectroscopy (FTIR), gel permeation chromatography (GPC) and
UV-Vis Spectroscopy.
References
[1] Dee K.C., Puleo D.A., Bizios R. (2002) An Introduction To TissueBiomaterial Interactions, Wiley-Liss Inc.
[2] Ratner B.D., Hoffman A.S., Schoen F.J., Lemons J.E. (2004)
Biomaterials science, Elsevier Academic Press.
[3] Bronzino J.D. (2000) The Biomedical Engineering Handbook:
Second Edition, Boca Raton: CRC Press LLC.
Pharmaceutical biotechnology
Lectures
L5.2
L5.1
How to pick the right pocket - the where

and how of ligand binding
Biosynthesis of delta-9-tetrahydrocannabinolic
acid from biotransformation studies
in Cannabis sativa L. to biotechnological
prodution in S. cerevisiae
Oliver Kayser
Technical University Dortmund
delta-9-Tetrahydrocannabinolic acid (THCA) is an important drug from Cannabis sativa L. with psychomimetic activities. THCA and related herbal products are
mostly misued and illegal use is dominating despite the
fact that THCA has a significant medicinal value (e.g. chemotherapy, multiple sclerosis, anorexia). Today, breeding
and cultivation of high content plants is not legal for extraction, but also organic synthesis is too expensive and
not efficient to obtain high amount of THCA and it corresponding decarboxylated THC derivative for industrial
production. Biotechnology may provide new avenues for
biosynthesis and production strategies, but the biosynthesis in planta is not fully eleucidated, why heterologous expression of biosynthetic genes is complicated. Herein we
provide an overview of recent developments on the elucidation of the pathway towards cannabinoids and updated
attempts for construction of a recombinant host for the
production of THCA and its main precursors CBGA and
GPP. In short plants genetics, transciptomics and metabolimcs of trichomes where biosynthesis is localised will be
discussed [1], principles of gene expression of the main
important genes (CBGA-Synthase, THCA-Synthase) will
be explained, and into basic concepts of bioengineering of
yeasts as potential hosts will be introduced [2].
Acknowledgements
This research was funded by Deutsche Bundesstiftung Umwelt (DBU,
Frderkennzeichen 13252) and Arbeitsgemeinschaft Industrielle Forschung (AIF). Further information on heterologous THC production
on www.pharma-biotechnologie.de.
References
[1] Happyana N. et al. (2013) Analysis of cannabinoids in laser
microdissected trichomes of medicinal Cannabis sativa using LCMS and
cryogenic NMR. Phytochermistry 87: 5159.
[2] Muntendam R et al. (2009) Perspectives and limits of engineering
the isoprenoid metabolism in heterologous hosts Appl. Microbiol
Biotechnol. 84: 10031019.
Anke C. Schiedel, Dominik Thimm, Sonja Hinz, Benjamin Seibt,

Farag Sherbini, Christa E. Mller
PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical
Chemistry I, An der Immenburg 4, 53121 Bonn, Germany
G protein-coupled receptors (GPCRs) are among the most

prominent drug targets and more than 30% of the drugs
on the market are targeting GPCRs. For the development
of new, highly potent, and subtype-selective drugs it is
of considerable interest to know the receptors binding
pocket and the exact binding modes of different ligands
as well as amino acids involved in subtype selectivity and
receptor activation. Although X-ray structures have recently been published for several GPCRs in complex with
agonists or antagonists a combination of homology modeling and mutagenesis studies is still a powerful approach
to facilitate rational drug design, especially since for the
majority of receptors and ligands structural data are not
available and will not be accessible in the near future.
In the present study we investigated the A2B adenosine
receptor (A2BAR), which plays a role in cancer, inflammation, and pain, and has been proposed as a new drug
target. We analyzed the A2BARs binding pocket, and the
subtype selectivity of several ligands versus its closest homolog, the A2AAR [1]. Using 3D homology models, based
on crystal structures of the A2AAR, we predicted several amino acids to be involved in ligand binding and/or
receptor activation [2]. In addition we analyzed the role
of the extracellular loop (ECL) 2, which contains four
cysteine residues [3, 4] The predicted amino acid residues were exchanged for alanine, or in case of cysteine,
for serine. The mutants were stably expressed in Chinese
hamster ovary (CHO) cells and characterized by radioligand binding and cAMP assays using three structural
classes of ligands: xanthine (antagonist), adenosine and
aminopyridine derivatives (agonists). Our studies revealed that diverse ligands have distinct, yet overlapping
binding pockets. Trp2476.48, Val2506.51, and Ser2797.42 were
shown to be important for binding of nucleosidic agonists, while non-nucleosidic agonists did not interact with
Ser2797.42 and showed weaker interactions with Trp2476.48
and Val2506.51. Leu813.28, Asn1865.42, and Val2506.51 were
found to be crucial for binding of the xanthine-derived
antagonist PSB-603.1,5 In addition, Leu813.28, which is not
conserved among the adenosine receptors, was found to
be important for subtype-selectivit [1]. We also investigated all four cysteine residues located in the ECL2 and
34
Eurobiotech 2013
were able to show that in the A2BAR only the highly conserved C3.25-C45.50 disulfide bond is essential for ligand
binding and receptor activation [4]. We additionally exchanged the whole ECL2 of the A2BAR for the ECL2 of
the A2AAR and demonstrated that the ECL2 is involved in
ligand binding and subtype selectivity and modulates agonist-bound receptor conformations, thereby controlling
signaling efficacy [3]. The interdisciplinary approach presented in this study using both, experimental data and
computational predictions, provides valuable information for the rational design of desired highly potent and
selective ligands, which are required to validate and exploit their therapeutic potential, and to further elucidate
the (patho)physiological roles of GPCRs.
References
[1] Thimm et al., Biochemistry, 2013, 52, 726740.
[2] Sherbiny et al., J. Comput. Aided Mol. Des., 2009, 23, 80728.
[3] Seibt et al., Biochem. Pharmacol., 2013, 85, 13171329.
[4] Schiedel et al., Biochem. Pharmacol., 2011, 82, 389399.
[5] Borrmann et al., J Med Chem, 2009, 52, 39944006.
L5.3
Monoclonal antibodies targeting
angiogenesis and lymphangiogenesis
in cancer therapies
Monika Bzowska, Magorzata Kulesza, Tomasz Klaus,
Karolina Ossysek, Renata Myk-Kope, Joanna Bereta
Faculty of Biochemistry, Biophysics and Biotechnology,
Jagiellonian University
Monoclonal antibodies (mAbs) are one of the fastest developing branches of the pharmaceutical industry. Due to
a very high affinity and selectivity of binding to antigens,
mAbs are used for specific targeting of certain molecules,
e.g. soluble cytokines or tumor-associated antigens expressed by cancer cells. Presently, therapeutic mAbs are
applied: (i) to prevent transplant rejection, (ii) to inhibit the progression of chronic inflammatory diseases and
(iii) to fight cancer (usually in combination with other
therapies). Apart from mAbs that directly target cancer
cells, another approach which aims at angiogenesis has
also been introduced to the clinic to inhibit tumor growth
and spreading (metastasis). However, long-term studies
indicate limited beneficial effects of this anti-angiogenic
approach. Since tumor cells can escape from the primary
site by entering not only blood- but also lymphatic vessels, the attempts to inhibit lymphangiogenesis in order
to hamper tumor metastasis seem reasonable [1].
The aim of our project is to generate mAbs able to bind
and inhibit VEGF-C, the major growth factor for lymphatic endothelium. We use a phagemid library Tomlinson I+J and a phage display method to select the set of
phages expressing anti-VEGF-C antibodies in a form of
scFv (single chain variable fragment). The procedure of
phage selection and obtaining of specific mAbs as well as
the evaluation of their inhibitory activity will be presented. The potential of anti-VEGF-C antibodies to limit tumor development and spreading will be discussed.
Acknowledgements
This work was supported by a grant from the Polish-Swiss Research
Programme (PSPB-057/2010 to JB).
References
[1] Antibody-based antiangiogenic and antilymphangiogenic therapies
to prevent tumor growth and progression. Bzowska M., Myk-Kope
R., Prchnicki T., Kulesza M., Klaus T., Bereta J., Acta Biochimica
Polonica, Paper in Press, No. 2013_481.
Eurobiotech 2013
L5.4
Bacterial cell wall assembly:
new antibiotics and industrial
biopolymers
Douglas Weibel2, 3, 4, George Auer3, Hannah Tuson4, Nate Cira4,
KC Huang5
Department of Chemistry, University of Wisconsin-Madison;
Department of Biomedical Engineering,
University of Wisconsin-Madison; 3Department of Biochemistry,
University of Wisconsin-Madison; 4Department of Bioengineering,
Stanford University
1
2
This presentation describes how we are using our discovery of bacterial cell wall assembly and maintanence to
develop new chemotherapeutics and produce new classes
of industrial biopolymers. This presentation builds a connection between two connected areas of research in our
lab that center on the bacterial cell wall: 1) identifying,
studying, and exploiting the machinery that bacteria use
to synthesize their polymer exoskeleton (the peptidoglycan, PG) as a new feedstock for industrial polymers; and
2) and developing new clinical antibiotics and that target
the bacterial cell wall and protecting these valuable biomedical resources.
In the first part of this presentation, I describe a gel-based
force clamp technique that we recently developed to measure the mechanical properties of live bacterial cells [1].
We recently used this technique to measure how single
gene deletions in bacteria alter cell stiffness [Auer et al.
in preparation]. Using this approach we have identified
machinery that is essential for regulation of the mechanical properties of the PG and are now engineering the
biochemistry of these proteins to tailor PG assembly as
asource of biopolymers.
In the second part of my talk, I introduce the cell wall machinery as the most important targets of clinical antibiotics developed to date. Protecting these valuable resources
requires providing health care workers with informed
data on how to best treat pathogens at the point-of-need.
To facilitate this process, we have developed a very simple, inexpensive, and small plastic cartridge, which we refer to as QuickChip that uses an isothermal, DNA-based
amplification technique to identify antibiotic resistance
elements in bacteria in clinical samples and determines
antibiotic susceptibility [24]. Integrating QuickChip
with a smart phone or tablet-powered manifold enables
its hands-free operation: the manifold heats the cartridge
and measures the fluorescence output from the isothermal assay, the app sends the data to the cloud for analysis and diagnosis, and the cloud returns information on
the bacterial strain and optimal antibiotic dosing to guide
the effective treatment.
We envision that the integration of these technologies
will have an important impact on biomaterials science
and clinical medicine by drawing new connections between the structure, assembly, and mechanical properties
of the bacterial cell wall.
35
Acknowledgements
This research was supported by the Aspen Center for Physics (NSF
grant 1066293), the NIH (grants T32 GM07215, DP2OD006466, and
DP2OD008735), DuPont, and the Alfred P. Sloan Foundation.
References
[1] Tuson H.H., Auer G.K., Renner L.D., Hasebe M., Salick M., Crone
W.C., Gopinathan A., Huang K.C., Weibel D.B. (2012) Measuring the
stiffness of bacterial cells from growth rates in hydrogels of tunable
elasticity. Mol. Microbiol. 84, 874891.
[2] Cira N.J., Dueck M.E., Weibel D.B. (2012) A self-loading microfluidic
device for determining antibiotic toxicity. Lab Chip 12, 10521059.
[3] Cira N.J., Weibel D.B., Self-loading microfluidic device and methods
of use. 13/303,982. Filed: November 23.2011
[4] Ho J., Cira N.J., Crooks J., Weibel D.B. (2012) Rapid identification of
bacterial pathogens in an autonomous microfluidic device. PLoS One.
7, e41245.
36
Eurobiotech 2013
L5.5
L5.6
Biocatalytic access tochiral intermediates

for -adrenergic receptorsantagonists
A plant-based oral vaccine against

Hepatitis B Virus from edible vaccine
to tablet formula
Janina Ewa Kamiska

Faculty of Biotechnology and Food Sciences, Institute
of General Food Chemistry, Lodz University of Technology
Numerous -adrenergic receptors antagonists used in the

treatment of hypertension, angina pectoris, other cardiac
diseases and glaucoma are derivatives of 1-amino-3-aryloxypropan-2-ol. Among -blockers of this structural
class (S)-enantiomer exhibits higher affinity for -adrenergic receptors than (R)-enantiomer e.g. (S)-propranolol
which is 130 times more active than its (R)-enantiomer.
In spite of that knowledge, some -blockers are still manufactured and used in therapy as racemates, even if there
is evidence that distomers may exhibit side-effects. For
instance (R)-propranolol may cause thyroid dysfunction.
Due to increased demand for more effective, safe medicines, and the recommendations of institutions responsible for registration of new drugs, the synthesis of single
enantiomers of chiral intermediates has become important for pharmaceutical industry. Kinetic resolution of racemic 3-aryloxy-1-halogenopropan-2-ols is convenient
approach to synthesis of -blockers new analogues with
varying aromatic substituent as well as amine part.
A significant decrease in prices of commercial enzyme
preparations made their industrial application economically attractive. However still prediction of enzyme enantioselectivity for a new substrate is doubtful and requires
experimental checking. We decided to investigate how
substitution pattern of aromatic ring and kind of halogen
in substrate affects enzyme activity and enantioselectivity. Therefore we obtained several novel racemic 3-aryloxy-1-halogenopropan-2-ols in two-step sequence by
treating phenols with epichlorohydrin following epoxide
opening with hydrochloric or hydrobromic acid.
Then resolution of 3-aryloxy-1-bromopropan-2-ols and
3-aryloxy-1-chloropropan-2-ols by lipase-catalyzed
acetylation was undertaken. From eleven enzyme preparations screened, two commercial lipases Lipozyme
TL and Novozym 435 we found effective for kinetic resolution of 3-aryloxy-1-halogenopropan-2-ols by acetylation with vinyl acetate in organic solvents. In preparative experiments enantioselectivity ratio E from 64 to 99
was achieved, yielding both enantiomers with 8999%
ee. Then both enantiomers we successfully applied as
chiral building blocks for preparation of new 1-alkylamino-3-aryloxyprop an-2-ols, by nucleophilic halogen
substitution with isopropylamine or tert-butylamine. The
activity of products as -adrenergic receptor antagonists
was evaluated in vitro.
Acknowledgements
The research was financially supported by Ministry of Science and
Higher Education (grant No. N N405 2514 33) and European Social
Fund and Polish State (Z/2.10/II/2.6/04/05/U/2/06).
Tomasz Pniewski1, Jzef Kapusta1, 2, Marcin Czy1,

Justyna Wojas-Turek3, Magdalena Milczarek3,
Elbieta Pajtasz-Piasecka3, Joanna Wietrzyk3, Piotr Bocig1,
Anna Kostrzak1, 4, Andrzej Pucienniczak2
Institute of Plant Genetics, Polish Academy of Sciences,
Poznan, Poland; 2Institute of Biotechnology and Antibiotics,
Warsaw, Poland; 3Institute of Immunology and Experimental
Therapy, Polish Academy of Sciences, Wroclaw, Poland;
4
Institut Pasteur, Paris, France
1
Hepatitis B is still one of the most common human infectious diseases, despite the fact that effective anti-HBV
subunit vaccines have been available for 30 years. Orally
administered plant-based vaccines have been considered
as alternatives or supplements for standard injection vaccines, due to assumed low-cost production and simplified
vaccination. First trials showed prospects of mucoso-intestinal immunisation. Unfortunately, despite the unquestionable milestone, edible vaccines were impractical or insufficiently effective. Professional anti-HBV plant-derived
vaccine would require an appropriate plant producer as
well as vaccine composition and administration protocol.
Herbicide-resistant lettuce lines expressing native HBV
antigens, HBsAg subunits (S, M, L) and HBcAg, at levels
5200 g/g FW were engineered. Vegetative propagation
of efficient plant lines enabled production scaling-up of an
antigen-bearing material for an oral vaccine preparation.
The prototype manufacture technology of the vaccine has
been developed, based on freeze-drying of plant material.
Powdered lyophilised tissue facilitated controlled antigen
delivery and enabled conversion into oral formulas, e.g.
tablets.
Exclusively oral delivery of lyophilised tissue containing
S-HBsAg induced systemic humoral response in mice at
the minimal protection level ( 10 mIU/ml of anti-HBs
antibodies) with higher S-IgA response in intestinal mucosa and immediate stimulation of Treg lymphocytes.
Combination of injection priming and low-dose oral
boosting triggered significant immune response in mice,
as high as classical three-dose intramuscular vaccination.
Titre of anti-HBs antibodies reaction reached hundreds
mIU/ml, as well as stimulation of specific lymphocyte
subpopulations and production of cytokines were clearly visible. In turn, tablets administered to humans previously immunised with the standard pattern, increased
anti-HBs level from 0 up to 100 mIU/ml or 2 3-fold
when pre-immune anti-HBs were detectable.
The study provides some basics regarding parenteral-oral
immunisation strategy using an efficacious and convenient
plant-derived formula which would serve as a booster vaccine for possible human vaccination against hepatitis B.
Acknowledgements
This study was supported by grants No. 2 P04B 001 27 and No. N N302
157837 from the Polish Ministry of Science and Higher Education and
under the auspices of Medana Pharma Inc., Sieradz, Poland.
Eurobiotech 2013
L5.7
Posters
New fluorescence methods of visualisation

of extracellular matrix in metabolically
active tissues
P5.1
Jerzy Dobrucki
Jagiellonian University, Faculty of Biochemistry, Biophysics
and Biotechnology, Laboratory of Cell Biophysics
Only a few microscopy methods of visualizing extracellular matrix (ECM) in live tissues in situ and in freshly
excised tissues are available. Collagen fibers polymerized
under laboratory conditions can be imaged in an optical microscope, using dark field, differential interference
(Nomarski), or polarization contrast, however these
methods are not ECM specific. Two sophisticated imaging techniques can image collagen detecting second
harmonic generation (SHG), and a coherent anti-Stokes
Raman scattering (CARS). There are no simple, inexpensive, or widely available techniques for 3-dimensional
imaging of collagen and elastin fibers in live animals, or
excised metabolically active tissues.
A new low molecular weight fluorescent probe, Col-F,
that exhibits affinity to collagen and elastin, was used
successfully in confocal imaging of extracellular matrix
in freshly excised animal tissues. The dye readily penetrates between live cells into tissues and binds to fibers of
collagen and elastin by a noncovalent mechanism. Col-F
provides a simple and convenient tool for fluorescence
three-dimensional imaging of intricate collagenous and
elastic structures in live and fixed animal tissues, as well
as in collagen-containing biomaterials.
Acknowledgements
This research was supported by a grant 2067/P01/2007/32 from Ministry
of Science and Higher Education in Warsaw.
37
The impact of immobilized metal affinity

chromatography (IMAC) resins on DNA
aptamer selection
Ewa Kowalska, Filip Bartnicki, Katarzyna Pels,
Wojciech Strzalka
Department of Plant Biotechnology, Faculty of Biochemistry,
Biophysics and Biotechnology, Jagiellonian University, Crakow,
Poland
DNA aptamers are single-stranded oligonucleotides which

can form various secondary and tertiary structures. They
can recognize a broad range of targets ranging from lowweight molecules, such as ions, vitamins, antibiotics, to
high-weight molecules, including enzymes and antibodies.
DNA aptamers are extensively studied as a potential source
of new pharmaceutical drugs due to their inexpensive synthesis, low immunogenicity and high specificity.
The classical DNA aptamer selection method called
SELEX (Systematic Evolution of Ligands by EXponential
enrichment) was described in the early 90-ties of the last
century. In the first selection cycle a target molecule is
exposed to a synthetic DNA aptamer library composed of
10141015 different oligonucleotides. The DNA fragments
bound to the target are amplified using PCR and the pool
of single-stranded DNA used for the next selection round
is prepared. Usually several selection rounds are sufficient
to obtain aptamers which are highly specific for the target
molecule.
There are many ways of immobilizing the target molecule
to perform the SELEX procedure. One of the commonly
used methods for protein/peptide targets is based on immobilized metal affinity chromatography (IMAC). There
is a broad range of commercially available resins which
can be used for IMAC. They are characterized by different
metal ions, linker types and bead material.
In this study we tested the impact of different IMAC resins on the DNA aptamer selection process during the
first four SELEX cycles. A histidine-tagged twenty-nine
amino acid peptide corresponding to the inter-domain
connecting loop fragment of human Proliferating Cell
Nuclear Antigen was used as a selection target. Agarose
resins containing identical linkers, but different metal
ions (Co2+, Cu2+, Ni2+, Zn2+) were studied. Simultaneously, different resin materials (agarose and polystyrene) containing the same metal ion (Co2+) were tested.
The results clearly demonstrated the impact of the metal
ion and resin material on the aptamer pool enrichment
level. Concluding, these data indicate that for successful
IMAC-based SELEX, the determination of the optimal
resin might be important.
Acknowledgements
This work was financially supported by The National Centre of Research
and Development grant LIDER/28/54/L-2/10/NCBiR/2011 to WS.
Eurobiotech 2013
38
P5.2
P5.3
Bcl-2 family gene expression on HeLa cells

treated with Physalis peruviana ethanol
extracts
Antibacterial and antioxidant activites

of Physalis peruviana
Ozgur akir1, Bilgin Candar akir2, Murat Pekmez1,

Kerem Fidan1
1
Istanbul University; Istanbul Kultur University
Murat Pekmez1, Fatma Elif epni1, Bilgin Candar akir2,

zgr akir1
1
Istanbul University; 2Istanbul Kultur University
As a member of Solanaceae family, Physalis peruviana

has great horticultural and economic importance due to
its high nutritional value, high vitamin content, minerals
and antioxidants. P. peruviana also has anti-inammatory, antimicrobial and anticancer properties, so we aimed
to investigate potential effects of Physalis peruviana
(goldenberry) ethanol extracts for cytotoxic activity and
apoptotic gene expression on human HeLa cells.
MTT (Tetrazolium blue) colorimetric assay was used to
determine the viability of cells in the presence of the extracts. HeLa cells were treated with 10, 25, 50, 100 and
200 microgram/ml concentrations of Physalis peruviana
shoot and leaf ethanol extract for 48 h. Furthermore, effects of extracts on apoptotic gene expression on Bcl-2,
Bax, Bak, Bcl-x, Bik, Mcl-1, Bfl-1 were determined by reverse transcriptase polymerase chain reaction (RT-PCR).
As a result of these assays, it was found that the shoot and
leaf ethanol extracts of Physalis peruviana had cytotoxic
effect on HeLa cell cultures when applied as 100 microgram/ml concentration. It was also determined that shoot
and leaf extracts caused significant changes on especially
antiapoptotic genes expressions.
Shoot and leaf extracts of Physalis peruviana is considered as a potential source of anticancer compounds. Future studies are needed to elucidate chemical characterization of the P. peruviana active compounds as potential
anticancer agents.
Medicinal plants are used in folk medicine for centuries due to biological activities of bioactive metabolites.
Physalis peruviana L. (Solanaceae) is one of the common
medicinal herb used for its properties as anticancer, antimicrobial, antipyretic, diuretic, and anti-inammatory
immunomodulator. For this reason we aimed to evaluate
in vitro antibacterial and antioxidant activites and total
phenolic content of P. peruviana ethanol extracts.
Disk diffusion assay was used to determine antibacterial
effect. In antibacterial activity maximum inhibition zone
was determined in Lactococcus lactis. The lowest MIC
value was 100 microgram/disc for Staphylococcus aureus
A950277, the highest MIC value was 700 microgram/disc
for Escherichia coli DH5-alpha. The antioxidant activity
was evaluated by Cuprac method using trolox as standard and extracts exhibit total antioxidant capacity. Total
phenolic content was measured as gallic acid equivalents.
The highest TEAC and total phenolic content values of
the leaf and the shoot extracts are 0.291 0.04 and 0.192
0.015 and 40.69 0.21 and 30.21 0.71, respectively.
These results indicate that P. peruviana ethanol extracts
may include effective compounds to be used as therapeutic agents and give a great interest for future reseach.
Eurobiotech 2013
P5.4
Arbutin production via biotransformation
of hydroquinone in in vitro cultures
of Aronia melanocarpa (Michx.) Elliott
Inga Kwiecie, Agnieszka Szopa, Kornelia Madej, Halina Ekiert
Chair and Department of Pharmaceutical Botany,
Jagiellonian University, Collegium Medicum, 9 Medyczna Street,
Arbutin (hydroquinone -D-glucoside) is a compound

of plant origin possessing valuable therapeutic (urinary tract disinfection) and cosmetic (skin whitening) properties, which can be obtained from in vitro
cultures of plants belonging to different taxa via biotransformation of exogenously supplemented hydroquinone [1, 2].
Preliminary studies demonstrated the ability of in vitro
cultures of Aronia melanocarpa established in our laboratory to perform -D-glucosylation of hydroquinone [3].
The aim of the present experiments was to optimize hydroquinone biotransformation, in particular, to establish
the effect of the dose and the method of precursor application on the course of the reaction.
The agitating cultures of Aronia melanocarpa were maintained on the Murashige and Skoog medium [4] containing growth regulators: the cytokinin BAP (6-benzylaminopurine), 2 mg/ml and the auxin NAA (-naphthaleneacetic acid), 2 mg/ml. The biomass was cultured
for 2 weeks and then hydroquinone was supplemented at
the following doses: 96, 144, 192, 288 and 384 mg/l either
undivided or divided into two or three portions added at
24-hour intervals. The content of the product of the reaction arbutin, was determined using an HPLC method
[5] in methanolic extracts from biomass and lyophilized
medium samples collected 24 hours after addition of the
last precursor dose.
Arbutin was accumulated mainly in the cultured biomass, but also in the medium when higher precursor
doses (288 mg/l and 384 mg/l) were supplemented. The
total contents of arbutin were very diverse, from 2.71 to
8.27 g/100 g d.w. Arbutin production rose with increasing
hydroquinone concentration. Its maximum content was
observed after hydroquinone addition at 384 mg/l divided into two portions.
Biotransformation yield also widely differed ranging
from 37.04%73.80%. The identity of the product arbutin, after its isolation and purification was confirmed by
spectral analysis (1H-NMR spectrum).
The obtained maximum content of arbutin was higher
than that required by the newest 9th Edition of the Polish Pharmacopoeia for Uvae ursi folium (7.00 g/100g
d.w.) [6], and is interesting from practical point of
view.
References
[1] Skrzypczak-Pietraszek E., Szewczyk A., Piekoszewska A., Ekiert H.
(2005) Acta Physiol. Plant. 27: 7987.
[2] Piekoszewska A., Ekiert H., Zubek Sz. (2010) Acta Physiol. Plant.
32: 223229.
39
[3] Kwiecie I., Majcher J., Ekiert H. (2013) 7th German-Polish

Symposium Interdisciplinary research for pharmacy, Gdask,
Abstracts, 136.
[4] Murashige T., Skoog F. (1962) Physiol. Plant. 15: 473497.
[5] tambergov A., Supikov M., Leifertov I. (1985) eskoslov Farm.
34: 179182.
[6] Uvae ursi folium. In: 9th Edition of the Polish Pharmacopoeia,
Vol. I, 1410-1411.
40
Eurobiotech 2013
P5.5
The accumulation of phenolic acids
in agitating cultures of Ruta graveolens L
Agnieszka Szewczyk, Marzena Surzyn, Halina Ekiert
Department of Pharmaceutical Botany,
Apart from many valuable secondary metabolites, like

alkaloids, flavonoids, coumarin compounds, volatile
oil, Ruta graveolens contains also phenolic acids. These
compounds exhibit very beneficial therapeutic properties, e.g. antioxidant, anticancer and immunostimulating
activities. These compounds occur in plant cells free and
bound as, for instance, glucosides or esters [1].
Our earlier studies demonstrated that stationary cultures
of Ruta graveolens maintained in our laboratory were capable of producing some free phenolic acids in the interesting amounts from practical point of view. The main
metabolite was protocatechuic acid [2]. The aim of the
present investigations was to assess productive capacity
of agitating cultures of this species.
The agitating cultures were maintained on four variants
of the Linsmaier and Skoog (L-S) medium [3] containing
growth regulators: the cytokinin BAP (6-benzylaminopurine) and the auxin NAA (-naphthaleneacetic acid)
in the concentration range from 0.1 to 3.0 mg/l. Biomass
cultured in vitro was collected after 4-week growth cycles
and extracted with methanol. These methanolic extracts
subjected or not to hydrolysis (2 M HCl, 2 h) were used
for determination of thirteen phenolic acids (cinnamic
and benzoic acid derivatives) and cinnamic acid using an
HPLC method [4].
None of the compounds under study was detected in unhydrolyzed samples. However, hydrolyzed extracts were
shown to contain eight phenolic acids: o-coumaric acid,
p-coumaric acid, ferulic acid, gallic acid, p-hydroxybenzoic acid, protocatechuic acid, syringic acid and vanillic
acid. Protocatechuic acid was accumulated at the highest levels in biomass cultured on all tested medium variants (19.633.6 mg/100g d.w.). Amounts of o-coumaric
acid (3.66.3 mg/100g d.w.) and p-hydroxybenzoic acid
(3.16.3 mg/100g d.w.) were much lower. Only small
levels of the remaining phenolic acids were confirmed.
The total content of phenolic acids ranged from 35.6 to
48.6mg/100g d.w. The maximum content was found in
biomass extract from the L-S medium supplemented with
2 mg/l BAP and 2 mg/l NAA.
The obtained results indicate that the phenolic acids under study occur in biomass of Ruta graveolens cultured
in agitating cultures only in the bound form. They also
demonstrate a significant concentration-dependent effect
of growth regulators on the accumulation of phenolic acids.
This type of Ruta graveolens in vitro cultures can be a potential rich source of protocatechuic acid.
References
[1] Ekiert H., Czygan F.Ch. (2007) In: Biotechnology Secondary
metabolites. Plants and Microbes (Ramawat K.G. and Merillon J.M.
ed.), Science Publishers, Enfield, New Hampshire, USA, 445482.
[2] Ekiert H., Szewczyk A., Ku A. (2009) Pharmazie 64: 694696.
[3] Linsmaier E.M., Skoog F. (1965) Physiol. Plant. 18: 100127.
[4] Ellnain-Wojtaszek M., Zgrka G. (1999) J. Liq. Chrom. Tech. 22:
14571471.
Eurobiotech 2013
P5.6
The influence of L-phenylalanine, methyl
jasmonate and sucrose concentration
on the accumulation of phenolic acids
in Exacum affine Balf. f. shoot culture
Ewa Skrzypczak-Pietraszek, Joanna Sota
Chair and Department of Pharmaceutical Botany,
Phenolic acids are an important group of plant secondary metabolites with different, valuable therapeutic
properties (e.g. antioxidant, antiphlogistic, immunostimulating, antiseptic) [1]. Besides plants growing in
the open air, tissue cultures can be an alternative source
of secondary metabolites. Yield of their accumulation in in vitro cultures can be increased by different
methods, including culture medium supplementation
with precursors, elicitors and changing the standard
amounts of the medium components. The purpose of
this study was to investigate the influence of precursor (L-phenylalanine), elicitor (methyl jasmonate) and
different sucrose concentration on phenolic acids accumulation in agitated shoot cultures of Exacum affine
Balf. f. (Gentianaceae).
Cultures were maintained in conical flasks with Murashige and Skoog medium [2] supplemented with plant
growth regulators: BAP (6-benzylaminopurine), 1 mg/l,
NAA (-naphthaleneacetic acid), 0,5 mg/l and GA3 (gibberelic acid), 0,25 mg/l. Variant A contained 3% of sucrose (standard amount) and the other six variants (A-F)
6% of sucrose. After two weeks L-phenylalanine (1,6g/l
of medium) and/or methyl jasmonate (two concentrations: 100 M or 800 M) were added to B-F variants.
Variants A and A were treated as references. Plant materials were collected after 1, 3 and 7 days after the addition
of the precursor and/or the elicitor. Control samples were
collected too. Phenolic acids were assayed in the collected
biomass before and after acid hydrolysis (2 M HCl, 2 h).
Qualitative and quantitative analysis of phenolic acids in
methanolic extracts from biomass were conducted by an
HPLC method [3].
Fourteen phenolic acids (protocatechuic, gallic, gentisic,
chlorogenic, p-hydroxybenzoic, vanillic, caffeic, syringic,
p-coumaric, ferulic, sinapic, salicylic, o-coumaric, rosmarinic) and cinnamic acid were found in all samples.
The total content of free phenolic acids increased from
approximately 0,368% to 0,838% (2,3-times) and the total
content of the whole phenolic acids (free and bound)
from 0,866% to 1,390% (1,6times) depending on the
MS medium variants. The studies show that the best variant contained 6% of sucrose (double amount of the standard), L-phenylalanine 1,6 g/l of medium and methyl
jasmonate 100 M.
Analysis of the results in the described experiment
showed that it is possible to increase the accumulation of
41
phenolic acids in Exacum affine shoot cultures adding

the precursor (L-phenylalanine), the elicitor (methyl jasmonate) and increasing the sucrose concentration.
References
[1] Khadem S., Marles R., Molecules, 15 (2010) 79858005.
[2] Murashige T., Skoog F., Physiol. Plant, 15 (1962) 473497.
[3] Ellnain-Wojtaszek M., Zgrka G., J. Liq. Chrom. & Rel. Technol., 22
(1999) 14571471.
42
Eurobiotech 2013
P5.7
P5.8
Biotechnology in cosmetology:
Introduction of Argireline an anti-aging
peptide. Study of cellular cytotoxicity
of Argireline solution
Antibacterial activity of essential oils

obtained from leaves and fruits of a native
plant from South America, Schinus areira
Marek Grosicki, Anna Cukier, Katarzyna Kie-Kononowicz

Jagiellonian University Medical College, Faculty of Pharmacy,
Department of Technology and Biotechnology of Drugs,
Argireline is an anti-aging peptide synthesized by company: Lipotec S.A. In cosmetology it is used as synthetic
cosmetic ingredient in form of a powder or more commonly 0,05% argireline solution. This acetyl hexapeptide-3 is mimicking the N-terminal end of SNAP-25 protein, that plays important role in formation of neuronal
SNARE complex. Argireline prevents formation of skin
lines and wrinkles in a very similar way as botulinum
toxin (Botox). It competes with SNAP-25 for a position
in the SNARE complex, destabilizing it. As a result, the
neuronal vesicles cannot release neurotransmitters from
the axon endings. In the end that causes attenuation of
skin muscle contraction, especially in the forehead and
around the eyes. It also inhibits overproduction of catecholamines, which can induce formation of wrinkles.
Argireline can be incorporated in cosmetic formulations
such as emulsions, gels or sera. Its effectiveness has been
confirmed in several anti-wrinkle tests performed in vitro
using chromaffin cells and in vivo on healthy volunteers
[1]. The aim of presented study is to elaborate the proper methodology of cytotoxity study of cosmetics active
ingredients. Tests were performed on argireline solution
in its declared by producer concentration. In order to estimate cytotoxity of the argireline solution MTS test was
used. This test allows to measure the efficiency of mitochondrial oxidative activity in living cells [2]. In the experiments both HEK293 cells and human fibroblasts were
treated with argireline solution. Examined were: short
time cytotoxic effect of argireline as well as its influence
on cellular proliferation features. Considered methods
result in dose dependent argireline cytotoxicity effect.
Argireline solution, dissolved more than six times from
original concentration decreases cellular proliferation
rate and metabolic activity. In conclusion it was shown
that the MTS test can be used in order to determine cytotoxic properties of cosmetics active ingredients.
Acknowledgements
We would like to thank: Przedsibiorstwo Produkcyjno-Handlowe
Ryszard Kaczmarek i Synowie Sp. z o.o. Spka Komandytowa company,
that is the only official distributor of Lipotec S.A. products in Poland,
for providing argireline solution sample, that was used in the presented
experiments.
This work was supported by K/ZDS/001915.
References
[1] Lipotec S.A. (2002) Neuronal exocytosis inhibiting peptides and
cosmetic and pharmaceutical compositions containing said peptides,
WO 00/64932.
[2] Cory J.G., Owen A.H., Barltrop T.C. (1991) Use of an aqueous
soluble tetrazolium/formazan assay for cell growth assays in culture.
Cancer Communications, 3(7), 207212.
Marta H. Alabrudzinska1, Liliana S. Celaya2, Ana C. Molina2,

Carmen I. Viturro2, Silvia Moreno3
Intercollegiate Faculty of Biotechnology, University of Gdansk
and Medical University of Gdansk, Gdansk, PC 80-822, Poland;
2
PRONOA Laboratory, F.I., National University of Jujuy,
S. S. de Jujuy PC 4600, Argentina; 3Plant Biochemistry
laboratory, Institute Leloir Foundation, IIBBA-CONICET.
Buenos Aires, PC 1405. Argentina
1
Essential oils (EOs) are aromatic components obtained

from different plant parts such as leaves, fruits and flower,
and are composed of secondary metabolites such as terpenes and terpenoids. These compounds act as a mechanism of defense against pathogen attack (microbes,
herbivores or competing plants). A great deal of essential
oils is used in different industries, mainly in perfumes
(fragrances), food (as flavoring and preservatives), pharmaceuticals (therapeutic action) and for centuries in traditional medicine [1]. It is well known that some essential
oils exert antimicrobial and antioxidant properties [2, 3].
Schinus is a genus belonging to the Anacardiaceae family.
This family comprises about 70 genera and 600 species.
They are used traditionally as a healing, stomachic and
antidiarrheal agent, due to the presence of tannins and oil
resins. In this family, S. molle L. (also known as California pepper and pink pepper) was introduced from South
America to most tropical and subtropical areas of the
world, as well as the Mediterranean. Another tree of this
genus, S. areira L. commonly called peppertree, molle
and aguaribay, is a native plant from South America and
nowadays it is distributed through Argentina, south-eastern of Brazil, Peru, Colombia, Ecuador, Uruguay and
widely distributed in Mexico, Central and Southern of
California and West Texas, United States. Several parts of
this tree are used in traditional medicine as antibacterial,
antifungal and antirheumatic [4].
Extensively studies regarding the characterization of EOs
isolated from the Schinus molle has been reported, however much less research has been done on the biological
properties of the oils of S. areira [5]. Moreover, significant variations in the chemical composition of the EOs of
S. areira have been reported in relation to the geographic
origin[5], although correlation between the presence and
content of specific compounds and the antimicrobial activity has not been deeply investigated.
The aim of this work was to characterize the antibacterial
activity of EOs isolated from leaves and fruits of S. areira
trees with different chemical profile. EOs were obtained
by distillation of dried leaves and fruits and their chemical composition were determined by gas chromatography
(GC)/FID and GC/MS as previously described [6]. A microplate bioassay for quick and sensitive determination of
antibacterial activity was used [7]. The antibacterial ac-
Eurobiotech 2013
tivity was assayed against the human pathogenic bacteria

Staphylococcus aureus ATCC 25923 and against amethicillin-resistant S. aureus strain (MRSA).
Results showed that the EO isolated from fruits and
leaves showed high antibacterial effect on S. aureus. The
inhibitory effect of both fractions on the bacterial growth
decreases with increasing dilutions, demonstrating direct proportionality between concentrations of components and the antibacterial activity. The EO of fruits have
a greater inhibitory effect than the leaves. Notably, both
the S. areira oil was able to inhibit the bacterial growth of
SAMR strain, which caused a large number of deaths in
hospitalized patients every year worldwide. The minimal
inhibition concentrations obtained for fruits and leaves
were from 3.2 L/mL to 15.0 L/mL. In conclusion, our
findings demonstrated the potential use of the oils against
Staphylococcal infections.
Acknowledgements
This research was supported by the National Agency of Scientific
and Technological Promotion, Argentina: Grant PICTO 00150 and
the National Council for Scientific and Technological Research
(CONICET), Argentina. S. M. is a member of the Research Scientific
Career from CONICET.
References
[1] Bakkali et al. (2008) Food Chem Toxicol 46 (2): 446475.
[2] Burt (2004) Int. J. Food Microbiol 94: 223253.
[3] Hosnia et al. (2011) Crops and Products 34: 16221628.
[4] Mendona Rocha et al. (2012) Molecules 17: 1202312036.
[5] Viturro C et al. (2010). Proyecto CYTED IV. 20, Edit. Universitria
da PUCRS. Chapter 10.
[6] Ojeda-Sana et al. (2013). Food Control 31: 189192.
43
P5.9
Cell wall proteins of Candida albicans
and non-albicans Candida species as the
binders for human proteins and potential
therapeutic targets
Justyna Karkowska-Kuleta1, Sylwia Kedracka-Krok2,
Karolina Seweryn1, Maria Rapala-Kozik1, Andrzej Kozik1
and Biotechnology, Department of Analytical Biochemistry;
2
and Biotechnology, Department of Physical Biochemistry
1
The development of infections caused by pathogenic fungi is inseparably connected with the specific properties
of pathogen cell surface. In the yeasts of genus Candida,
comprising a number of species pathogenic for humans,
the cell wall is composed of -1,3- and -1,6-glucans, chitin, mannoproteins anchored to the polysaccharide scaffold through various types of linkages, loosely attached
moonlighting proteins and small amount of lipids.
As is well known, the surface-connected proteins of
C. albicans, the most extensively characterized fungal
pathogen, are often involved in the adhesion to host proteins and cells, contributing to yeast virulence, whereas so
far nothing is known about the adhesins of non-albicans
Candida species, particularly two emerging pathogens
C. tropicalis and C. parapsilosis.
In this work, with the use of affinity chromatography
and tandem mass spectrometry coupled with high-performance liquid chromatography (LC-MS/MS), we were
able to isolate and identify fungal proteins responsible for
tight binding of a human serum protein, high molecular
mass kininogen (HK), a component of kinin-generating
system involved in the regulation of many physiological
and pathological processes such the coagulation and fibrinolysis, inflammation, blood pressure control, neovascularization and apoptosis.
In the case of unicellular, yeast-like forms of C. albicans,
the interaction with HK occurred mainly via the moonlighting proteins such as glycoamidase (Png2p), enolase
(Eno1p), phosphoglycerate mutase (Gpm1p) and triosephosphate isomerase (Tpi1p). In C. tropicalis and C. parapsilosis, in their most adhesive hyphal forms, the typical
adhesins, agglutinin-like sequence proteins Als3p and
Als7p, respectively, were identified as the key elements
that strongly bound HK. Because of multifunctionality of
HK, HK-binding proteins which occur on the pathogen
surface can be considered as potentially useful targets for
new therapeutic approaches.
Acknowledgements
This work was supported in part by the National Science Centre, Poland
(the grant No. 2012/07/B/NZ1/02867 to A.K.)
44
Eurobiotech 2013
P5.10
P5.11
Quercetin decreases myelosuppression

and oxidative DNA damage induced by
etoposide in bone marrow of rats
Application of immobilized
ethylbenzene dehydrogenase and
whole-cell recombinant phenylethanol
dehydrogenase system for synthesis
of chiral alcohols
Monika A. Papie
Department of Cytobiology, Faculty of Pharmacy,
Jagiellonian University Medical College, Crakow, Poland
A common side effect of etoposide is myelosuppression,

which limits the use of this anticancer drug. A few percent
of patients treated with high-dose of etoposide develop
treatment-related acute myeloid leukemia (t-AML). The
leukemogenic action of etoposide may correspond to oxidative damage to DNA in bone marrow precursor cells
caused by phenoxyl radicals of this drug. The aim of this
study was to determine the effect of antioxidant quercetin
on myelosupression and oxidative DNA damage induced
by etoposide in bone marrow cells of rats.
Brow Norway rats were treated with: 1) quercetin
(100 mg/kg bw by gavage) for two weak, 2) etoposide
(50mg/kg bw, i.p.) for three consecutive days. 3) quercetin + etoposide, and 4) vehicle for examined compounds.
The rats were decapitated two hours after the last dose of
quercetin and/or one hour after administration of etoposide. Bone marrow smears were prepared and stained
with May-Grnwald-Giemsa stain. Oxidative DNA damage was analyzed by comet assay using formamidopyrimidine DNA glycosylase, which cuts oxidised purine bases.
Etoposide administration caused a significant decrease in
the percentage of myeloid precursors and erythroid nucleated cells. Etoposide also induced significant increase
in oxidative DNA damage in comparison to the control.
Pretreatment with quercetin, followed by the coadministration of etoposide, led to significant increase in the percentage of myeloid precursors and nucleated erythroid
cells compated to group treated with etoposide alone.
Quercetin also significantly diminished the level of oxidative DNA damage induced by etoposide. The obtained
results indicate that quercetin could be a useful object in
the complementary treatment.
Maciej Szaleniec1, Agnieszka Dudzik1, Mateusz Tataruch1,

Wojciech Snoch1, Joanna Opaliska-Piskorz1, Jolanta Bryjak2,
Magorzata Witko1, Johann Heider3
Jerzy Haber Institute of Catalysis and Surface Chemistry PAS,
Niezapomianjek 8 St., 30-239 Crakow, Poland; 2Department
of Bioinorganic Chemistry, Wroclaw Technical University,
Wybrzeze Wyspianskiego 27, 50-370 Wroclaw, Poland;
3
Laboratory of Microbial Biochemistry, Philipps-University
of Marburg, Karl-von-Frisch Strasse 8, D-3504 Marburg,
Germany
1
Chiral building blocks are valuable synthons for the

preparation of biologically active compounds. They find
its application as precursors of a range of pharmaceutically active compounds (e.g. (S)-fluoxetine, duloxetine).
Up to date two biocatalytic strategies found its way into
industry: kinetic resolution of alcohol racemates (particularly with use of lipases) and stereoselective reduction of
prochiral carbonyl compounds (catalyzed by alcohol dehydrogenases). However, both strategies have their own
weakness such as the maximum 50% yield for the former
or a need to recover NADH/NADPH cofactor and problem of reduction process reversibility in case of the latter.
The reversibility of ketones reduction process results with
establishing of an equilibrium which decreases the final
alcohol yield especially for compounds more thermodynamically stable in the keto form than in the alcohol
form.
Fortunately, the synthesis of chiral alcohols can also be
achieved with the stereospecific hydroxylation of achiral
hydrocarbons. The oxidation reaction is thermodynamically irreversible, which removes limitation imposed by
reaction reversibility. Such reaction can be catalyzed by
ethylbenzene dehydrogenase (EBDH), a molybdenum
bacterial enzyme stereoselectively hydroxylating more
than 30 different ethylbenzene derivatives [1]. The enzyme immobilized on a functionalized solid support in
the optimal reaction conditions can carry on conversion
of alkylaromatic and alkylheterocyclic compounds to
their secondary (S) alcohols and is able to achieve substantial substrate conversions. Moreover, the enzyme immobilization enables an easy separation of the biocatalyst
from the reaction media.
The performance of EBDH will be compared with an
outstanding alcohol dehydrogenase, phenylethanol dehydrogenase (PEDH) [2], that is overexpressed in E.coli. PEDH immobilized in bacterial cells can be directly used in batch reactors as a biocatalysts. Its amazing
tolerance for organic solvents, wide substrate spectrum
and resistance to deactivation makes PEDH an ideal industry catalyst.
Eurobiotech 2013
The presentation will compare these two complementary

approaches to synthesis of chiral alcohols and will discuss
strong and weak points of each method in light of their
potential for application in fine chemical industry.
Acknowledgements
Authors acknowledge financial support of the project
Biotransformations for pharmaceutical and cosmetics industry No.
POIG.01.03.01-00-158/09-04.
References
[1] Knack D., Hagel C., Szaleniec M., Dudzik A., Salwinski A., Heider J.,
Appl. Environ. Microb., 78 (2012) 64756482.
[2] Hffken H.W., Duong M., Friedrich T., Breuer M., Hauer B.,
Reinhardt R., Rabus R., Heider J., Biochemistry, 45 (2006) 82.
45
P5.12
Studies on sintering process
of bone-derived hydroxyapatite
Dagmara Malina, Kamila Biernat, Agnieszka Sobczak-Kupiec
Institute of Inorganic Chemistry and Technology,
Crakow University of Technology, 24 Warszawska St.,
Being the main inorganic constitution of hard tissues

(bone and teeth), calcium phosphates have been attractive in medical and dental applications in hard tissue
repair. Hydroxyapatite (HAp) ceramics have attracted
attention because of their excellent osteoconductive and
bioactive properties. Such materials are biocompatible,
bioactive, osteoconductive, nontoxic, noninflammatory
and have an ability to form strong bonds with the living
hard tissue. Although hydroxyapatite exhibits excellent
biological properties in tissue environment, possibilities
of HAp applications are limited due to its poor mechanical properties. Thermal treatment is one of many routes
to improve mechanical parameters of hydroxyapatite.
The aim of this research was to investigate the sintering
process of different hydroxyapatite materials. The HAp
derived from animal bones was calcined for 3 h in different temperatures (700C, 800C and 900C respectively)
in order to obtain three different types of hydroxyapatite
powder. HAp powders were then uniaxially pressed and
sintered at different temperatures ranging from 800C to
1400C. The physical properties of as-produced sintered
bodies and raw HAp powders were investigated.
The results show that there is a difference in sintering
behavior of natural hydroxyapatites depending on temperature. The main differences refer to the loss of mass,
shrinkage, changes in porosity and density of the investigated materials.
Acknowledgements
The research was supported by The National Centre for Science in
Poland.
46
Eurobiotech 2013
P5.13
P5.14
Sugar analogs as a novel therapeutic

agents against cancer
Fluorometric assay for screening

the new multidrug-resistant strains
of Pseudomonas aeruginosa with efflux
pumps overexpression
Anna Czubatka1, Pawel Tokarz2, Zbigniew Witczak3,

Tomasz Poplawski1
Department of Molecular Genetics, University of Lodz,
Lodz, 90-236, Poland; 2Department of Organic Chemistry,
University of Lodz, Lodz, 91-403, Poland; 3Department
of Pharmaceutical Sciences, Nesbitt School of Pharmacy,
Wilkes University, Wilkes-Barre, PA 18766, USA
1
Understanding the biological function of sugars that

occur naturally in cells resulted in the development of
new ways of research aimed at the use of carbohydrates
as drugs in the therapy of various human pathologies.
Further development of carbohydrate chemistry synthesis requires the development of new compounds of this
group and analysis of their properties. Small alterations
in chemical structures of the sugars functional group(s)
or in a sugars ring can change their chemical character
or/and their biological activity. For example sugars after
the introduction of the sulfur atom into their molecule
have a significant smaller susceptibility into chemical and
enzymatic degradation. Thats why sugar analogs could
be valuable compounds used in the therapeutic strategies
for human diseases including cancer.
The aim of this study was to investigate the effect of sugar analogs: 6-thio--D-fructopyranose, 5-thio-D-glucose
and 1-epoxy-myo-inositol on two chosen cancer cell
lines: LoVo (human colon adenocarcinoma cell line) and
A549 (human lung adenocarcinoma epithelial cell line).
Potential cytotoxic properties of sugar analoges were
tested through incubation specific cell line with a chosen
sugar analog and then determining the number of living
cells by using colorimetric assay Cell Counting Kit-8
(CCK-8). This assay showed that all tested sugar analogs caused a decrease in cancer cells viability between
2080% at a concentration of 8mM, depending on the
compound. Comet assay in various versions reveal genotoxic potential of sugar analogs. As we found our tested
compounds induced broad spectrum of DNA damage by
introduction single and double strand breaks, alkali-labile sites and oxidative modifications into DNA. Comet
assay also shown increase of the number of DNA damage
generated in the cell lines with increasing time of incubation with studied sugars. But the cancer cell lines were
able to repair more than 50% DNA damages in 120 min.
The results suggest that the sugar analogs may act as
acyto- and genotoxic agents on cancer cells. It is possible
that sugar derivatives will be a novel therapeutic agents
against various types of cancer.
Acknowledgements
This work was supported by the The National Science Centre, decision
No. DEC-2011/01/B/NZ4/03391.
Gniewomir Latacz, Zuzanna Rowiska, Anna Matys,

Katarzyna Kie-Kononowicz
Jagiellonian University Medical College, Crakow, Medyczna 9,
Pseudomonas aeruginosa is a Gram-negative bacteria

which is responsible for 10% of all hospital-acquired
(nosocomial) infections. It is now well understood that
nosocomial infections caused by those organisms are often hard to treat because of the constitutive expression
of AmpC -lactamase and efflux pumps, combined with
a low permeability of the outer membrane (intrinsic resistance of the species). Additionally, the bacterial mutations leading to overexpression of Resistance Nodulation
Division (RND) efflux pumps eliminating several classes
of antibiotics may cause one of the major antibiotic resistance mechanisms in Pseudomonas.
The previously reported fluorescence agar-based method was shown to be simple and useful for the detection
of efflux activity among multidrug-resistant Gram-negative and Gram-positive clinical isolates [1][2]. Within
this study we modified this method by using fluorescence plate reader to determinate fluorescent dye acridine orange (AO) retention in bacterial cells. Control
and clinical Pseudomonas aeruginosa isolates were cultured overnight in Mueller-Hinton Bulion (MHB) broth
with the presence of AO. The OD of the cultures was
adjusted to the value 0,6. The fluorescence of cultures
as well as media were measured and RFU were calculated. Internal fluorescence of each bacterial strain were
also measured in MHB broth without AO to estimate
the presence of pyoverdine the yellow-green and fluorescent pigment secreted by some Pseudomonas species.
Additionally, the effect of efflux pump inhibitor PAN
on control and clinical strains was examined at the concentration of 0,1 mM.
The method was applied to one control strain ATCC
27853 and two clinical MDR Pseudomonas strains with
described resistance on several classes of antibiotics:
P4600/01/09 (named P1) and P3768/03/08 (named
P2). We observed that the clinical strains P1 and P2
showed generally lower RFU value after incubation
with AO in compare to control ATCC 27853. It suggests lower retention of fluorescent dye AO and higher efflux activity of P1 and P2 strains. The increased
RFU value of P1 (increased AO retention) was also
observed after incubation with efflux pomp inhibitor
PAN. The high effect of PAN on ATCC 27853 strain
was also confirmed.
Acknowledgements
The work was partly supported by UJ CM grant: K/DSC/001407.
Eurobiotech 2013
References
[1] Martins A., Amaral L. (2012) Screening for Efflux Pump Systems
of Bacteria by the New Acridine Orange Agar Method, In vivo 26:
203206.
[2] Martins M., Viveiros M., Couto I., Costa S.S., Pacheco T., Fanning S.,
Pags J.-M., Amaral L. (2011) Identification of Efflux Pump-mediated
Multidrug-resistant Bacteria by the Ethidium Bromide-agar Cartwheel
Method, In vivo 25: 171178.
47
P5.15
Production of triterpenoids with cell
and tissue cultures
Magdalena Malinowska, Elbieta Sikora, Jan Ogonowski
Crakow Univeristy of Technology
Triterpenes are group of biologically active compounds

which can be found mainly in resins or oils of various
plants. The compounds structure is based on isoprene
units and there are about 30,000 identified compounds.
The biological activity of triterpens are very diverse
and many studies have already confirmed the following
therapeutic effects: anti-inflammatory, analgesic, antimicrobial, antiviral, antimycotic, immunomodulatory
and hepatoprotective [1]. Synthesis of triterpenes is often quite problematic and it requires many complicated
stages and hard reaction conditions. These compounds
can be also extracted from plant material, however the
process very often does not meet all the pharmaceutical
requirements.
As an alternative, triterpens biosynthesis can be performed by enzymatic and chemical reactions, whereas
biotransformation is generally more specific and eliminates unwanted side products and the molecule alterations. Furthermore, modification of the triterpenes
structure to enhance their pharmaceutical application
can be efficiently carried out using biotransformation
processes. These kind of synthesis processes use microorganisms or isolated enzymes. Apart from that, plant
cell and tissue cultures as methods of the triterpen compounds production are also popular.. The biotechnological methods eliminate some disadvantages of classic
methods, such as extract variability and instability of
the obtained compound, moreover they ensure high reproducibility, optimal regio- and enantioselectivity and
the environmentally friendly reaction conditions [24].
Sometime the microbial transformation is the only way
to obtain desired product. During the last years, in the literature there are several studies describing the microbial
transformation as a useful tool to improve the structural
diversity of triterpenoids [5, 6]. The attention is mainly
paid on looking for an optimal organism. The most widely used technique is still classical screening of a series
of microbial strains. The studies on triterpene biotransformation give various information of new synthesized
compounds and let predict the metabolism mechanism
of the compounds [7]. It should be pointed out that plant
in vitro production of biologically active metabolites are
cultivated in bioreactors under sterile conditions, due to
the substances are completely insulated from adverse environmental factors, both biotic (e.g., diseases and herbivores) and abiotic [8, 9]. The disadvantage of the mentioned method is that the amounts of desired metabolites
are often lower than the contents in plants [10]. The strategies to enhance the yields of the metabolites in plant in
vitro cultures include media and hormone optimization,
48
Eurobiotech 2013
improvement of bioreactor designs and operation modes

and the use of various techniques such as immobilization
and genetic modification [1113].
References
[1] Dzubak P., Hajduch M., Vydra D., Hustova A., Kvasnica M.,
Biedermann D., Nat Prod Rep, 2006, 23, 394.
[2] Mufflera K., Leipolda D., Schellera M.C., Haasb C., Steingroewerb
J., Bleyb T., Neuhausc H.E., Miratad M.A., Schraderd J., Ulbera R.,
Process Biochem., 2011, 46, 11.
[3] Cheng Z.-H., Yu B.-Y., Cordell G.A., Qiu S.-X., Org Lett, 2004, 6,
3163.
[4] Chen Q.-H., Liu J., Zhang H.-F., He G.-Q., Fu M.-L., Enzyme
Microb. Tech., 2009, 45, 175.
[5] Qian L.-W., Zhang J., Liu J.-H., Yu B.-Y., Tetrahedron Lett, 2009,
50, 2193.
[6] Parra A., Rivas F., Garcia-Granados A., Martinez A., Mini-Rev. Org.
Chem., 2009, 6, 307.
[7] Chatterjee P., Kouzi S.A., Pezzuto J.M., Hamann M.T., Appl Environ
Microb, 2000, 66, 3850.
[8] Vanisree M., Lee C.Y., Lo S.F., Nalawade S.M., Lin C.Y., Tsay H.S.,
Bot Bull Acad Sin, 2004, 42, 1.
[9] Ramachandra Rao S., Ravishankar G.A., Biotechnol Adv., 2002, 20,
101.
[10] Wink M., Charlwood B.V., Rhodes M.J.C. (Eds.), Clarendon Press,
Oxford, 1990.
[11] Pavlov A., Georgiev M., Bley T., Z Naturforsch C, 2007, 62, 439.
[12] Drnenburg H., Process Biochem, 2004, 39, 1369.
[13] Lee M., Jeong J., Seo J., Shin C., Kim Y., J. In, Plant Cell Physiol,
2004, 45, 976.
P5.16
Determination of antioxidant activity of
black tea extract
Magdalena Malinowska, Kamil Kurleto, Grzegorz Kurowski,
Barbara Laskowska, Elbieta Sikora, Otmar Vogt
Crakow Univeristy of Technology
Tea has been consumed all over the World for over
two thousand years and now it is the most popular caffeine-containing beverage. The first written records of the
tea can be found in VIII century BC, where a brews of the
tea leaves were treated as a medicine. Over the centuries,
tea plantations have been established and the methods of
tea collection and processing have been improved [14].
The tea is not only important because of its popularity
but also due to its beneficial influence on human health.
Therapeutic effects of tea have been extensively examined
in many in vitro and in vivo studies. It was confirmed
that tea leaves ingredients has antibacterial, antifungial,
antiviral properties, they also prevent cell mutations and
they inhibit progress of heart diseases. Moreover, tea can
stimulate neural system and regulate its functions. Positive effect of the tea drinking is associated mainly with
high content of polyphenols, mainly catechins that act as
an antioxidants. Their amount in tea infusion depends
not only on the type of tea, but also on brewing process
[519].
The subject of this work was to investigate the influence
of the brewing conditions: temperature, time and degree
of leaf fragmentation, on antioxidant activity of black tea.
The total antioxidant activity (TAA) of the tea infusions
was studied using three different test methods: DPPH
free radical reduction, Folin-Ciocalteu and thiocyanate.
UV-VIS spectrophotometer was applied to measure the
absorbance of the samples.
The obtained results showed that brewing conditions
have a significant effect on the TAA of the black tea infusions. The brewing temperature is the most important parameter influencing on polyphenols content in black tea
infusions.. Results of all three applied methods showed
that the highest antioxidant activity was characterized
the infusions obtained at the 90 and 95C. Brewing time
also determines TAA of the tea infusions and the optimal
brewing time was 5 to 10 minutes. Recommended 23
minutes of brewing time is not sufficient to obtain the
maximum antioxidant activity of the tea extracts. Also
the leaf fragmentation has a positive impact on TAA of
the tea infusions.
References
[1] uczaj W., Skrzydlewska E., Prev. Med., 2005, 40, 910.
[2] Bykbalci A., Sedef Nehir E., Plant Foods Hum Nutr., 2008, 63, 27.
[3] Caprari M., Herbata, Warszawa 2009.
[4] Sharangi A.B., Food Res. Intern., 2009, 42, 529.
[5] Gramza A., Korczak J., Amarowicz R., Pol. J. Food Nutr. Sci., 2005,
3, 219.
[6] Wei K., Wang L., Zhou J., He W., Zeng J., Jiang Y., Cheng H., Food
Chem., 2011, 125, 44.
[7] Leung L.K., Su Y., Chen R., Zhang Z., Huang Y., Chen Z.Y., J. Nutr.,
2001, 131, 2248.
Eurobiotech 2013
[8] Wright L.P., Biochemical analysis for identification of quality in
black tea, University of Pretoria, Pretoria, 2002.
[9] Friedman M., Mol. Nutr. Food Res., 2007, 51, 116.
[10] Davies M.J., Judd J.T., Baer D.J., Clevidence B.A., Paul D.R.,
Edwards A.J., Wiseman S.A., Muesing R.A., Chen S.C., J. Nutr., 2003,
133, 3298.
[11] Ferrazzano G.F., Amato I., Ingenito A., de Natale A., Pollio A.,
Fitoterapia, 2009, 80, 255.
[12] Robak J., Gryglewski R.J., Pol. J. Pharmacol., 1996, 48, 555.
[13] Saha P., Das S., Asian Pac. J. Cancer Prev., 2002, 3, 225.
[14] Cicho Z., Miniakiewicz M., Zesz. Nauk. AE Krak., 2005, 678,
103.
[15] Yang D.J., Hwang L.S., Lin J.T., J. Chromatogr. A, 2007, 1156, 312.
[16] Gramaza A., Korczak J., Trends Food Sci. Tech., 2005, 16, 351.
[17] Thanaraj S.N., Seshardi R., J. Sci. Food Agric., 1990, 51, 57.
[18] Wei K., Wang L., Zhou J., He W., Zeng J., Jiang Y., Cheng H., Food
Chem., 2011, 125, 44.
[19] Obanda M., Owuor P.O., Mangoka R., Food Chem., 2004, 85, 163.
49
P5.17
Homology Modeling of Steroid C25
Dehydrogenase
Agnieszka Rugor1, Stefan Mordarski2, Jakub Staro2,
Andrzej Bojarski2, Maciej Szaleniec1
Jerzy Haber Institute of Catalysis and Surface Chemistry, PAS,
Niezapominajek 8, 30-239 Crakow, Poland;
2
Instytute of Pharmacology, PAS, Smetna 12, 31-343 Crakow,
Poland
1
Steroid C-25 dehydrogenase (S25DH), a new EBDH-like

molybdenum enzyme isolated from denitrifying Sterolibacterium denitrificans Chol-1ST [1], catalyzes regioselective hydroxylation at the C-25 tertiary carbon atom
of the aliphatic side chain in cholesterol and its derivatives [2]. Up to date the structure of the enzyme is still
unknown. However, the S25DH amino acids sequence is
highly similar to other enzymes from DMSO reductase
family such as ethylbenzene dehydrogenase (EBDH, PDB
code 2IVF) from Aromaticum aromatoleum EbN1 [3]
(identity 40%, similarity 96%).
Based on the sequence and topological similarities of the
active site, the homology model of S25DH was generated
with MODELLER 9v7. The best homology structure was
selected based on the induced fit docking results of the
known S25DH substrates (cholest-4-en-3-one, cholest4,6-dien-3-one, choles1,4-dien-3one, cholest-5-en-3-ol,
cholesteryl hemisuccinate and cholesteryl sulphate). The
docking results allowed detailed analysis of interactions
between amino acid residues and substrates. It also provided an insight into the active site structural features
which can be used in the elucidation of S25DH reaction
mechanism. The results suggest a strong vdW stabilization of the sterane ring system of the substrate as well as
indicated involvement of the Asn531 residue in electrostatic interaction with C3 oxo/hydroxyl substituent of the
substrate.
Moreover, the structural analysis of the MGD binding
mode showed strong electrostatic and van der Waals
interactions of the cofactor with a range of highly conserved residues, both in case of S25DH homology model
and the EBDH. This results suggests that the structural
features surrounding the molybdenum cofactor were correctly modelled by our protocol.
Acknowledgements
The authors acknowledge the financial support of the National Center
of Science under the SONATA grant UMO-2012/05/D/ST4/00277
The mechanism of regioselective oxidation of cholesterol derivatives
by a novel molybdenum enzyme, steroid 25-OH dehydrogenase from
Stereolibacterium denitrificans.
References
[1] Dermer J., Fuchs G., J. Biol. Chem. (2012) 287, 3690536916.
[2] Chiang Y. et al., J. Bacteriology (2008) 190, 905914.
[3] Szaleniec M. et al., Biochemistry (2007) 46, 7637764.
50
Eurobiotech 2013
P5.18
P5.19
Bacterial steroid C-25 dehydrogenase

a novel biocatalyst for regioselective
hydroxylation of steroid compounds
Screening of porous carriers for covalent

immobilization of pepsin and laccase
Natalia Zawada, Agnieszka Rugor, Mateusz Tataruch,

Maciej Szaleniec, Daniel Knack
Jerzy Haber Institute of Catalysis and Surface Chemistry, PAS,
Niezapominajek 8, 30-239 Crakow, Poland
Steroid C25 dehydrogenase (S25DH) from Sterolibacterium denitrificans is a member of the DMSO reductase
family of molybdenum enzymes with a bis-MGD cofactor in its active site. It catalyzes the oxygen independent
hydroxylation of the tertiary C25 atom of the side chain
of cholesterol and other steroid compounds to the respective tertiary alcohols [1].The ability of S25DH to introduce hydroxyl groups into the C25 atom of cholesterol
and its derivatives has the potential for application in the
production of pharmaceuticals such as activated vitamin
D3 or 25-hydroxycholesterol [2].
Up to date, S25DH was purified by a multi-step anaerobic
protocol developed by Dermer and Fuchs. However, it has
been shown that EBDH-like enzymes can be stabilized in
aerobic conditions by addition of a suitable electron acceptor (e.g. ferrocenium (III) ions) [3]. In the poster we
present the shortened anaerobic and aerobic enzyme purification procedures that delivers an enzymatic formulation with defined biological activity.
To confirm the application potential of S25DH, it was
used as a catalyst in the synthesis of 25-hydroxy-cholest4-en-3-one. The synthesis was carried out in a batch reactor system with homogenous and immobilized enzyme.
Admittedly, the S25DH purification from S. denitrificans
is still not efficient enough for industrial purposes. Therefore, the experiments aiming at the development of a heterologous expression system were undertaken. An artificial operon containing all the three S25DH subunits and
the chaperone gene was generated and the genes overexpressed in E.coli. The poster will discuss the method used
for construction of the artificial operon and its efficiency
in production of the enzyme.
Acknowledgements
The authors acknowledge the Polish National Center of Research and
Development under grant LIDER/33/147/L-3/11/NCBR/2012.
References
[1] Dermer J., Fuchs G. (2012) J. Biol. Chem. 287: 3690536916.
[2] Chiang Y.R., Ismail W., Mller M., Fuchs G. (2007) J. Biol. Chem.
282: 1324013249.
[3] Szaleniec M. et al. (2007) Biochemistry, 46 7637-764653: 10851091.
Katarzyna Szaapata1, Monika Osiska-Jaroszuk1,

Kamila Wlizo1, Jolanta Bryjak2, Anna Jarosz-Wilkoazka1
Maria Curie-Sklodowska University, Department
of Biochemistry, Lublin, Poland; 2Wroclaw University
of Technology, Department of Bioorganic Chemistry, Wroclaw,
Poland
1
Pepsin (EC 3.4.23.1) is one of the principal enzymes in

the digestive system and it belongs to the acidic protease family. Laccase (EC 1.10.3.2), a copper-containing
oxidase produced mainly by fungal strains, requires
an oxygen as one of the substrates during oxidation of
broad spectrum of substrates about different chemical
structure. Thanks to catalytic activity of these enzymes
they are widely used in many industries. Their potential
applications, for example in food, textile or biomedical
industries, become more common and replace many of
the traditional methods. This situation makes it necessary
to increase the stability of biocatalysts in order to higher
profitability of their use, which is possible thanks to enzyme immobilization.
In this work pepsin from hog stomach (Fluka) and laccase (Cerrena unicolor) were covalent immobilized onto
typical enzyme carriers. Controlled pore glass, acrylic
beads, silica-gel and cellulose-based carriers were functionalized by the presence of various chemical groups
(NH2, COOH and OH). We used three different
cross-linkers for their activation: glutaraldehyde, carbodiimide and divinyl sulphone. After the immobilization
we tested the storage stability for each carrier. For the immobilized pepsin the stability under different conditions
of pH and temperature were also measured. The results
obtained in this experiment allowed to select the optimal
carrier for each enzyme.
Eurobiotech 2013
51
P5.20
P5.21
Poly(AA-co-MMA) micro- and nanoparticles

for controlled drug delivery
Effect of conjugated linoleic acid (CLA)

on serum lipid profile and markers of liver
function in rats fed different dietary fat
sources
Katarzyna Bialik-Ws, Boena Tyliszczak,

Krzysztof Pielichowski
Department of Chemistry and Technology of Polymers,
The modern medicine and pharmacy require more and

improved drug delivery systems targeting only on the
areas covered by the diseases. Such conditions can fulfill
the micro-and nanoparticles based on polymers, pH-sensitive hydrogels, biopolymer based microgels/nanogels
(biomicrogels/bionanogels). Furthermore, polymer and
copolymer nanoparticles are characterized by high stability in contact with biological fluids [13]. The controlled
drug delivery systems are truly important in treatment
of human diseases due to their therapeutic advantages in
improving bioavailability and minimizing systemic side
effects [4].
In this study, poly(AA-co-MMA) micro- and nanoparticles with feed molar ratios AA:MMA = 7:3 were used for
metronidazole loading. Metronidazole (MET) 2-(5-nitro-2-methylimidazol-1-yl)-ethanol is very well known
antimicrobial agent and commonly used in clinical medicine for > 45 years. Mainly it is applied for anaerobic
infections and for treatment of giardiasis, trichomoniasis and amoebiasis [5,6]. The reaction for preparation of
poly(AA-co-MMA) micro- and nanoparticles was carried out at 80C under nitrogen for 8h, whereas MET was
dispersed in distilled water and mixed with micro- and
nanoparticles as an aqueous suspensions. The complexation was conducted for 24h at room temperature.
The drug release behavior of MET-loaded poly(AA-coMMA) micro- and nanoparticles was evaluated in water and phosphate buffered saline (PBS, 0,9% NaCl) at
37C. Introduction of MET into poly (AA-co-MMA)
micro-and nanoparticles allow to gradual and controlled
release of the active substance. Furthermore, structural
analysis using FT-IR (ATR) and 1H NMR, as well as surface morphology assessment by SEM, were performed.
Acknowledgements
This research was supported by Ministry of Science and Higher
Education project C-4/257/2013/DS-M.
References
[1] Bajpai A.K., Shukla S.K., Bhanu S., Kankane S., Prog Polym Sci 33
(2008) 10881118.
[2] Liu Z., Jiao Y., Wang Y., Zhou C., Zhang Z., Adv Drug Deliv Rev 60
(2008) 165062.
[3] Agnihotri S.A., Mallikarjuna N.N., Aminabhavi T.M., J Control
Release 100 (2004) 528.
[4] Nafea E.H., Marson A., Poole-Warren L.A., Martens P.J., J Control
Release 154 (2011) 110122.
[5] Lofmark S., Edlund Ch., Nord C. E., Clin Infect Dis 50 (2010) 1623.
[6] Herculano R.D., Alencar de Queiroz A. A., Kinoshita A., Oliveira
O.N. Jr., Graeff C.F.O., Mat Sci Eng C 31 (2011) 272275.
Magdalena Franczyk-arw1, Edyta Malak2,

Renata B. Kostogrys1
Department of Human Nutrition, Faculty of Food Technology,
Agricultural University of Crakow, Balicka 122,
30-149 Crakow, Poland; 2Jagiellonian Centre for Experimental
Therapeutics (JCET), Jagiellonian University, Bobrzynskiego 14,
Crakow, Poland
1
Over the years, various components of dietary fat have

been studied to examine their effect on serum lipids. According to knowledge about oil, butter and margarine
consumption on human health, the current study was designed to determine the effects of conjugated linoleic acid
(CLA) on serum lipid profile and markers of liver function as well as oxidative stress markers concentrations in
rats fed different dietary fat sources.
Thirty six male Wistar rats were divided into six groups
(n = 6) and fed the following diets: control AIN-93G diet
contained soybean oil (O) and diets with modification
of fat source: butter (B) and margarine (M). The experimental diets were supplemented with 1% of conjugated
linoleic acid (O+CLA, B+CLA, M+CLA). After 21 days
the blood was collected and lipid profile, glucose, liver
enzymes (AST and ALT), malonic dialdehyde (MDA) as
well as lipid hydroperoxide (LPO) were analyzed.
The dietary treatments had no effect on body weight
of animals. Whereas, liver weight was significantly increased in B+CLA and M+CLA groups compared to
control (O). Total cholesterol (TCh), LDL+VLDL cholesterol, MDA and AST concentrations did not differ significantly between experimental groups. Concentration
of HDL cholesterol was significantly increased only in B
and M groups compared to O. However, triacylglycerol
(TAG) level significantly increased in animals fed M diet
and decreased after CLA supplementation (M+CLA).
Concentration of plasma glucose was slightly decreased
in all CLA groups, but significant difference was observed
in rats fed O+CLA diet compared to O group. Serum ALT
levels were increased in all experimental groups, but significant differences were observed only in B+CLA and
M+CLA groups compared to O group. Nethertheless, lipid hydroperoxide (LPO) level was significantly increased
in all CLA groups.
In conclusion, CLA supplementation in margarine fed
rats shows positive effect on TAG and glucose concentration. Moreover, in rats fed M and B diets, HDL level was
increased.
Acknowledgements
This work was supported by the subsidy grant for Young Scientists
from the resources of the Agricultural University (grant No.
BM-4738/KCz/2013).
52
Eurobiotech 2013
P5.22
P5.23
Novel class of primosomal protein B

from Clostridium thermocellum
The antioxidant quercetin modifies

etoposide acton in HL-60 cells
Marta pibida, Marta Marszakowska, Marcin Olszewski
Monika Papie
Gdansk University of Technology, Department of Microbiology,

Narutowicza 11/12 St., 80-233 Gdansk, Poland
Department of Cytobiology, Faculty of Pharmacy,

PriB is a primosomal protein that catalyzes DNA replication in Procaryota. The replication pathway starts with
PriA protein the initiator protein that binds to a DNA
replication fork, unwinds double-stranded DNA and role
of PriB is to stabilize PriA on the DNA. However there are
many biochemical differences in replication mechanism
in bacteria and only some of them use PriB proteins.
A few of PriB proteins were published and only three
structures of them were resolved (Escherichia coli, Klebsiella pneumoniae and Neisseria gonorrhoeae). All upto-date known PriB proteins have one OB domain per
monomer and they are homodimers in solution.
Recently, we have published the crystal structure of PriB
protein from Thermoanaerobacter tengcongensis that
represents new class of PriB with two oligonucleotide/
oligosaccharide-binding domain (OB) per monomer that
means it exists as monomer in solution.
The aim of this study is identification and characterization
of the primosomal protein B (PriB) from an anaerobic,
thermophilic bacterium Clostridium thermocellum (CthPriB). This PriB protein consisting 238 amino acid residues and a calculated molecular mass is 27,5 kDa. What is
more CthPriB protein contains two single-stranded DNA
binding domain (OB-fold) and functions as monomer
like PriB protein from bacterium Thermoanaerobacter
tengcongensis.Therefore, our studies suggest that we discovered new classes of PriB which werent published recently.
The cells possessing high activity of myelopeoxidase

(MPO) are particularly sensitive to the compounds which
increase oxidative stress, such as etoposide. The aim of
this study was to determine the effect of antioxidant quercetin, on the activity of etoposide in HL-60 cell line showing constitutively high MPO activity.
HL-60 cells were incubated for 24 hours in the presence
of etoposide and/or quercetin at concentrations range
1100 uM. Apoptosis was dectected using annexin V
(Becton Dickinson) and propidium iodide staining. The
level of free radicals was measured using the CellRox kit
(Life Technologies). The cells were analyzed by flow cytometry.
Etoposide increased apoptosis of HL-60 cells in a dose-dependent manner. This anticancer drug at lower concentrations of 520 uM increased the level of ROS, whereas
higher concentrations of this drug showed antioxidant
activity. Quercetin reduced ROS generated by low concentrations of etoposide. The investigated polyphenol at
concentrations of 110 uM protected HL-60 cells against
apoptosis induced by etoposide. However, the amount of
apoptotic cells was significantly higher after treatment
with both quercetin and etoposide in comparison to the
control. Higher concentrations of quercetin increased
apoptosis induced by etoposide.
The observed influence of quercetin and etoposide is
dose-depend. Quercetin modifies the activity of etoposide in myeloid leukemia cells with high MPO activity by
antioxidant property.
Acknowledgements
This study was supported by the Ministry of Science and Higher
Education (K/ZDS/003318).
Eurobiotech 2013
P5.24
Salvia lavandulifolia from spain: aromatic
profile by enantioselective gas
chromatography-mass spectrometry
Ana Belen Cutillas5, Alejandro Carrasco1, Vanessa Ortiz1,
Ramiro Martinez-Gutierrez3, Francisco Javier Martinez4,
Mariano Sanchez5, Virginia Tomas2, Jose Tudela1
GENZ-Grupo de Investigacion Enzimologia (www.um.es/genz),
Departamento de Bioquimica y Biologia Molecular-A, Campus
de Excelencia Internacional Regional Campus Mare Nostrum,
Universidad de Murcia, Murcia, Spain; 2Departamento
de Quimica Analitica, Universidad de Murcia; 3NOVOZYMES
SPAIN S.A. (www.novozymes.com); 4Esencias Martinez-Lozano
S.A. (www.esenciaslozano.com); 5Europermanent S.L.
1
Objectives. The identification and determination of biochemicals of the essential oil of Salvia lavandulifolia,
grown from organic farming in Murcia (Spain). The oil
has been eco-extracted, by steam distillation with a portable still, next to cultivation, and with a boiler feed with
biomass, from plants previously distilled.
Methodology (www.um.es/genz/e00605/serv03.htm).
Fast Gas Chromatography on a non-polar fast column
(SLB-5ms) 15 m 0.1 mm 0.1 m, and Enantioselective
Gas Chromatography on a chiral column Chiraldex
B-DM 30m x 0.25 mm 0.12 m, were carried out on
a GC-chromatograph (Agilent 7890), with hydrogen as
gas carrier (PDH). The Mass Spectrometry Detector used
an electronic impact ionizer (70 eV), and a single quadrupole analyzer (Agilent 5975 MSD). Sandwich split/
splitless injections, with hexadecane as inner standard,
and calibration straigths with standards (Gerstel MPS2XT autosampler).
Results. The chromatographic results showed that the
S. lavandulifolia oil from Murcia is especially rich in
some biomolecules like (mM concentration, % enantiomers): Camphor (4068, +76, 24), Eucalyptol (1299),
Camphene (811, +26, 74), -Pinene (483, +52, 48),
-Pinene (365, +42, 58), Limonene (333, +81, 19),
Borneol (319, +34, 66), Myrcene (233), Linalool
(142, +4, 96), Linalyl acetate (138), -Terpineol
(96, +8, 92), -Terpinyl acetate (66, +6, 94), Bornyl acetate (61, +4, 96), and other minor components.
Conclusions. Salvia lavandulifolia oil has a very high
concentration of Camphor, Eucalyptol and Camphene,
about 1020 times higher than that of many other
biomolecules. Near pure ()-enantiomers are present for Linalool, -Terpineol, -Terpinyl acetate and
Bornyl acetate. There are mainly (+)-Camphor and
(+)-Limonene, whereas there are mainly ()-Camphene
and ()-Borneol. Furthermore, -Pinene and -Pinene
have similar proportions of both enantiomers. This biochemotype is markedly different than that of Salvia lavandulifolia oil, with similar cultivation and extraction
procedures in Castilla-La Mancha (Spain). The essential
oil from Murcia has higher concentrations than that
stated in the corresponding ISO standard for Camphor,
Eucalyptol and Limonene. This oil is a good source of
53
biomolecules for pharmaceuticals, cosmetics, fragrances

and food industries.
Acknowledgements
This work has been partially supported by grants from several Spanish
organizations. Projects BIO2009-12956 (MINECO, Madrid) and
08856/PI/08 (Fundacin Seneca, CARM, Murcia). AC has a fellowship
from Esencias Martinez Lozano S.A. (Murcia). VO has a FPU fellowship
(AP2010-4300).
54
Eurobiotech 2013
P5.25
P5.26
Permeation of iodine from iodine-enriched

yeast through porcine intestine
Novel class of primosomal protein B

from Clostridium thermocellum
Florian Ryszka1, Barbara Doliska1, 2, Micha Zieliski1, 2,

Dagmara Chyra1, Zbigniew Dobrzaski3
Marta pibida, Marta Marszakowska, Marcin Olszewski
Pharmaceutical Research and Production Plant Biochefa,

Sosnowiec, Poland; 2Department of Applied Pharmacy and
Drug Technology, Medical University of Silesia, Sosnowiec,
Poland; 3Department of Animal Hygiene and Animal Welfare,
Wroclaw University of Environmental and Life Sciences,
Wroclaw, Poland
1
Iodine deficiency is a common phenomenon, threatening

the whole global human population. Recommended daily
intake of iodine is 150 g for adults and 250 g for pregnant and breastfeeding women. About 50% of human
population can be at risk of moderate iodine deficiency.
Due to this fact increased iodine supplementation is recommended, through intake of iodized mineral water and
salt iodization.
The aim of this study was to investigate permeation and
absorption of iodine from iodine bioplex (experimental
group) in comparison with potassium iodide (controls).
Permeation and absorption processes were investigated
in vitro using a porcine intestine. The experimental model was based on a standard Franz Diffusion Cell(FD-Cell).
The iodine bioplex was produced using Saccharomyces cerevisiae yeast and whey powder: iodine content
388g/g, total protein 28,5%, total fat 0,9%., glutamic
acid 41,2%, asparaginic acid 29,4%, lysine 24,8%;
purchased from: F.Z.N.P. Biochefa, Sosnowiec, Poland.
Potassium iodide was used as controls, at 388 g iodine
concentration, which was the same as in iodine-enriched
yeast bioplex.
A statistically significant increase in iodine permeation
was observed for iodine-enriched yeast bioplex in comparison with controls potassium iodide.
After 5h the total amount of permeated iodine from iodine-enriched yeast bioplex was 85%, which is ~ 2-fold
higher than controls 37%.
Iodine absorption was by contrast statistically significantly higher in controls 7,3%, in comparison with 4,5% in
experimental group with iodine-enriched yeast bioplex.
Presented results show that iodine permeation process
dominates over absorption in case of iodine-enriched
yeast bioplex.
Acknowledgements
Original article with the same title will be submitted to Acta Biochimica
Polonica. Submitted abstract and article are a part of Prof. Florian
Ryszkas contribution.
Gdansk University of Technology, Department of Microbiology,

Narutowicza 11/12 St., 80-233 Gdansk, Poland
PriB is a primosomal protein that catalyzes DNA replication in Procaryota. The replication pathway starts with
PriA protein the initiator protein that binds to a DNA
replication fork, unwinds double-stranded DNA and role
of PriB is to stabilize PriA on the DNA. However there are
many biochemical differences in replication mechanism
in bacteria and only some of them use PriB proteins.
A few of PriB proteins were published and only three
structures of them were resolved (Escherichia coli, Klebsiella pneumoniae and Neisseria gonorrhoeae). All upto-date known PriB proteins have one OB domain per
monomer and they are homodimers in solution.
Recently, we have published the crystal structure of PriB
protein from Thermoanaerobacter tengcongensis that
represents new class of PriB with two oligonucleotide/
oligosaccharide-binding domain (OB) per monomer that
means it exists as monomer in solution.
The aim of this study is identification and characterization of the primosomal protein B (PriB) from an anaerobic, thermophilic bacterium Clostridium thermocellum
(CthPriB). This PriB protein consisting 238 amino acid
residues and a calculated molecular mass is 27,5 kDa.
What is more CthPriB protein contains two single-stranded DNA binding domain (OB-fold) and functions as
monomer like PriB protein from bacterium Thermoanaerobacter tengcongensis.Therefore, our studies suggest
that we discovered new classes of PriB which werent
published recently.
Eurobiotech 2013
55
P5.27
P5.28
A novel DNA polymerase from

hyperthermophilic bacterium Thermovibrio
ammonificans: gene cloning, expression,
and characterization
Screening of carriers for lipase

immobilization suitable for reactions
in water, biphasic and pure organic solvent
systems
Marta Marszakowska, Sandra Zakrzewska, Marta Spibida,

Marcin Olszewski
Zofia Hrydziuszko1, Agnieszka Dmytryk1, Paulina Majewska1,

Katarzyna Szymaska2, Jolanta Liesiene3, Jolanta Bryjak1
Department of Microbiology, Faculty of Chemistry,

Gdansk University of Technology, Narutowicza 11/12 St.,
80-233 Gdansk, Poland
Thermostable DNA polymerases are one of the most

important bio-tools widely used in molecular biology.
They are able to catalyze synthesis of DNA by addition
of mononucleotide units derived from deoxynucleoside
5 triphosphates (dNTP) to the 3 hydroxyl terminus of
the template DNA under extreme conditions [1]. However, in molecular diagnostics, which is fundamental field
of modern medicine, conventional thermostable DNA
polymerases are often inhibited by chemical compounds
present in biological samples from blood or soil.
In order to find thermostable DNA polymerase that
shows novel properties we decided to conduct expression
of type A polymerase from hyperthermophilic bacterium
Thermovibrio ammonificans. This bacterium is one of
three discovered to date representants of newly described
genus Thermovibrio [2]. Gene coding Thermovibrio
DNA polymerase shows significant sequence homology
to commonly used polymerase from Thermus aquaticus
(TaqPol) [3], however it lacks 3 to 5 exonuclease domain. As a result of cloning and expression we obtained
protein with molecular weight of 99,5 kDa, which was
purified from Escherichia coli TOP10 strains expressing
the cloned genes. Now we are trying to characterize activity of Thermovibrio DNA polymerase under different
conditions (pH, temperature, inhibitors from blood and
soil).
References
[1] Lehman I.R., Discovery of DNA Polymerase, J. Biol. Chem. (2003)
278: 3473334738.
[2] Vetriani C., Speck M.D., Ellor S.V., Lutz R.A., Starovoytov V.,
Thermovibrio ammonificans sp. nov., a thermophilic, chemolitotrophic,
nitrate-ammonifying bacterium from deep-sea hydrothermal vents, Int.
J. Syst. Evol. Microbiol. (2004) 54: 175181.
[3] Huang J.-P., Ito J., DNA Polymerase C of the Thermophilic Bacterium
Thermus aquaticus: Classification and Phylogenetic Analysis of the
Family C DNA Polymerases (1999) J. Mol. Evol. 48: 756769.
Wroclaw University of Technology, Faculty of Chemistry,

Department of Bioorganic Chemistry, Poland; 2Silesian
University of Technology, Department of Chemical Engineering,
Poland; 3Kaunas University of Technology, Faculty of Chemical
Technology, Lithuania
In biotransformations, lipases encompassed a wide range

of reactions, such as esterification, transeserification (alcoholysis) and aminolysis (amide synthesis). Trans-esterification and amide synthesis are preferably performed in
anhydrous organic solvents whereas glycerolysis of commercial oils is practiced at a large scale in a solventless
system. Third system is composed of water immiscible
organic solvent and water. The choice of the system is dictated by substrate/product solubility and/or by preferred
reaction direction (synthesis over hydrolysis). Regardless
reaction system, lipases are used in immobilized form
rather to ensure biocatalyst evenly distributed suspension
in water restricted media and/or to ensure easy separation after being reused for several times in all reaction
systems.
The aim of this study was to select an appropriate enzyme-carrier preparation for carrying out hydrolysis reaction in aqueous medium or biphasic systems and transesterfication in organic solvent. For this purpose Candida
rugosa lipase (CRL) was bound by adsorption or covalent
attachment into array of carriers having different structure, porosity and kind of functional groups. Selection of
proper carrier and method of immobilization was based
on measured activity, thermal stability and operational
stability in successive batch processes.
For immobilization studies 15 carriers (silica gel (Z),
acrylic copolymer (A), cellulose beads (G), mesoporous
cellular foams (MCF)) were used. Obtained 25 enzyme-carrier preparations were tested for activity in reaction of 4-nitrophenyl palmitate hydrolysis in water. To
test thermal stability, 4 h incubation at 60oC was applied
and then the best preparations were subjected to operational stability in 20 batch processes. Selected preparations of each carrier group were used for hydrolysis of
ethyl (1-butyryloxyethyl)-phenylphosphinate in biphasic
system, and after drying (5 days, 30oC) in ethyl (1-hydroxyethyl)-phenyl-phosphinate transesterification in
organic solvent.
Activity of obtained enzyme-carrier preparations varied from 20 (G) to 5100 U/mL (MCF). The most stable
preparations were those bound by adsorption or covalent attachment to NH2-Z and COOH-A carriers (activity over 90%). The highest conversion in reaction of hydrolysis in biphasic system was obtained for NH2-MCF,
56
Eurobiotech 2013
NH2-Z and OH-A carriers. Moreover, lipase immobilized

by adsorption and covalent attachment onto NH2-Z and
OH-A carriers maintained almost 100% of initial activity
after drying and these preparations were used in transesterfication reaction. It was found that in biphasic and
non-aqueous systems hyperactivation took place. Taking
into account hydrolysis, similar substrate conversion at
the same reaction time was achieved when several times
higher concentration of native CRL was used than immobilized preparations. What was more, no progress in
transesterfication was observed when native CRL was
applied.
For industrial applications operational stability is more
important than enzyme activity. Taking into account reaction of hydrolysis in water, CRL covalently bound to
NH2-Z preserved 50% of initial reaction rate in 23 batch
processes. Satisfactory hydrolysis in biphasic system was
obtained for preparations with CRL bound to OH-A
(90%) or to NH2-Z (76%). In the case of transesterification, the best results were obtained when CRL was adsorbed on NH2-Z carrier. Thus, NH2-Z carrier can be
considered as an universal matrix for CRL immobilization in all reaction systems tested.
Acknowledgements
This study was supported by the project Biotransformations for
pharmaceutical and cosmetics industry No. POIG.01.03.01-00158/09-03 partly financed by the European Union within the European
Regional Development Fund.
P5.29
The effect of new hydantoin derivatives
on the increase of ciprofloxacin efficacy
in drug-resistant E. coli
Anna Matys, Ewa Otrbska, Jakub Mazurkiewicz,
Daria Studnicka, Beata Mastek, Jadwiga Handzlik,
Katarzyna Kie-Kononowicz
Jagiellonian University, Collegium Medicum, Faculty
of Pharmacy, Crakow, Poland
The aim of the study was to assess a series of hydantoin

derivatives in terms of their activity against two E. coli
strains: HEMEC 10 (a clinical drug-resistant strain overexpressing efflux pumps) and E. coli ATCC 25922 (a reference strain). The compounds were tested in combination
with ciprofloxacin to see whether they enhance its action
by exerting an inhibitory effect on efflux pumps for example. The chemical families which exhibited an interesting chemosensitizing effect in previous microbiological
assays conducted on drug-resistant E. aerogenes were selected for the study [1]. Three groups of compounds were
tested: piperazine derivatives of 5-arylidenehydantoin,
piperazine derivatives of 5-arylideneimidazolone and derivatives of 5--naphtylmethylhydantoin. The activity of
the compounds in concentrations no higher than 1/2 of
their minimum inhibitory concentrations was examined
by assessing the effect of the compounds on reduction
of minimum inhibitory concentration of ciprofloxacin
in the E. coli strains. The compounds did not show high
activity most of them reduced the MIC of ciprofloxacin only 2-4-fold. The most active compound was A10
(p-chlorobenzylidene derivative of hydantoin), which reduced the MIC of ciprofloxacin in the E. coli ATCC 25922
reference strain 32-fold. However, its effect in the E. coli
HEMEC10 strain was much lower (2-fold).
Acknowledgements
The authors wish to thank Professor Leonard Amaral and Professor
Isabel Couto (Grupo de Micobacterias, Unidade de Microbiologia,
Instituto de Higiene e Medicina Tropical, Universidade Nova de
Lisboa, Lisbon, Portugal) and dr hab. Anna Biaecka (Centrum
Bada Mikrobiologicznych i Autoszczepionek, Cracow, Poland) for
providing the strains used in this study. The study was financed from
the programme K/ZDS/001915.
References
[1] Handzlik J., Szymaska E., Alibert S., Chevalier J., Otrbska E.,
Pkala E., Pags J.-M., Kie-Kononowicz K., Search for new tools to
combat Gram-negative resistant bacteria among amine derivatives of
5-arylidenehydantoin. Bioorg. Med. Chem. 21 (2013) 135145.
Eurobiotech 2013
P5.30
Laccase activity and stability in the
presence of menthol-based ionic liquids
Joanna Feder-Kubis1, Jolanta Bryjak2
Wroclaw University of Technology, Faculty of Chemistry,
Department of Chemical Engineering; 2Wroclaw University
of Technology, Faculty of Chemistry, Department of Bioorganic
Chemistry
1
In the last years, room temperature ionic liquids (ILs)

have gained increasing importance in enzymatic catalysis and they are claimed as green alternative for volatile
organic solvents. ILs can be used in monophasic (pure
ILs or water-miscible ILs as co-solvent) or biphasic systems. ILs are viscous salts that causes strong reaction rate
reduction, thus interest is directed towards biphasic systems. Apart from better preservation of reactivity, these
systems provided better conformational stability. In the
case of reactions with oxidases, additional advantages are
easiness of oxygen dissolution and/or unchanged diffusion rates of redox species in water phase. Oxidases are
a very important group of enzymes because they use oxygen as the only oxidant without need of cofactor (most
of oxidoreductases) or hydrogen peroxide (peroxidases).
Laccases (EC 1.10.3.2) attract considerable attention due
to their potential for manufacturing pharmaceutical intermediates and specialty chemicals from a wide array of
phenolic and non-phenolic substrates that are sparingly
soluble in water.
The main aim of this work was to find ILs suitable for
Cerrena unicolor laccase. Rehmann et al. (Green Chem.
2012, 14, 725) reported that performances of Trametes
versicolor laccase depended strongly on the anion nature and showed that bis(trifluoromethanesulfonyl)imide supported the highest enzyme activity. For that five
water-immiscible ILs with this anion were synthesized.
The cations described here contained natural alcohol
(1R,2S,5R)-()-menthol and varied in the structure: (I)
3-butyl-1-[(1R,2S,5R)-()-menthoxymethyl] imidazolium, (II) 1-[(1R,2S,5R)-()-menthoxymethyl]-3-heptylimidazolium, (III) 1-[(1R,2S,5R)-()-menthoxy-methyl]-3-methylpyridinium, (IV) heptyl[(1R,2S,5R)-()menth-oxymethyl]dimethylammonium, and (V) decyl
[(1R,2S,5R)-()-menthoxymethyl]dimethyl-ammonium.
Laccase activity was tested in buffer saturated with ILs.
Special attention was paid towards stability tests in biphasic systems, lasting 5 days. Kinetic parameters of inactivation models were estimated using nonlinear regression
(OriginPro 8.0).
Correctly chosen ILs can provide at least good conditions
for an enzyme activity expression and to be concomitant
with its stability not lower than in the buffer solution. It
was shown that all but one ionic liquids tested did not
significantly alter laccase activity (90123% respective
to the buffer. The best laccase-friendly ILs were no. I
and IV whereas IL II decreased reactivity in 20%. This,
perhaps, depends on weak influence of ions activity on
57
proteins structure even at their very low concentrations

in the water phase. Stability tests showed that no laccase
activity was observed in all ILs phases and that ILs I, IV
and V, with the highest reactivity, allowed to maintain
the enzyme stability even more than in the buffer alone.
Exceptional stabilization of laccase was observed in
presence of ILs I and IV. In the contrary, ILs II and III
caused destabilization of protein structure, probably via
defolding and/or changing water molecules organization
in the protein micro-environment. Another factor,
that has been discussed was the possibility of laccase
stabilization by preventing of microorganisms growth by
ILs molecules during 5-days incubation. Destabilization
caused by enzyme contact with the interface between
the aqueous and ILs phases and air water phases was
discussed as well. For mathematical formalization of
inactivation courses, isoenzyme model was applied and
kinetic parameters were evaluated. It was stated that this
model allowed to fit experimental data accurately only for
sets obtained in the buffer (control) and in the presence
of ILs II. In the other cases, first-order reaction model
reflected experiments very well with concomitant good
statistical validation. This shows that ILs, even at very
low concentrations, influence conformational stability
of proteins and that it depends on the cation structure.
In general, the imidazolium and ammonium salts with
shorter alkyl chain, tested in this study, supported laccase
activity and stability.
Acknowledgements
This work was supported by the Polish State Committee for Science and
Research under grant N N209 119337, 20092012.
58
Eurobiotech 2013
P5.31
P5.32
Antiradical capacity of fruits of Polish

raspberry and blackberry cultivars
an electron paramagnetic resonance
spectrometry study
Development of novel cell line, expressing

human histamine H4 receptor and its use
in affinity testing of potent receptor ligands
Anna Kostecka-Gugaa, Aleksandra Mech-Nowak,

Pawe Kaszycki

Departament of Biochemistry, Institute of Plant Biology

and Biotechnology, Faculty of Horticulture, University
of Agriculture in Crakow, al. 29 Listopada 54, 31-425 Crakow,
Poland
The world production of raspberry fruits increases over

the recent years and in 2011 Poland was the second
raspberry producer. It is known that Rubus (raspberry and
blackberry) fruit antioxidant activity is highly correlated
with the presence of phenolic compounds. We examined
differences in antiradical capacity of fruits of several
Polish raspberry cultivars in relation to the content and
composition of main polyphenols in 80%-methanol
extracts.
Among the tested cultivars were: summer red-fruited
raspberry Benefis, black-fruited Litacz and autumn
(primocane-fruiting) red-fruited Polka, yellow-fruited
Poranna Rosa, and a blackberry cultivar Ruczaj. All the
specimens were collected in 2011 and 2012 at The Friut
Experiment Station in Brzezna, Southern Poland. Antiradical activity was measured with the L-band (1.2 GHz)
electron paramagnetic resonance spectrometry which enabled to directly detect the DPPH free-radical quenching
reaction by fruit antioxidants. The analyses of phenolic
compounds were performed using high performance liquid chromatography and supplemented with a standard
Folin-Ciocalteau spectrophotometric assay.
The results of the study show that black fruits of the raspberry Litacz were distinguished by the largest antiradical
capacity, higher than that observed for blackberry fruits.
The typically coloured, red raspberry fruits revealed moderate antiradical capacities and the differences in the obtained activities were found insignificant for fruits of the
summer and autumn (primocane-fruiting) cultivars. Relatively smaller antiradical potential was observed for the
yellow fruits of raspberry. The great antiradical properties
of the black-fruited raspberry Litacz were determined by
a high amount of polyphenols, mainly flavonoids. Litacz
was found to be rich in rutin, catechins, anthocyanins
(especially cyanidin derivatives) as well as chlorogenic
and p-hydroxybenzoic acids. The above characteristics
make this Polish black-fruited cultivar a valuable and
recommendable source of active antiradical compounds.
Acknowledgements
The work was supported by a research grant of Polish National Science
Centre No. N N310 306139.
Tadeusz Karcz, Katarzyna Kie-Kononowicz
Recombinant receptors systems are pharmacological

tools widely utilized in search for novel G-protein coupled receptors (GPCRs) ligands. Contrary to historically
used native receptors systems, they can be involved in
studies on poorly characterized receptors, for which selective ligands are not known and low expression levels
are observed in native cells.
Recombinant proteins can be obtained in various expression systems. In studies on GPCRs, mammalian cell
lines are predominantly used. These models guarantee
satisfactory grade of posttranslational protein modifications and enable cellular membrane localization of recombinant proteins. Moreover mammalian cells contain
whole range of necessary intracellular signaling pathway
elements [1, 2].
Human histamine H4 receptor, being a biological target
of presented studies is, by far, the last histamine receptor discovered. Its gene has been cloned in 2000, independently by seven groups [3]. Histamine H4 receptor
expression on basophils, eosinophils, mast cells and dendritic T cells suggests its role in immunological answer
[4]. Therefore its ligands could provide promising drugs
for immuno-based diseases [5], however none of the potent ligands was brought to the market so far. Thus, histamine H4 receptor remains an important pharmacological
target in drug development.
The aim of current work was to develop a novel cell line,
expressing human histamine H4 receptor, with use of
retroviral expression system. Following the molecular
biology experiments, which led to obtaining of pQCXIN-hH4R plasmid, containing human histamine H4
receptor sequence, transduction of CHO cells was performed with use of retroviral particles produced in GPenv+AM12 packaging cells. Resultant cell line was tested for
its utility in radioligand binding assays.
First, recombinant receptor was characterized in saturation binding assay with use of [3H]histamine as a radioligand. Homological histamine competition binding
was employed for comparison of obtained cell lines properties with membranes preparation from commercial
source. Competition binding experiment for estimation
of unknown Ki values of tested compounds was validated
against the insect cells system used for the same purpose
by our scientific collaborators, proving the usefulness of
developed cell line in testing of novel compounds affinities towards histamine H4 receptor.
Acknowledgements
Work supported by Polish National Science Center, project Preludium I,
No. UMO-2011/01/N/NZ4/01126.
Eurobiotech 2013
References
[1] Siehler S., Cell-based assays in GPCR drug discovery. J Biotech, 2008,
3: 471483.
[2] Hermans E., Generation of model cell lines expressing recombinant
G-protein-coupled receptors. Methods Mol Biol, 2004, 259: 137153.
[3] Oda T. et al., Molecular cloning and characterization of a novel type
of histamine receptor preferentially expressed in leukocytes. J Biol Chem,
2000, 275: 3678136786.
[4] Nguyen T. et al., Discovery of a novel member of the histamine
receptor family. Mol Pharmacol, 2001, 59(3): 42733.
[5] Jablonowski J., Carruthers N., Thurmond R., The histamine H4
receptor and potential therapeutic uses for H4 ligands. Mini Rev Med
Chem, 2004, 4: 9931000.
59
P5.33
The effect of egg yolk lipids enriched with
CLA on cytotoxicity of human melanoma
cell line WM793
Dominik Domagaa1, Aneta Koronowicz1, Jarosaw Oczkowicz1,
Elbieta Sikora1, Piotr Laidler2, Teresa Leszczyska1
University of Agriculture, Department of Human Nutrition,
Crakow, Poland; 2Jagiellonian University Medical College,
Crakow, Poland
1
Conjugated linoleic acids (CLAs) are a family of at least

28 isomers of linoleic acid found mainly in the meat and
dairy products derived from mammals called ruminants.
Among these compounds two isomers cis9,trans11 and
trans10,cis12 have received considerable attention. In recent study we have demonstrated that fatty acids extract
obtained from egg yolks enriched naturally with the CLA
isomers may exhibit a strong tendency to inhibit proliferation and induce cytotoxicity in melanoma WM 793 cell
lines.
Cytotoxicity in cells increases with higher concentrations
of CLA, however it was still lower than one caused by fatty acids without addition of CLA, which correlated with
decreased level of cell proliferation, which most probably is caused by cell apoptosis. Apoptotic mechanism was
proved by staining cells with annexin and propidium iodide.
These results demonstrated that fatty acids of CLAenriched egg yolks may induce apoptosis in carcinoma
cells. The received data suggests that CLA may be used in
future to prevent cancerogenesis, however it still require
deeper insight into molecular mechanisms which are involved into that process.
Material and Methods: Experiments were performed
using melanoma cancer cell line WM 793 treated with
fatty acids of CLA-enriched egg yolks. Proliferation
tests were performed with use of BrdU proliferation kit
(Roche). Cytotoxicity was measured by LDH Detection
kit (Roche). Apoptosis assay was conducted with use of
Annexin-V-FLUOS Staining Kit (Roche).
Plant molecular breeding
Lectures
L6.2
L6.1
Pea resistance genes to Pea Seed-borne

Mosaic Virus (PSbMV), from basic science
to applications in breeding
Engineering of a retrotransposon
for insertion mutagenesis in plants
Nikola Winter, Eneda Xhikaj, Lilian Nehlin, Andrea Tramontano,
Andreas Bachmair
Dept. of Biochemistry and Cell Biology, Center for Molecular
Biology, Max F. Perutz Laboratories, Univ. of Vienna,
Dr. Bohr Gasse 9, A-1030 Vienna, Austria
New methods allow acquisition of sequence data for

many plants of agricultural interest. However, insight
into gene function by mutant analysis in crop plants has
not kept pace with sequence data accumulation, due to
the lack of efficient mutagenesis tools. We are studying
the plant retrotransposon Tto1 with the goal to generate
a next generation mutagenesis tool for plants. Retrotransposons have been used for insertion mutagenesis in
plants, but optimization of this process is difficult due to
the limited understanding of the retroelement life cycle.
In order to facilitate experimental analysis of plant retrotransposon Tto1, we replaced the natural promoter of
Tto1 by an artificial, inducible one. The engineered element can transpose in the model plant Arabidopsis thaliana after induction [1]. Sequences of the long terminal
repeat region were investigated for their role in transposition [2], with the goal to reduce the length of sequence
repeats as potential inducers of silencing. In order to optimize insertion frequency, we are currently testing different inducible promoters and integrase versions for their
impact on transgenerational transposition events. Based
on insights obtained in the model plant, Tto1 constructs
are generated for transposon tagging in the agriculturally important plants bean (Phaseolus vulgaris) and barley
(Hordeum vulgare). Mutant plants can eventually serve
for analysis of gene function, and in breeding programs
for crop improvement.
Acknowledgements
The authors work is supported by the Austrian Science Foundation
FWF (grant TRP14-B12).
References
[1] Bhmdorfer et al. (2010) Syst Synth Biol 4, 133138.
[2] Tramontano et al. (2011) Virology 412, 7582.
Petr Smkal
Department of Botany, Slechtitelu 11, Palacky University
in Olomouc, 783 71 Oolomouc, Czech Republic
Pea seed-borne mosaic virus (PSbMV) belongs among the

most frequent viral pathogens causing severe losses in field
pea and other legumes. These losses might be prevented
by growth of resistant varieties. Resistance to the common
P1 strain of PSbMV is conferred by a single recessive gene
(eIF4E), localized on LG VI (sbm-1 locus), while to lentil L1
strain is localized on LGII at sbm-2 locus. We have analyzed
variation in the eIF4E genomic sequences from pea lines,
reported as donors of resistance. Subsequently, gene-specific single nucleotide polymorphism and co-dominant
amplicon length polymorphism markers were developed
(Smkal et al. 2010). All markers were reproducibly amplified across a broad spectra of pea varieties and breeding lines and found to be 100% accurate when compared
to symptomology and ELISA testing. Hence, application
of these DNA markers substantially speeds-up resistance
breeding processes. From the comparison of genotype
verses phenotype for selection of resistance/susceptibility in F2 plants, there was a 26% discrepancy between the
PCR and ELISA-based assays with potentially susceptible
heterozygote plants missed (Smkal et al. 2010). The novel eIF4E alleles were detected in wider pea germplasm and
TILLING mutants (Dalmais et al. 2008) are tested for various potyviruses. The series of identified allelesprovide the
basis for the testing of various potyviruses and pathotypes
to reveal possible co-evolution of potyviruses and its pea
host. Durability of host based resistances in relation to genetic plasticity of viral pathogens is an issue for practical
breeding, however as seen in the case of PSbMV, ancient
alleles are still effective in pea resistance. It can be expected
that information gain in this study might be extended to
other legume species, particularly to economically important species of tribus Vicieae or even to subfamily Papilionoideae such as chickpea, lentil, faba bean or soybean where
Potyviruses cause problems. The implications for marker
assistend breeding in legumes will be presented.
Acknowledgements
The work was supported by NAZV QI91A229 project.
Reference
[1] Smkal P., afov D., Navrtil M., Dostalov R. (2010) Mol
Breeding 26: 425438.
[2] Dalmais M., Schmidt J., Le Signor C. et al. (2008) Genome Biol 9:
R43.
Eurobiotech 2013
L6.3
High through output phenotyping and
genotyping stepsfor drought tolerance
improvement in barley
Marcin Rapacz1, Magdalena Wjcik-Jaga1, Anna Fiust1,
Bartomiej Kozera1, Mirosaw Tyrka2
University of Agriculture in Crakow; 2Rzeszow University
of Technology
1
Water deficit limits crop production all over the world.

Also in Poland periodic droughts cause serious decrease
in agricultural production. Spring barley production is
especially affected, mainly due to short vegetation period and development schedule, in which critical periods
of water demand occurs often at the time of periodic
droughts. Because of the limitations of classic breeding
methods in drought tolerance improvement biotechnology seems to be the only solution. Either the development
of marker systems or genetic modifications are taken into
consideration. Despite many attempts made all over the
world the progress in this field is, however, limited. The
main problems seems to be 1) a very complex drought response of plants with hundreds of genes involved in, and
2) different drought response mechanisms desired at different environments, eg. in Middle East, Mediterranean
area or in Poland. We hope that the solution is possible
and very simple we must take the advantage from the
above mentioned disadvantages.
Two mapping populations based on Polish breeding materials of barley well adapted to local conditions and varied in their drought tolerance were used for QTL analysis
of the traits describing their short-time drought response
at the seedlings stage. High throughput genotyping
(DArT markers) and phenotyping techniques, including
many physiological characteristics important for high
yielding potential of plants affected by periodic droughts
were performed. The results showed either a high genetic
diversity of studied populations, enabling the creation of
high density linkage maps, as well as a high diversity in
the physiological response of studied breeding materials.
The analysis revealed in total 23 QTLs for 9 physiological characteristics. Three QTL regions were common for
both mapping populations.
This analysis was a start point for further work of either
applied and basic research character.
68 DArT markers from QTLs regions were successfully converted into SSR and STS polymorphic markers.
During the further analysis of breeding strains different in their drought tolerance we confirmed that 20
markers were effective in selection for drought tolerance and these markers are used now for a pilot large
scale selection in plant breeding. The same sequences
were used for sequence analysis which reveals connections between fenotype and certain genes, protein
activity or signaling pathways. Results give significant
sight into candidate genes which may be involved in
different drought responses observed among Polish
61
breeding materials of barley and may be useful for genetic modifications.

It seems that the creating of mapping populations from
current breeding materials, the use of high throughoutput genotyping and phenotyping methods based on deep
physiological knowledge may be a key strategy for improving not only drought tolerance but also other complex agronomic traits.
Acknowledgements
The research were supported by Polish National Research and
Development Center (PBZ-2/3/2006/17 and GENMARK PBS1/
A8/1/2012).
62
Eurobiotech 2013
L6.4
L6.5
In vitro induced variation

in Triticale cv. Bogo
Conversion of a DArT marker

differentiating wild and cultivated carrots
to a codominant CAPS marker
Joanna Machczyska1, Piotr Bednarek1, Janusz Zimny2

Department of Plant Physiology and Biochemistry, Plant
Breeding and Acclimatization Institute-National Research
Institute, Radzikow, 05-870 Blonie; 2Department of Plant
Biotechnology and Cytogenetics, Plant Breeding and
Acclimatization Institute-National Research Institute, Radzikow,
05-870 Blonie
1
In vitro culture plant regeneration is a widespread method

used to obtain genetically uniform plants double haploids (DH) that should be identical to their donor plants.
However, in vitro culture induces genetic and epigenetic
variation that may be observed in regenerants. Genetic
changes include translocation and transposition as well as
sequence mutations, while epigenetic alterations concern
gene silencing, gene regulation and chromatin condensation.
The metAFLP approach is a valuable tool for the analysis
of the quantitative characteristics of (epi)genetic changes
affecting restriction sites (Acc65I & KpnI) and their vicinity within the whole genome. Moreover, it enables quantification of unaffected sites. MetAFLP assumes utilization
of two isoschizomes Acc65I and KpnI. Acc65I and KpnI
recognize the same restriction sequence but differ in their
sensitivity towards DNA methylation of their recognition
sequences; the former is sensitive to methylation of the
restriction site and adjacent sequences whereas the latter
is not. Comparison of the molecular data obtained with
the Acc65I/MseI and KpnI/MseI metAFLP platforms for
donor plants and their regenerants allows qualification
and quantification to distinct variation types (sequence
changes, de novo methylation and demethylation).
Plant DNA methylation occurs on CpG sites (symmetric
methylation) as well as on CpXpG (X denoting A, C or
T) and CpXpX (asymmetric) sites. In our study specially
designed primers were used to distinguish between symmetric and asymmetric methylation types present within
restriction sites.
Triticale is important for Europe as it uses less good soil,
less fertilizer, exhibits better disease tolerance, high yield
potential, grain quality, resistance to pathogens, favourable amino acid composition. Hexaploids gained prominence in Poland as feed grains. However triticale is
apolyploid species, which demonstrates high genetic and
epigenetic instability.
In our research 4 DH lines of triticale cv. Bogo were used
to evaluate the level of in vitro induced variation. Regenerants obtained via androgenesis in anther culture and
shed-microspore culture and via somatic embryogenesis
in immature embryo culture. Our results demonstrate
that triticale is not genetically stable during tissue culture
manipulations. Average total tissue culture induced variation amounted to 28%, where sequence variation constituted of 18%, de novo methylation reached up to 4,5%
and demethylation equaled to 5,3%.
Alicja Macko-Podgrni, Krzysztof Smka, Dariusz Grzebelus

Department of Genetics, Plant Breeding and Seed Science,
University of Agriculture Crakow, al. 29 Listopada 54,
1
Carrot is one of the most important vegetables grown

in the world, mostly due to high content of -carotene
vitamin A precursor. Wild carrot (Daucus carota subsp. carota) is an ancestor of cultivated carrot (D. carota
subsp. sativus), and Central Asia is a likely region where
the biennial cultivated carrot was domesticated. Cultivated and wild carrots spread from their centre of origin
to distant geographical regions. Both forms can easily
hybridize, thus a considerable increase of genetic variation is exhibited both among and within each subspecies.
Even though carrot diversity was extensively studied using a variety of molecular marker techniques, until now
no handy markers allowing determination between cultivated carrots and wild populations was available.
Recent results of investigation on genetic diversity of cultivated and wild carrots using Diversity Arrays Technology (DArT) allowed identification of a DArT marker differentiating wild and cultivated accessions. We sequenced
the polymorphic region and identified a 16 bp-long indel
containing a PstI restriction site and causing the DArT
polymorphism. We designed site-specific primers anchored in regions flanking the indel and converted the
DArT marker into an inexpensive, efficient, codominant
cleaved amplified polymorphic site (CAPS) marker for
routine detection of wild/cultivated variants present in
that locus.
The CAPS-based assay was used to screen 72 cultivated
and 16 wild accessions of different origin, each accession
being represented by three individuals. All wild accessions were characterized by presence of the allelic variant
associated with the wild phenotype. In all plants from 60
accessions of cultivated carrot the expected variant typical for cultivated carrot was observed. In the remaining
12 cultivated accessions the wild variant was present in
at least one plant. Most of them were old primitive landraces from Turkey and Iran, likely poorly monitored for
cross-pollination with wild carrots. The result suggests
that the identified cultivated variant, rare in wild germplasm, was enriched in the cultivated germplasm in the
course of domestication.
Eurobiotech 2013
Posters
P6.1
New sources of phenolic compounds
and anthocyanins for biotechnolgy
Nijol Anisimovien1, Jurga Jankauskien1, Milda Jodinskien1,
Vidmantas Bendokas2, Vidmantas Stanys2,
Tadeuas iknianas2
Institute of Botany of Nature Research Centre;
Institute of Horticulture, Lithuanian Research Centre for
Agriculture and Forestry
1
2
Introduction. One of the current problems in biotechnology is a search for plant original bioactive compounds
important due to their benefits to human health (He,
Guisti, 2010; Oancea, Oprean, 2011). Anthocyanins from
berries have received considerable interest as dietary
antioxidants among the different bioactive substances
in human health (Tsuda, 2012). These compounds are
being able to capture reactive oxygen species, delay the
initiation or propagation of oxidative chain reactions.
The prevention role of anthocyanins in cardiovascular
disease, cancer, diabetes and other chronic diseases was
indicated (Pascual-Teresa, Sanches-Ballesta, 2008; Shipp,
Abdel-Aal, 2010; Tsuda, 2012).
The growing interest in impact of antioxidants on human
health has triggered our study of berries from different
Ribes and Prunus species. Also, complex interspecific hybrids were created in order to obtain plants with higher
phenolic compound and anthocyanin amount, as well
as clarification of anthocyanin composition in order to
identify cultivars or hybrids with highest antioxidant activity.
Material and Methods. Four different Ribes species:
R. nigrum L. Ben Tirran (black berries), R. aureum
Au Gs5 (yellow berries) and Corona (black berries),
R. petreum Jonkher van Tets (red berries), R. uva-cripsa
Liai (red berries) and iornyj negus (black berries)
and four interspecific Ribes hybrids were studied. Anthocyanin and phenol compound content, and antioxidative
activity were evaluated in P. avium, P. cerasus cultivars,
P. machii wild form and 4 their interspecific hybrids, too.
All berries were collected at technical maturity phase
and immediately frozen at 70C. Anthocyanins and
other phenolics from frozen berries were extracted by
90% aqueous methanol, acidified by HCl to 0.1 N, at ratio 1:20 (g/ml). Total phenolics content in extracts was
determined by Folin-Ciocelteu method, using galic acid
as standard. Antioxidant activity (capacity) was evaluated spectrophoretically, by radical scavenging assay,
according DPPH test (Anisimovien et al., 2009). The
anthocyanin content was determined using a spectrophotometric differential pH method (Wrolstad et al., 2005)
and calculated using a molar extinction coefficient of cyanidin-3-glucoside 29600 (Cy-3-Glu). The composition
of anthocyanins was determined by HPLC procedure
(Durst, Wrolstad, 2001; Liobikas et al., 2009).
63
Results. Large anthocyanins content differences (up to

38 times) were identified in berries of tested Ribes species. It ranged from 16 mg 100 g1 FW to 615 mg 100g1
fresh weight (FW), and the amount of total phenolic
compounds ranged from 172 mg 100 g1 FW to 672 mg
100g1 FW. The highest content of anthocyanins was obtained in R. aureum Corona and R. nigrum Ben Tirran
berries. The lowest was identified in R. aureum Au Gs-5
and gooseberry Liai. The antioxidative activity of all
tested Ribes berry extracts was higher than 65%. All studied interspecific hybrids were characterized as possessing
high biological activity values of tested indexes matched
or outfaced antioxidant activity of R. nigrum Ben Tirran,
which berries accumulate high anthocyanin content in
Lithuanian climatic conditions. The highest content of
anthocyanins was identified in berries of R. nigrum x
R. petraeum and R. nigrum x R. aureum hybrids. Delphinidins were predominant (68.6%) in berries of blackcurrant Ben Tirran, cyanidins dominated in gooseberry
berries independently on berry colour and amount of
anthocyanin and it constituted 75.890.8% of total anthocyanins.
Results of quantitative and qualitative analysis of anthocyanins and phenolic compounds in fruits of tested
Prunus species cultivars and interspecific hybrids revealed, that anthocyanin and phenol compound contents
varied, too. Anthocyanin content in fruits of P. cerasus
cultivars ranged from 45 to 147 mg 100 g1 of FW, and
total phenol 241430 mg 100 g1 of FW. 126192 mg 100
g1 FW of anthocyanins and 251323 mg 100 g1 FW of
total phenols was identified in P. avium cultivars. The
highest phenolic compound content 568 mg 100 g1 FW
was found in berries of P. padus, while amount of anthocyanins was similar to P. cerasus and P. avium. Anthocyanin amount in fruits of interspecific Prunus hybrids
ranged from 156 to 333 mg 100 g1 FW; phenolic compounds varied from 302 to 484 mg 100 g1 FW. Anthocyanin amount in fruits of interspecific hybrid P. cerasus x
P. maximovitchii 2n was 2 times higher than in fruits
of P. cerasus cultivar Rechta and P. avium cultivar Kitajanka with high anthocyanin content, compared to other
sour and sweet cherry cultivars. The antioxidative activities of Prunus were 20% lower, in comparison with Ribes.
Cyanidins were the most abundant anthocyanin class in
fruits of most studied Prunus species and interspecific
hybrids, however, in fruits of Irema BS and P. cerasus x
P. maximovitchii 2n cyanidins amount was significantly
lower, and the most abundant anthocyanins were pelargonidins.
Conclusion. The newly created interspecific hybrids of
Ribes and Prunus species are proposed to be perspective
new sources of anthocyanins.
Acknowledgements
Research was funded by a grant No. SVE-01/2011 from the Research
Council of Lithuania.
References
[1] Anisimovien, N., Rubinskien M., Vikelis P., Stackeviien E., Stanys
V., iknianas T., Jankovska, E., Sasnauskas A. (2009) Anthocyanins
in currants, cherries, blueberries, and antioxidative activity of berry
extracts. Zemdirbyste=Agriculture, 96(3): 158167.
64
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[2] Durst R.W., Wrolstad R. (2001) Separation and characterization

of anthocyanins by HPLC. p. F1.3.1F1.3.13. In: R.E. Wrolstad (ed.),
Current Protocols in Food Analytical Chemistry. Wiley, New York.
[3] He J., Guisti M.M. (2010) Anthocyanins: natural colorants with
health-promoting properties. Annual Review Food Sci. Technol., 1:
163187.
[4] Liobikas J., Trumbeckait S., Bendokas V., Baniulis D., Majien
D., Kopustinskien D.M., iknianas T., Anisimovien N. (2009)
Pro-apoptotic effect of black currant berry extracts on rat heart
mitochondria. Zemdirbyste=Agriculture, 96(3): 149157.
[5] Pascual-Teresa S., Sanchez-Ballesta M.T. (2008) Anthocyanins: from
plant to health. Biochemistry Reviews, 7: 281299.
[6] Oancea S., Oprean L. (2011) Anthocyanins, from biosynthesis in
plants to human health benefits. Acta Uninversitatis Cibiniensis. Series
E: Food Technology 15: 315.
[7] Shipp J., Abdel-Aal El-S.M., Food application and physiological
effects of anthocyanins as functional food ingredients. The Open Food
Science Journal, 4, 2010, 722.
[8] Tsuda T. (2012) Dietary anthocyanin-rich plants: Biochemical
basis and recent progress in health benefits studies. Mol. Nutr. Food
Research, 56: 159170.
[9] Wrolstad R.E., Durst R.W., Lee J. (2005) Tracking color and
pigment changes in anthocyanin products. Trends in Food Science and
Technology, 1: 423428.
P6.2
Selection efficiency of converted DArT
markers in spring barley breeding for
drought tolerance
Magdalena Wjcik-Jaga1, Anna Fiust1, Marcin Rapacz1,
Mirosaw Tyrka2
Department of Plant Physiology, University of Agriculture
in Crakow; 2Department of Biochemistry and Biotechnology,
Rzeszow University of Technology
1
21 DArT markers converted into STS and SSR markers

were tested on 24 spring barley genotypes that differed in
their response to drought and all proved to be polymorphic. Selection efficiency was determined on the basis of
correlation of the presence of a certain allele and values
of physiological traits associated with drought tolerance
(Rapacz i in. 2010 [1]). Among the markers tested, several seem to be suitable for discarding of the susceptible
to drought genotypes during the selection process. The
chosen markers were most efficient in differentiating the
genotypes in terms of gas exchange, especially net photosynthesis, which is the most informative parameter for
assessment of plant growth ratio in drought. Two of the
tested markers appear to be potentially most suitable for
marker-assisted selection (MAS) of drought-tolerant Polish spring barley genotypes. They differentiated the tested
genotypes in terms of several physiological parameters,
including net photosynthesis. Average net photosynthesis
stress index (ASI) of the genotypes that did not contain
those markers was only ca. 70% of the average ASI for all
of the tested genotypes
Acknowledgements
This study was supported by the by Polish National Research and
Development Center [PBZ-MNiSW-2/3/2006/17].
References
[1] Rapacz M., Kocielniak J., Jurczyk B., Adamska A., Wjcik M. (2010)
Different Patterns of Physiological and Molecular Response to Drought
in Seedlings of Malt- and Feed-type Barleys (Hordeum vulgare). J.
Agronomy & Crop Science 196: 4854.
Eurobiotech 2013
65
P6.3
P6.4
Studies on micropropagation of Polish

garlic (Allium sativum L.) varieties
Analysis of mitochondrial transcripts

associated with cytoplasmic male-sterility
in cauliflower (Brassica oleracea L. var.
botrytis)
Alicja Chuda
University of Agriculture in Crakow, Faculty of Horticulture,
Institute of Plant Biology and Biotechnology, Department
of Genetics, Plant Breeding and Seed Science,
al. 29 Listopada 54, 31-425 Crakow, Poland
Garlic is one of the most important Allium vegetable crop

widely cultivated throughout the world. Due to sexual sterility commercial varieties of garlic are propagated
vegetatively. Unfortunately, this system favors the accumulation of viruses in plant material and thereby causes
significant yield losses. Tissue cultures, especially meristem cultures and shoot tip cultures have been applied
successfully to generate virus-free garlic plants. Moreover, through somaclonal variation in vitro cultures can
generate new forms at the phenotypic, cytological and
molecular levels. The somaclones can be used in breeding
programmes designed for increasing the genetic variability within Allium sativum species. However, due to the
low propagation rates plant tissue cultures require optimization associated with the selection of donor varieties,
type of explants and regeneration media. This study was
conducted to provide an efficient protocol for micropropagation of polish garlic varieties. In the experiment the
influence of variety: Huzar, Jarus, Mega and Ornak and
medium: LS [Linsmaier and Skoog 1965], MS [Murashige
and Skoog 1962], LIK (LS+0.2 mg/l IAA+ 2 mg/l kinetin) and MIK (MS+0.2 mg/l IAA+ 2 mg/l kinetin) on the
development of garlic plantlets in in vitro cultures were
investigated.
Acknowledgements
The research project was funded by the Polish Ministry of Science and
Higher Education, decision No. 202914/E/377/M/2013.
References
[1] Linsmaier E., Skoog F. (1965) Organic growth factor requirements
for tobacco tissue cultures. Physiol. Plant., 18: 100127.
[2] Murashige T., Skoog F. (1962) A revised medium for rapid growth
and bio assays with tobacco tissue cultures. Physiol. Plant., 15: 473497.
Gabriela Machaj, Marek Szklarczyk

Dapartament of Genetics, Plant Breeding and Seed Science,
University of Agriculture in Crakow, al. 29 Listopada 54, Crakow,
Poland
When transferred to cauliflower (Brassica oleracea L. var.

botrytis) the cytoplasm of black mustard (Brassica nigra
L.) causes cytoplasmic male sterility (CMS). In sterile
plants stamens are substituted by petal-like organs and
as a result pollen is not produced. CMS in plants is determined by specific sequences of the mitochondrial genome. The present work was aimed at identification of
mitochondrial transcripts which were associated with
the sterilizing effect of the nigra cytoplasm in cauliflower
plants. The analysis was based on the use of northern hybridization, RT-PCR, real-time RT-PCR and long PCR.
The obtained results indicate that CMS-nigra is associated with altered length of the high-molecular ccb206 transcript and lowered accumulation of the low-molecular
atp9 and ccb206 transcripts. The lowered accumulation
of the ccb206 mRNA was confirmed by RT-PCR and real-time RT-PCR analyses. Moreover, the northern data
suggest co-transcription of the atp9 and ccb206 sequences. Their physical vicinity in the cauliflower mitochondrial genome was proved with the use of long PCR. These
experiments also indicate altered organization of the atp9
and ccb206 loci in the nigra and oleracea cytoplasm. Furthermore, long PCR and hybridization anlyses showed
increased expression of the rrn26 gene in male-sterile
plants.
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P6.5
P6.6
The role of nitric oxide in calamine plant

species response to lead stress during
in vitro culture
Evaluation of the nucleotide sequences of

DNA rearrangements in wheat-rye hybrids
using ISSR-PCR, IRAP-PCR, REMAP-PCR
and ITAP-PCR
Ewa Muszyska, Ewa Hanus-Fajerska

Department of Botany and Plant Physiology,
University of Agriculture in Crakow
Abiotic stresses such as increased concentration of heavy

metal ions results in reduction of rate plant growth and
development. Therefore, the searching of effective methods increasing the probability of plant survival in harsh
conditions is very important task. The results of some
recent experiments point at protective role of nitric oxide in species subjected to the various stress factors. For
this reason, the aim of our work was to evaluate the effect of exogenous nitric oxide application on the seed
germination ability and further growth and development
of plants cultivated in vitro under lead stress. For experiment seeds of Dianthus carthusianorum and Gypsphilla
fastigiata (Caryophyllaceae) representing calamine populations from the Olkusz Ore bearing Region (Southern
Poland) were used. Seeds were treated with nitric oxide
applied as sodium nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine (SNAP) in concentration of 50,
100 and 150M. Afterwards they were surface sterilized
and placed on germination medium. Cultures were established on modified MS medium using 10 mm fragments
of obtained seedlings shoots. Three concentration of lead
nitrate were tested: 0,1 mM, 0,5 mM, 1,0 mM. After eight
weeks of culture, micropropagation efficiency was evaluated on the basis of number and length of regenerated
organs (shoots and roots) and their fresh and dry matter. Vitality of plants was also determined through the
measurements of several physiological parameters. The
chlorophyll a fluorescence with using Handy-PEA (Hansatech, UK) spectrofluorometer was provided and photosynthetic functional parameters (Fv/Fm and PI) were calculated. Additionally, the content of photosynthetic pigments according to Wellburn (1994) and the content of
total phenolic compounds according to the protocol proposed by Fukumoto and Mazza (2000) were investigated
with using UV-Vis spectrometry. The obtained results
will be presented and discussed during poster session.
Acknowledgements
Support of experimental work by the Ministry of Science and Higher
Education of Republic of Poland (4562/2013) is gratefully acknowledged.
References
[1] Fukumoto L.R., Mazza G. (2000) Assesing antioxidant and
prooxidant activities of phenolic compounds. Journal of Agricultural
and Food Chemistry, 4: 35973604.
[2] Wellburn A.R. (1994) The spectral determination of chlorophylls
a and b, as well as total carotenoids, using various solvents with
spectrophotometers of different resolution. J. Plant Physiol, 144:
301313.
Izabela Szuko, Stanisawa Maria Rogalska

Chair of Cell Biology, University of Szczecin, Waska 13,
PL-70415 Szczecin, Poland
Early generations of wheat-rye hybrids are characterized

by cytogenetic, genetic and physiological instability. This
is manifested by a significant percentage of aneuploids
in next generations of the hybrid, the occurrence of new
genotypes that are difficult to predict and the diversified development of hybrid plants. Many literature data
confirm a significant changes of nucleotide sequences in
DNA hybrids in generations F1-F4. These changes were
most commonly identified as the elimination of a sequence and the emergence of new DNA sequence motifs
which were not present in the parental genomes. We conducted a study on changes at the DNA sequences level
in order to show whether similar changes occur in our
hybrids material in the F2 and F3 generation.
The aim of this study was to investigate the occurrence
of the nucleotide sequences of DNA rearrangements in
wheat-rye hybrids based on a combination of four marker systems (ISSR, REMAP, IRAP and ITAP).
The material consisted of F2 and F3 generations of wheatrye hybrids derived from the Plant Breeding Company
Strzelce, agency Mayszyn. They were obtained by crossing three hexaploid wheat varieties (Triticum vulgare L cv
Zyta, Tonacja, Ostka Strzelecka and two families (STHN
5067, 1002/1003) with the widely cultivated rye variety
Dakowskie zote (Secale cereale L).
Six primers were used for the IRAP analysis. Primers were
designed based on sequences of the retrotransposons derived from cereals of the Secale genus (Bilby, Cassandra,
Sukkula), Triticum (Angela) and Hordeum (Nikita, Sabrina). For the ISSR analysis, 16 arbitrary primers were selected. For REMAP analysis, 15 combinations of primers
were used. Each pair consisted of outward-facing retrotransposon primer and a second primer from a microsatellite sequence. Five primers were used to the ITAP
method, primers were designed on the basis of the transposons sequences from the Secale cereale (Rev) and Triticum (Sher, Mut, Jude, Caspar) genomes.
The results obtained from the IRAP, REMAP, ISSR and
ITAP methods enabled evaluation of the nucleotide
sequence rearrangements. In addition, the value of each
marker system was assessed. Marked efficiency index
method (EMR), diversity index (DI) as the average value
of the PIC system and marker index (MI) as the product
of EMR and DI were counted. In the analysis, the presence or absence of band was treated as a single feature
and assigned as 1 or 0. Results of ISSR, IRAP, REMAP
and ITAP methods showed changes in the structure
Eurobiotech 2013
of specific DNA nucleotide sequences. Disappearance of

certain types of rye bands was mainly observed, and the
percentage of wheat-specific bands which disappeared
was lower. A new sequence types, absent in the parent
species were observed less frequently. This indicates possible rearrangements or elimination of sequences of wheat
and rye. These results suggest that the early generations
of wheat-rye hybrids undergo many genetic changes. It is
worth noticing, that the DNA elimination (5-14% of the
genome) increases the differences between the diploid
genomes contained in the same nucleus in allopoliploid
which is triticale. Through such differentiation the possibility of homoelogous chromosome pairing is reduced,
and the result may be a diploid-like meiosis behaviour,
affecting polyploidy plant growth and fertility. Disomic
inheritance leads to stabilization of progeny plants what
makes them potentially useful for crop breeding.
The values of the coefficients showing the quality of the
individual marker systems were similar, indicating that
each of these markers could be used separately to evaluate
changes in the genomes of wheat-rye hybrids, however,
using the whole set makes the analysis much deeper and
more precisely shows the degree of rearrangement of the
DNA nucleotide sequence variations in wheat-rye hybrids.
Acknowledgements
This work is co-financed by the European Union within the European
Social Fund, Investment in knowledge as a driving force for
development of innovation in the region II edition implemented
within the framework of Subaction 8.2.2 Regional Strategies of
Innovation SOP HRD 20072013.
67
P6.7
Intergenic Spacer length variability
in cultivated, weedy and wild rye species
Lidia Skuza, Ewa Filip, Izabela Szuko
Chair of Cell Biology, University of Szczecin, Waska 13,
PL-70415 Szczecin, Poland
Ribosomal DNA (rDNA) in plants is organized in tandem

arrays with a high copy number (Arnheim 1983). Each repeating unit contains sequences coding for 18S, 5.8S and
25/26/28S rRNA genes (25S is found in most of the plants;
26S is found in wheat and 28S in mammals) and three
spacer regions, i.e. internal transcribed spacers (ITS1and
ITS2) and the intergenic spacer (IGS). Because there are
highly conserved portions (18S, 26S, 5.8S)(Gerbi 1985;
Gerbi et al. 1987) as well as less conserved (ITS) and more
variable (IGS) regions (Appels et al. 1980; Beech and Strobeck 1993; Borisjuk et al. 1994), this area has been used for
both phylogenetic and genetic diversity analyses. Sequencing of this region has also been used to detect and quantify
variability in some species (Chou et al. 1999).
The non-coding rDNA spacers (IGS) and the internal
transcribed spacer (ITS) could be substantially variable
in size due to differences in the number of repetitive elements among the closely related species. The repetitive
and highly variable nature as well as the fast evolution
rate allowed the rDNA spacers to be considered valuable
alternative molecular marker systems. Three pair of universal primers were used for amplification of non-coding regions of ribosomal (rRNA) IGS. The IGS amplified
products obtained from 19 Secale accessions, consisting
of cultivated and non-cultivated rye, and represent three
species and four subspecies of rye genus, showed a high
level of polymorphism. The material was obtained from
several world collections (Center for Biological Diversity
Conservation in Powsin Warsaw, Poland, Nordic Genetic
Resource Center, Sweden and National Small Grains Germplasm Research Facility, National Small Grains Collection, Plant Germplasm Inspection Stadion Agricultural
Research Sernice United States Department of Agriculture, USA).
The primers for IGS PCR resulted in multiple bands
(2 13), different size (59.9bp-3249.7bp) and polymorphism average 81,3%. The genetic similarity of the 19 Secale accessions ranged from 8.4 to 72.2. Cluster analysis
by the Neighbor-Joining method in the Molecular image
Gel DocTM XR (Bio-Rad) program on the basis of the
Dices coefficient of genetic similarity indicated a division of the species studied into three groups of similarity.
S. vavilovii (Hungary) and S. vavilovii (Russia) made
aseparate group. The second group includes nine species:
S. strictum (Turkey), S. strictum (Australia), S. ancestrale
(Japan), S. afganicum (Armenia), S. segetale (Azerbaijan),
S. anatolicum (Canada), S. ancestrale (Turkey), S. cereal
(Macedonia), S. cereal (USA). The third the remaining
species of rye.
68
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The variation in the total size of the IGS among the species detected in this work could be due to dissimilarity in
the sequence of the repetitive elements or in their tandem
repeat number (Saghai-Maroof et al. 1984; Barker et al.
1988; May and Zhiyong 1996). Highly inter specific polymorphisms for rDNA IGS region suggesting the IGS will
be a useful molecular marker for studies of Secale species.
Acknowledgements
This work is supported by the State Committee for Scientific Research
grant No. N N310 435498 Degree of relatedness within the genus
Secale using non-coding chloroplast and mitochondrial sequences, and
nuclear rDNA IGS regions.
P6.8
Intra-population genetic diversity
of cultivated carrot (Daucus carota L.)
assessed by analysis of microsatellite
markers
Anna Maksylewicz, Rafal Baranski
University of Agriculture in Crakow, Institute of Plant Biology
and Biotechnology, Unit of Genetics, Plant Breeding and Seed
Science, al. 29 Listopada 54, 31-425 Crakow, Poland
Cultivated carrot (Daucus carota L. ssp. sativus) is one of

the most important vegetable crops grown worldwide. It
has spread from the centers of origin to remote geographic regions that contributed to large diversity of accessions
at the genetic level. The use of molecular markers, including microsatellites (SSRs), allowed for differentiation of
Asiatic and European gene pools (Baranski et al. 2012),
yet the molecular evidence for intra-population diversity
has not been evaluated.
The aim of the present study was to assess intra-population variation of cultivated carrot using SSR markers.
The study was performed on a collection of 18 carrot accessions originating from Europe, Asia, Japan and USA.
Seeds were obtained from gene bank collection, breeding
companies and research institutes. The accessions included hybrids (F1), open pollinating varieties (OP) and landraces (LR) that differed in root color. Diversity of carrot
accessions represented by 9 to 20 single plants was evaluated using 27 codominant SSR markers detected after
separation in a capillary sequencer.
In total 253 alleles were identified with a mean 9.4 and
range from 2 to 18 alleles per locus. Most of the alleles
(73.5%) had frequencies below 0.1 and only 3.95% alleles
occurred with frequencies above 0.6. Rare alleles (frequency < 0.05) were detected in all except two loci and
they represented 60.5% of all alleles identified. In 23 loci
64 unique alleles were detected (25,3%). The observed
heterozygosity (Ho = 0.39) was lower than the expected
heterozygosity (He = 0.6), which is consistent with previous results on polymorphism of SSR markers in carrot
(Baranski et al. 2012), and the fixation index was higher than zero indicating excess of alleles in the homozygous state, probably due to the use of advanced cultivars
in the analysis. AMOVA results showed that 29% of the
observed variation was due to differences among populations and 71% within the populations that is often noticed
for cross-pollinated species having greater diversity within population than between populations. The first three
PCoA coordinates visualized 61% of total variability. Two
OP cultivars (Yellow Belgian and Shima Ninjin) of the
lowest intra-population variation clearly differed from
the remaining accessions. Accessions originating from
continental Asia and Europe had more allelic variants
(allelic richness 1.47 and 1.42, respectively) than those
from Japan and USA (1.30 and 1.32, respectively). Also
allelic richness in landraces (1.47) was higher than in F1
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hybrids (1.40) and OP cultivars (1.39). In consequence,

accessions from Asia and Europe were more diverse
(He = 0.47 and 0.42, respectively) than those from Japan and USA (He = 0.26 and 0.33, respectively). Landraces were more diverse (He = 0.47) than F1 hybrids
(He = 0.40) and both types were more diverse than OP
cultivars (He = 0.30). It should be however noted that all
accessions from Asia were landraces and those from Europe were either OP or F1 cultivars so greater variability
in the Asian gene pool can be partially explained by the
fact that it was represented by landraces only.
Acknowledgements
The research was supported by the Ministry of Science and Higher
Education grant No. NN310782440.
References
[1] Baranski R., Maksylewicz-Kaul A., Nothnagel T., Cavagnaro P.F.,
Simon P.W., Grzebelus D. (2012) Genetic diversity of carrot (Daucus
carota L.) cultivars revealed by analysis of SSR loci. Genet Resour Crop
Evol 59: 163170.
69
P6.9
Detection of Plasmodiophora brassicae
in soil by PCR method
Anna Czubatka, Jozef Robak, Agnieszka Czajka,
Wojciech Szczechura, Miroslawa Staniaszek
Research Institute of Horticulture, Konstytucji 3 Maja 1/3 St.,
96-100 Skierniewice, Poland
Plasmodiophora brassicae Wor. fungus is the cause of

clubroot one of the most dangerous and most frequent
diseases of brassicas. Resting spores of this pathogen are
able to remain viable in soil even up to 8 years. The most
effective way to protect cultivations against this disease
is introduction resistant plants varieties and analysis of
fields in the presence of this pathogen before planting
brassicas. Detection of P. brassicae relied on bioassays are
long and require large amount of infected soil and space
in greenhouse. Molecular methods use in the detection
of this pathogen should be more sensitive and faster
than traditional plant bioassays. Furthermore, application real-time PCR method could allow detect spores of
P. brassicae and specify their amount in soil samples. Development of reliable and sensitive method to detect this
pathogen is necessary to protect cruciferous crops cultivation against clubroot.
The aim of this work was compare traditional bioassays
and PCR methods as a tools for the detection of P. brassicae in soil. Soil samples containing series of different
concentrations of studied pathogen between 0 to 108
spores per 1 g1 were analysed using bioassay on Chinese
cabbage cv. Granaat, nested PCR and real-time PCR. Genomic DNA for PCR reactions were isolated from soil
samples using commercial kit.
In a biological test P. brassicae was detected in the soil
when the resting spores concentration was 106 of spores
per 1 g1. In nested PCR the specific DNA fragment was
amplified in soil in which the concentration of spores was
higher than 104 spores per 1 g1. Preliminary results indicate that real-time PCR detects 103 spores per 1 g1, which
gives a chance to use it as a sensitive method for the detection and quantification of P. brassicae in soil. However,
this method still requires further work to become a routine diagnostic test replaces the biological assays in detection of P. brassicae in soil.
Acknowledgements
The study was performed in the frame of Multi-annual Programme
financed by Polish Ministry of Agriculture and Rural Development.
70
Eurobiotech 2013
P6.10
P6.11
Chromosomal distribution of repetitive

DNA sequences and karyotyping in
Cucumis metuliferus
The effect of temperature on growth,

and several parameters of Pheodactylum
tricornutum in batch culture
Kohei Yagi, Ewa Siedlecka, Zbigniew Przybecki,

Stefan Malepszy, Plder Wojciech
Monika Bojko1, Klaudia Brzostowska1, Paulina Kuczyska1,

Dariusz Latowski1, Monika Olchawa-Pajor1,
Weronika Krzeszowiec2, Kazimierz Strzaka1
Department of Plant Genetics, Breeding and Biotechnology,

Faculty of Horticulture and Landscape Architecture, Warsaw
University of Life Sciences SGGW, Nowoursynowska,
Warsaw, Poland
1
Cucumis metuliferus (African cucumber, African horned

cucumber, African horned melon, Jelly melon, kiwano) is cultivated species in Cucumis genus like C. sativus. Cucumis metuliferus has become economically and
medically important species in Cucumis genus. It has
attracted attention as a genetic resources for cucumber
and melon. Genomic base information about C. metuliferus will be necessary for grifting or gene introduction
to the major Cucumis crops in the future. Cytological
and molecular cytogenetic studies of C. metuliferus have
been performed by some researcher (Dane and Tsuchiya
1976, Yadava et al. 1984, Nijs 1985, Ramachandran and
Narayan 1990). Most of the these studies reported only
chromosome number (2n = 24) by basic staining (Dane
and Tsuchiya 1976, Yadava et al. 1984, Nijs 1985). Giemsa C-banding has been reported by Ramachandran and
Narayan (1990). They also isolated repetitive DNA, and
indicated that it hybridized to 12 sets of inter phase and
6 heterochromatin knob of pachytene chromosomes.
But distribution patterns and chromosomal locations are
still unclear due to small chromosome size. In this study,
we analyzed the early-metaphase chromosomes of C.
metuliferus by means of 4-6-diamidino-2-phenylindole
(DAPI) and chromomycin A3 (CMA) fluorescence staining and found the location of the 45S rDNA (18S rDNA),
5S rDNA genes, telomere sequence and species-specific
tandem repeats using fluorescent in situ hybridization
(FISH).
Department of Plant Physiology and Biochemistry, Faculty

of Biochemistry, Biophysics and Biotechnology, Jagiellonian
University, Gronostajowa 7, 30-387 Crakow, Poland;
2
Biophysics and Biotechnology, Jagiellonian University,
Gronostajowa 7, 30-387 Crakow, Poland
1
The effect of different temperatures on the kinetics of

diatoms growth in batch cultures was examined. The
experiments were performed using Pheodactylum tricornutum CCAP/1055/1 strain as a model diatom with
known genome sequence. The elucidation of significance
of temperature as an important factor for growing of diatoms is crucial for planning all experiments with these
algae in in vitro systems. The studies compared the effect
of temperature on several parameters important for cultivation of the diatoms in culture medium. These parameters were: the concentration of photosynthetic pigments,
proteins and Fv/Fm, cells size, RNA and DNA content as
well as the optical density (OD405, OD600) of the culture.
The diatoms were grown at two optimal temperatures:
15C and 20C and under temperature stress conditions
at: 12C and 23C with photoperiod 10/14h and light intensity of 40 Em1s1. The above-mentioned parameters
were registered for 14 days, individually for each culture
at different temperatures.
The analysis of the obtained results demonstrated that
temperature had significant effects on the growth kinetics of Pheodactylum tricornutum. Two phases of culture
growth were observed. The faster phase was detected
between first and 5th day after inoculation, The second
one was slower and was recorded from 6th till 14th day,
In cultures growing at 20C and 23C the highest chlorophylls and proteins concentration as well as OD were
measured at the last day of observation. The analysis
of the relation between investigated parameters shows
that in first 5 days after inoculation there are significant
positive correlation between OD increase, chlorophyll
a, chlorophyll c and protein concentration, and Fv/Fm
at different temperatures. The pattern and strength of
correlations varied with temperatures. Several days after
inoculation (from 6th till 14th day) chlorophylls and proteins concentration as well as OD were increasing continually at all growth temperature. On the other hand
the Fv/Fm parameter achieved maximum level at 6th or
7th day and then decreased to the values registered on
the first day of observation. At the highest temperature
(23C) the values of this parameter were the lowest. Total level of RNA and DNA as well as cell size of Ph. tricornutum were additional parameters tested during two
weeks culture of diatoms. Genetic material undergoes
Eurobiotech 2013
gradual degradation 10 days after inoculation. Size of

the cells of Ph. tricornutum was invariable and independent of the time and growth temperature.
All analyzed parameters demonstrated that the optimal
time of the diatoms culture is the period between 4th and
7th day after inoculation.
Acknowledgements
This work was supported by project No. 2011/01/M/NZ1/01170.
71
P6.12
DNA markers as a tool for selection
of tomato plants with resistance to
Fusarium oxysporum f.sp. radicis-lycopersici
Mirosawa Staniaszek, Wojciech Szczechura, Elbieta Kozik,
Marzena Nowakowska, Hanna Habdas
Research Institute of Horticulture, Department of Genetic
Breeding and Biotechnology Vegetable Plants,
96-100 Skierniewice, Poland
Tomato is one of the most important and popular vegetable grown in many world regions. In modern breeding of
this species more frequently are using molecular markers,
especially based on the polymerase chain reaction (PCR).
These methods allowing to selection of plants with specific trait such as resistance to fungal pathogens. Fusarium
oxysporum f.sp. radicis-lycopersici (Forl) is an important
tomato pathogen. This pathogen causes fusarium crown
and root rot, one of the most destructive disease of tomato in crops under cover. Resistance to Forl is determined
by single dominant gen, named Frl, located on the long
arm of chromosome 9 tomato genome. In studies undertaken in Research Institute of Horticulture, Skierniewice,
CAPS C2-251100 marker was identified which is linked to
Frl gene. The amplification products was digested by XapI
restriction enzyme to obtain a DNA polymorphism. This
marker can be useful in marker assisted selection in tomato breeding programs.
This work was performed in the frame of Multi-annual
Programme Development of sustainable methods of
horticultural production to ensure high biological and
nutritional quality of horticultural products and to preserve the biodiversity of the environment and to protect
its resources, finance by Polish Ministry of Agriculture
and Rural Development; Task 6.6.
72
Eurobiotech 2013
P6.13
Effect of different conditions for cell
immobilization and phytosulfokine
supplementation on somatic
embryogenesis in protoplast cultures
of Daucus species
Katarzyna Makowska, Ewa Grzebelus
University of Agriculture in Crakow, Dept. of Genetics,
Plant Breeding and Seed Science
Somatic embryogenesis more or less follow the pattern

of zygotic embryo development and therefore is an important pathway of plant regeneration from cell culture
systems. Additionally, in comparison to often applied organogenesis, plant production through embryogenesis is
much faster and effective. Embedding of the cells in solid
matrices such as alginate may significantly enhance efficiency of that process by inducing asymmetrical divisions
of the cells promoting further polar organization of the
developing structures and in consequence giving rise to
compact embryos. Phytosulphokine (PSK), a peptidyl
plant growth factor found lately in both monocotyledonous and dicotyledonous plants, also has been shown to
stimulate somatic embryogenesis. The aim of the present
study was to evaluate influence of different conditions for
protoplasts immobilization in alginate gel and PSK application on somatic embryogenesis in Daucus species.
Protoplasts were isolated from shoot cultures of seven
Daucus accessions including D. carota subsp. sativus,
D. carota subsp. azoricus, D. carota subsp. drepanensis,
D. carota subsp. maritimus, D. aureus, D. montevidensis,
and D. pusillus. Tissue digestion and protoplast cultures
were performed as described by Grzebelus et al. (2012)
with some modifications: isolated cells were embedded
in filter- or autoclave-sterilized alginate solution as thin
alginate layer (TAL) or extra thin alginate film (ETAF)
and cultured in the presence of 100 nM PSK. Culture
time points both for first cell division and microcolonies
up to 70 m and 150 m in size were recorded. Moreover
frequency of protoplasts undergoing asymmetric cell division, number of macrocolonies (above 500 m in size),
presence of somatic embryos, and number of regenerating plants were estimated.
First cell divisions were observed between fourth and
fifth day of culture for D. carota subsp. sativus, D. carota subsp. drepanensis, D. carota subsp. maritimus, and
D. aureus while for the remaining accessions around 7-8th
day of culture. Cell colonies up to 70 m and 150 m in
size were microscopically visible in 8-16-day-old cultures
and 14-20-day-old cultures, respectively. Both method of
sodium alginate sterilization, culture technique and addition of PSK to the culture medium did not influenced
on the time of the first cell division and cell colonies
formation. Percentage of asymmetric cell divisions was
accession-dependent and varied from 91% to 98%. Protoplasts of D. c. subsp. maritimus and D. montevidensis
most frequently in comparison to the other accesisons

underwent this type of division. After 2 months of culture macrocolonies above 500 m in size were found and
their number varied from 13 (D. c. subsp. azoricus) to 574
(D. c. subsp. sativus). For D. montevidensis and D. pusillus
such macrocolonies were not observed. Additionally, at
that time in the cultures of all D. carota subspecies and
D. aureus somatic embryos in torpedo-stage were found.
After transferring of macrocolonies and embryos to a solid hormone-free medium regeneration into plants were
occurred. The number of morphologically normal plants
varied from 8 for D. c. subsp. maritimus to 73 for D. aureus. Application of the TAL technique for protoplasts
immobilization and supplementation of the cultures with
100 nM PSK increased frequency of plant regeneration.
Acknowledgements
This work was supported by the Polish Ministry of Science and Higher
Education, grant No. N N310 440238.
References
[1] Grzebelus E., Szklarczyk M., Baranski R. (2012) An improved protocol
for plant regeneration from leaf- and hypocotyl-derived protoplasts of
carrot. Plant Cell, Tissue and Organ Culture, 109: 101109.
Eurobiotech 2013
P6.14
P6.15
Characterization of wheat mRNAs

expressed during grain development
Molecular and cytogenetic verification

of the F1 and F2 hybrids between Betula
nana and Betula utilis Doorenbos
Magdalena Simlat, Micha Nowak, Patrycja Wieczorek,

Maria Mo
Department of Plant Breeding and Seed Science,
University of Agriculture, Lobzowska 24, 31-140 Crakow,
Poland
The study was performed using wheat grains harvested at

different developmental stages (from 10 to 45 days after
pollination, DAP) from cultivars Piko and Sawa susceptible and resistant to pre-harvest sprouting (PHS),
respectively. The RNAs isolated from these grains were
used for differential display (DDRT-PCR) analysis of
gene expression. Transcript derived fragments (TDFs)
which were specific for one or more grain developmental
stages were cut out from the gel, re-amplified, T/A-cloned
and sequenced. BLAST analysis revealed homology for
22 from the 25 analyzed fragments. The identified homologies included sequences coding for VP1 transcription
factor, SNF/SWI2 family transcription factor, protein
kinase PK3, flowering locus C (FLC) protein, starch synthase I and germin-like proteins (GLP). Seven fragments
showed high similarity to transposon sequences e.g. the
gene coding for the putative gag-pol poliprotein. Differential expression of the identified sequences will be verified using real-time PCR.
73
Magorzata Czernicka1, Jarosaw Pawiak1, Piotr Muras2

Department of Genetics, Plant Breeding and Seed Science,
31-425 Crakow, Poland; 2Department of Dendrology and
Landscape Architecture, University of Agriculture in Crakow,
al. 29 Listopada 54, 31-425 Crakow, Poland
1
The birch (Betula sp.) is very popular as garden and

park tree. Most of birch species and varieties grow rapidly and for this reason they are not appropriate enough
to cultivate in gardens. In the research, the breeding
program was conducted in order to obtain the interspecific hybrid (Nanabos) which could be characterized by
slow growth rate and interesting shape of purple leaves.
Crossing and selection were done in Tomaszkowice
near Krakow in 2001 while the female inflorescences of Betula nana were manually pollinated by Betula
utilis Doorenbos pollen. In 2002 the total number of
seedlings was about one hundred and only several ones
have survived up to date. The research purpose was the
verification of the hybid character of F1 and F2 putative progeny derived from the interspecific cross i.e.
B. nana x B. utilis Doorenbos. The verification was based
on the RAPD-PCR method and the ploidy level analysis
which were supported by morphological results. Plant
material were: parental taxa, 8 putative F1 hybrids, 8 putative F2 hybrids, 3 cultivars of B. pendula (Doopurea,
Purpurea, Dalecarlica). For molecular research, DNA
was extracted from young leaf tissue using DNeasy
Plant Mini Kit (Qiagen). RAPD analysis was performed
using twenty 10-mer random primers and RAPD-PCR
products were analyzed using agarose gel electrophoresis. For karyotype, mitotic chromosomes were isolated
from young leaf buds using protoplast dropping method
of Anmthawat-Jnsson (2003). The phenotypic analysis
suggested that most hybids had leaves similar to maternal species and they were characterized by unbranched,
columniform growth pattern with tall straight trunk,
that could be inherited from the paternal form. However, the main branches showed spiral growth what was
typical for B. nana.One specimen from the hybrid F1
population was intermediate in phenotype (relative to
parental genotypes) i.e. columniform but low growth
and size of leaves was higher in comparison with the rest
of hybids. Molecular markers were obtained and adendrogram was worked out, which illustrated the level of
the genetic diversity among the parental taxa and F1 and
F2 hybrid plants. Classification of the hybrid plants by
ploidy level proved that most of the putative hybrids indicated that they were diploid (2n = 28) and no triploid
as expected.
Acknowledgements
The research was supported by Polish Ministry of Science and Higher
Education grant for Young scientists (Project No. 4554/2013).
74
Eurobiotech 2013
References
[1] Anamthawat-Jnsson (2004) Preparation of chromosomes from
plant leaf meristems for karyotype analysis and in situ hybridization.
Methods in Cell Science 25(3-4): 9195.
Environmental biotechnology
Lectures
L7.2
L7.1
Bioaugmentation with engineered

endophytic bacteria improves
phytoremediation in the field
Metallophytes: a biodiversity
and phytotechnological resource for
soil clean-up, phytomining and mine site
restoration
Alan J M Baker
School of Botany, The University of Melbourne, Australia;
Centre for Mined Land Rehabilitation, The University
of Queensland, Australia; Department of Animal and Plant
Sciences, The University of Sheffield, UK
Metallophytes plants that have evolved on metal-enriched soils have key values that must drive research
on their unique properties, and ultimately their conservation. The ability of metallophytes to tolerate extreme metal
concentrations commends them as the optimal choice for
phytostabilization and ecological restoration of mineral
wastes and metal-contaminated soils. Metallophytes, and
in particular metal-hyperaccumulating plants, have also
spawned several novel phytotechnologies, including phytoremediation, phytoextraction and phytomining. Other
new potentials for exploiting their unique properties are
emerging. The last decade has seen an ever-increasing
interest in metal-tolerant and metal-accumulating plants
both from an academic standpoint and in developing their
potentials in phytotechnologies. Few studies have highlighted the need to conserve these species. This presentation identifies future research needs for the conservation
and utilization of the global metallophyte biodiversity.
Jaco Vangronsveld, Panagiotis Gkorezis, Sofie Thijs,

Nele Weyens
Hasselt University, Environmental Biology,
Centre for Environmental Sciences, Agoralaan Building D,
B-3590 Diepenbeek, BelgiumL 7.5L.7.6
Phytoremediation is a promising technology: driven by

solar energy, plants are able to pump contaminations to
their rhizosphere and even take them up. In the rhizosphere and during its transport throughout the plant, the
present plant-associated micro-organisms can take care
of the organic contaminants degradation.
Successful application of phytoremediation was demonstrated in 2 field cases (BTEX and oil contamination). On
these sites, poplar trees were planted in the contamination plume and groundwater concentrations and possible
evapotranspiration to the atmosphere were monitored.
Despite the above mentioned and other successful field applications, phytoremediation is not yet applied on alarge
scale due to some constraints. At first, plants should tolerate the occurring contaminant levels. Further, the degradation capacity of the plant-associated micro-organisms must be high enough to prevent phytotoxicity and
evapotranspiration. To solve these constraints, adiversity
of interesting characteristics of plant-associated bacteria
can be exploited.
The availability/uptake of many organics can be stimulated by bacteria producing e.g. surfactants, siderophores
and organic acids. Bacteria with the appropriate degradation pathway(s) can strongly improve the degradation
efficiency.
In a third field case, this concept of endophyte-enhanced phytoremediation was successfully applied on
aTCE-contaminated site. Poplar trees were planted and
in situ inoculated with TCE-degrading endophytic bacteria. Three months after inoculation, a 90%-reduced TCE
evapotranspiration was observed. Further investigations
revealed that this reduction was not only achieved by an
enrichment of the inoculated strain, but also by of transfer of the degradation genes from the inoculated strain to
bacteria from the natural abundant population.
76
Eurobiotech 2013
L7.3
L7.4
Support of Plant Growth by Selected Soil

Microbes and Manipulation of Soil Texture
in Metal Contaminated Soil
Highland plants under extreme conditions

and their phytoremediation potential
I. Lichtscheidl1, S. Wernitznig1, 2, W. Adlassnig1, A.R. Sprocati3,

K. Turnau4, A. Neagoe5, C. Alisi3, S. Sassmann1, A. Nicoara5,
V. Pinto3, C. Cremisini3
UNASAM, Huaraz, Peru
University of Vienna, Core Facility Cell Imaging and

Ultrastructure Research, Althanstrae 14, A-1090 Vienna,
Austria; 2Medical University of Graz, Institute of Cell Biology,
Histology and Embryology. Harrachgasse 21, A-8010 Graz,
Austria; 3Italian National Agency for New Technologies, Energy
and Sustainable Economic Development. Via Anguillarese,
I-301-00123, Rome, Italy; 4Jagiellonian University, Institute
of Environmental Studies, Gronostajowa 7, Pl-30387 Crakow,
Poland; 5University of Bucharest, Research Center for
Ecological Services. Splaiul Independentei 9195, Sector S,
Ro-050089 Bucureti, Romania
1
Establishment of a vegetation cover on mine waste substrate is one of the crucial but also one of the most difficult parts of the remediation process. This study tests
two different approaches to facilitate plant growth on
toxic and oligotrophic substrates, i.e., the addition of
coarse mineral particles and the inoculation with putative
growth supporting bacteria.
Extremely fine grained mine waste from Ingortosu/Italy
containing high amounts of Zn, Cu, Cd and other toxic elements was supplemented with a mixture of sand
and volcanic clay, with a bacterial consortium (Bacillus
cereus, Curtobacterium flaccumfaciens and eight other
strands) or with a combination of both, a control was left
untreated. On these substrates, Euphorbia pithyusa, Helianthus annuus, Agrostis capillaris, Deschampsia flexuosa
and Festuca rubra were grown in pots under greenhouse
conditions.
The bacteria established well and reduced the extractability of most metals. Their effect on plant growth, however, was ambiguous and usually negative. The addition of
sand and volcanic clay, on the other hand, had a positive
effect on all plant species except E. pithyusa. The effects
of a double treatment with both bacteria and sand and
volcanic clay were insignificant or negative.
It is concluded that manipulations of the soil texture of
mine substrates may play a key role in the process of
bioremediation.
Acknowledgement
This study was supported by funding of the the EU project Umbrella
(EU 226870), of the Austrian Ministery for Science and Research, and
the Appear project Biorem. We acknowledge the use of the green
house facility of the University of Vienna and we are thankful for the
help of the gardeners, Thomas Joch and Andreas Schrfl. The soil from
Ingurtosu/Sardinia was kindly provided by Giovanni de Giudici from
the University of Cagliari/I.
Edwin J.P. Cadenas
Eurobiotech 2013
L7.5
L7.6
X-Ray Microanalysis of Copper and Zinc

Uptake in the Moss Physcomitrella patens
Hedw
Bioremediation by Phormidium Biofilms

a Case Study from a Historic Mine Site
S. Sassmann, M. Weidinger, W. Adlassnig, I. Lichtscheidl,

I. Lang
University of Vienna, Core Facility Cell Imaging
and Ultrastructure Research, Althanstrae 14, A-1090 Vienna,
Austria
Mosses are frequently regarded as tolerant to toxic metals and as accumulators of such metals regardless of their
original habitats. The reasons for this tolerance are poorly
understood. Unlike vascular plants, mosses absorb nutrients via the entire surface, and do not possess exclusion mechanisms like an exodermis or an endodermis. In
preliminary experiments, we confirmed an exceptionally
high zinc tolerance of moss gametophytes from heavy
metal contaminated mining sites, and also for Physcomitrella patens Hedw. (Funariaceae) which avoids metal
enriched habitats in nature.
Gametophytes of P. patens were cultivated on sterile
agar plates. We offered zinc (as Na2ZnEDTA, ZnCl2 and
ZnSO4) and copper (as Na2CuEDTA, CuCl2 and CuSO4).
After five weeks of cultivation the moss samples were
washed, air-dried, mounted and carbon-coated. During
sample preparation, great attention was paid to avoid
contamination with the metal rich growth medium. Copper and zinc uptake of P. patens leaves were semi-quantitatively analysed by X-raymicroanalysis in a scanning
electron microscope.
We observed differences in tolerance and uptake of copper and zinc. A gradient elevation of the heavy metal content of leaf tissues corresponding to the added amount of
zinc and copper in the growth media could be observed.
Furthermore, our results indicate strong differences in
uptake and resistance depending on the anion. E.g., metals coordinated with EDTA proved to be significantly less
toxic for P. patens. The relevance of coordinating agents
for metal toxicity is discussed.
Acknowledgements
This research was supported by the Gesellschaft zur Frderung der
Pflanzenwissenschaften, the Hochschuljubilumsstiftung der Stadt Wien
(grant H-1939/2008), the University of Vienna (Forschungsstipendium
2010 to S.S.) and Appear (Biorem)
77
W. Adlassnig1, N. Prez2, S. Sassmann1, S. Reipert1,

F. Hofhansl3, I. K. Lichtscheidl1
University of Vienna, Core Facility Cell Imaging and
Ultrastructure Research, Althanstrae 14, A-1090 Vienna,
Austria; 2Lule Tekniska Universitet, Institutionen fr
Samhllsbygnad och Natururser, Campus Lule, F Huset,
SE-97187 Lule, Sveden; 3University of Vienna, Department
of Microbiology and Ecosystem Science, Althanstrae 14,
A-1090 Vienna, Austria
1
Mining and ore processing frequently results in the formation of metal rich drainage water, representing a serious threat for people and environment. The remediation
of such waters is difficult and expensive, and alternatives
to conventional water treatment are intensively investigated.
At a historic mining site in the Austrian Alps, a Cu contaminated creek is remediating via constant binding of
metals to microbial mats. This study deals with the localisation of the absorbed copper, as well as the isotopic
signature and species composition of the microbial community.
The biofilm is almost exclusively dominated by the Cyanobacterium Phormidum sp. and contains 3.9 1.8%
Cu. The constant absorption from the water reduces
the Cu content of the creek from 0.6 0.1 mg l1 to
0.2 0.2 mg kg1 after only 300 m, thereby exceeding
abiotic Cu precipitation by far. Ancient dead layers of biofilm covered by humus but still rich in Cu indicate that the
immobilisation is permanent. In spite of the variable Cu
content of the biofilm, it exhibits a remarkably constant
65Cu of 0.50.1 which is significantly higher than in
the contaminated water (0.90.2) making uptake of
the Cu into the living cell improbable. This is confirmed
by the ubiquitous occurrence of electron dense mineral
particles in the gelatinous sheath of Phormidium, partly
identified as the secondary Cu mineral sampleite.
Fixed mats of Phormidium have been frequently used
for metal absorption under laboratory conditions. The
spontaneous self-remediation of a creek by Phormidium,
however, is a novel feature and suggests new strategies for
the application of Cyanobacteria in the field of bioremediation.
Acknowledgements
We are grateful to Prof. Dr. A. Beran (University of Vienna) for the
identification of sampleite. Thanks are due to Forstmeister R. Schilcher,
who made scientific research in the Schwarzwand possible. This study
was supported by the EU project Umbrella (EU226870), the Appear
project Biorem and the OEAD project Promote.
78
Eurobiotech 2013
L7.7
FISH detection of autochthonous bacteria
of ceramic clayey raw materials
Paulina Supel1, Pawe Kaszycki1, Joanna Brzeszcz2,
Piotr Kapusta2, Piotr Wyszomirski3
Departament of Biochemistry, Institute of Plant Biology and
Biotechnology, Faculty of Horticulture, University of Agriculture
in Crakow; 2Department of Microbiology, Oil and Gas Institute,
Crakow, 3AGH University of Science and Technology, Faculty
of Materials Science and Ceramics, Crakow
1
Contamination of clay raw materials with organic compounds causes severe problems in ceramic industry. Occurrence of organic fraction leads to lowered aesthetic
value, profoundly reduced mechanical strength, and
thus decreases the overall industrial suitability. Therefore, quality requirements along with increasingly strict
environmental standards give strong reason to elaborate
innovative methods of beneficiation of ceramic clays.
Among these, biotechnological methods based on microbiological enzymatic activities seem to be the most challenging and efficient. The main principle of these methods is to degrade undesirable organic admixtures by heterotrophic microorganisms capable of bioremediation of
an organic fraction as well as to remove iron compounds
by bioleaching.
Autochthonous microorganisms appear as the best natural candidates for biological improvement of ceramic
industry clay materials. If present, they are best fitted
and adapted to the clayey microenvironment and can
possibly utilize material organic impurities as a source
of carbon.
The aim of the study was to prove the occurrence of
autochthonous bacteria in three selected ceramic clay
materials, obtained from different areas and differing in
the content and form of an organic fraction: (1) Lower
Jurassic (Liassic) clay from arnw, with a low organic substance content, (2) Tertiary material form Turw,
characterized by a moderate level of carbon compounds, especially the so called fine-dispersed brown
coal substances, obtained from a site adjacent to brown
coal deposits, and (3) a clay from the Radzymin deposit,
belonging to the relatively young, Quaternary materials,
used mainly in building ceramics due to the high content of organic compounds.
Standard microbiological plating techniques showed the
occurrence of autochthons in materials (2) and (3). Identification of several colony morphotypes indicated high
biological diversity of the strains. The FISH (fluorescence
in situ hybridization) technique enabled to reveal even
greater biodiversity, including clay (1). The visualisation
method was based on several specific molecular probes
directed at conservative genes 16S rRNA (EUB338I, II,
III-Cy3, ALF968-Cy3) and 23S rRNA (BET42a-Cy3,
GAM42a-Cy3, HGC69a-Fluos). The above sets of oligonucleotide probes were constructed to identify bacteria
belonging to the following systematic groups: -Prote-
obacteria (ALF 968), -Proteobacteria (BET42a), -Proteobacteria (GAM42a), Actinobacteria (HGC69a), and
Eubacteria (a mix of three probes EUB 338I, II, III.)
The above data bring for the first time the information
of bacterial consortia inhabiting unique environments of
clayey minerals. We claim the research that we have just
launched will bring interesting and valuable applications
in terms of treatment of ceramic industry materials prior
to their processing.
Acknowledgements
The work was financially supported by the grant for scientific research
No. 3500, approved by the Polish Ministry of Science and Higher
Education.
Eurobiotech 2013
L7.8
Posters
Phytoremediation crops for sustainable

biofuels scope and limitations
P7.1
Prasad M.N.V.
Department of Plant Sciences, University of Hyderabad,
Hyderabad 500 046, India
Energy and environmental security are the crux of the

sustainable development. Natures cure using plant resources (= Phytoremediation) is a sustainable solution for
environmental decontamination. As of now about 18000
articles have been published on various aspects of using
biological resources for environmental cleanup starting
with only 11 in 1989. Often policy and decision makers,
academia and civic governance think that phytoremediation is a temporary solution of transferring the pollutants and contaminant are transferred from one place to
another. Scientists and academia are no exception to this
feeling. Regulators have expressed apprehensions about
phytoremediation due to lack of knowledge of contemporary knowledge of environmental sustainability. Often
it is generally believed, that this kind of environmental
decontamination is a temporary solution. How to dispose
of the contaminated phytomass is a puzzling question by
environmental managers and regulators.
Plants are assisted by soil microbes and mediate wide
array pollutants and are able to decontaminate the toxic substances, thus contribute significantly to pollution
abatement. Phytoremediation is the use of plants to accumulate, remove or render harmless toxic compounds
contaminating the environment. Plants absorb/adsorb/
exclude, translocation, store or detoxify inorganic and organic contaminants. Thereby they contribute significantly
to the fate of these contaminants of the biosphere. Biofuel
production is other side of the coin of the Phytoremediation crops.
Keywords: bioremediation, contaminants, energy and environment
security, geoenvironment, inorganics, natures cure, natural resources,
organics, phytoremediaiton, pollutants, rhizoremediation
Acknowledgements
The author is grateful to the Ministry of Environment and Forests,
GOI, New Delhi For the award of Pitamber Pant National Environment
Fellowship (MoEF Ref. No. 17/3/2010-RE Dt 29-2-2012).
79
The influence of phytase addition during

the enzymatic starch hydrolysis process
with the use of basic amylolytic enzymes
on yield and the course of alcoholic
fermentation process
Dawid Mikulski, Grzegorz Kosowski
Phytic acid (myo-inositol hexa-phosphoric acid, IP6)

is the major phosphorus storage compound of most
cereal grains. Phosphorylated form of inositol due to
ionized phosphates groups has strong chelating properties. Phytic acid creates complexes, the so-called phytin
complexes with divalent cations (Mg2+, Ca2+, Fe2+, Zn2+),
with proteins and polysaccharides, i.e. starch [1]. These
complexes limit the availability of the divalent cations,
proteins and starch for organisms which do not produce the phosphohydrolases enzymes. The applications
of phytase, the enzyme hydrolysing phytin complexes
during the preparation of the fermentation medium,
should result in the increase of availability of biogenic
compounds [2]. The aim of the study was to determine
the influence of phytin hydrolysis before and after starch
hydrolysis process present in high gravity maize mashes
(HG mashes) on yield and the course of alcoholic fermentation process. The raw material used in preparation of HG mashes according to pressureless liberation
of starch technology (PLS technology) was milled maize
grain (starch concentration 53.34%, phytic acid concentration 2.76 mg/g, total phosphorus concentration
2.93 mg/g). The process of enzymatic starch hydrolysis
was conducted with heat-stable -amylase (Termamyl
SC DS preparation) and glucoamylase (Saczyme preparation) produced by Novozymes company. Phytin complexes hydrolysis was conducted with the use of Phytase
10000L preparation produced by Erbsloh company.
During the alcoholic fermentation the basic technological indicators (alcohol concentration, productivity, yield
and fermentation energy) and indicators of the physiological condition of yeast (number, viability, phosphorus
concentration in yeast cells) were determined. Alcoholic
fermentation volatile by-products were determined with
capillary gas chromatography (GC method). The analysis of variance (significant level 3/100kg of starch was
observed in the variant with phytin hydrolysis before
starch hydrolysis process. In the initial fermentation
phase irrespectively of the research variants the viability
of yeast was ca. 98%. In subsequent phases the increase
of ethanol concentration caused the decrease of viability
and number of yeast cells in variants with addition of
phytase in relations to the control variant. The highest
80
Eurobiotech 2013
concentration of acetaldehyde (524.125.6mg/L) and

higher alcohols (4144.2154.0mg/L) was observed in
the distillate obtained in the fermentation medium with
phytin hydrolysis before hydrolysis of starch.
Acknowledgements
The study financed from the science resources in the years 20132014, as
an experimental study of the National Science Centre (No. 2012/05/N/
NZ9/02436).
References
[1] Garcia-Estepa R.M., Guerra-Hernndez E., Garcia-Villanova B.
(1999) Phytic acid content in milled cereal products and breads. Food
Research International 32, 217221.
[2] Lei X.G., Porres M. (2003) Phytase enzymology, applications, and
biotechnology. Biotechnology Letters, 25, 17871794.
P7.2
The Kinetic reduction of Cr(VI) by yeast
cells of Saccharomyces cerevisiae,
Phaffia rhodozyma and their protoplasts
Jarosaw Chwastowski, Henryk Kooczek
Crakow University of Technology, Department of Inorganic
Technology and Environmental Biotechnology
Contamination with heavy metals is a serious environmental problem. Pollution by chromium originating
from several industrial processes such as leather tanning, electroplating, textile industries, nuclear power
plant, water cooling, pulp production, ore and petroleum refining processes is a reason for deterioration of
midland water quality. In trace amounts, chromium is
beneficial to living organisms (M. Pas et al., 2004). However, at higher concentrations it is extremely toxic, causing allergies, egzema, and respiratory track disorders. It
is also a strongly mutagenic and cancerogenic agent, especially in its oxidized form, Cr(VI) (Barceloux, 1999).
Once inside the cell Cr(VI) is being reduced to the
Cr(V) and can react with H2O2 what leads to the production of hydroxyl radicals (OH) via the Fenton-like
reaction. Cr(V) causes DNA breaks and various mutations in chromosomes.
Chromium on the sixth oxidation state can enter easily
into the living cells because Cr(VI) ion has tetrahedral
symmetry (same as SO42 and HPO42, unlike Cr(III)
which has octahedral symmetry) and can enter the cell
membrane through non-specific sulfate transporters by
facilitated diffusion.
The adverse health effects and diverse cellular and molecular reactions make the studies on chromium toxicology and metabolism very crucial in terms of environmental protection and clinical medicine. Such
studies are performed using eukaryotic organisms,
mainly yeast, plants, mammalian cells and transgenic
mice (Cervantes et al., 2000). Among these, yeast has
proved to be a very suitable model for the research of
eukaryotic cell response to chromium stress and bioremediation pathways (Ksheminska, Koloczek et al., 2001,
2005). Microbiological bioremediation of chromium
contamination is performed by means of the reduction
of chromate to the less toxic Cr(III) form which can be
easily removed by precipitation. The mechanisms of
chromate reduction and its transport to the yeast cells
are the subject of the studies.
The kinetic reduction of Cr(VI) presented in this study
is determined in intact yeast cells and their protoplasts.
The study evaluates the behavior of yeasts: Saccharomyces
cerevisiae and Phaffia rhodozyma cells and their isolated
protoplasts to reduce Cr(VI) to lower oxidation states.
The viability of yeast cells was carried out using the methylene blue staining method and the plate method (after
the incubation with different concentration of Cr(VI),
which varied from 0,5 mM to 10 mM).
Eurobiotech 2013
Chromate-reducing activities are monitored by means of

Cr(V) free radical form determination using the L-band
(1.2 GHz) EPR (electron paramagnetic resonance) spectrometer.
Each reduction process was carried out in 120 minutes
and it was measured in vivo in the EPR resonator. Both
strains of yeast displayed substantial reduction of 4 mM
Cr(VI). However, the cells of P.rhodozyma showed about
3.5 times higher reduction than S.cerevisiae. The Cr (VI)
concentration equal to 4 mM was the sub-lethal concentration for S.cerevisiae while for P. rhodozyma didnt have
any negative effect on the viability. The data showed that
Phaffia rhodozyma is far more resistant to higher concentrations of chromium than Saccharomyces cerevisiae. The
obtained result is confirmed by others (H.I Nechay et al.,
2009).
The protoplasts of the above mentioned strains were isolated using the method developed and optimized by the
authors with the use of the Zymolyase enzyme produced
by a culture of Arthrobacter luteus. Protoplasts of both
strains showed couple times faster reduction of chromium compared to intact cells which suggests the high
influence of the cell wall on uptake and/or inhibition of
the reduction process of Cr(VI). Moreover, P. rhodozyma
protoplasts have demonstrated greater Cr(VI) reduction
than S. cerevisiae protoplasts. The observed effect of chromium reduction could be connected with more sufficient
production of metabolites like glutathione and cysteine
which also can be the factors responsible for its resistance
for high concentration of toxic chromium.
Acknowledgements
This work was supported by grant of Ministry of Science and Higher
Education, Poland, No. NN 304326136.
81
P7.3
The citricacid-modified enzyme-resistant
dextrin from potato starch
as potentialprebiotic
Katarzyna liewska1, Renata Barczyska2, Janusz Kapuniak2
Institute of Fermentation Technology and Microbiology,
Faculty of Biotechnology and Food Sciences, Technical
University of Lodz, 171173 Wolczanska Street, 90-924 Lodz,
Poland; 2Institute of Chemistry, Environmental Protection and
Biotechnology, Jan Dlugosz University, 13/15 Armii Krajowej
Avenue, 42-200 Czestochowa, Poland
1
The objective of the study was to determine whether

dextrin obtained as a result of heating starch with citric
acid (patent claim no. 392895) is a substance with prebiotic properties. The enzyme-resistant dextrin, prepared
by heating of potato starch in the presence of hydrochloric (0.1% dsb) and citric (0.1% dsb) acid at 13C for
3h (CA-dextrin), was tested as the source of carbon for
probiotic lactobacilli and bifidobacteria cultured with
intestinal bacteria isolated from faeces of three heathy
70-year old volunteers. The dynamics of growth of bacterial monocultures in broth containing citric acid
(CA)-modified dextrin was estimated. It was also investigated whether lactobacilli and bifidobacteria cultured
with intestinal bacteria in the presence of resistant dextrin would be able to dominate the intestinal isolates.
Prebiotic fermentation of resistant dextrin was analyzed
using prebiotic index (PI). Fermentation products were
determined by HPLC. It was shown that all of the tested bacteria were able to grow and utilize CA-modified
dextrin as a source of carbon, albeit to varying degrees.
In co-cultures of intestinal and probiotic bacteria, the
environment was found to be dominated by the probiotic strains of Bifidobacterium and Lactobacillus, which is
abeneficial effect.
Acknowledgements
The study was supported by the Polish Ministry of Science and Higher
Education, Grant No. N N312 3353 39.
82
Eurobiotech 2013
P7.4
Comparison of ARDRA and ARISA
fingerprinting patterns analysis
as a methods in early bulking activated
sludge symptoms detection
Dagna Sotysik, Daria Matczyska, Ilona Bednarek
Department of Biotechnology and Genetic Engineering, Medical
University of Silesia in Katowice, Narcyzow 1,
41-200 Sosnowiec, Poland
Microbial community very often is characterized on the

basis of ribosomal DNA analysis. Presence in this part of
genome highly variable and conserved regions allow, not
only to differentiation and classification microorganism
species, but also, to observe permanent transformation
of bacterial population. Appropriate balance between
microorganisms from different taxonomy group is critical for proper wastewater treatment process. Using fingerprinting pattern method gives the possibility to detect
those changes and to analyze activated sludge microbial
community. In this study two molecular methods were
used: amplified ribosomal DNA restriction analysis (ARDRA) and automated ribosomal intergenic spacer region
analysis (ARISA) to differentiate activated sludge samples
concerning microbial qualitative fluctuation between
bulking and non-bulking activated sludge.
Active sludge samples (collected from September 2007
to February 2009, twice per month) were derived from
waste water treatment plant in aziska Grne. Each
sample was physicochemical and microscopic analysis
conducted. As a template to molecular investigation genomic DNA was used. To obtain sludges DNA fingerprints by ARDRA technique, 16S rRNA genes fragments
(amplify by use universal primers 27F and 1492R) was
digested by chosen endonucleases. To receive samples
genetic profile by ARISA primers complementary to
conservative regions of 16S and 23S rDNA were used.
Numbers and position of bands in generated genetic
populations profiles (poliacrylamide electrophoresis
gel for ARDRA, capillary electrophoresis results for
ARISA) were comprised basis to created dendrograms
using the Wards algorithm.
As a results fingerprinting patterns for each sample were
generated. Results from ARISA as well as ARDRA allow
to clustered all samples into two groups. Arrangement of
sludge samples in a tree diagrams were similar for both
ARISA and ARDRA method (differences in distribution
concerning 11 sludge samples). Most normal sludge samples were grouped together suggesting alike microbial
composition assurance correct activated sludge condition. Detailed analysis of dendrograms (especially normal
sludge samples assigned to bulking sludge samples cluster) and comparison obtained results with physicochemical and microscopic observations allow to detect subtle
transformations in activated sludge bacterial population
leading to bulking process. It is very important, especial-
ly in the fact, that recognition of bulking process early

symptoms plays important role in prevention processes
which are less expensive and easier than fully developed
bulking syndrome elimination.
Acknowledgements
This project was financially supported by research grant
KNW-1-110/P/2/0 and KNW-1-095/N/3/0, subsidized by Medical
University of Silesia in Katowice.
Eurobiotech 2013
P7.5
P7.6
Phytofiltration laboratory prototype

of uranium-contaminated waters
Biological activity of lipopolysaccharide

from cyanobacterium Synechococcus
PCC 7002
Paulo Favas1, 3, Joo Pratas2, 3
83
School of Life Sciences and the Environment, University

of Trs-os-Montes e Alto Douro, Portugal; 2Department of Earth
Sciences, Faculty of Sciences and Technology, University
of Coimbra, Portugal; 3IMAR-CMA Marine and Environmental
Research Centre, University of Coimbra, Portugal
Agnieszka Rombel-Bryzek1, Anna Krop-Wtorek1, 2
This study presents the results of a laboratory experiments, including a phytofiltration prototype system, to
reduce the uranium (U) concentration in contaminated
waters. The species Callitriche stagnalis Scop., Potamogeton natans L. and Potamogeton pectinatus L. of the native plant community of the uraniferous region of Beiras
(Central Portugal) were selected because they are autochthonous and they show high accumulation levels and/or
high biomass production. Fluorometry was adopted for
the determination of the U content in the water and plant
samples using a Fluorat-022M analyzer (Lumex, Russia). The installed prototype consists of a closed circuit
of channels. The system was initially contaminated with
500 g/L of U as uranyl. The performance of this system was very effective. The U concentration in the water
dropped to 220 g/L in 24 hours and after two weeks it
had decreased to 72.3 g/L, thus representing an efficiency of 85.5%. The U concentration in C. stagnalis increased
from 0.98 to 1567 mg/kg, in P. natans increased from 3.46
to 271 mg/kg and in P. pectinatus increased from 2.63 to
1588 mg/kg. The results show the effectiveness of these
plants to remove U from the water.
Lipopolysaccharide (LPS, endotoxin) is an integral component of the outer membrane of the cell envelope of
Gram-negative bacteria as well as some cyanobacteria.
LPS is the major virulence factor of bacteria and a strong
stimulator of innate immunity in diverse of eukaryotic
species. LPS massively released by pathogenic bacteria infecting the organism, induces overproduction of inflammatory mediators, leading to the systemic inflammatory
response (sepsis) and potentially death. Cyanobacterial
lipopolysaccharides, compared with pathogenic bacterial LPS such as Vibrio cholerae, Neisseria meningitidis,
Yersinia pestis, Plesiomonas shigelloides, have been less
studied [1, 2].
The purpose of this study was to investigate biological activities of lipopolysaccharide isolated from cyanobacterium Synechococcus PCC 7002. LPS was tested by the chromogenic kinetic Limulus amebocyte lysate assay (LAL
test) and by cell culture assay the induction of proinflammatory cytokines, tumor necrosis factor a (TNF-a)
and interleukin 6 (IL-6) by mouse macrophage cell line
J774A.1. Lipopolysaccharide Synechococcus PCC 7002,
activated of the clotting factors released by amebocytes
Limulus and has the ability to induction of the inflammatory cytokines in immunocompetent cells. However LPS
Synechococcus PCC 7002 was less active than this from
the pathogenic Escherichia coli O55.
Department of Biotechnology and Molecular Biology, University

of Opole, Opole, Poland; 2Ludwik Hirszfeld Institute
of Immunology and Experimental Therapy, Polish Academy
of Sciences, Wroclaw, Poland
1
References
[1] ukasiewicz J., ugowski C. (2003) Post. Hig. Med. Dosw. 57: 3353.
[2] Bernardova K. et al. (2008) J. Appl. Toxicol. 28: 7277.
84
Eurobiotech 2013
P7.7
Influence of imidazolium ionic liquids
on activated sludge process
Dorota Gendaszewska1, Ewa Liwarska-Bizukoj1,
Cedric Maton2, Chris V. Stevens2
Technical University of Lodz, Institute of Fermentation
Technology and Microbiology, Lodz, Poland; 2Ghent University,
Department of Sustainable Chemistry and Technology, Ghent,
Belgium
1
Ionic liquids (ILs) have attracted great interest in scientists and industrial centers during the last decade. This
compounds due to their specific features could be a green
alternative for organic solvents. Unfortunately, their
properties like high chemical and thermal stability and
solubility in water can affect on the quality and quantitative composition of the population of activated sludge.
As a consequence, biological wastewater treatment can
be difficult, not fully effective and cause a threat for
aquatic environment. Therefore, it is important to carry
out research on the impact of these compounds on biological wastewater treatment. The aim of the study is to
investigate the effect of the ILs on activated sludge process. To achieve it, the selected imidazolium liquids were
tested with respect to their influence on morphology of
activated sludge flocs, biomass concentration, degrees
of ILs and COD removal. Four different ionic liquids
were tested:1-ethyl-2H-3-methyl-4,5-dimethylimidaziolium iodide (IL 1), 1-ethyl-2-methyl-3-methyl-4,5-dimethylimidazolium iodide (IL 2), 1-ethyl-2-isopropyl-3-methyl-4,5-dimethylimidazolium iodide (IL 4),
l-hexyl-2H-3-methyl-4,5-dimethylimidazolium iodide
(IL 6). The activated sludge was taken from the aeration
chamber at the Combined Wastewater Treatment in Lodz
(Poland). All experiments were conducted in the shake
flasks (300 ml) under aerobic conditions. The following
concentrations of ILs were applied: 1, 5, 25 and 50 mg/l.
The flasks were incubated at 200,5C in a thermostated rotary shaker at 130 min1 for 24 hours. For each ILs
test was performed in triplicate. In addition, control tests
without ionic liquid were conducted. The concentration
of ILs, Chemical Oxygen Demand (COD) and volatile
suspended solids (VSS) were determined at the beginning
and at the end of the tests. Moreover, three independent
slides from each sample were prepared and approximately 40 images were taken. They were processed and analysed using the automated image analysis procedure. As
a result the basic morphological parameters of the flocs
were measured. Ionic liquids influenced on activated
sludge flocs morphology, particularly with regard to flocs
size. The mean projected area of flocs for each sample was
lower than the control, especially at the high initial concentrations of ILs in wastewater (25 and 50 mg/l). The
smallest flocs were found in the tests with IL 6 (50 mg/l).
Also, the presence of ILs in wastewater at higher concentrations caused to the decrease of the concentrations of
activated sludge biomass. The above mentioned observa-
tions can be explained by the fact that ILs having higher

number of longer substituents are more hydrophobic. As
a consequence, ILs at higher concentrations can inhibit
the growth of biomass and cause the formation of small
flocs. Moreover, the degree of removal of ionic liquids
decreased with the increase of the initial concentration
of ILs in wastewater. The highest degree of removal was
observed for IL 6 with the longest alkyl chain. Also, at the
higher initial concentrations of ILs, the degree of COD
removal decreased. This probably resulted from poor
biodegradability of ILs. The obtained results shown that
the influence of imidazolium ionic liquids on activated
sludge depends on their concentration in wastewater and
associates with their chemical structure, particularly the
chain length of alkyl substituents and number of substituents.
Eurobiotech 2013
P7.8
Treatment of fibreboard wastewater
by combined ozone, membrane bioreactor
and activated carbon process
Dorota Krzemiska, Magdalena Madea, Ewa Neczaj
Czestochowa University of Technology, Institute
of Environmental Engineering, Brzeznicka 60a,
42-200 Czestochowa, Poland
The aim of this work was to estimate the influence of ozonation pretreatment of fibreaboard wastewater (FBM) on
anaerobic treatment efficiency in membrane bioreactor
(MBR). Moreover, granular activated carbons (GAC)
were used as a tertiary treatment step.
The influent fed into the anaerobic bioreactor was
collected from the raw wastewater generated in fibreboard company (Silesian Region, Poland). Ozone
gas was generated from air using an ozone generator
(CH-KTB-3G with a power on 0,075 KW). The gas
mixture of air and ozone was fed into the glass tank
containing wastewater via a porous diffuser. Batch experiments were conducted with the use of 1 L wastewater with natural pH and temperature room (2023C).
A 23,5 mg/L of ozone dose in different time was applied for ozonation experiments with an air ow rate of
0,18 m3/h. Anaerobic treatment experiment was conducted by means of 12 L capacity laboratory scale submerged membrane reactor with the capillary ultrafiltration module. The reactor was filled up with granular
sludge from industrial anaerobic wastewater treatment
plant (UASB) from brewing industry. Experiments
were performed under temperature of 37oC with granular sludge concentration of 10 g/l. After acclimatization period reactor was fed with mixture of synthetic
wastewater (80%, v/v) and FBM wastewater (20%, v/v).
Membrane bioreactor was operated at organic loading
rates (OLR) of 1.51 and 1.24 kg COD/m3d and hydraulic retention time (HRT) of 2,4d. In the next step
of this investigations a two types of activated carbons:
WG-12 and Picabiol with different characteristic were
used as a post-treatment step.
The results of our investigations indicated that ozonation irradiation improves the treatability of fibreaboard
wastewater resulting in a higher percent COD removals
and biogas yield. Under the optimal condition, the investigated dose of 19,5 mgO3/L/h and reaction time of
10min, anaerobic biodegradability of FBM wastewater in
MBR reactor was increased by 19%. Moreover the higher
biogas production (0.750.78 m3/kgCODrem.d) was also
observed under ozonation pretreatment. Therefore ozone
pretreatment of fibreboard wastewater is a promising
method to enhance fermentation efficiency. Moreover,
the results obtained showed that GAC can be used in the
removal of COD from fibreboard wastewater. The higher
reduction of COD (67%) was obtained for carbon WG-12
with adsorbent dose of 6 g/l.
85
Keywords: fibreboard wastewater, ozonation, activated carbon,

anaerobic membrane bioreactor
Acknowledgements
This work was supported by the Faculty of Environmental Protection
and Engineering (Czestochowa University of Technology) BS/MN-401315/11 and BS/PB-401-303/11.
86
Eurobiotech 2013
P7.9
Treatment of fibreboard wastewater by
combined fentons process, membrane
bioreactor and activated carbon process
Dorota Krzemiska, Magdalena Madea, Ewa Neczaj
The present study was aimed to treat the fibreaboard

wastewater (FBM) by Fentons process on anaerobic
treatment efficiency in membrane bioreactor (MBR).
Moreover, granular activated carbons (GAC) were used
as atertiary treatment step.
The rst part of this study examined the effect of operating
conditions on Fentons process pretreatment of industrial wastewater. The effectiveness of the AOP pretreatment
was assessed by evaluating wastewater biodegradability
enhancement (BOD5/COD), as well as monitoring major
pollutant concentrations (COD) with reaction time. Anaerobic treatment experiment was conducted by means of
12 L capacity laboratory scale submerged membrane reactor with the capillary ultrafiltration module. The reactor was filled up with granular sludge from industrial anaerobic wastewater treatment plant (UASB) from brewing
industry. Experiments were performed under temperature of 37C with granular sludge concentration of 10 g/l.
After acclimatization period reactor was fed with mixture
of synthetic wastewater (80%, v/v) and FBM wastewater
(20%, v/v). Membrane bioreactor was operated at organic
loading rates (OLR) of 1.54 and 1.25 kg COD/m3d and
hydraulic retention time (HRT) of 2,4 d.
In the next step of this investigations a two types of activated carbons: WG-12 and Picabiol with different characteristic were used as a post-treatment step. The sorption
process was carried out by the static method. The main
difference between those carbons is bulk density. The carbon WG-12 has a reported density of 26,1 lb/cu ft. The
carbon Picabiol is lighter and has reported density of
14,54 lb/cu ft. Taking into consideration capillary disintegration, it was possible to affirm, that WG-12 is typical
agent of microporous carbons, however carbon Picabilo
macroporous carbons.
The results of our investigations indicated that the optimum dose Fe2+ and H2O2 was found to be 1,0 and 1,0 g/L,
respectively. In a single biological treatment the average
removal efficiencies of COD, and BOD were 69%, and
86%, respectively. Integration of Fentons process and biological treatment resulted in 91% removal of COD and
96% BOD from the fibreaboard wastewater. However, the
biogas production was on the same level in both processes (0.540.56 m3/kgCODrem.d). The results indicated
that the combined process would be a promising alternative for the treatment of fibreaboard wastewater. Moreover, the results obtained showed that granular activated
carbons can be useful in the removal of COD from fibreboard wastewater after MBR treatment process. It was
found that carbon WG-12 has better adsorption capacity

as compared with carbon Picabiol. The higher reduction
of COD (54%) was obtained for carbon WG-12 with adsorbent dose of 6 g/l.
Keywords: fibreboard wastewater, Fentons process, activated carbon,

anaerobic membrane
Acknowledgements
This work was supported by the Faculty of Environmental Protection
and Engineering (Czestochowa University of Technology) BS/MN-401315/11 and BS/PB-401-303/11.
Eurobiotech 2013
P7.10
P7.11
Alga Spirogyra sp. biosensor of surface

water contamination with heavy metals
Kinetics and equilibria of heavy metal

sorption by aqueous plants Elodea
canadensis L. and Myriophyllum
spicatum L.
Magorzata Rajfur1, Magorzata Anna Jwiak2, Andrzej Kos1

Chair of Biotechnology and Molecular Biology, Opole University,
kard. B. Kominka 6 St., 45-032 Opole, Poland,
phone +48 77 401 60 42, fax: +48 77 401 60 50,
2
Department of Environment Protection and Modelling
of the Jan Kochanowski University, Swietokrzyska 15 St.,
25-406 Kielce, Poland, phone +48 41 349 64 34,
fax +48 41 349 64 18
1
Algae, due to their presence in various conditions and

high resistance to physicochemical factors, are the pioneers which inhabit new environments, and their sorption properties are used in water biomonitoring. The capacity of heavy metal sorption by algae used for in situ
study depends on the abiotic factors, eg. water pH and
the presence of other cations naturally present in water
ecosystems.
In order to assess the algae sorption properties and the
effect of other factors on the sorption equilibria, the
sorption properties od freshwater alga Spirogyra sp. were
studied in the laboratory conditions. Saline solutions of
the following heavy metals: Mn, Cu, Zn and Cd were
used for the study. The heavy metals affinity, determined
on the basis of the conduced analyses, with the thallus
of alga Spirogyra sp. increases in the following sequence:
Cd2+ < Mn2+ Zn2+ < Cu2+. It was reported that the sorption competitiveness of the cations naturally present in
the algae habitat in relation to Mn2+ ions changes in the
following series: Na+ < Ca2+ < H+ determined for concentrations referred to ion unit charge.
The study results were compared with the results of the
analyses carried out on dried marine algae Palmaria palmata.
The conducted study is a part of the scientific project,
the aim of which is to develop classification method of
surface waters on the basis of the chemical composition
analysis of algae thalli.
Keywords: alga Spirogyra sp., sorption, ions, heavy metals, metals
affinity series
Acknowledgements
The Project received financial assistance from the funds of the National
Science Centre granted by force of the decision No. DEC-2011/03/D/
NZ9/00051.
87
Pawe Krems, Dominik Jerz, Magorzata Rajfur, Andrzej Kos

phone +48 77 401 60 42, fax: +48 77 401 60 50
In the laboratory conditions, the sorption of the following

ions: copper, cadmium, zinc and lead was carried out by
the chosen species of aqueous plants: Elodea canadensis
L. and Myriophyllum spicatum L. Sorption of the studied heavy metals in solutions was conducted under static
conditions and under the conditions of a dynamic solution flow. The process kinetics, equilibrium parameters,
the effect of pH and the manner of preparing the chosen
species on the heavy metal ions sorption were studied.
Process kinetics was described with the pseudo-first and
pseudo-second order reaction models, while the equilibrium was described with the Langmuir isotherm model.
The affinity series of the studied heavy metals was determined for Elodea canadensis L. and Myriophyllum spicatum L. It was found out that the equilibrium between
biosorbent and solution, with which it was in contact, is
attained after approximately 2030 min. It was also stated
that heavy metal sorption processes restrict the presence
of hydrogen cations in solution and that preparation of
the studied plants has effect of their sorption properties.
Keywords: heavy metals, aqueous plants, sorption kinetics and
equilibrium, Langmuir isotherm
88
Eurobiotech 2013
P7.12
P7.13
Heavy metal sorption by moss Pleurozium

shreberi and lichen Hypogymnia physodes
Preparative scale production

of immobilized lipase preparations for
continuous bioconversion processes
Dominik Jerz, Magorzata Rajfur, Andrzej Kos

phone +48 77 401 60 42, fax: +48 77 401 60 50,
The conducted study regarded the assessment of sorption properties of moss Pleurozium shreberi and lichen
Hypogymnia physodes in reference to heavy metal cations: Mn2+, Ni2+, Cu2+, Zn2+, Cd2+ and Pb2+. The aim of the
study was to confirm the thesis that moss reveals better
sorption properties regardless of the type of sorbed cations. The sorption process of the studied analytes was
conducted under static conditions (at changing analytes
concentration in solution) and under dynamic conditions (at constant analytes concentration in solution). To
describe the equilibrium, the Langmuir isotherm model
was applied.
On the basis of the conducted study, it was found out that
the equilibrium is attained after approximately 30min. In
the experiment conditions, 9095% of metals are sorbed
within the first 10 min. It was also stated that moss
Pleurozium shreberi is characterised by better sorption
properties as compared to lichen Hypogymnia physodes,
and that they sorb heavy metals proportionally to their
amount in the solution, in which they were immersed.
On the basis of the conducted study, the affinity of the
studied cations with moss and lichen sorption structures
was determined.
Keywords: lichen Hypogymnia physodes, moss Pleurozium shreberi,
heavy metals, sorption kinetics and equilibrium, Langmuir isotherm
Lukasz Stanczyk, Katarzyna Struszczyk-Swita,

Miroslawa Szczesna-Antczak, Tadeusz Antczak
Institute of Technical Biochemistry, Faculty of Biotechnology
and Food Sciences Lodz University of Technology,
Stefanowskiego 4/10, 90-924 Lodz, Poland
Currently, bioconversion and biotransformations are

important processes in pharmaceutical and chemical industries. Evidence of their significance is provided by the
growing tendency to replace classical chemical manufacturing technologies by purely biotechnological processes
or chemical operations coupled with bioconversion and
biotransformations that are named chemo-enzymatic
processes. Applications of biocatalysis are becoming fully
competitive compared to chemical catalysis and constitute one of fundamental elements of green chemistry.
Among approximately 4000 various enzymes which have
hitherto been known only around 200 ones have been
used in practice. This group has been dominated by hydrolases including lipases.
Lipases are versatile enzymes of industrial importance.
They can catalyze various reactions thereby giving diverse products that are used in many branches of industry: cosmetic, pharmaceutical, production of fuels, food
processing etc.
The potential of lipase has been only partially exploited, mainly because of high costs of lipase preparations
caused by their purification. New and inexpensive lipolytic preparations with excellent catalytic properties under technological conditions, high stability, catalytic efficiency, and molecular and kinetic characteristics, have
been still prospected for. The cheapest biocatalysts were
found to be whole-cell biocatalysts.
The main objective of this project is the development of
a method of preparative scale production procedure of
microbial lipase preparations, e.g. from lipolytic Mucor
circinelloides and Mucor racemosus strains from pure
culture collection at ITB TUL, in a large laboratory scale.
1. Mycelium of Mucor filamentous fungi immobilized in
a porous carrier in the form of uniform thin foams with
open porosity and the large internal surface.
2. Whole-cell preparations Mucor, dehydrated, ground
(particles with a diameter close to 3 m) and additionally
stabilized.
Processes of production of immobilized preparations of
Mucor lipases was optimized in terms of: culture medium composition, method of agitation in a fermentor,
type of porous carrier (pore size, shape and dimensions
of the porous carrier), conditions of de-fatting and de-hydration of mycelium (including pH of lipase microenvironment and water activity (aw) of preparation). It is also
planned to develop a quick method enabling analysis
of the degree of colonization of the porous material by
Eurobiotech 2013
filamentous fungi based on digital techniques of image

analysis. One of objectives of this task is also the development of a method of long-term storage and activation of
immobilized preparations of Mucor lipases.
Acknowledgements
Project Nr POIG.01.03.01-00-158/09 Biotransformations useful in
pharmaceutical and cosmetic industries.
Project partially financed by the European Regional Funds within the
scope of Operation Program Innovative Economy.
89
P7.14
Changes in dynamics of ascorbateglutathione metabolism in response
to trace metal stress in Pisum sativum
Aneta Piechalak1, Arleta Malecka1, Agnieszka Kutrowska1,
Anetta Hanc2, Danuta Baralkiewicz2, Barbara Tomaszewska1
Department of Biochemistry, Faculty of Biology,
Adam Mickiewicz University, Umultowska 89, 61-614 Poznan,
Poland; 2Department of Trace Element Analysis by
Spectroscopy Method, Faculty of Chemistry, Adam Mickiewicz
University, Umultowska 90, 61-614 Poznan, Poland
1
Heavy metals are common pollutants in ecosystems and

due to anthropogenic activities its concentration steadily
increase each year. They are not biodegradable and are
thus persistent in the environment. Many heavy metals
(such as cadmium, lead but also high amount of copper
and zinc) are toxic for plants. They can reduce photosynthesis, cause water imbalance, disturb nutrient accumulation and induce oxidative stress. The toxicity of heavy
metals is generally thought to be due to inactivation of
enzymes and/or functional proteins by directly binding
to them or caused their oxidation by reactive oxygen
species (ROS) generated in the presence of heavy metals, such as hydrogen peroxide (H2O2), hydroxyl radicals
(OH) and superoxide radicals (O2). These can react
very rapidly with DNA, lipids, pigments and proteins and
cause lipid peroxidation, membrane damage and inactivation of enzymes (Singh et al. 2006). To minimize the
harmful effects of ROS, plants activate defense system including both enzymatic antioxidants and non-enzymatic
antioxidants that participate in scavenging ROS (Malecka
et al. 2009, 2011). Among the antioxidant defense system,
ascorbate (AsA), glutathione (GSH) and related enzymes
(such as ascorbate peroxidase, glutathione reductase and
glutathione peroxidase) play a pivotal role in direct or
indirect scavenging of ROS from plant cells (Noctor and
Foyer 1998, Aravind and Prasad 2005).
In order to elucidate the response of the ascorbate-glutathione (ASC-GSH) cycle to trace metal stress, the activities of antioxidant enzymes and the levels of molecules
involved in the ASC-GSH metabolism were studied in
Pisum sativum L. plants subjected to copper, zinc, cadmium or lead. Compared to the control, the contents of
H2O2, ascorbate (ASC), dehydroascorbate (DHA), and
glutathione disulfide (GSSG) increased in metal-treated
plants, whereas the reduce glutathione content decrease
due to GSH role as antioxidant and substrate to phytochelatin biosynthesis. Further more, the ASC/DHA and
GSH/GSSG ratios decreased in the metal ions presence.
The activities of ascorbate peroxidase (APX), glutathione
reductase (GR) and gamma-glutamylcysteine synthetase
(ECS) were up-regulated compared to control. These data
indicate that fluctuations in the ASC-GSH metabolism
observed during treatment may have a positive effect on
metal stress mitigation in P. sativum.
Acknowledgements
This research was supported by grant MNiSWN N310 107840.
90
Eurobiotech 2013
P7.15
P7.16
Biological denitrification of wastewater:

Metabolic acticity in a microbial
consortium
Influenceof radical reactions

on the kinetics of PAH biodegradation
in aqueous media
Agnieszka Piotrowska-Cyplik1, Wojciech Juzwa2,

Zuzanna Szczepaniak1, Justyna Staninska2, Jakub Czarny3,
Roman Marecik2
Justyna Staninska1, Zuzanna Szczepaniak2, Pawe Cyplik1
Department of Biotechnology and Food Microbiology, Poznan

University of Life Sciences, Wojska Polskiego 48,
60-627 Poznan, Poland; 2Institute of Food Technology of Plant
Origin, Poznan University of Life Sciences, Wojska Polskiego 31,
60-624 Poznan, Poland
The wastewater originating from explosives manufacturing plants are characterized by a high concentration of
nitrates, sulphates and low pH. The determined composition of treated wastes was: 3200 mg N L1, 48 mg L1
chlorides, 1470 mg L1 sulfates. The DNC6 consortium
bacterial cell samples collected during denitrification of
industrial wastewater in the bioreactor were subjected
to separation with the use of the FACS Aria III flow cytometer (Becton Dickinson, USA). Cytometric analysis
of the metabolic activity, expressed as the red-ox potential, allowed for the evaluation of specific single microbial
cells. Separation of cells allowed for the isolation of pure
monocultures, which were used for sequencing and subsequent genetic identification. The microorganisms present in the bacterial population used for the denitrification
were identified at the first, second, fourth and sixth day
of the process. The combination of flow cytometry and
genetic identification allowed for determination of bacterial species present in both areas of metabolic activity.
The low metabolic activity area accounted for 32% of the
total cell count, while the high metabolic activity area
constituted for 19% of the total cell count. In the area of
highest activity P. stutzeri accounted for 43% of total cell
count, while P. putida accounted for 39%. The remaining 18% included freundi, P. alcaligenes and A. xylosoxidans. The composition of cells present low activity area
was more diverse, with 79% accounting for four main
species O. intermedium, R. rubber, S. multivorum and
S. maltophila). The highest difference in metabolic activity between these two areas was observed during the
second day of the denitrification process. The microorganisms in the high activity area exhibited a fluorescence
intensity at 12500, while the value for microbes in the low
activity area was at 960.
The industrial development and expansion of urban agglomerations, which have been constantly progressing
since the 18th century, contribute to the accumulation
of compounds from the group of polycyclic aromatic
hydrocarbons (PAH) in different areas of environment
and to their subsequent deposition into surface waters.
The scale of the phenomenon is evidenced by numerous
exceeding of standards recorded during the diagnostic
monitoring of priority substances conducted under the
State Environmental Monitoring. Since it was found that
the prolonged effects of PAH on living organisms are
highly unfavorable, there has been an increasing need to
develop technologies of their effective degradation. This
study aimed to determine the effect of ozonation-induced
radical reactions on the biodegradation kinetics of selected PAH compounds by a microbial consortium isolated
from soils contaminated with organic substances. The
degradation was carried out under aerobic conditions at
25C for 240 h in aqueous mineral solutions containing
naphthalene, phenanthrene or pyrene at concentrations
of 50 mg/dm3. The ozonation was performed before microbial inoculation and lasted 60 minutes (at an efficiency of 25 g ozone/h). Measurement of PAH removal was
performed using the photoluminescence method with
prior hexane extraction. It was found that ozonation had
no lethal effects on the microbial consortium, reflecting
the lack of toxicity of radical reactions products. In addition, the biodegradability of particular PAH in ozonated
systems showed similarity to the reference tests. The increase in the number of aromatic rings was related with
a decrease in the degradation rate, which was associated
with a decrease in water solubility and lower bioavailability of the studied compounds. Assessment of biodegradation kinetics (based on the comparison of PAH degradation half-times) showed that ozonation increased the
biodegradation efficiency by approx. 25%. These results
demonstrate the possible application of combined techniques: ozonation and biodegradation in environment
protection.
Institute of Food Technology of Plant Origin, Poznan University

of Life Sciences, Wojska Polskiego 31, 60-624 Poznan, Poland;
2
Department of Biotechnology and Food Microbiology, Poznan
University of Life Sciences, Wojska Polskiego 48,
60-627 Poznan, Poland; 3Institute of Forensic Genetics,
al. Mickiewicza 3/4, 85-071 Bydgoszcz, Poland
Acknowledgements
This work was financially supported by the Ministry of Science and
Higher Education, Poland (Grant No. N N523 419 837).
Eurobiotech 2013
P7.17
Different patterns of SOD generation
and scavenging in barley SSD lines
during drought
Renata Bczek-Kwinta1, Magorzata Borek2,
Janusz Kocielniak3, Katarzyna muda4
University of Agriculture in Crakow, Faculty of Agriculture and
Economics, Plant Physiology Department; 2University
of Agriculture in Crakow, Faculty of Agriculture and Economics,
Plant Physiology Department; 3University of Agriculture
in Crakow, Faculty of Agriculture and Economics, Plant
Physiology Department; 4University of Agriculture in Crakow,
Faculty of Agriculture and Economics, Plant Physiology
Department
1
Breeding lines obtained via SSD technology provide the

benefits of using 56 rounds of crossing-over during the
breeding process [1]. The aim of the POLAPGEN project
is to supply breeding companies with some tools useful
in generation of new cultivars of barley resistant to water
scarcity, new methods for assessing plants resilience, and
to develop plant ideotype for drought resistance. One of
the elements of such resistance is the activity of antioxidant system [2]. Therefore, the intensity of superoxide
anion generation (O2; histochemical method) and superoxide dismutase activity (SOD, spectrophotometric
determination by cytochrome c) in leaves of plants of
barley SSD lines subjected to drought during their seedling stage was studied. For the measurement and analyses the 3rd leaf (counting from the base) was collected.
For a thorough evaluation of the results of histochemical
analyses, an image analysis software (WinDIAS 3, Delta
T, United Kingdom) was used.
It was found that the intensity of the O2 production varied widely in both the control plants and these subjected
to drought. Drought caused an increased generation of
O2 in leaves of 69, a reduced in 20 lines, and 12 lines
showed no changes when compared to their controls.
SOD activity revealed an increase in 43, and a drop of
18 lines. The direction of changes in O2 generation and
SOD activity was consistent in 43 per 101 studied cases.
While analysing the pattern of staining the tissue against
O2, which can provide information about small injuries
and/or leaf structure, in 5 cases an extremely weak O2
generation, resulting from tissue drying, was noticed. The
type of the staining was also very differentiated, from only
marginal (leaf blade not dyed), mottled and/or localised
along the veins, through the patchy one to the complete
coverage.
Protein content was genotypically differentiated, but
a strong stimulating impact of drought was noticed, as
in the most of the lines an increase was triggered (even
7-fold in relation to the control). In 4 cases no effect was
observed, but also a drop in 10 cases, including a 3-fold in
leaves of two lines, was established.
The results reveal a very weak antioxidant activity of
plants of some studied lines, and suggest the high metabolic activity of these genotypes, in which both intense
91
generation and scavenging of O2 was accompanied by

an increase in protein level during drought. The comparison of the results with these obtained in the other tasks of
the project will improve the phenotyping of barley lines
tested for antioxidant efficiency and signalling processes.
Acknowledgements
Project Biotechnological tools for breeding cereals with increased
resistance to drought (POLAPGEN-BD) is co-financed by European
Union from European Regional Development Fund in a framework of
the Innovative Economy Operational Programme 20072013.
References
[1] Surma M., Adamski T., Kaczmarek Z., Czajka S. (2003) Zmienno
cech ilociowych w populacjach linii DH i SSD jczmienia. Biuletyn
IHAR 226/227/1, 277283.
[2] Bczek-Kwinta R., Borek M., Kocielniak J., muda K. (2013)
Does the increased SOD activity reflects drought resistance of barley?
BioTechnologia 94(2), 163.
92
Eurobiotech 2013
P7.18
P7.19
Influence of additional carbon sources

on pentachlorophenol transformation
The influence of bioaugumentation and

biosurfactant addition on bioremediation
efficiency of diesel-oil contaminated soil:
feasibility during field studies
Magdalena Kurek, Edyta Burdzik, Joanna Surmacz-Grska

Faculty of Energy and Environmental Engineering, The Silesian
University of Technology, Konarskiego 18A, 44-100 Gliwice,
Poland
The influence of additional carbon and energy sources

(phenol and glucose) on pentachlorophenol transformation by microorganisms of activated sludge, in batch
reactors, was investigated. Pentachlorophenol is a highly toxic, cancerogenic and resistant to biodegradation
compound, which has been widely used as a wood preservative and biocide. Obtained results showed, that the
degradation of 15 mg/l of pentachlorophenol as a only
carbon and energy source, with unacclimated activated
sludge is inefficient. The presence of phenol, glucose or
both, phenol and glucose, brought about the increase of
pentachlorophenol degradation rate. The highest removal
of pentachlorophenol (62%) was gained for both growth
substrates (phenol and glucose) together. Furthermore,
the presence of readily growth substrates, resulted in increase of biomass concentration, correlated with pentachlorophenol and growth substrates degradation. When
pentachlorophenol was only carbon and energy source
the decrease in biomass concentration was observed.
Acknowledgements
This work was financially supported by National Science Centre (NCN),
PRELUDIUM project No. 2012/05/N/ST8/03804.
Alicja Szulc, Daria Pziak, Aleksandra Piotrowska,

Marta Woniak, Mateusz Sydow, ukasz awniczak,
Anna Parus, ukasz Chrzanowski
Institute of Chemical Technology and Engineering,
Poznan University of Technology, Poznan, Poland
The study focused on assessing the influence of bioaugmentation and addition of rhamnolipids on diesel oil biodegradation efficiency under environmental conditions.
Initial laboratory studies (measurement of emitted CO2
and dehydrogenase activity) were carried out in order
to select the consortium for biougmentation as well as
to evaluate the most appropriate concentration of rhamnolipids. The selected consortium consisted of following
bacterial taxa: Aeromonas sp., Alcaligenes sp., Gordonia sp., Pseudomonas fluorescens, Pseudomonas putida,
Rhodococcus sp., Stenotrophomonas maltophilia, Xanthomonas sp. It was established that the application of the
rhamnolipids at 150 mg/kg of soil was most appropriate
in terms of dehydrogenase activity. Based on the obtained
results, four treatment methods were designed and tested
during long-term field studies: I) natural attenuation; II)
addition of biosurfactants III) bioaugmentation; IV) bioaugmentation and addition of biosurfactants. It was observed that bioaugmentation contributed to the highest
diesel oil biodegradation efficiency, whereas the addition
of rhamnolipids did not notably influence the treatment
process.
Acknowledgements
This work was supported by the grant number 32-400/2013 DS-MK.
Eurobiotech 2013
P7.20
P7.21
Contributions of rhamnolipids to natural

or induced bioremediation
Biodegradation of ionic liquids

by microbial consortia in aqueous media
Alicja Szulc1, Marta Woniak1, Mateusz Sydow1, Piotr Lisiecki1,

Grzegorz Framski2, Roman Marecik3, Kamila Myszka3,
ukasz Chrzanowski1
Joanna Wojtera-Kwiczor3, Anna Gielnik3, ukasz awniczak1,

ukasz Chrzanowski1
Institute of Chemical Technology and Engineering, Poznan

University of Technology, Poznan, Poland; 2Institute
of Bioorganic Chemistry, Polish Academy of Sciences, Poznan,
Poland; 3Department of Biotechnology and Food Microbiology,
University of Life Sciences in Poznan, Poznan, Poland
1
Surface-active compounds of biological origin have attracted much attention and their popularity seems to
steadily increase during recent years. Like every other
natural substance, biosurfatants have numerous advantages compared to their synthetic counterparts. They
are equally effective as synthethic surfactants in terms of
solubilization and emulsification, however they are readily biodegradable, less toxic and more environmentally
friendly. Moreover, biosurfactants can be obtained from
waste materials, which renders their production favorable on account of economics. All these relevant traits
contribute to a high applicability of biosurfactants, which
currently stems to several branches of industry as well
as remediation. This review summarizes the recent revelations in the field of biosurfactant amended bioremediation, focusing mainly on a critical approach towards
potential limitations and causes of failure, while studying
the effects of biosurfactants on the efficiency of biodegradation and phytoextraction processes.
Acknowledgements
93

of Chemistry, Poznan University of Technology, Poznan,
Poland; 3Institute of Molecular Biology and Biotechnology,
Adam Mickiewicz University in Poznan, Poznan, Poland
1
Ionic liquids are specialized chemical compounds which

have gained much attention due to their broad range
of properties. Since the applicability of ionic liquids is
rapidly expanding, it is imperative to evaluate their influence on the environment. Due to their high biological activity and potential toxicity, these compounds have
been considered as major xenobiotics. In particular, the
biodegradability of ionic liquids has been recognized as
a topic of high environmental relevance. In order to assess their recalcitrance and develop a potentially efficient
removal method, a set of biodegradation studies was carried out with the use of a defined microbial consortium
which displayed a high catabolic potential towards numerous pollutants. The studies focused on investigation
of the biodegradation efficiency of selected ionic liquids
by the microbial consortium as well as on the determination of the qualitative and quantitative changes in the
microbial consortium during the biodegradation of ionic
liquids. The following compounds were selected for biodegradation studies: trihexyl(tetradecyl)phosphonium
chloride, trihexyl(tetradecyl)phosphonium bromide,
trihexyl(tetradecyl)phosphonium benzotriazolate, trihexyl(tetradecyl)phosphonium 3-amino-1,2,4-triazolate,
trihexyl(tetradecyl)phosphonium 1,2,4-triazolate, tetradecyl(trihexyl)phosphonium (dicyano)imide, tetradecyl(trihexyl)phosphonium bis(2,4,4-trimethylpentyl)
phosphinate, tetradecyl(trihexyl)phosphonium bis(trifluoromethylsulfonyl)imide, Benzalkonium chloride,
benzalkonium 3-amino-1,2,4-triazolate, 1-Butyl-1-methylpiperidinium bromide, 1-Butyl-1-methylpyrrolidinium bromide, 1-dodecyl-3-methylimidazolium bromide,
1-butyl-3-methylimidazolium bromide, Didecyldimethylammonium bromide, Didecyldimethylammonium
3-amino-1,2,4-triazolate. The biodegradation rates were
assessed via HPLC-MS as well as the DBAS (disulphine
blue active substances) method while the quantitative
and qualitative changes in bacterial community dynamics were determined by employing real-time quantitative
PCR with the ddCt approximation method. The obtained
results suggest that phosphonium-based ionic liquids
were much more biodegradable, whereas the ammonium-based compounds were toxic, recalcitrant and contributed to distinguishable changes in the abundance of
specific consortium members.
Acknowledgements
This work was supported by the grant number 2011/03/B/NZ9/00731
from The National Science Centre.
94
Eurobiotech 2013
P7.22
P7.23
Influence of 1-alkoxymethyl-2-methyl5-hydroxypyridinium chloride homologues

on biodegradation of diesel fuel by
a microbial consortium
Biodegradation of diesel/biodiesel blends

in sandy soil
Jakub Idkowiak2, Bogdan Wyrwas2, Agnieszka Zgoa-Grzekowiak2, Anna Syguda1, Joanna Wojtera-Kwiczor3,
Anna Gielnik 3, ukasz awniczak 1, ukasz Chrzanowski 1
of Chemistry, Poznan University of Technology, Poznan,
Poland; 3Institute of Molecular Biology and Biotechnology,
Adam Mickiewicz University in Poznan, Poznan, Poland
1
Fast development of ionic liquids is gaining more and

more attention. New formulations and application of ionic liquids may result in contamination in the presence of
hydrophobic compounds, such as petroleum mixtures.
We hypothesize that in the presence of diesel fuel, the
low-water-soluble ionic liquids may become more toxic
to hydrocarbon-degrading microorganisms. In this study
the influence of 1-alkoxymethyl-2-methyl-5-hydroxypyridinium chloride homologues (side chain length from
C3 to C18) on biodegradation of diesel fuel by a bacterial consortium was investigated. The tests performed
for the consortium cultivated on disodium succinate
showed that toxicity of the investigated ionic liquids decreased with increase in side-chain length, only higher
homologues (C8C18) caused a decrease in diesel fuel
biodegradation. Additionally, the modification in cell
surface hydrophobicity was observed as a result of exposure to toxic compounds (MATH). Disulphine blue
active substances method was employed to determine
the partitioning index of ionic liquids between the water
and diesel fuel phases, which varied from 1.1 to 51% for
C3 and C18 homologues, respectively. We conclude that
in the presence of hydrocarbons acting as a solvent, the
increased bioavailability of hydrophobic homologues is
responsible for the decrease in biodegradation efficiency
of diesel fuel.
Acknowledgements
Piotr Lisiecki1, Daria Pziak1, Aleksandra Piotrowska1,

ukasz awniczak1, Anna Parus1, Grzegorz Framski2,
Jacek Staniewski1, ukasz Chrzanowski1
of Bioorganic Chemistry, Polish Academy of Sciences, Poznan,
Poland
1
This study was aimed at evaluation of the biodegradation

rates of aromatic and aliphatic hydrocarbon fractions in
saturated sandy soil spiked with diesel/biodiesel blends
(D, B10, B20, B30, B40, B50, B60, B70, B80, B90 and
B100, where D is commercially available petroleum diesel
fuel and B is commercial rapeseed biodiesel). The bacterial consortium of petroleum degraders previously isolated from crude oil contaminated site in the Carpathian
mountains was applied as a biological agent. The biodegradation kinetics were evaluated using measurement of
the amount of CO2 evolved within 578 days. The residual
aromatic and aliphatic fractions of diesel fuel were determined by GC-FID and GCGC-TOF-MS techniques.
Furthermore, the influence of biodiesel amendment on
the community dynamics was assessed using Real-Time
PCR. The obtained results suggest that there was no preferential degradation of aliphatic fraction of diesel fuel due
to amendment with biodiesel since the ratio of aliphatic/
aromatic hydrocarbons remained constant regardless of
the biodiesel concentration. Our observation leads to the
conclusion that biodiesel does not present many of the
previously described environmental advantages.
Acknowledgements
Eurobiotech 2013
P7.24
P7.25
The influence of biodiesel

on the biodegradation of hydrocarbons
in aqueous microcosms
Preferential degradation of Triton

X-100 and polyethylene glycols during
biodegradation of diesel fuel
Piotr Lisiecki1, Marta Woniak1, Mateusz Sydow1, Anna Parus1,

Joanna Wojtera-Kwiczor2, Roman Marecik3, Kamila Myszka3,
ukasz Chrzanowski1
ukasz awniczak1, Daria Pziak1, Aleksandra Piotrowska1,

Marta Woniak1, Mateusz Sydow1, Alicja Szulc1,
Bogdan Wyrwas2, ukasz Chrzanowski1

of Molecular Biology and Biotechnology, Adam Mickiewicz
University in Poznan, Poznan, Poland; 3Department
of Biotechnology and Food Microbiology, Poznan University
of Life Sciences, Poznan, Poland
Biodiesel is considered as an attractive alternative to petroleum derived diesel fuel. However its environmental impact has not been fully investigated yet. Many researches concerning the emission of green-house gases
and exhaust emission have been conducted, but little attention has been paid towards the fate of biodiesel fuels
in case of an environmental spill. As the production and
consumption of biodiesel grows, the threat of its spills
into the environment increases as well. In fact, biodiesel
is mainly used in the form of blends with petro-diesel.
Therefore simultaneous presence of fatty acids alkyl esters
and petroleum hydrocarbons in the fuel contaminated
site is highly probable. Although it has been confirmed
by several authors that biodiesel is more readily biodegraded than petroleum diesel in general, the impact of
biodiesel on the biodegradation of hydrocarbons remains
unclear. Some authors suggest that the presence of a readily available carbon source may stimulate the growth of
microorganisms and hence increase the dissipation of
hydrocarbons. On the other hand, other researchers do
not confirm this hypothesis. The aim of this work was
to investigate the influence of biodiesel on the biodegradation of hydrocarbons in the water environment. For
this purpose, a set of aqueous microcosms was prepared.
Mixtures of model hydrocarbons paraffins, aromatics
and naphthenes served as a sole carbon source in one
half of the microcosms. The other half was supplemented with 20% addition of biodiesel. During the 3 weeks
lasting experiment, sets of samples were sacrificed periodically for chromatographic determination of carbon
source residues. The results of the research show that
the applied bacterial consortium isolated from crude oil
polluted site exhibits great abilities to decompose various
chemical groups of hydrocarbons and biodiesel, although
branched alkanes and naphthenes were degraded in lesser extent. On the other hand, the most readily degraded
compounds were fatty acids methyl esters and straight
chain alkanes, which were dissipated in a similar rate.
There was no significant change of hydrocarbons biodegradation rate in the presence of biodiesel. This suggests
that biodiesel addition to diesel fuel will not affect the
biodegradation of the petroleum fraction of such fuels.
Acknowledgements
95

Poznan University of Technology, Poznan, Poland;
2
Institute of Chemistry, Poznan University of Technology,
Poznan, Poland
Biological methods based on biodegradation processes

mediated by anionic or nonionic surfactants have recently received much scientific attention. Surfactants may
intensify the bioprocesses through solubilization and
mobilization of water-immiscible hydrocarbons. In the
concept, hydrophobic droplets are enclosed in micelles
and transported to the aqueous phase. On the other hand,
the negative influence of addition surfactants, especially
synthetic, on biodegradation process was also frequently reported. These compounds can have a toxic effect on
microorganisms able to degrade petroleum compounds.
It was also described that molecules of surfactants may
interact with cell surface and decrease the adhesion to
organic phase. Moreover, it is possible that hydrocarbon
droplets entrapped in micelles are not available for bacteria. It is also noted, that some surfactants can be preferential carbon sources for degrading bacterial species. Triton
X-100 is an intensively studied nonionic surfactant with
application in bioremediation processes. Therefore, the
hypothesis regarding preferential biodegradation of Triton X-100 applied for enhancement of microbial removal
of hydrocarbons was studied. The microbial degradation
of Triton X-100 as a sole carbon source as well as with
co-degradation of surfactant and diesel hydrocarbons by
a soil-isolated bacterial consortium was confirmed under
both full and limited aeration with nitrate as an electron
acceptor.
Acknowledgements
96
Eurobiotech 2013
P7.26
P 7.27
Biological treatment of spent metalworking

fluids by non-autochthonous bacterial
consortia
Bioavailability of hydrocarbons to bacterial

consortia during Triton X-100-mediated
biodegradation in aqueous media
ukasz awniczak1, Daria Pziak1, Aleksandra Piotrowska1,

Marta Woniak1, Mateusz Sydow1, Alicja Szulc1,
Roman Marecik2, ukasz Chrzanowski1
Daria Pziak1, Aleksandra Piotrowska1, Marta Woniak1,

Mateusz Sydow1, Alicja Szulc1, Jakub Idkowiak2,

2
Poznan University of Life Sciences, Poznan, Poland
Metalworking fluids (MWF) are commonly used in the

metalworking industry as coolants and shear reducing
media. The metalworking fluids which are currently in
use are mostly oil-in-water emulsions, which contain
several chemical additives (surfactants, stabilizing and
anticorrosive agents, biocides etc.). Due to rapidly progressing industrialization, the MWF market is constantly
developing. The most popular types of MWF (emulsified
oil, semi-synthetic and synthetic fluids) are usually used
as 5% aqueous solutions. Constant dilution the metalworking plants contributes to the generation of immense
wastewater volumes, which must be subjected to treatment.
A fundamental shift in the interest regarding the microbiology of metalworking fluids could be observed over
the recent years. While the previous approach was mainly
focused on suppressing microbial growth in operational
media, presently, a need to explore the principals of colonization and biodegradation of waste MWF has been recognized. This effect is clearly associated with the growing
popularity of biological remediation methods.
The results of previously conducted studies suggest that
populations which inhabit metalworking fluids are characterized by low biodiversity and high similarity in terms
of prevalent taxa, even when comparing samples of fundamentally different origin. Additionally, several scientific reports indicate, that the native microorganisms often
display virulence, pathogenic traits and the tendency to
resort to antagonistic interactions (i.e. secretion of toxins).
With this in mind, the aim of this study was to evaluate
the ability of non-autochthonic bacterial consortia originating from petroleum-contaminated soil to colonize and
biodegrade spent metalworking fluids. Several crucial
process factors were studied (temperature, pH, carriers
for cell immobilization, addition of nitrogen and phosphorous) in order to evaluate the optimal biodegradation
conditions. The biodegradation efficiency was assessed
by determining the decrease of chemical and biochemical oxygen demand. Additionally, the biodegradability of
selected MWF components was also studied by employing GC-MS and HPLC-MS. The obtained studies confirm
that the use of non-autochthonic bacterial consortia may
contribute to several advantages for biological treatment
of metalworking fluids.
The aim of our study was to investigate the effect of Triton X-100 on the biodegradation efficiency of hexadecane and phenanthrene carried out by two bacterial consortia isolated from soil. It was established that the tested
consortia were not able to directly uptake compounds
entrapped in micelles, since biodegradation extents of
both hydrocarbons were limited by the presence of Triton
X-100. In micellar systems, the nonionic synthetic surfactant was preferentially degraded (the degradation efficiency of Triton X-100 after 21 days was 70% of the initial
concentration 500 mg/l), followed by a lesser decomposition of hydrocarbon released from the micelles (30% for
hexadecane and 20% for phenanthrene). In comparison,
when hydrocarbons were used as the sole carbon source,
70% of hexadecane and 30% of phenanthrene were degraded. Those results confirmed limited bioavailability of
hydrocarbons entrapped in micelles. In conclusion, one
can observe that Triton X-100 was the preferred carbon
source compared to hydrocarbons. The degradation of
the surfactant did not contribute to notable shifts in bacterial community dynamics, as determined by Real-Time
PCR. The obtained results suggest that if surfactant-supplementation is to be used as an integral part of a bioremediation process, then possible bioavailability decrease
due to entrapment of the contaminant into surfactant
micelles should also be taken into consideration, as this
phenomenon may have a negative impact on the biodegradation efficiency. Surfactant-induced mobilization of
otherwise recalcitrant hydrocarbons may contribute to
the spreading of contaminants in the environment and
prevent their biodegradation.
Acknowledgements

2
Poznan, Poland
Acknowledgements
Eurobiotech 2013
P7.28
P7.29
Rhamnolipids increase biodegradation

of diesel oil in the presence
of chlorophenols
Simultaneous degradation of phenol

and alkanes by unconventional yeast
strains
Daria Pziak1, Aleksandra Piotrowska1, Grzegorz Framski2,

Bogdan Wyrwas3, Roman Marecik4, Kamila Myszka4,
Agnieszka Piotrowska-Cyplik5, ukasz Chrzanowski1
Aleksandra Piotrowska1, Daria Pziak1, Marta Woniak1,

Mateusz Sydow1, Alicja Szulc1, Jakub Idkowiak2,

2
Institute of Bioorganic Chemistry, Polish Academy of Sciences,
Poznan, Poland; 3Institute of Chemistry, Poznan University
of Technology, Poznan, Poland; 4Department of Biotechnology
and Food Microbiology, University of Life Sciences in Poznan,
Poznan, Poland; 5Institute of Food Technology of Plant Origin,
University of Life Sciences in Poznan, Poznan, Polan
Surfactant-mediated treatment increases hydrocarbon

solubilization and is widely considered as being responsible for potentially facilitated biodegradation. This
changes dramatically when toxic co-contaminants inhibiting microbial activity are present in the hydrocarbon
mixture. This study was aimed at assessing the effect of
rhamnolipids on the performance of a bacterial consortium degrading diesel fuel employed as a model hydrocarbon-rich effluent, co-contaminated with toxic phenol,
4-chlorophenol (4-CP) or 2,4-dichlorophenol (2,4-DCP).
This approach led to the unexpected finding that rhamnolipids reduced the toxicity of 4-CP and 2,4-DCP to the
hydrocarbon-degrading cells. The facts that rhamnolipids
decreased diesel fuel/water partition coefficient (KFW) of
both 4-CP and 2,4-DCP and modified the aggregate size
distribution profiles of the dispersed diesel fuel/chlorinated phenols solutions, suggest the existence of specific
interactions between rhamnolipids and the co-contaminants. Due to the polar nature of both 4-CP and 2,4-DCP,
possible explanations involve adsorption of 4-CP and
2,4-DCP on the surface of biosurfactant aggregates. This
property of rhamnolipids might potentially be of interest
to all those using biosurfactants for microbial treatment
of hydrocarbon-rich wastewaters co-contaminated with
toxic compounds.
Acknowledgements
97

2
Poznan, Poland
This study was focused on assessing the modification of

the yeast cell surface hydrophobicity in the presence of
two diametrically different carbon sources, n-alkanes
(represented by a mixture of dodecanehexadecane) and
phenol. Degradation efficiency of either sole hydrocarbons or hydrocarbons in the mixture with phenol as well
as phenol itself by unconventional yeast species Candida maltosa, Yarrowia lipolytica and Pichia guilliermondii
were investigated. Simultaneously, the formation of biomass was also monitored. The cell surface hydrophobicity
was assessed by microbial adhesion to hydrocarbons test
(MATH). Phenol degradation was assessed using GCSPE technique, whereas hydrocarbons were extracted
prior to gravimetric determination. The obtained results
suggest that the hydrophobic/hydrophilic nature of the
carbon source had a significant influence on the yeast cell
surface hydrophobicity. The observed dependence between hydrophobicity and biodegradation efficiency may
lead to a conclusion, that the more hydrophobic cells are,
the better the biodegradation efficiency of hydrocarbons
they express. A similar relation is observed for hydrophilic compounds like phenol. Higher biomass formation
during biodegradation of both contaminants present in
a mixture, followed by changes in hydrophobicity and
possible correlation between them, attracts our attention
and this phenomenon would become an interesting subject of further research in the future.
Acknowledgements
98
Eurobiotech 2013
P7.30
P7.31
Biodegradation of Triton X-100 by

a bacterial community isolated from
activated sludge
Biodegradation of diesel and biodiesel

fuels under various aeration conditions
Aleksandra Piotrowska1, Daria Pziak1, Marta Woniak1,

Mateusz Sydow1, Grzegorz Framski2, Bogdan Wyrwas3,
Agnieszka Zgoa-Grzekowiak3, ukasz Chrzanowski1
2
Poznan, Poland; 3Institute of Chemistry, Poznan University
of Technology, Poznan, Poland
1
Triton X-100 is a commonly used non-ionic surfactant

which has a branched hydrocarbon chain with an aromatic ring and a hydrophilic part consisting of ethylene
oxide chain. Through the past few years it has found several industrial, commercial and household applications,
such as paper manufacturing, metal processing and production of detergents. However, with increasing production of Triton X-100 the environmental contamination
with this surfactant has been rising continuously. The
result of primary biodegradation of this compound is the
shortening of the ethoxyl chain and formation of stable
metabolites, which can be toxic and capable of bioaccumulation in aquatic organisms. Therefore, it is important
to create a strategy focusing on biological reduction of
alkylophenoletoxylates from the environment. Most of
the previous biodegradation studies concerning Triton
X-100 were carried out with the use of pure bacterial cultures. However, the aim of this study was to isolate amicrobial community capable of utilizing this surfactant
from activated sludge biomass, employed to treat plant
wastewaters of different origin. In this study the complex
structure and biodiversity of the activated sludge allowed
for selection of a bacterial community, which could be
potentially applied as a biosorbent for treatment of wastewater with ahigh surfactant concentration.
Acknowledgements
Mateusz Sydow1, Daria Pziak1, Aleksandra Piotrowska1,

Marta Woniak1, Piotr Lisiecki1, ukasz awniczak1,
2
1
Biodiesel, as renewable fuel, is becoming the alternative

to commonly used petroleum diesel. It has a documented advantage over diesel associated with a more suitable
environmental behavior. Biodiesel easily undergoes aerobic biodegradation and it was observed that its addition
facilitated the utilization of petroleum diesel. Therefore,
supplementation with biodiesel has been offered as the
remediation technic to accelerate the biodegradation of
diesel. However, the microbiological consortium capable
of utilizing diesel with the addition of biodiesel is characterized by considerable diversity and complex structure
of mutual interactions between the degrading species,
which may change during the biodegradation process.
The aim of this study was to investigate the degradation
rates of diesel fuel, pure biodiesel and B20 blend under
different aeration conditions by a seven-member bacterial consortium. It allowed to demonstrate the changes
in bacterial community structure depending on the presence of biodiesel in petroleum diesel or changes in aeration during biodegradation process.
Acknowledgements
Eurobiotech 2013
99
P7.32
P7.33
Biodegradation of rhamnolipids
and its effect on bacterial community
composition during diesel oil degradation
Environmental fate of selected ionic liquids

introduced into terrestrial microcosms
Marta Woniak1, Mateusz Sydow1, Alicja Szulc1, Piotr Lisiecki1,

Anna Parus1, Katarzyna Mucha2, Roman Marecik3,
ukasz Chrzanowski1
2
Faculty of Materials Science, Technology and Design,
Kazimierz Pulaski University of Technology and Humanities
in Radom, Radom, Poland; 3Department of Biotechnology
and Food Microbiology, University of Life Sciences in Poznan,
Poznan, Poland
1
Susceptibility of rhamnolipids towards microbial degradation during biosurfactant-enhanced biodegradation of

diesel and B20 (20% biodiesel and 80% diesel v/v) fuels
was evaluated under both aerobic and anaerobic conditions. Rhamnolipids were supplied at 150 mg/l, which is
approx. double the value of critical micelle concentration
(CMC). Rhamnolipid-induced changes in community dynamics were assessed by employing real-time PCR
and the ddCt method for relative quantification. The obtained results confirmed that rhamnolipids were readily
degraded by a soil-isolated consortium of hydrocarbon
degraders in all samples. Mathematical modelling of the
GC results allowed us to present the following order of
microbial preference regarding carbon sources: biodiesel>rhamnolipids>diesel. The presence of rhamnolipids
proved to be effective only for B20 however it did not influence the biodegradation rate of pure diesel. No effect
associated with rhamnolipids supplementation was observed under anaerobic conditions. The biodegradation
of rhamnolipids did not favor the growth of any specific
consortium taxa, which proved that the employed biosurfactant did not interfere with the microbial equilibrium
during diesel/biodiesel biodegradation.
Acknowledgements
ukasz Chrzanowski1, Daria Pziak1, Aleksandra Piotrowska1,

Bogdan Wyrwas2, Agnieszka Zgoa-Grzekowiak2,
Anna Syguda1, Joanna Wojtera-Kwiczor3, ukasz awniczak1
2
Poznan, Poland; 3Institute of Molecular Biology and
Biotechnology, Adam Mickiewicz University in Poznan, Poznan,
Poland
1
Ionic liquids (IL) are considered as a novel and promising group of chemical compounds which exhibits
vast structural diversity as well as high applicability in
several branches of the industry (i.e. organic synthesis,
phase transfer catalysis, electrochemistry, formation of
functional polymers, textile softening and production
of hygiene-related products). These peculiar chemicals
are composed of an ionic pair, in which both the cation
and the anion may exhibit certain properties, enabling
the synthesis of highly specific agents. Ionic liquids have
gained immense popularity over the past decades, mostly due to their abundant applications and high efficiency, hence their high-scale production and introduction
into commonly used products is becoming a fact. While
there are certainly many advantages of employing ionic liquids for every-day use, there are also numerous
environmental concerns regarding these compounds.
Certain IL are characterized by a high biological activity, which is why the presence of such xenobiotics may
contribute to negative interactions with micro and macroorganisms in case of environmental contaminations.
Furthermore, several types of IL are excellent pesticides
and herbicides such applications correspond to a direct introduction of these compounds into the environment. Considering the fact, that IL exhibit surface active
properties and may therefore easily penetrate the soil
matrix and transport via groundwater, it should be clear
that assessment of their influence of the environment is
a priority.
The aim of this study was to investigate the biodegradation
rate of selected ionic liquids (ammonium and phosphonium-based IL) by autochthonous soil microorganisms and
determine the qualitative changes in bacterial soil populations as a response to the presence of such compounds
during long-term (300 days) soil microcosm studies. Biodegradability of ionic liquids was assessed by employing
HPLC-MS and the Disulphine Blue Active Substances
(DBAS) method as well as respirometric measurements
(determination of emitted CO2). Total bacterial DNA
was extracted from the soil prior to the experiments and
upon finishing the experiments. Subsequent molecular
analyzes (16s rRNA) allowed for identification of residual
dominant autochthonic species, which were most likely
involved in the biodegradation of the selected ionic liquids. Interestingly, the obtained results suggest that the
100
Eurobiotech 2013
toxicity and biodegradability of the studied compounds

is associated with their structure, and that biotransformation and sorption into the soil matrix play a crucial role
for the fate of IL in the terrestrial environment.
Acknowledgements
P7.34
Phytotoxic effect of rhamnolipids
supplementation during biodegradation
of diesel oil in soil
Anna Parus1, Marta Woniak1, Mateusz Sydow1, Alicja Szulc1,
Katarzyna Mucha2, Joanna Wojtera-Kwiczor3, Anna Gielnik3,
ukasz Chrzanowski1
2
Faculty of Materials Science, Technology and Design,
Kazimierz Pulaski University of Technology and Humanities
in Radom, Radom, Poland; 3Institute of Molecular Biology and
Biotechnology, Adam Mickiewicz University in Poznan, Poznan,
Poland
1
This study aimed at assessing the influence of rhamnolipids on the phytotoxicity of diesel oil-contaminated soil
samples. Standard tests evaluating the seed germination
and growth inhibition of alfalfa, sorghum, mustard and
cuckooflower were carried out at rhamnolipids concentrations ranging from 0 to 1.200 mg/kg (wet soil). Diesel
oil content ranged from 0 to 25 ml/kg of wet soil. Obtained results confirmed that the sole presence of rhamnolipids may be phytotoxic at various levels, which is especially notable for sorghum (the germination index decreased to 41%). The supplementation of diesel oil-contaminated soil samples with rhamnolipids contributed
to a significant increase of their phytotoxicity. The most
notable toxic effect was observed after a rhamnolipids-supplemented diesel oil biodegradation, carried out
with the use of a hydrocarbon degrading bacteria consortium. The supplemention of rhamnolipids (600 mg/kg
of wet soil) resulted in a decrease of seed germination of
all studied plant species and an inhibition of microbial
activity, which was measured by the 2,3,5-triphenyltetrazolium chloride (TTC) tests. These findings indicate that
the presence of rhamnolipids may considerably increase
the phytotoxicity of diesel oil. Therefore, their use at high
concentrations during in situ bioremediation processes
should be carefully planned in a terrestrial environment.
Acknowledgements
Eurobiotech 2013
101
P7.35
P7.36
Removal of atrazine in the presence

of wetland plant species
Utilisation of betaine in vinasse stillage by

a mixed culture of aerobic bacteria under
non-controlled pH
Anna Parus1, Daria Pziak1, Aleksandra Piotrowska1,

Alicja Szulc1, ukasz awniczak1, Joanna Wojtera-Kwiczor2,
2
Institute of Molecular Biology and Biotechnology,
Adam Mickiewicz University in Poznan, Poznan, Poland;
3
1
This study focused on assessing the phytoremediation

potential of wetland plants toward atrazine in an aquatic environment. Changes in plant biomass and atrazine
content were investigated for three plant species: sweet
flag, broadleaf cattail, and narrow-leaf cattail. Atrazine
removal and shifts in plant biomass were assessed. Two
mathematical models were built to describe atrazine toxicity toward the studied plant species and fate of atrazine
during long-term phytoremediation. Sweet flag exhibited the highest tolerance toward atrazine as well as the
most efficient atrazine removal rate. The average atrazine
half-life was significantly reduced from about 400 days
to 5 days. The highest studied initial concentration of
atrazine (20mg/l) was reduced by more than 99% after
40days.
Acknowledgements
Agnieszka Ryznar-Luty, Edmund Cibis, Magorzata Krzywonos

Wroclaw University of Economics, Department of Bioprocess
Engineering
The aim of this work was to examine how temperature

and the initial pH influence the efficiency of an aerobic
biodegradation of betaine in vinasse stillage. Betaine accounted for as much as 37.6% of total organic carbon. In
the study two series of batch biodegradation processes
were carried out in a stirred tank reactor by a mixed culture of Bacillus bacteria. The temperatures applied were
27, 36, 45, 54 and 63C. The pH was not controlled, and
the initial pH of the culture medium amounted to 6.5
in first series and 8.0 in the second series. Efficiency of
biodegradation was expressed in terms of reduction in
SCODsum, which is a sum of SCOD (soluble chemical oxygen demand, i.e. COD determined after suspended solids separation) and theoretical COD of betaine. The high
biodegradation efficiency obtained in the processes was
attributable to the assimilation of betaine by the bacterial
strains used in the study. Maximal reduction in SCOD(85.41%), BOD5 (97.91%) and TOC (86.32%) was
sum
achieved during biodegradation at 36C and pH0 = 8.0.
Under the same temperature and pH regime, betaine was
removed completely, while biodegradation proceeded at
the fastest rate (1.1684 g O2/(lh) for SCODsum removal).
During biodegradation the main organic pollutants were
removed at the same time, but with diverse rate of removal. An exception to this rule was the assimilation of betaine, which intensified only after the medium was lacking
in easily available carbon sources.
Acknowledgements
The study was financed by the Polish Ministry of Science and Higher
Education under Project No. 2P06T 045 30.
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Eurobiotech 2013
P7.37
P 7.38
Bacterial changeability monitoring

of activated sludge bacterial communities
in SBR bioreactors treating reject water
with PCR-DGGE
Semiquantitative ICP-MS analysis

as a tool in the study of new
hyperaccumulators
Aleksandra Ziembiska, Grzegorz Cema, Anna Meresta,

Agata Karo, Lesaw Ponka
Geochemistry and the Environment Div., Institute of Chemistry,

Jan Kochanowski University, 15G Swietokrzyska St.,
25-406 Kielce, Poland
The Silesian University of Technology, Faculty of Environmental

Engineering and Energy, Environmental Biotechnology
Department, Akademicka 2, 44-100 Gliwice, Poland
The microbial community and physiochemical processes were monitored in two SBR systems differ in operational parameters. PCR-DGGE of total bacterial community and Anammox bacteria together with sequencing of the dominant Anammox genotypes were used as
a tool for bacterial biocenoses analysis. Both SBRs were
treating reject water with high ammonia concentration
(> 600 mg/l) for over 370 days. After this period of time
the ammonia concentration decreased to 250300 mg/l.
PCR-DGGE monitoring enabled to present the biodiversity shifts linked with community rearrangement and
bacterial biocenoses qualitative change. The analysis revealed that the drastic decrease of ammonia concentration in the influent to adapted biocenoses caused slight
qualitative change in community composition, mainly
in low GC part of the bacteria genotypes but biodiversity index slightly responded to such a change. It was
also stated that bacteria which DNA was amplified with
Anammox-bacteria targeted primers and underwent sequencing were not identified as Planctomycetes probably
because this group of bacteria are still not well known and
Genbank still does not possess enough 16S rRNA gene
sequences belonging to these microorganisms. Difference
in the SBRs working scheme can be the reason that the
community structure differs but with no drastic influence
on its performance.
Acknowledgements
This work was supported by Ministry of Science and Higher Education.
Grant number N523751740.
Karina Krzciuk
The interest in the study of hyperaccumulation increases from year to year. Natural plant species that are able
to take up and accumulate high concentrations of trace
elements are called hyperaccumulators [1]. Hyperaccumulators have found their application in environmental
biotechnology (phytoremediation, phytomining) [2], as
well as in nanotechnology [3]. According to the recently published data, about 577 plant species show hyperaccumulative properties [4]. Most of them, about 78%,
are hyperaccumulators of Ni. Other known species hyperaccumulate Cu, Co, Se, Pb, Zn, Mn, As and Cd [4].
However, there is a need to widen the scope of data for
plants accumulating other elements, including anthropogenic pollutants (Cr, Pb, B) or elements of commercial
value (Cu, Au, rare earth elements). Semiquantitative
mode in inductively coupled plasma mass spectrometry
(ICP-MS) is a good tool for the search of new hyperaccumulators. Semiquantitative analysis is usually used for
arapid screening before quantitative multi-element analysis of unknown samples and it can be used for preselection of the samples with high-concentrations of elements.
This approach may help both for identification of new hyperaccumulators and for pinpointing a larger number of
elements accumulated by plants. The reliability of semiquantitative analysis by ICP-MS is high due to its good
accuracy (typically 3050%) and reproductibility.
Acknowledgements
This work was supported by project from the program Knowledge and
Economics a development of scientific and business competence for
the growth of regional economy competitiveness.
References
[1] Baker A.J.M., Brooks R.R., Terrestrial higher plants which
hyperaccumulate metallic elements A review of their distribution,
ecology and phytochemistry. Biorecovery 1987; 1: 81126.
[2] Brooks R.R., Plants that hyperaccumulate heavy metals. Their role in
phytoremediation, microbiology, archaeology, mineral exploration and
phytomining. CAB International: Wallingford, 1998.
[3] Qu J., Luo C., Cong Q., Yuan X., Carbon nanotubes and CuZn
nanoparticles synthesis using hyperaccumulator plants. Environmental
Chemistry Letters 2012; 10: 153158.
[4] Van der Ent A., Baker A.J.M., Reeves R.D., Pollard A.J., Schat H.,
Hyperaccumulators of metal and metalloid trace elements: Facts and
fiction. Plant Soil 2013; 362(1-2): 319334.
[5] Chen H., Dabek-Zlotorzynska E., Rasumssen P.E., Hassan N.,
Lanouette M., Evaluation of semiquantitative analysis mode in ICP-MS.
Talanta 2008; 74: 15471555.
Eurobiotech 2013
103
P7.39
P7.40
Supporting the process of biosorption
Effect of tannic acid on Acidithiobacillus

ferrooxidans biofilm formed on the concrete
Paulina Olesiak, Longina Stpniak

Politechnika Czstochowska, Wydzia Inynierii rodowiska
i Biotechnologii, Instytut Inynierii rodowiska
Weronika Dec, Beata Cwalina, Pawe Stachura, Joanna Michalska
Microorganisms are used in environmental processes

from antiquity, but until recently unconsciously. Originally it was believed that the presence of undesirable microorganisms in the process. Only current trends prevailing in the second half of the twentieth century as
amatter of microorganisms include the desired changes
in the processes of substance. They allow you to speed
up or initiate many decomposition or synthesis. They
are used widely in many industries, but research continues them what time the extensive use of the following
processes. The microorganism used on a large scale are
mainly bacteria in the treatment and purification of waste
water and sewage. The object of this study was to present
the process of biosorption as one of the methods used
currently in the process of water treatment. This process
brings many benefits, but for the purpose of even greater
intensification apply modifications such as ozonation or
sonication.
The subject of the research presented in the work was
to evaluate the effectiveness of the use of biological deposits for water from organic substances. As an enabler,
said process used ultrasonic field towards a solution
subjected to sorption on the activated carbon bed. For
this purpose, used ultrasonic disintegrator Sonics Vibra
Cell (750 W, 20 kHz). The substrate studies were humic
compounds. Biological filter (in imitation of a natural
colonization by microorganisms in the Reservoir Water
Treatment Stations), was prepared using biopreparation,
or biota sludge. The filtration was carried out in columns,
with a constant flow rate. Samples of effluent were collected each day. To evaluate the effectiveness of the ongoing
process of the following parameters were analyzed: UV254
absorbance, the amount of dissolved organic carbon
DOC, oxidizability, as well as color, turbidity and pH. In
the case of biofilters also evaluated the overall number of
microorganisms and the dissolved oxygen level in order
to determine the ratio S providing the advantages of the
process occurring on the bed (sorption / biodegradation).
Rapid biodeterioration of concrete in sewage collection

systems has a major impact on health and environmental safety, especially in countries with warm climate. Microorganisms that produce sulfuric acid accelerate the
destruction of concrete sewer pipes in a process termed
Microbially Induced Concrete Corrosion (MICC).
Amongst many microorganisms participating in the materials deterioration, the acidophilic, sulphur-oxidizing
bacteria of Acidithiobacillus genus (especially of A. ferrooxidans species) are especially important. Tannic acid
(TA) belongs to tannins (a type of polyphenol) which are
considered to be bacteriostatic or bactericidal agents.
The aim of this work was to assess the growth and activity of A. ferrooxidans biofilm formed on concrete in the
presence of TA.
Experiments were carried out in the laboratory. Test samples of materials were placed into 6-well microtiter plates
containing liquid medium 9K (of Silverman & Lundgren), then inoculated with A. ferrooxidans. A control
samples were the sterile systems without bacteria. Stock
solutions of TA (1, 5, 10, 50, 100 mg l1) were prepared in
liquid medium 9K. The biocide was introduced into the
system at 48 h of incubation (when mature biofilm was
obtained). The plates were incubated at 28C at agitation
speeds 120 rpm under appropriate pH conditions.
Biofilm quantification has been carried out using following methods:
crystal violet staining, which is one of the more expedite methods to quantify biofilm biomass and may be
used directly, without disruption of the biofilm;
protein concentration assay in the medium;
XTT assay; XTT is yellow salt that is converted to dark
blue formazan in the presence of metabolic activity (respiratory chain in cells) without disruption of the biofilm;
extraction of extracellular polymeric substances (EPS)
from biofilms;
scanning electron microscopy (SEM) observations.
The results of investigation showed, that TA inhibits
A. ferrooxidans biofilm formation. The concentration of
10 mg l1 inhibits biofilm activity by 27% but the highest
activity of biofilm was observed in the media with 1 mg
l1 (compared with control). Significant effect of TA on
concentration of the extracellular polysaccharides and
proteins was observed whereas this substance did not influence significantly the biofilm biomass.
Keywords: biosorption, ultrasound, humic substances

Acknowledgements
The study was supported by Czestochowa University of Technology
internal grant BS/MN-401-321/11. The autor Paulina Olesiak received
a grant for the project DoctoRIS Scholarship program for innovative
Silesia co-financed by the European Union under the European Social
Fund.
Silesian University of Technology
Acknowledgements
The work is realized with financial support from Polish National Science
Center, grant No. NN523614839, using equipment purchased in the
project: BIO-FARMA Silesia. Centre for Biotechnology, Bioengineering
and Bioinformatics, co-financed by ERDF OP IG, 20072013.
W. Dec is a scholar in the Project SWIFT (Supporting Innovative Grants
Technology Forum).
104
Eurobiotech 2013
P7.41
P7.42
Textile dyes synthesis by alkalic laccase

from soil-dwelling fungus Rhizoctonia
praticola
Immobilization of cellobiose
dehydrogenase from Cerrena unicolor
to silica beads supports
Kamila Wlizo, Jolanta Polak, Anna Jarosz-Wilkoazka
Justyna Sulej, Monika Osiska-Jaroszuk, Jerzy Rogalski
Department of Biochemistry, Maria Curie-Sklodowska

University, Akademicka 19 St., 20-033 Lublin, Poland
Department of Biochemistry, Maria Curie-Sklodowska

University, Akademicka 19 St., 20-033 Lublin, Poland
Laccase, (EC 1.10.3.2) is multi-copper-containing enzyme which catalyses oxidation of a wide range of substrates including phenolic and aromatic compounds
which may be transformed to potentially textile dyes.
First of all, laccase is widespread in ligninolytic fungi,
especially in white -rot strains of Basidiomycota which
produce extracellular laccase during the growth on media about low pH value, optimal for the activity of ligninolytic enzymes. Nevertheless there is an exception like
asoil-dwelling and plant-pathogenic fungus Rhizoctonia
praticola. This species is well known because of producing a large amount of extracellular laccase which is active
in alkaline conditions.
The aim of presented work was the screening of different organic precursors to obtain the best substrates for
transformation by alkalic laccase to coloured products.
The process was conducted on solid growing medium
with addition one or mixture of two different phenolic
compounds as dyes precursors. During the transformation the dyes appearance and the growth of the mycelium
were measured. As it came out not every phenolic precursor may be an suitable substrate for laccase from R. praticola. Some of the precursors could not be transformed to
dyes probably because of their structure. Additionally, the
efficiency of the transformation and mycelium growth
were different for each precursor or their mixture. What
is more interesting the process conducted only in places
where the ground was covered by the mycelium.
This study allowed qualify which precursors may be used
in further studies of the transformation by laccase from
R. praticola and define potentially what kind of structure
is decisive in this process.
Cellobiose dehydrogenase (cellobiose:acceptor 1-oxidoreductase, EC 1.1.99.18) is the only currently known

extracellular flavocytochrome produced by various species fungi under cellulolytic culture conditions [1]. CDH
comprises two distinct domains, one containing FAD
and one containing b-type heme as cofactor, connected
by aflexible linker region consisting a hydrophilic polypeptide [2]. The flavoprotein domain of CDH catalyzes
the oxidation of cellobiose (Glc--1,4-Glc) and other
-1,4-linked disaccharides or oligosaccharides at the C-1
position to the corresponding lactones [3]. The exact biological role of CDH has not been fully elucidated yet, but
our current knowledge points to its participation in the
degradation and modification of polymers such as cellulose, hemicellulose, and lignin by generating hydroxyl
radicals via the Fenton reaction [4].
Due to the potential applications of CDH in biotechnology, for example, detection of soluble cellodextrins
as biosensors, monitoring cellulose reactions, production of lactobionic acid or bioremediation, the effective
production of CDH has become an important. Current
advancements in biotechnology have promoted the usage of immobilized enzymes for a wide range of applications not only in the field of biotechnology, but also
in pharmaceutical, environmental, food and biosensor
industries.
The purpose of the present study was to isolate, purify
and immobilization the novel extracellular cellobiose
dehydrogenase from the basidiomycete fungus Cerrena
unicolor. CDH was covalent immobilized to the silica
gel and porous glass beads activated by either silanization (APTES) with immobilization yield ranging from
4 to 98 %. The activity of unbound and immobilized to
silica beads supports CDH was determined. The pH-activity and temperature-activity profiles were evaluated.
Affinity changes, exhibited by kinetic parameters, pH
and thermal stability and operational stability were also
tested.
Acknowledgements
This work was partially supported by the National Science Centre
(NN 302 633040).
References
[1] Zamocky M., Ludwig R., Peterbauer C.K., Hallberg B.M.,
Divne C., Nicholls D., Haltrich D. (2006) Cellobiose dehydrogenase
A flavocytochrome from wood-degrading, phytopathogenic
and saprotropic fungi, Current Protein and Peptide Science, 7 (3):
255280.
[2] Stoica L., Dimcheva N., Haltrich D., Ruzgas T., Gorton L. (2005)
Electrochemical investigation of cellobiose dehydrogenase from new
fungal sources on Au electrodes, Biosensors and Bioelectronics, 20 (10):
20102018.
[3] Desriani, Ferri S., Sode K. (2010) Functional expression of
Phanerochaete chrysosporium cellobiose dehydrogenase flavin domain
in Escherichia coli, Biotechnology letters, 32: 8559.
Eurobiotech 2013
[4] Harreither W., Sygmund C., Augustin M., Narciso M., Rabinovich
M.L., Gorton L., Haltrich D., Ludwig R. (2011) Catalytic properties and
classification of cellobiose dehydrogenases from Ascomycetes. Appl
Environ Microbiol 77: 18041815
Acknowledgements
This research was supported by the National Science Centre in
Poland (Grant No. 2011/01/N/NZ1/03458) and the research program
BS/UMCS.
105
P7.43
The use of Miscanthus gigantues
and Phalaris arundinacea in the process
of phytoremediation of soils
Karolina Rosiko, Magorzata Kacprzak
The introduction of vegetation in degraded areas of heavy

metal effect allows their remediation, or restoration of
the utility and value of nature. Degraded land as they are
excluded from food crops for consumption, ideal for the
cultivation of energy crops such as Miscanthus giganthus
(miscanthus) and Phalaris arundinacea (reed canary
grass). These plants have the ability to phytoremediation,
collect and accumulate contaminantion in their tissues,
mainly heavy metals. Depending on the plant species as
well as the absorbed of metal, they can be accumulated in
large quantities in the aerial parts or roots. In addition,
both miscanthus and canary through a well developed
root system can prevent soil erosion, and therefore serve
as both energy and reclamation.
This paper present the influence of various factors, such
as plant species, type of absorbed metal, fertilizer applied
and the growing season, in the process of phytoremediation of Cd, Ni and Zn.
Research show that after first year of vegetation Cd
(0.344 mg kg1 d.m.) and Zn (29.47 mg kg1 d.m.) were
the most absorbed metals by the biomass of miscanthus
grown on soil fertilized with municipal sludge (OW).
However, in the case of the reed canary grass, the correlation can be attributed to the Ni (3.76 mg kg1 d.m.)
and Zn (122.6 mg kg1 d.m.). In turn, after the second
year of vegetation all of the tested metals are characterized by alower absorbtion as compared to the first year.
In the case of reed canary grass Cd and Zn content in the
biomass after the second year of vegetation was similar
to the control, while the Ni concentration is was lower in
relation to the control. In the case of miscanthus, it was
observed that the concentration of Ni and Zn in the biomass after the second year of vegetation was similar to the
control, while the Cd content varied in dependence on
the applied fertilizer.
On soils with high metal concentration of both reed
canary grass and miscanthus are able to collect metals,
which can result in quality biomass (unfavorable from the
point of view of energy).
Acknowledgements
The study was supported by Czestochowa University of Technology
internal grant BS/MN401317/11.
The author Karolina Rosiko received a grant for the project DoktoRIS
Scholarship program for innovative Silesia co-financed by the European
Union under the European Social Fund.
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Eurobiotech 2013
P7.44
P7.45
Identification of lipid derivatives

in HepG2 cells
Removal heavy metals from wastewaters

by yeast biomass
Joanna Gdula-Argasinska1, Aneta Garbacik2,

Malgorzata Tyszka-Czochara1, Michal Wozniakiewicz2
Sawomir Wierzba, Adam Lataa
Jagiellonian University Medical College Faculty of Pharmacy

Department of Radioligands; 2Jagiellonian University Faculty
of Chemistry Laboratory for Forensic Chemistry
1
Polyunsaturated fatty acids (PUFA) are highly susceptible

to oxidative stress produced hydroperoxide species. Besides non-enzymatic lipid peroxidation, enzyme-induced
peroxidation, cyclooxygenases and lipoxygenases is
aprocess that leads to hydroperoxides specifically transformed into a series of more stable metabolites eicosanoids or docosanoids from C18 to C22 PUFA.
The aim of our study was to assessed the effect of PUFA
supplementation with added of PAHs on the HepG2 cells.
Our study describes a rapid and simple technique for the
analysis of PGF2a, PGF3a from cultured cells using UHPLC/MS-TOF. This method permits detection of selected
individual prostaglandins derived either from arachidonic acid (AA) or eicosapentaenoic acid (EPA) from cell
extracts.
Acknowledgements
This project was possible through the support given by National
Science Center, Poland to the author Joanna Gdula-Argasinska
DEC-2011/01/B/NZ7/00038.
Opole University, Chair of Biotechnology and Molecular Biology
Conventional methods for removing metals from aqueous solutions include chemical precipitation, chemical
oxidation or reduction, ion exchange, filtration, electrochemical treatment, reverse osmosis, membrane technologies, and evaporation recovery. These processes may
be ineffective or extremely expensive;especially when
the metals in solution are in the range of 1100 mg/l,
the production of toxic chemical sludge and its disposal/
treatment becomes a costly affair and is not ecofriendly.
Therefore, removal of toxic heavy metals to an environmentally safe level in a costeffective and environment
friendly manner was of great importance. The use of biosorbents with microbial origin especially bacteria, algae,
fungi and yeasts is a considerable alternative to the existing methods because of their good performance, low cost
and large availability. This work presents some results
on the use of Saccharomyces cerevisiae cells biomass for
removal of lead, zinc, nickel, cadmium and copper ions
from contaminated industrial wastewater. Yeast biomass
was immobilized in the process of flocculation by using
calcium chloride and hydrogen peroxide. Process conditions: pH 5.0, temperature 20C, the concentration of
biomass, 1.0 g/l, contact time 30 minutes of flocculation,
aeration and continuous operation. The initial content of
metal ions ranged from 2.2 mg/l (cadmium) to 83.1 mg/l
(lead). Analysis of microbiological and physicochemical
industrial wastewaters showed high efficiency the methods immobilization of insoluble aggregates of cells with
calcium chloride and hydrogen peroxide in removing
heavy metals. Reduction of concentration of Pb(II) was
98.0%, Cd (II) 96.0%, Cu (II) 95.9%, Zn (II) 95.2%
and Ni (II) 92.7%. The results indicate the possibility of
using the aggregation of cells in flocs during flocculation
in wastewater treatment from heavy metal ions.
Eurobiotech 2013
107
P7.46
P7.47
Bacillus spp. and their metabolites present

ecofriendly alternative to synthetic
chemicals for plant growth enhancement
in many different applications.
Extensive research has demonstrated
that these microorganisms and their
metabolites could have an important role
in agriculture and horticulture in improving
crop productivity
Traditional methods of plant protection

employing chemical substances may
be replaced with a biological alternative
based on non-pathogenic microorganisms.
Investigations into properties of such
microorganisms in order to find the most
suitable bacterial strains brought the
attention to the activity of Pseudomonas
fluorescens
Katarzyna Grata, Magorzata Nabrdalik
Magorzata Nabrdalik, Katarzyna Grata
Department of Biotechnology and Molecular Biology,

University of Opole, Opole, Poland
Department of Biotechnology and Molecular Biology,

University of Opole, Opole, Poland
Bacillus spp. and their metabolites present ecofriendly

alternative to synthetic chemicals for plant growth enhancement in many different applications. Extensive research has demonstrated that these microorganisms and
their metabolites could have an important role in agriculture and horticulture in improving crop productivity.
The aim of the research was to assess a potential biological activity of cell-free supernatants (CFS) obtained from
4, 6, 8, 10 and 24-hour culture of Bacillus subtilis against
4 pathogenic strains of Rhizoctonia solani marked as R1,
R2, R3 and R4. The bacterium was isolated from soil fertilized with urea phosphate while R. solani strains were
isolated from field cultivation of sugar beet. The antagonistic properties of bacterial egzometabolites were determined by the dual-culture technique on Czapek-Dox medium, where the carbon source was sucrose. Plates were
incubated at 26C for 46 days and the fungal growth was
measured every day and compared to control. The influence of metabolites produced by B. subtilis on the growth
of R. solani was evaluate on the basis the growth rate index .
On the basis of obtained results, it has been proved that
fungistatic activity of B. subtilis is varied and depends ob
the age of the bacterial culture and susceptibility of the
fungus. Taking into consideration all the analysed parameters, R. solani R1 and R4 were the most sensitive but R3
lest sensitive to the metabolites produced by B. subtilis.
The highest decrease, amounting 84%, in the value of
the growth rate index of R. solani R4 was obtained for
10-hour culture of B. subtilis. The growth rate index of
R. solani R1 and R3 was also strongly inhibited (amounted 82%), when were applied 6-hour and 4-hour culture
of B. subtilis, respectively. Moreover, on the basis of obtained results, it shows that the most favourable is to apply young 8-10-hour bacterial cultures to inhibit the linear growth of R. solani. B. subtilis may find a wide range
of application, in the process of plant protection against
diseases caused by R. solani.
Traditional methods of plant protection employing

chemical substances may be replaced with a biological
alternative based on non-pathogenic microorganisms.
Investigations into properties of such microorganisms in
order to find the most suitable bacterial strains brought
the attention to the activity of Pseudomonas fluorescens.
The aim of conducted research was to determine the
influence of metabolites of Pseudomonas fluorescens on
the growth of 4 pathogenic strains of Rhizoctonia solani
marked as R1, R2, R3 and R4 infesting sugar beet. The
antagonistic properties of metabolites were assessed after
4, 6, 8, 10 and 24 hours of culturing of Pseudomonas fluorescens with a culture-plate method on PDA medium.
The bacterial strains were cultured at 25C for 47 days.
Fungistatic activity of Pseudomonas fluorescens was determined on the rate of mycelial growth inhibition.
Obtained results have proved that the strains of Rhizoctonia spp. under study were both sensitive and resistant
to Pseudomonas fluorescens metabolites. The highest
inhibition of the linear growth of fungi was noted for
Rhizoctonia solani R1 and R4. In both cases the highest
inhibition of the growth rate, amounting almost 60%,
was obtained after 4 hours of culturing and the lowest,
amounting around 30%, was noted after 24 hours of culturing. Whereas the following strains of Rhizoctonia solani R2 and R3 were resistant to metabolites produced by
Pseudomonas fluorescens regardless of the culturing time
length.
Conducted research confirmed fungistatic properties of
Pseudomonas fluorescens strains against Rhizoctonia solani strains. However, due to the fact that differences in the
mycelial growth inhibition have been recorded among
strains of Rhizoctonia solani it allows to assume that the
sensitivity to metabolites is characteristic for individual
strains. The tests also showed that growth inhibition of
the mycelium depends not only on the type of metabolites produced by a specific bacterial strain but also on the
length of culturing.
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Eurobiotech 2013
P7.48
P7.49
Isolation of biosurfactant producing

strains from environmental samples
Substrate preferences in an environmental

microbial consortium degrading diesel oil
Marta Woniak1, Kamila Myszka2, Mateusz Sydow1,

Alicja Szulc1, ukasz awniczak1, Aleksandra Piotrowska1,
Daria Pziak1, Anna Parus1, ukasz Chrzanowski1
Mateusz Sydow1, Daria Pziak1, Aleksandra Piotrowska1,

Marta Woniak1, ukasz awniczak1, Anna Parus1,

2
In this study we carried out research on aerobic bacteria

selected from cow, chicken and pig feces as well as from
manure. According to the literature reports, the main isolation sources of microorganisms capable of biodegrading petroleum hydrocarbons are oil-contaminated soil,
water tanks or sediments. Both livestock feces and manure are a rich source of gram-negative bacteria, which
have a high potential to biosynthesize biosurfactants. In
the last few decades, the ability to employ biosurfactants
in bioremediation processes of petroleum contamination
has attracted a lot of scientific attention. Therefore, from
this point of view, the research has shown a pioneering
approach to the subject of isolation of microorganisms
capable of growth on hydrocarbons from uncommon research materials.
Using 4 screening techniques, we selected a bacterial consortium consisting of three strains: Enterococcus faecalis,
Pseudomonas aeruginosa and Escherichia coli. The current
literature reports concerning the bioremediation and the
role of biosurfactants in these processes has focused primarily on the use of monocultures, occasionally mixed
cultures. The present study underlines the impact of bacterial consortia, which increase the efficiency of biodegradation in comparison with monocultures. Pseudomonas
aeruginosa isolated from the tested consortium had the
ability to secrete compounds that reduced surface tension.
Both the consortium, as well as the isolated Pseudomonas
aeruginosa strain, were capable of growth on hydrocarbons
as hydrophobic carbon sources. The intensity of secretion
of surface active compounds by Pseudomonas aeruginosa
species can be correlated with the respective sequence of
hydrocarbons metabolization. The influence of strains in
bacterial consortium caused stimulation of Pseudomonas
aeruginosa strain to an increased biosurfactants secretion
during cultivation on less available carbon sources. In addition, some diesel components inhibited secretion of surface-active agents by the consortium and stimulated their
synthesis by the Pseudomonas aeruginosa strain. During
cultivation in the bioreactor, the consortium delayed the
release of surface active compounds by the Pseudomonas
aeruginosa strain. Most likely, it was a result of preliminary
competition among the microorganisms for nutrients in
the new environment. Ultimately, the consortium stimulated Pseudomonas aeruginosa in terms of more efficient
production of surface-active agents.
The microbial populations inhabiting soil or aquatic environments are composed of diverse, synergistic or antagonistic communities. The presence of multiple bacterial
species in an environmental community allows for an
efficient removal of a wide range of compounds. Largescale pollution due to man-made chemical substances is
the current global concern. From the viewpoint of man
and his impact on the environment, the most important form of pollution are oil spills. Diesel oil is still the
worlds first volume-produced fuel. The composition of
this mixture comes close to 3,000 different hydrocarbons.
Among them are aliphatic, cycloaliphatic and aromatic
compounds. The straight-chain alkanes fraction is the
easiest to decompose by bacteria, whereas cyclic and aromatic compounds are degraded over a relatively long period of time. Moreover, if the pollution consists of many
chemical compounds, as in the case of diesel oil, gradual
changes in mixture composition are observed.
Relatively little is known regarding the determinants of
microbial population dynamics in soil contaminated
with complex hydrocarbon mixtures such as diesel oil
or petroleum. The ability to metabolize oil is displayed
by many different microorganisms and some of them are
more versatile than others. Many bacterial species prefer
specific carbon sources over complex mixtures of compounds. Other bacteria are capable of using many carbon
sources.
The aim of this study was to evaluate quantitative and
qualitative changes within the composition of a microbial consortium resulting from selective cultivation on
the chosen diesel oil fractions and to assess the impact
of such changes on the biodegradation efficiency of commercial diesel oil. During the research, specific substrate
preferences as well as quantitative and qualitative changes within the microbial consortium were determined by
using relative quantitative real-time PCR method. Moreover, all mineralization efficiencies of diversified consortia were compared to utilization capacity of the initial
bacterial community with the use of mathematical modeling methods. The obtained results demonstrate that cultivation on specific carbon sources did not significantly
affect diesel oil biodegradation efficiency in soil.
Acknowledgements

2
Acknowledgements
Eurobiotech 2013
109
P7.50
P7.51
Biodegradation of diesel/biodiesel blends

in urban soil
Identification and phylogenetic analysis

of Rhizopus spp. based on 28S rRNA gene
ukasz Chrzanowski1, Piotr Lisiecki1, Grzegorz Framski2,

Anna Syguda1, Roman Marecik3, Kamila Myszka3,
Jacek Staniewski1, ukasz awniczak1
Adam Kuzdraliski, Sylwia Kowalczyk, Elwira Komo,

Agnieszka Glibowska, Jakub Wyrostek, Zdzisaw Targoski

2
Poznan, Poland; 3Department of Biotechnology and Food
Microbiology, University of Life Sciences in Poznan, Poznan,
Poland
1
Biodiesel has gained a lot of scientific attention over the

last few years. It is commonly believed that the addition
of biodiesel to diesel fuel may potentially enhance the
biodegradation of specific hydrocarbon fractions present
in diesel. Except for assumptions that fatty acid methyl
esters are very similar to alkanes and this similarity would
accelerate degradation of the aliphatic fraction of diesel,
no hard proof has been published so far. Thus, the aim of
our study was to evaluate the long-term biodegradation
rate of both aromatic and aliphatic hydrocarbon fractions in urban soil spiked with diesel/biodiesel blends.
Additionally, we tried to evaluate the effectiveness of
bioaugmentation with a previously isolated (non-autochthonous) bacterial consortium of high affinity towards
hydrocarbons. GCxGC-TOF-MS studies of residual hydrocarbons as well as respirometric studies were extensively applied during this project. The obtained results
suggest that all diesel/biodiesel blends were degraded in
the extent of > 95% within 450 days. Moreover, no effect
of the externally added bacterial consortium was noticed.
The most prominent observation is that biodiesel did not
affect the biodegradation rate of aromatic and aliphatic
fractions.
Acknowledgements
University of Life Sciences in Lublin, Poland
Background. Many species of genus Rhizopus are very

important due to their biotechnological relevance. These
fungi are becoming increasingly important for biotech
industry due to their ability of production of a wide
spectrum of metabolites such as ethanol and lactic and
fumaric acids. The correct identification and phylogeny
of fungi is very important in plant pathology, biotechnology, clinical settings and environmental studies.
Objective. We aimed to establish the molecular phylogeny of the Rhizopus oryzae, R. stolonifer and R. microsporus belonging to the genus Rhizopus, based on the sequencing of the large subunit gene (28S) and to compare
the results with other classification studies of the genus.
We have also established a PCR-based method for screening this fragment of the large subunit gene.
Methods. Isolates were obtained from the Leibniz Institute DSMZ-German Collection of Microorganisms and
Cell Cultures, Agricultural Research Service Culture Collection, Biological Resource Center Culture Collection
and from southeastern Poland from different substrates
such as cereals, bread, soil, water and apples. We developed a direct DNA extraction method, which offers high
efficiency of DNA isolation from fungi. We designed PCR
primers by comparing Rhizopus species sequences published in the NCBI GenBank database. They were intended to be specific for genus Rhizopus and to flanking the
most polymorphic region of 28s rRNA gene. The amplification products were extracted and purified from agarose
gel. The sequencing of both DNA strands was performed
and DNA sequences were edited and aligned. All fungal
DNA sequences were initially aligned using the ClustalW.
The molecular diversity and its variance were estimated.
Consensus sequences were constructed for each species.
Phylogenetic analyses of the aligned sequences were performed with PAUP 4.0 b10.
Results. A partial sequence of 28s rRNA gene has been
amplified by PCR using our primers in all isolates belonging to the genus Rhizopus. The amplified region of the
28S rRNA gene included 5 haplotypes. Analysed sequences of R. oryzae represented 2 haplotypes, 2 haplotypes of
R. stolonifer, and 1 haplotype in case of R. microsporus.
R. stolonifer sensu lato has proven to be most intraspecies variable fungus. Species of R. oryzae, R. delemar and
Amylomyces rouxii showed high similarity, as expected.
R. oryzae had the highest number of parsimony-informative and variable sites but only if we divided R. stolonifer
sensu lato into separate clades accordingly to previous
intraspecies alignment. Comparative alignment of this
clade might also suggest that these isolates belonged to
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Eurobiotech 2013
one of the two varieties of R. stolonifer, i.e. lyococcus or

reflexus. Based on the alignment data of partial consensus sequences of 28s rRNA gene, the phylogeny analysis
was carried out separately for all species and for both
clades of R. stolonifer. Occurrence of separate clades in
R. stolonifer group revealed that genetic polymorphism
of R. stolonifer sensu lato is caused by the presence of
genetically distant varieties. Genetic variation of both
R. stolonifer clades is lower in comparison to R. oryzae
and R. microsporus species. All clades except for R. stolonifer (with R. stolonifer var. lyococcus and reflexus) appeared monophyletic.
Conclusions. We postulate a division of R. stolonifer
var. stolonifer and R. stolonifer var. lyococcus and reflexus
into separated species. Phylogeny analysis of 28s rRNA
gene of R. oryzae, R. delemar and A. rouxii showed limited potential of discrimination. Domesticated species of
R. oryzae and R. microsporus formed a polyphyletic clusters.
Acknowledgements
This study was prepared within the framework of the project PO IG
01.01.02-00-074/09, co-funded by The European Union from The
European Regional Development Fund within the framework of the
Innovative Economy Operational Programme 20072013.
P7.52
Metal cross-talk in Indian mustard results
in induced uptake of Cd and Pb when
supplemented with Zn
Agnieszka Kutrowska1, Aneta Piechalak1, Arleta Maecka1,
Anetta Ha2, Danuta Barakiewicz2, Barbara Tomaszewska1
Department of Biochemistry, Faculty of Biology,
Adam Mickiewicz University, Umultowska 89,
61-614 Poznan, Poland; 2Department of Trace Element Analysis
by Spectroscopy Method, Faculty of Chemistry,
Adam Mickiewicz University, Umultowska 90, 61-614 Poznan,
Poland
1
Trace metal toxicity towards plants varies with each

metal considered. Comparison of two essential (zinc
and copper) and two non-essential elements (lead and
cadmium) results in following generalized sequence of
metal toxicity Cd > Pb > Cu > Zn (Singh 2008). Detrimental influence of metals depends on the plant species, nature of an element and correlates with metal
bioavailability in soil solution (affected by pH, organic
matter and cation exchange capacity of the soil). For
instance, phytoextraction of lead is difficult due to its
low bioavailability and solubility (Jadia and Fulekar
2009). However, trace metals in soil solution are never
present as separate ions. The mixture of different elements in varying concentrations stimulate cross-talk
both before and after uptake by plant. Resultant effect
can comprise of decrease in metal toxicity and concentration in planta, e.g. inhibited uptake after competition for metal transporters or, conversely, increased
import due to stimulation of transporters (Rascio and
Navari-Izzo 2011). Therefore, metal cross-talk could
be used for improvement of phytoextraction, if its effect led to increased metal transport to aboveground
plant tissues.
In order to assess interactions between four trace metals: Zn, Cu, Cd, Pb, we have measured metal uptake and
distribution in seedlings of Indian mustard (Brassica
juncea), a known hyperaccumulator. Plants were grown
hydroponically and subjected to trace elements in following concentrations: 50 M of Zn2+, Cu2+, Cd2+ or Pb2+
of each, or mixtures of two metal ions (variants ZnCd,
ZnCu, ZnPb, CuCd, CuPb, CdPb) with 25 M of each
metal. With use of two methods, Inductively Coupled
Plasma Mass Spectrometry (ICP-MS) and Laser Ablation Mass Spectrometry (LA-MS), we have determined
i.a. that in variants ZnCd and ZnPb concentration of Cd
and Pb were significantly higher than in seedlings treated
with single metals, respectively. Our results suggest that
presence of zinc ions could improve efficiency of phytoremediation. Additionally, because Zn is an essential metal,
often exogenously supplied to increase crop productivity, its use in phytoextraction of Cd and Pb in controlled
quantities should not pose additional threat to the environment.
Acknowledgements
This research was supported by grant MNiSW N N305 381138.
Eurobiotech 2013
References
[1] Jadia C.D., Fulekar M.H., Phytoremediation of heavy metals: Recent
techniques. African Journal of Biotechnology, 2009, 8(6): 921928.
[2] Rascio N., Navari-Izzo F., Heavy metal hyperaccumulating plants:
How and why do they do it? And what makes them so interesting? Plant
Science, 2011, 180: 169181.
[3] Singh V.P., Metal Toxicity and Tolerance in Plants and Animals.
Sarup & Son, 2008.
111
P7.53
High density polyethylene as a carbon
source for Achromobacter xylosoxidans
Anna Kowalczyk1, Marek Chyc2, Przemysaw Ryszka3,
Kazimierz Strzaka1, Dariusz Latowski1
Department of Plant Physiology and Biochemistry,
Jagiellonian University, Gronostajowa 7, 30-387 Crakow,
Poland; 2Silesian Environmental Doctoral Study, Plac Gwarkow 1,
40-166 Katowice, Poland; 3Institute of Environmental Sciences,
Faculty of Biology and Earth Sciences, Gronostajowa 7,
1
Disposal of waste is one of the most important current

issues of environmental protection. Particularly burdensome waste are polymers, including high density polyethylene (HDPE), strongly resistant to degradation. Used
HDPE is disposed to landfill, often burned, which leads
to contamination of the air with toxic compound, such
as polycyclic unsaturated hydrocarbons. Less frequently
the HDPE is recycled, which is still costly method of utilization.
The present study deals with degradation of the chemical
structure of HDPE with the use of microorganisms, as it
is resistant to degradation in environment.
The biological material was isolated from the soil, and
the substance to be degraded was HDPE in the form of
foil. Disinfected with ethanol foil samples were subjected
to the action of microorganisms suspended in a liquid
Davis Minimal Broth medium for 6 weeks. The chemical
composition of the medium was modified. There were
2assays carried simultaneously. First of them contained:
Davis Minimal Broth medium with 30% of recommended content of glucose to enhance the growth of microorganisms, foil samples and previously isolated microorganisms. The second one contained: Davis Minimal
Broth medium without glucose, foil samples and microorganisms, so that the only carbon source for microorganisms was polyethylene. Part of foil samples in both
assays were irradiated with UV light, to accelerate HDPE
degradation. Control assays included foil (also samples
irradiated) suspended in proper medium with no microorganisms. The populations of microorganisms were
controlled by measurement of OD650 every second day
throughout the experiment. After 6 weeks the foil samples were analyzed with Attenuated Total Reflectance
Fourier Transform Infrared Spectroscopy (ATR-FTIR)
method and the results revealed degradation of chemical
structure of HDPE polyethylene in the microorganisms
supplemented media. FTIR research showed formation
of carbonyl groups in structure of HDPE, which probably induces formation of another groups, such as hydroxyl or ether groups and also skeletal vibrations. The
study also demonstrated, that in assays with glucose
addition microorganisms growth is improved and the
degradation of foil samples is more effective. There was
showed no correlation between irradiating with UV and
the extent and HDPE degradation.
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Eurobiotech 2013
The microorganisms were identified by analysis of the

16S ribosome subunit coding sequences. They were diagnosed as Achromobacter xylosoxidans. This is the first
communication showing that these bacteria can degrade
HDPE, although it was documented that some chemical
substances like benzoate, bisphenol A, ethylbenzene or
p-nitrophenol could be metabolized by these microorganisms.
P7.54
The influence of vermicomposting
on heavy metals contained in sewage
sludge
Hanine Suleiman1, Agnieszka Rorat2, Barbara Pytycz3,
Magorzata Kacprzak2, Franck Vandenbulcke1
Univ Lille Nord de France, LGCgE, EA 4515, Univ Lille 1,
EcoNum-Ecotox, F-59655 Villeneuve dAscq, France;
2
Institute of Environmental Engineering, Czestochowa
University of Technology, Czestochowa, Poland;
3
Institute of Zoology, Jagiellonian University, Crakow, Poland
1
Vermicomposting is a biotechnological method used to

convert organic wastes to fertilizer using earthworms.
Sewage sludge produced in wastewater treatment plants
is a rich waste in organic matter. Sewage sludge may be
valued in agriculture as fertilizer but its heavy metal content presents a constraint.
The aim of this study was to evaluate the changes in sewage sludge (its composition and mainly its heavy metals
content) after vermicomposting. Two earthworms species were used (Eisenia fetida and Eisenia andre) separately and in mixture. Three sewage sludges differ mainly
in their heavy metals content were used. Each sewage
sludge was mixed with added material (commercial compost) by a ratio (1:3). The process was observed after three
time periods (3, 6 and 9 weeks) by analyzing of heavy
metals as a main stress factor and of its total carbon, total nitrogen and total phosphorus content to evaluate its
value as fertilizer.
Eurobiotech 2013
P7.55
The effect of phosphate starvation
and mycorrhizae on the gene expression
of phosphate transporters in greenhouse
tomato
Iwona Kowalska1, Marzena Wiska-Krysiak2, Anna Konieczny1
Department of Soil Cultivation and Fertilization of Horticultural
Plants, Faculty of Horticulture, University of Agriculture
in Crakow, al. 29 Listopada 54, 31-425 Crakow, Poland;
2
Laboratory of Basic Research in Horticulture, Warsaw
University of Life Sciences SGGW, Nowoursynowska 159,
02-776 Warsaw, Poland
1
The symbiosis of arbuscular mycorrhizal fungi (AMF)

allows for the water and mineral supply of a plant to be
enhanced, as well as creates increased toleration for heavy
metals, salt, water and thermal stress. In soilless cultivation, including the hydroponics, an increase in the effectiveness of nutrient and water uptake by the roots system
developed in the limited area is especially important.
Current studies suggest that in this kind of cultivation
there is a diversified level of the colonization of roots by
AMF as a result of both the presence of easily accessible
forms of P in the culture, and of weak aeration of the root
environment. The hypothesis of the experiment assumes
that through the colonization of AMF on the tomato
roots, it is possible to decrease the concentration of P in
the nutrient solution while maintaining a similar feeding
status in accordance with the recommended levels.
An increased P uptake by the plant as a result of mycorrhizis will enhance the gene expression of P transporters
in a plant. Above hypothesis was verified in a tomato
cultivation on two different kinds of medium, since the
development of AMF may depend on the physical and
biological characteristics of environments, such as air
access.
Tomato plants cv. Admiro F1 were grown hydroponically in cultivation rows located in the foil tunnel, in 2012
year. The experimental factors were the concentration
of P in the nutrient solution (15 or 50 mg dm3), type of
substrate (rockwool or coconut fiber) and root inoculation by AMF. AMF colonization and gene expression of
P transporters (LePT1-LePT5) were determined in two
periods (I fruit setting on the 6rd cluster, II the end
of cultivation). Plant material was lyophilized and RNA
was isolated by the method of Gasic et al. (2004). RT-PCR
was performed with primers designed for amplifications
of genes LePT1-LePT5 and 18S rRNA in tomato (Balestrini et al. 2007). Synthesis of cDNA and RT-PCR reactions
were carried out using a Reverse Transcription System
Kit (Promega, USA).
Inspection of microscopic preparations of the roots revealed the presence of mycorrhiza in the roots of inoculated plants, as well as the influence of the substrate type
and level of P in the medium on the presence of AMF.
Expression of LePT depended mainly on the concentration of P in the medium, the presence or absence of AMF,
113
stage of growth of tomato plant as well as the type of substrate.

The level of transcripts was strongest for LePT4 and the
weakest for LePT1. Gene expression LePT2 and LePT4
had a similar profile. These genes were strongly expressed
in plants growing with AMF, regardless of other factors
studied. Gene expression LePT3 in plants growing on
coconut fiber and rockwool after application of 15 mg
P dm3 in the medium and AMF was higher than in the
plants growing on the same medium, but without the
AMF. A similar expression profile was obtained for the
LePT5 gene.
Acknowledgements
The research studies were financed by the National Science Centre
within the Project No. N N310 725040.
References
[1] Balestrini et al. (2007) Mol Plant Microbe Interaction 9: 10551062.
[2] Gasic et al. (2004) Plant Mol. Biol. Report 22: 437a437g.
114
Eurobiotech 2013
P7.56
P7.57
Effect of composting proces

on the changes of some organic
compounds
Isolation and characterization of plant

growth-promoting rhizobacteria (PGPR)
from isolated from Arabidopsis thaliana (L.)
Heynh. and Morus alba L. grown on heavy
metals rich acid soi
Marcin Milczarek, Ewa Neczaj, Dorota Kusal

Sewage sludge is a product of wastewater treatment processes; nowadays, about 8 million tones are produced
each year in the EU member states. A preferred method
of sewage sludges utilization is its application in soil fertilization and reclamation. The above has an unfavorable
impact on the environment, it opens the gates to environment pollution by organic and inorganic compounds and
pathogens. Sewage sludge pollution with polycyclic aromatic hydrocarbons is common. Their content in sewage
sludge can range across very wide borders. The standards
valid in the European Union forbid the usage of sewage
sludge with the sum content of 11 PAHs exceeding 6 mg
kg1. This article presents results of many authors, who
composted organic wastes such as sewage ludge, green
wastes, organic fraction of municipal solid wastes. Studies
have shown (Semple et al., 2001; Antizar-Ladislao et al.,
2004) that composting of soils polluted with PAH results
in a significant reduction of PAH content. Some recent
studies (Lazzari et al., 2000; Amir et al., 2005; Oleszczuk,
2006) showed also that sewage sludge composting can
significantly reduce PAH content in this material. The
above issue should be considered not only in the case of
sewage sludges in which valid norms are exceeded but
also in the case of sludges and organic fraction of municipal solid waste in which the content level of these compounds is increased. Such an approach will allow for the
limiting of PAH accumulation in fertilised soil and, at the
same time, influence the improvement of sewage sludge
fertilising properties. Microbial degradation represents
the major route suitable for the ecological recovery of
PAH-contaminated sites, with better results obtained
for the degradation of low molecular weight PAHs (24
rings). The refractory nature of some PAHs is related to
their limited water solubility, to their diverse and complex structure and to the requirement of providing oxygen to start degradation. Environmental conditions
which can affect the biodegradation rate are: high temperature which increases the rate of enzymatic reactions
and water solubility as well, good availability of nutrients,
high moisture level, good oxygenation, presence of alternative substrates required for fulfilling co-oxidation reactions, increased microbial variety and pH near neutrality.
It seems that composting process is very good to remove
some organic compounds from wastes or change them to
less toxic form.
Acknowledgements
This article was financed by the projects own Minister of Science and
Higher Education BS/MN-401-313/11.
Anna Grobelak, Anna Napora, Anna Grosser,

Magorzata Kacprzak
of Technology, Brzeznicka 60a, Czestochowa 42-200, Poland
Plant growth-promoting rhizobacteria (PGPR) are naturally occurring soil bacteria and benefit plants by providing growth promotion. This study was designed to
isolate and characterize PGPR bacteria from Arabidopsis
thaliana (L.) Heynh. and Morus alba L. (white mulberry). Plants were grown under extreme soil contamination with heavy metals (Zn 980 mg/kg, Pb 1400 mg/kg,
Cd 16 mg/kg) near zinc smelter area in Silesia region.
A large number of bacteria were isolated from roots with
three time levels of surface sterilization (0, 2, 10 min.)
using ethanol. For enrichment and isolation of root-associated bacteria the following media were used: Congo
Red agar and nitrogen-free base (NFb) media to isolate
afree-living diazotrophic bacteria, Luria agar (LA) to isolate nutritionally demanding bacteria, and yeast extract
mannitol agar (YEMA) to isolate Rhizobiaceae bacteria.
Isolates were grown until exponential growth phase to
evaluate the atmospheric nitrogen fixation, phosphate
solubilization, siderophores, indole-3-acetic acid production, as well as antifungal, protease, and 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity. A total
number of 26 for A. thaliana and 25 for mulberry isolates
were obtained from the enrichment cultures and tested.
All bacteria were able to grow and to produce IAA, and
the highest concentrations were found for bacteria isolated from mulberry plants. Only few of them were able to
produce siderophores, and solubilize phosphate. ACC deaminase activity was positive solely for three strains. The
growth of Alternaria alternta, Fusarium oxysosporum
and F. culmorum was inhibited over 50% for eight bacteria strains. To determine and confirm the plant growth
promoting potential, some bacteria strains were chosen
for plant inoculation in the growth chamber experiment.
Acknowledgements
The project was supported by National Science Centre grant
UMO-2011/ 03/N/ NZ9/ 02034.
Eurobiotech 2013
115
P7.58
P7.59
Biogenic catalysis in sulphide minerals

weathering processes and acid mine
drainage genesis
Isolation of saponins from plants and their

influence on hydrocarbon bioremediation
Maria Kusnierova1, Maria Prascakova1, Anna K. Nowak2,

Zbigniew Wzorek2
Institute of Geotechnics, Slovak Academy of Sciences,
Watsonova 45, SK-04353 Kosice, Slovak Republic;
2
Institute of Chemistry and Inorganic Technology,
Crakow University of Technology, Warszawska 24,
1
From a great group of environmental processes participating on the natural material circle is possible to use
mainly the processes of bioleaching and biogenesis in
raw materials processing. The bio-oxidation reactions are
the main basis for bioleaching procedures often parallel
participating in the leaching processes. During leaching
processes of the polycomponent sulphide substrates also
the factor of processes selection plays an important role
being in a direct relation to the electric properties and
galvanic effect occurring between the individual components of the leaching substrate.
This work gives summary of results from research focused on possibilities of using biotechnological procedures for Slovak sulphides ores treatment. The object of
the research is an extraction of valuable metals, undesirable admixtures and degradation of crystal lattice of
sulphides for subsequent chemical leaching processing of
precious metals.
Further, the results of experiments on existence of biogenic processes in situ on waste dumps from exploitation
containing residual sulphides are presented. Outcome of
this processes is acid mine drainage waters generation.
These waters are strongly mineralized and of low pH
thats why they are very aggressive. The heavy and toxic
metals contents as well as Cu, Zn, Fe, As, Cd etc. in out
flowed waters from old mines loadings are high over the
lawful limits.
Acknowledgements
This work was supported by the Operational Programme Research and
Development through the project: Centre of Excellence for Integrated
Research of the Earths Geosphere (ITMS: 26220120064), which is
co-financed through the European Regional Development Fund,
SGA project No. VEGA-2/0086/10 and within the SRDA project No.
APVV-0252-10.
Zuzanna Szczepaniak1, Justyna Staninska2,

Andrzej Olszanowski3
Institute of Food Technology of Plant Origin, Poznan University
of Life Sciences, Poznan, Poland; 2Department of Biotechnology
and Food Microbiology, Poznan University of Life Sciences,
Poznan, Poland; 3Institute of Chemical Technology
and Engineering, Poznan University of Technology, Poznan,
Poland
1
A very dynamic development of the industry in recent

years is the major cause of water and soil pollution. The
most common sources of contamination are hydrocarbons, especially oil derivatives. Increasing environmental
public awareness has led to development of many remediation methods technologies aimed at removing contamination from soil and groundwater. Apart from traditional, physicochemical techniques, there is a big interest
in biological methods that lead to degradation of hazardous compounds carrying out by plants (fitoremediation),
or microorganisms (bioremediation). Unfortunately, low
solubility of hydrophobic compounds in water results in
very limited bioavailability of these substances for the microorganisms, and thus significantly reduces the biodegradation process. In addition, the high molecular mass of
toxic compounds, such as polycyclicaromatic hydrocarbons (PAH) leads to even lower solubility. The aim of this
study was to isolate saponins natural surfactants occur
in plants - from soup nut shells and ivy leaves, as well as
the characteristics of the obtained extracts and their effect on diesel oil biodegradation efficiency. Moreover the
study was aimed at determination of bacterial cell surface
properties, such as zeta potential and cell hydrophobicity. It allowed to demonstrate that saponins isolated from
plants are not a toxic factor. On the contrary, they are
an easily assimilable carbon source. It has been proven,
that addition of saponins in most cases results in a higher
efficiency of diesel oil biodegradationa. Among all tested concentrations of saponins (120, 240, 360, 400mg/l)
in most cases, the lowest concentration of saponins
(120mg/l) did not significantly affect the efficiency of oil
biodegradation, whereas the concentration of 240 mg/l
turned out optimal. This is probably not related to the
change hydrophobicity cells, since no strict relationship
between the degree of bacterial adhesion to hydrocarbon, and the efficiency of hydrocarbon biodegradation
has been proven. These results indicate the possible application of saponins as natural surfactants isolated from
plants in biodegradation process.
116
Eurobiotech 2013
P7.60
P7.61
Biodegradation of soot components

by soil bacteria
Biodiesel-derived crude glycerol

as a substrate for fumaric acid production
by fungus of the genus Rhizopus
Barbara Kalicka1, Marek Chyc2, Monika Bojko1,

Kazimierz Strzaka1, Dariusz Latowski1
Poland; 2Silesian Environmental Doctoral Study,
Plac Gwarkow 1, 40-166 Katowice, Poland
1
Reducing the level of soot released into the atmosphere

should be one of the most important challenges aimed
at reducing emissions of air pollutants. It has been suggested that the soot particles in the air contribute to the
enhanced global warming effect to a degree comparable
to the greenhouse gases such as methane, halocarbons,
volataile organic compounds (VOCs) and tropospheric
ozone. In this study were compared biodegradation of
two types of soot: unmodified (obtained only from burning hard coal in a central heating boiler) and soot SAS
(obtained from burning coal with an adequate amount of
new marketed fuel additive labelled SAS (an acronym for
Safety And Strong). SAS is an innovative and effective fuel
additive, obtained mainly from a waste material, based on
iron oxide and inorganic salts. This invention reduces the
emmision of particulate matter, CO, CH4 and unburned
VOCs exhaled with the flue gases into the atmosphere.
SAS maintains the surface of the fireside heat exchanger
free of soot thereby increasing the thermal efficiency of
the heating system.
6.00% w/v solutions of soot in basic salt medium (BSM)
where soot was the only carbon source were autoclaved
and inoculated with one of two types of bacteria: Pseudomonas aeruginosa or Bacillus subtilis. Results obtained
after 4 weeks of incubation of soot samples (30C, RPM
150) exposed to bacteria were compared to the samples
stored under the same conditions but without the participation of microorganisms by static headspace gas
chromatography with mass spectrometry detection
(HS-GC-MS) of the soot residue. Biodegradation was
concluded by analysing sum of 4 aromatic compounds
(phenol, naphthalene, phenanthrene, anthracene). The
highest level of biodegradation (about 80%) was obtained
for soot SAS samples with Pseudomonas cultures. From
the results it can be concluded that soot obtained from
carbon modification with SAS fuel additive will be more
quickly removed from the environment. Unmodified
soot, SAS and four other types of soot were also tested for
microbial content. The microorganisms were present in
all analysed kinds of soot. This suggests that soot can be
treated as source of microorganisms potentially useful in
biodegradation of soot components. All analysed types of
soot were non-toxic for Pseudomonas and Bacillus strains
applied in our studies, even with high contents of soot in
medium.
Elwira Komo, Jakub Wyrostek, Adam Kuzdraliski,

Zdzisaw Targoski
University of Life Sciences in Lublin
Crude glycerol is the waste byproduct of biodiesel production process and its conversion mainly to organic
acids brings significant economic benefits. Fungus of the
genus Rhizopus are well known producers of key metabolites such as lactic acid, fumaric acid and ethanol as
co-products. Most of researches was focused on simple
carbon sources like glucose or fructose to study metabolic profiles of this fungus. Previous cultures that were
conducted with supplementation of pure glycerol showed
minimal or no consumption of this carbon source by investigated fungi, which affected on low fumaric acid production. Crude glycerol contains lots of inorganic salts
and MONG (matter organic non glycerol) which can be
used for enhancing biomass growth and modify metabolic profiles.
In this study ability to utilization of crude glycerol by six
strains of Rhizopus oryzae was studied. Preselected cultures showed that optimal amount of crude glycerol in
fermentation medium is 30 g/L. Each of selected fungus
were grown in shaking flask culture in triplicate for 168h.
To avoid excessive acidification of cultures sodium carbonate as a neutralization factor was added to reach 2%
as the final concentration. After HPLC analysis of broth
samples the glycerol consumption rate was observed to
reach 0,16 g/L/h. Best result of final amount of fumaric
acid was observed in R oryzae ATCC 20344 culture, that
concentration reached 15 g/L. Some of studied fungus
showed weak biomass growth in crude glycerol supplemented medium. HLPC analysis of those samples proved
that neither significant utilization of propanetriol nor fumaric acid production was occurred.
Acknowledgements
01.01.02-00-074/09, co-funded by The European Union from The
Eurobiotech 2013
P7.62
Effectiveness of eco-friendly,
natural products in in vitro rooting
of Prunus domestica L. microshoots
Alina Wiszniewska, Barbara Nowak, Anna Koton
Faculty of Horticulture, University of Agriculture in Crakow
Development of novel, effective chemical preparations

biostimulators, that could enhance plant production,
plays an important role in sustainable agriculture. Recent
studies confirm that natural biopreparations may induce
plant defense reactions and stimulate development of
plant organs, with limited influence on the environment
(Pacholczak et al. 2012). Biotechnological approach allows to test the effectiveness of biostimulators that are
composed of environmentally friendly, natural products, such as algae extracts, plant extracts and cell exudates. Therefore the aim of our study was to evaluate the
root-promoting activity of two natural products, i.e. pineapple pulp and conditioned medium containing green
algae exudates, during in vitro rooting of plum (Prunus
domestica L.) microshoots. The long term purpose of the
research is to limit the level of synthetic growth regulators exploited during rooting phase in micropropagation
of this fruit tree.
In vitro shoots of two Polish plum varieties, Wgierka
Zwyka and Wgierka Dbrowicka, were used as plant
material. Root induction was conducted for 7 days on
fourculture media: 1) control medium WPM (Lloyd and
McCown 1981) containing 5 mg/l IAA and 2 mg/l IBA
(according to Nowak and Miczyski 1996); 2) WPM
medium containing 10 ml/l of dialyzed pineapple pulp
(low-molecular weight fraction Dialyzate and conditioned medium were produced and kindly provided by
Professor Z. Tukaj and Dr K. Grabski, from the Department of Plant Physiology, University of Gdask, Poland.
After one-week long induction, shoots were transferred
to hormone-free MS medium (Murashige and Skoog
1962). Root formation was observed for 46 weeks and
effectiveness of rooting was evaluated on the basis of biometric measurements.
The study also aimed at determination of optimal time
needed for rooting induction on tested medium variants.
Peroxidase activity and phenolic profile were determined
using 5-mm long shoot bases. Peroxidase activity was analyzed during 7 days, starting from 1. day (24 h) after culture establishment. Phenolic profile was determined after
7 days of induction phase.
Results revealed that rooting ability in plum depends
strongly on the genotype and applied organic medium supplements. In Wgierka Dbrowicka the highest
rooting percentage was obtained on medium containing
dialyzate of pineapple pulp. Microshoots of Wgierka
Zwyka rooted decidedly less effectively,on every tested
medium. Moreover, the results of physiological analyses
117
suggested that rooting protocol for Wgierka Zwyka

could be modified in order to achieve higher percentage
of rooted shoots. Details concerning obtained results will
be presented and discussed during poster session.
Acknowledgements
The study was financed by Polish Ministry of Science and Higher
Education.
References
[1] Lloyd G., McCown B. (1981) Commercially-feasible micropropagation of mountain laurel, Kalmia latifolia, by use of shoot tip culture.
Comb Proc Intl Plant Prop Soc 30, 421437.
[2] Murashige T., Skoog F. (1962) A revised medium for rapid growth
and bioassay with tobacco tissue cultures. Physiol Plant 15, 473497.
[3] Nowak B., Miczyski K. (1996) Regeneration capacity of Prunus
domestica L. cv. Wgierka Zwyka from leaf explants of in vitro shoots
using TDZ. Folia Hort 8/2, 4149.
[4] Pacholczak A., Szydo W., Jacygrad E., Federowicz M. (2012)
Effect of auxins and the biostimulator Algaminoplant on rhizogenesis
in stem cuttings of two dogwood cultivars (Cornus alba Aurea and
Ellegantissima). Acta Sci Pol, Hortorum Cultus 11(2), 93103.
118
Eurobiotech 2013
P7.63
P7.64
Monoaromatic hydrocarbon degradation

by selected bacterial strain
The improvement of fumaric acid

production trough coutilization of glycerol
and pectin by Rhizopus oryzae ATCC 20344
Przemysaw Petryszak, Pawe Kaszycki

of Agriculture in Crakow, al. 29 Listopada 54, 31-425 Crakow,
Poland
Dynamic industrial expansion leads to overproduction of

various toxic and environmental nuisance compounds.
The anthropogenic contamination with aliphatic and aromatic hydrocarbons is caused by a variety of petroleum
processing technologies and results mostly from improper use, transport and storage of diesel fuel and oil-derived
substances. This leads to the release of huge amounts of
toxic xenobiotics such as low- and highmolecular nalkanes as well as aromatic hydrocarbons, especially those
belonging to the BTEX group (benzene, toluene, ethylbenzene, xylene), naphthalene and many other chemical
derivatives. Since the above pollutants tend to negatively
affect the natural soil and water environments, the
development of effective and relatively cheap decontamination methods is necessary.
In search for optimized bioremediation strategies, bacterial decomposition of aromatic hydrocarbons utilized
as carbon and energy sources was studied. The possibility and conditions for effective biodegradation of BTEX
substances (applied as single xenobiotics or in a mixture)
were evaluated. Various strains of the bacterial culture
collection of Biochemistry Department, earlier isolated from hydrocarbon-contaminated soil and belonging
mainly to the genus Pseudomonas, were tested. The selected strains showed high level of tolerance for xenobiotic action, which was expressed either as a growth inhibition-rate index, or as an effectiveness of 2,3,5-triphenyltetrazolium chloride reduction. Bacterial biodegradation efficiency was proved by gas chromatography analyses of monoaromatic hydrocarbon content in culture
media. Biodegradation kinetics of the most recalcitrant
xenobiotics were presented. The possibility of adaptation
of the selected strains to toxic environmental conditions,
i.e. to sub-lethal concentrations of monoaromatics, was
shown employing qualitative and quantitative analyses of
fatty acid methyl esters. SDS-PAGE electrophoretic profiles obtained upon biodegradation processes were compared to reveal protein changes as induced by the presence of xenobiotics.
Acknowledgements
The work was financially supported by the University of Agriculture
Research Grant for Young Scientists no. BM/2012/4519 and Grant for
Scientific Research no. 3500, approved by the Polish Ministry of Science
and Higher Education.
Jakub Wyrostek, Elwira Komo, Adam Kuzdraliski,

Zdzisaw Targoski
University of Life Sciences in Lublin
Fumaric acid is commercially important element of the

components of food, medicines and industrial materials,
it is used for food acidification and the production of synthetic resins. Rhizopus oryzae is a zygomycete filamentous fungus, well-known as a saprobe ubiquitous in soil
and as a pathogenic/spoilage fungus able to overproduce
fumaric acid, lactic acid and ethanol.
The effects of substrats and their mixtures on the fumaric
acid production by Rhizopus oryzae ATCC 20344 were
studied, with the aim of improving the efficiency of fumaric acid production. The amount of fumaric acid produced on mixtures of the carbon sources glycerol and
pectin was different from the produced on each of those
substrates separately. The fumaric acid amount was observed to reach 1,96 and 1,2 g/L for glycerol and pectin
supplemented medium, respectively. After cofermentation in different variants of glycerol-pectin medium with
pectin after enzymatic hydrolysis and with non-hydrolyzed pectin the amount was 4,2 and 19,6 g/L. The present
study aimed to put forward a theory for glycerol/pectin
cofermentation.
Acknowledgements
01.01.02-00-074/09, cofunded by The European Union from The
Eurobiotech 2013
P7.65
Biostimulation of xenobiotic-degrading
bacterial consortia with oxygen-releasing
compo
Pawe Kaszycki, Paula Bana, Przemysaw Petryszak
of Agriculture in Crakw
Bioremediation is a powerful method to deal with environmental pollution. Particularly toxic and recalcitrant
xenobiotics can be biodegraded employing microbial
communities that reveal specialized metabolic pathways. Biochemical processes leading to the reduction of
total load of chemical pollutants such as oily products
are very complex. In general, the maximum contaminant removal efficiency is obtained at aerobic conditions where oxygen serves as the final electron acceptor
in biooxidation reactions. Since water and soil are main
targets for hydrocarbon discharge by industrial activities, oxygen level often becomes a limiting factor hampering biodegradation activity of autochthonous and/or
allochthonous microorganisms. To overcome the bottle-neck effect of oxygen consumption some methods of
soil bioremediation suggest microflora biostimulation
with oxygen-releasing compounds. Wastewaters can be
treated using consortia aerated with air-compressors,
however this approach may not applicable for in-situ
cleanup projects.
In this study a microbial consortium ZB-01 was tested
for its susceptibility for aerobic metabolism stimulation with two variant oxygen-supplementing commercial products, differing in the mechanism and intensity of oxygen release. The ZB-01 is a highly specialized
biocenosis constructed at Biochemistry Department
of University of Agriculture in Crakow. It consists of
anumber of strains able to biodegrade aliphatic and aromatic hydrocarbons, both in water and soil environments. The two sources of oxygen were: an EHC-OTM
preparation (Adventus Americas Inc.), applied predominantly for reclamation of soil environment, and oxygen
tablets, OxyTABS (JBL GmbH & Co. KG), used mainly
to maintain proper aeration for live in small ponds and
fish tanks. For both substances, optimized concentrations were established as determined by bacterial survivability experiments. The preparations showed a stimulating influence on microorganisms metabolism measured with triphenyltetrazolium chloride (TTC) used to
reveal dehydrogenase activity. Also, bacterial frequency
determinations proved proliferation of microbial cells
under oxygen supplementation conditions. Oxygen level was monitored as a DO (dissolved oxygen) parameter.
Based on the comparative tests the best effect was found
for EHC-OTM applied at 0.1%.
It is concluded that the use of oxygen-releasing compounds to provide aerobic conditions in aqueous media
appear favorable in terms of both, short-time bacterial
119
metabolism stimulation prior to application at contaminated sites, and optimization of long-term consortia
storage.
Acknowledgements
The work was financially supported by the grant for scientific research
No. 3500, approved by the Polish Ministry of Science and Higher
Education.
120
Eurobiotech 2013
P7.66
P7.67
Analysis of physico-chemical properties

of porous carriers for bacteria
immobilization
The cultivation of endophytic

methanotrophs isolated from different
species Sphagnum as perspective
for environmental bioengineeringSesja:
Environmental Biotechnology
Dagmara Leniak, Paulina Worsztynowicz

Poznan University of Life Science, Department of Food
Biotechnology and Microbiology
Introduction. Using crude glycerol from a biodiesel production process for producing 1,3-PD is a good solution
from the economical as well as ecological point of view.
Biotechnological production of 1,3-PD from waste biomass is a promising and attractive alternative to the traditional chemical synthesis. The productivity of 1,3-PD
can be improved through the application of fermentation
with cell immobilization on porous carriers in packed
bed reactor (PBR).
AIM. The aim of this study was to investigate the influence of physico-chemical properties of porous carriers
used in PBR to immobilize Clostridium butyricum cells.
Materials and methods. An analysis of the surface properties was made using scanning electron microscopy
(SEM) and energy-dispersive X-ray spectroscopy (EDS).
SEM is a type of electron microscope that produces images of a sample by scanning it with a focused beam of
electrons used for analysis of samples surface topography
and composition. EDS is an analytical technique used for
the elemental analysis or chemical characterization of
a sample. All samples were cleaned of any organic residues (300oC for 3h), cut to appropriate size, and mounted
on a specimen holder for viewing in the SEM.
Results. Using the signal of secondary electrons (accelerating voltages of 515 kV), image of: pumice, expanded
clay (LCA), Sera Siporax Mini, Eheim Substrat Pro and
coral gravel were made. Images allowed for assessment
of topography and porosity of each carrier, which are related to the adsorption of bacterial cells to the surface.
EDS analysis have shown that most common element was
silicon (Si).
Conclusions. The area of porous carriers available for the
adhesion of microorganisms, affect the amount of immobilized biomass, which in turn affects the efficiency of the
bioconversion process. Impact on the bacterial adhesion
efficiency has a content of such elements as magnesium
and calcium, and any scratches and surface irregularities.
Acknowledgements
This work was funded within the framework of project No. 01.01.0200-074/09 co-funded by The EU from the European Regional
Development Fund within the framework of the Innovative Economy
Operational Programme 20072013.
Zofia Stpniewska, Agnieszka Kuniar

The John Paul II Catholic University of Lublin, Department
of Biochemistry and Environmental Chemistry,
Konstantynow 1I St., 20-708 Lublin, Poland
Enriched cultures of microorganisms are essential step in

the production of inoculum of these organisms for biotechnology and bioengineering. A potential application
of methanotrophs is widely studied as microorganisms
for the removal of methane produced from landfills, coal
mines as well as biodegradation of toxic compounds.
Therefore, searching for new sources of methanotrophs
can contribute to increase the possibilities of biotechnology and bioengineering.
Enrichment cultures of endophytic methanotrophs were
initiated in NMS medium as most widely used medium
for cultivation of methanotrophic bacteria from various
environments is that proposed in 1970 by Whittenbury.
Incubation was carried out at 10, 20, 30 and 37C with
vigorous shaking on a shaker (180 rpm). The source of
carbon and energy for endophytes were methane at the
concentrations range between 120%. The medium NMS
were inoculated homogenized Sphagnum sp. materials
that originated from Moszne peatbog (the Poleski National Park, Eastern Poland). It have been demonstrated
that in natural conditions exist methanotrophic bacteria
in cooperation with other microorganisms and their in
pure cultures are unstable for extended periods of time
(Hoefman et al., 2010). Therefore, in these studies we investigated consortium of whole microorganisms communities. During the incubation, growth of the cultures and
dynamics of gases were controlled, respectively by the optical density measurements at the wave length of 600 nm
and concentration of gases in headspaces with the use of
gas chromatograph (SIMADZU, GC 2010).
It appeared that the consortium of endophytic bacteria
grew only at 20 and 30oC temperature which is characteristic for mesophilic methanotrophs. We determined
that the length of phase adaptation of endophytes was
ranged from 5 to 12 days. During these time the bacteria adapted to the new conditions in the environment.
Then, there was rapid growth of endophytic population
(phase logarithmic) that it was reflected by an increase
of optical density in the range from 0.3 to 2.0, depending on methane concentration (120%). After phase
logarithmic, it have been observed the lack of carbon/
energy sources and/or increase of waste products concentration to the level of harm for methanotrophic consortia. These time was called as a stationary phase of
enrichment cultures.
Eurobiotech 2013
During the culture endophytes, the measurements of

gases concentration showed a steady loss of methane and
oxygen, as well as the accumulation of carbon dioxide as
a CH4 oxidation product.
The use of FISH has made possible characterization of
endophytic consortia. I turned out that the population of
endophytic consist with type I and II methanotrophs as
well as associated non methanotrophic bacteria.
Furthermore, we determined the potential activity of examined bacteria for methane oxidation that it was ranged
up to 4,7 MCH4 per ml population of endophytes and
day.
The interest in methanotrophs has largely been due to
their biotechnological potential for the production, in
particularly single cell protein, methanol, osmoprotectants (for example ectoine) vitamins and other biotechnological products.
Acknowledgements
This work was supported by the National Science Centre grant in Poland
(No. 2011/01/N/NZ9/06811).
121
P7.68
The physiological response of perennial
grasses grown on heavy-metal polluted
soilSesja:
Krystyna Rybka1, Grzegorz urek1, Marta Pogrzeba2,
Jacek Krzyak2, Kamil Prokopiuk1
Plant Breeding and Acclimatization Institute IHAR-PIB,
Radzikow, 05-870 Blonie, Poland; 2Phytoremediation Team,
Institute for Ecology of Industrial Areas, 40-844 Katowice,
Poland
1
Phytoremediation requires plants that maintain good

fitness on contaminated soils in parallel with the highest
possible concentration of pollutants in aerial parts. For
evaluation of species and varieties as potential phytoremediators in breeding programs, low-cost, easy to carry
and high throughput techniques are required. Chlorophyll a (Chl a) fluorescence measurements fulfill those
conditions and in parallel with detection of heavy metal
(HM) ions concentration represent a good screening tool
for evaluation of perennial grasses suitability to grow on
lands of long history of industrial emission.
Our experiment was located on contaminated agricultural soil of Silesia, southern Poland. Plots were established
in the vicinity of a closed-down cadmium/lead/zinc/ ore
mine and processing plant, which operated for more than
100 years and had significant impact on local soils. Reference site (uncontaminated soil) was located in a distance
of 25 km North from above, on agricultural land with
similar forage crop history. HM ions amount in polluted
soil exceeded by almost 2 orders of magnitude the concentration of Cd and Zn ions and about 50 times of Pb
ions when compared to reference place.
To detect the effect of HM soil contamination on selected
perennial grass cultivars: plant height, generative shoots
abundance, biomass yield as well as total Pb, Cd and Zn
ions concentration were measured in parallel with Chl
a fluorescence transient. The HM ions negatively influenced fluorescence in all listed points: F0 (minimal), FM
(maximal), FV (variable), F1F5 (at times [ms]: 0.05, 0.15,
0.30, 2.0, 30) as well as total complementary area (Area)
of Chl a fluorescence induction curve and forward electron transport measured as (1-VJ)/(VJ)). Tall wheat grass
cv. Bamar and tall oat grass cv. Wiwena were characterized by unreduced yield, whereas rescuegrass cv. Broma.
and smooth bromegrass cv. Brudzyska significantly reduced the biomass yield as well as F0, FV and FM. The highest concentration of Cd and Zn ions in the biomass of cv.
Rahela was not associated with yield reduction.
All parameters detected during fluorescence measurement could be used for plant physiological state description, but sometimes it is difficult to highlight those best
suiting for data interpretation. One of the method of parameter number reduction is PCA. Grouping of cultivars
calculated on bases of 2 principal components, which explained 85% of differentiation, gave logical results. Cultivars were grouped in sub-classes as polluted and refer-
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ence. Cultivars Bamar and Wiwena were characterized

by positive correlation with component 2. Both principal
components: 1st and 2nd, have all sub-components positively correlated, which means that if increasing values
were recorded, the position of scatter would move towards higher values on axes. The other statistical method,
presented by Goltsev et al. (2012) is neutral network.
To compare the dynamics of electron transfer chain in
chloroplasts Strasser et al (2004) proposed double normalized curves enabling specification of the best stress
tolerating cultivar (Gururani et al. 2012; Bussotti et al.
2011; Oukaroum et al. 2007). The usage of such an approach pointed out cv. Rahela as outstanding cultivar
under the HM ion stress. Contrary to expectation, cv. Rahela which sequestrated the highest concentration of HM
ions in the aerial part, did not show PSII antennas grouping under the stress, as other cultivars did. All cultivars
except cv. Rahela accelerated metabolism, which is a typical physiological response characteristic for plants slightly resistant or non-resistant to the stress. Resistant plants
typically launch additional defense mechanisms and rebuild its metabolism (Foyer et al. 2012, Zagdaska 1995).
Such cultivars, which survive the stress by accelerating
photosynthesis, as well as the whole metabolism, could be
less resistant in front of additional environmental stresses
such as i.e. drought or cold and could react more rapidly
on them. Cultivar Rahela in fact deals the best with the
stress. Its antennas were not damaged, might be due to
redox guard in the form of ascorbate and proline which in
other cultivars seemed not to be induced strongly enough
to be reflected on duble normalized graphs.
Acknowledgements
The work was financed by the Ministry of Agriculture and Rural
Development, as PW3-3-00-0-03. KR and G are considered to be the
first author.
References
Goltsev et al. (2012) Biochim Bioph Acta 1817: 14901498.
Strasser et al. (2004) Kluwer.
Gururani et al. (2012) Plant Physiol Bioch 58: 182194.
Bussotti et al. (2011) Envir Exp Bot.
Oukarroum et al. (2007) Envir Exp Bot 60: 438446.
Foyer et al. (2012) JXB 63: 1637166.
Zagdaska (1995) Physiol Plant 95: 428436.
P7.69
Utilisation of betaine in vinasse stillage by
a mixed culture of aerobic bacteria under
non-controlled pH
Agnieszka Ryznar-Luty, Edmund Cibis, Magorzata Krzywonos
Wroclaw University of Economics, Department of Bioprocess
Engineering
The aim of this work was to examine how temperature

and the initial pH influence the efficiency of an aerobic
biodegradation of betaine in vinasse stillage. Betaine accounted for as much as 37.6% of total organic carbon.
In the study two series of batch biodegradation processes were carried out in a stirred tank reactor by a mixed
culture of Bacillus bacteria. The temperatures applied
were 27, 36, 45, 54 and 63C. The pH was not controlled,
and the initial pH of the culture medium amounted to
6.5 in first series and 8.0 in the second series. Efficiency
of biodegradation was expressed in terms of reduction
in SCODsum, which is a sum of SCOD (soluble chemical
oxygen demand, i.e. COD determined after suspended
solids separation) and theoretical COD of betaine. The
high biodegradation efficiency obtained in the processes was attributable to the assimilation of betaine by the
bacterial strains used in the study. Maximal reduction in
SCODsum (85.41%), BOD5 (97.91%) and TOC (86.32%)
was achieved during biodegradation at 36C and
pH0 = 8.0. Under the same temperature and pH regime,
betaine was removed completely, while biodegradation proceeded at the fastest rate (1.1684 g O2/(lh) for
SCODsum removal). During biodegradation the main organic pollutants were removed at the same time, but with
diverse rate of removal. An exception to this rule was the
assimilation of betaine, which intensified only after the
medium was lacking in easily available carbon sources.
Acknowledgements
Eurobiotech 2013
123
P7.70
P7.71
Use of biotechnological methods

in reforestation of degraded land
Pilot study on feasibility of application

of gas chromatography for the assessment
of acrylamide concentration in sewage
sludge
Rafa Wany1, Katarzyna Turnau1, 2

Jagiellonian University, Malopolska Centre of Biotechnology,
Gronostajowa 7, 30-387 Crakow; 2Jagiellonian University,
Institute of Environmental Sciences, Gronostajowa 7,
30-387 Crakow
1
Presence of beneficial organisms in degraded soil is usually limited, especially in soil polluted with heavy metals
or in soil previously used for agricultural purposes. The
main obstacle is usually the lack of well adapted mycorrhizal fungi. Bioremediation and reforestration can be
supported by inoculum production of mycorrhizal fungi.
The introduction of the particular strain should be preceded, however, by proper knowledge of fungal species in
a given habitat, known receptivity of the soil understood
as the ability of the fungus to survive under given condition and to establish long lasting existence. Further, the
succession of fungal taxa should be estimated and controlled. In such studies it is not possible to rely only on
conventional studies but the use of molecular tools is necessary. The research carried out on Abies alba seedlings
regenerating in forest sites established on agricultural
land are presented. We aimed to determine whether the
fungal communities of seedlings from degraded land differ from fungal communities of seedlings growing in natural forest and whether there was the need for some treatments like inoculation. Mean mycorrhizal colonization
of seedlings was over 90%. A total of 49 ectomycorrhizal
taxa were identified, 36 of which were identified in natural forest and 23 in forest on degraded area. According to
the analysis of similarity, the ECM fungal communities
were different between these forests. Only 20% symbionts
common to both sites suggests that succession of ECM
fungus specific to fir regenerating on post-arable soil is
still in progress. The use of mycorrhizal inocula offers the
possibility to improve the reforestation process.
Elbieta Wodarczyk1, Marta Prba1, ukasz Wojtal2

Czstochowa University of Technology Faculty of Engineering
and Biotechnology Institute of Environmental Engineering
Brzenicka 60A St., 42-200 Czstochowa, Poland;
2
Center for Research and Environmental Control,
Owocowa 8 St., 40-158 Katowice, Poland
1
Background. Acrylamide (2-propenamide CH2=CH

CONH2) is a low molecular weight organic compound
containing in its structure conjugated double bonds and
a part of the amide. Due to the presence of characteristic
functional groups, acrylamide is polar compound. It is
well soluble in water and polar solvents such as methanol or ethanol. It is produced on an industrial scale for
60 years by catalytic hydrolysis of acrylonitrile. Currently,
it is used, for the synthesis of modified polyacrylamides,
which are used in industry, in the production of plastics,
dyes, adhesives, cosmetics, mortar, as well as a coagulant
for treatment of water, wastewater or sewage sludge conditioning.
Objectives. Aim of this study was to determine the possibility of using gas chromatography to measurement of the
acrylamide concentration in sewage sludge.
Methods. Determination of acrylamide by gas chromatography is based on the standard: EPA Method 8032A
Acrylamid by gas chromatography. To mark acrylamide
in the sludge elementary test should be performed with
ratio of liquid to solid part equal 10l/kg [mg/kg dry
weight].
The method permits for the determination of acrylamide
(2-propenamide) in water in the range of 0.040 mg/l to
2.0 mg/l.
It consists of: bromination reaction of the compound in
the presence of dibromopropendial derivative, triple extraction with ethyl acetate, concentration of eluate sample
up to the 1 ml volume, and analysis by gas chromatography using an electron capture detector (ECD).
According to the manual of chromatograph software calibration curve was made (calibration using the internal
consistency of the NIST standard (Lot No. 51011).
Calibration is considered valid if the correlation coefficient is r
Results. To ensure the quality of test results for each
test series were performed: a blank sample (checking
the purity of the reagents, water) and a control sample (50 mg/l-checking the precision and stability of the
measurement system) and a desired sample of sewage
sludge water extract. Samples were performed twice
(the difference between the results is not greater than
10%), and the average value of the two samples was
1.62 mg/ l.
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Conclusions. The presence of acrylamide in sewage

sludge has been confirmed. Therefore, the use of polyacrylamide materials, containing acrylamide, is becoming wider, more and more of the compound enters the
natural environment (through sewage and sediments into
groundwater and surface water). Acrylamide, as the toxic
substance is not indifferent to human health. The need of
further research in this area was highlighted.
Keywords: acrylamide, polyelectrolyte, sewage sludge, gas chromatography
Acknowledgements
BS/MN 401-307/12.
Plant genetic engineering
Lectures
L8.2
L8.1
Biotechnology of Flax
The ins and outs of multigene engineering

of complex biosynthetic pathways inplants
Changfu Zhu1, Teresa Capell1, Paul Christou1, 2
Departament de Produccio Vegetal i Ciencia Forestal,
Universitat de Lleida-AGROTECNIO Center, Lleida, Spain,
2
Institucio Catalana de Recerca i Estudis Avanc ats, Barcelona,
Spain
1
The simultaneous transfer of multiple genes into plants

enables researchers to study and modulate entire metabolic pathways, express multimeric proteins or protein
complexes, and study complex genetic control circuits
and regulatory hierarchies. Technical hurdles limiting the
number of genes transferred to plants have introduced
a significant bottleneck to progress in plant biotechnology for the past three decades. Early transformation
methods for plants were developed with the implicit intention to introduce one or two genes (usually a primary transgene and a selectable or screenable marker) and
have been optimized on that basis. Although multigene
transfer (MGT) can be achieved using such methods, they
operate under the law of diminishing returns. The more
transgenes are included, the less likely all of them will be
integrated and expressed. Larger and larger populations
of plants must be screened to identify rare individual lines
with the sought-after genotype.
More recently, researchers have attempted to address these
limitations by developing new transformation methods
that recognize the desire to introduce multiple transgenes
into plants and express them in a coordinated manner.
Essentially all these methods aim to achieve the creation
of aSMART locus, i.e. one containing Stable Multiple Arrays of Transgenes. We will discuss the most recent strategies for MGT in plants using case studies to illustrate how
these approaches have been applied in the dissection and
modulation of complex metabolic pathways.
Anna Kulma, Jan Szopa

Wroclaw University, Linum Foundation
The development of molecular biology, especially the sequencing of nucleic acid, resulted in the stream of data
on gene structure. Study of gene structure forced the development of research on identification of their function.
For evaluation of gene function, the most effective and
actually the only method is the generation of an organism
with overexpression or/and repression or/and complementation of repression/overexpression of the gene with
the subsequent analysis of modified organism. GM plants,
generated for over three decades, provided the valuable
information of gene function, and simultaneously indicated the advantages of transgenic plants.
According to the European Commission, the challenge
for biotechnology, as a scientific discipline, is searching
for tools for agriculture and industry development and
providing such a plant productivity, that will satisfy growing food demand.
To rise this challenge, the generation and application of
GM plants is justified. At the turn of 2010/2011 the European Commission released a study summing up the tenyear (20012010) research on GM plants (A decade of
EU-funded GMO research). The aim of the study was to
estimate the influence of transgenic plants on the broadly- taken environment, study of the risks connected with
their release into the environment, determination of the
putative horizontal gene transfer between GMO and nonGMO, study of the safety of food produced from transgenic plants, etc. 50 projects realised for over 200 million
of Euro by over 400 research groups were summarised.
The main conclusion summarizing these research is, that
the cultivation, processing and using of GM plants is not
more risky than those of conventional cultivation. Even
though, another study released by the European Commission (Europeans and Biotechnology in 2010) says,
that over 60% (6190% depending of the country) of the
society does not accept GMO.
From two decades we (Linum Foundation, www.leczenielnem.pl) have been generating GM potatoes and nowadays
GM flax. At first, the aim was to identify the genes function
and their significance for plant productivity and metabolism. The most attention was given to those genes, that
are putatively key genes for plant infection resistance, are
crucial for regulation of the synthesis of the compounds
of biomedical functions (phenolic acids, flavonoids, terpenes) or are applicable in industry (fatty acids) and in the
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environment protection (biodegradable polymers). We

generated plants of elevated resistance to pathogen infection, of increased accumulation of phenylpropanoids and
terpenoids, of altered synthesis and accumulation of biopolymers (pectins, lignins, cellulose, hemicellulose) and
biodegradable polymers (polyhydroxybutyrate) and flax
of altered fatty acids profile. We estimated the influence
of GM flax cultivation on soil ecosystem and we revealed
the intensified and beneficial interaction of mycorrhized
organisms on flax root system. We showed the positive
influence of GM flax seeds consumption on the broad
range of parameters describing health condition of laboratory animals (mice, rats). We generated a few preparations applicable in the prevention and treatment of
people, including certified flax dressing LENPLAST for
chronic wounds treatment, certified preparations of antiinflammatory and antibacterial function (LINFIX) and
of skin regenerating function (OILFIX), diet supplement
LINACTIVE beneficial in antiinflammatory and anticancer prevention, admitted to the trade by GIS and we offer
flax oil of the characteristics of ideal oil and other preparations of antibacterial function (see www.pkblinum.pl).
The favourable characteristics of GM flax and the preparation generated from GM flax cannot be produced on
the largescale due to the difficulties in obtaining agreement on GM plants release into the environment for
production. Although unjustified but still strong social
reluctance to GMO and the restrictive regulations forced
the elaboration of new technology using the knowledge
resulting from GM plants analysis and using it to the
generation of favourable altered plants omitting the introduction to their genome the heterologous genes. Twoyear experiments led to the elaboration of alterGMO
technology resulting in the profitable changes in plants
without their modification by the vector transgenesis.
Briefly, the first stage of the method comprises the induction of the changes in the endogenous gene by its methylation/demethylation or the changes in accumulation
of gene derived products (mRNA). In the second stage,
the plants are selected by molecular markers derived
from the analysis of GM plants. In the third stage, the establishment of introduced changes by the exposition of
plants to the factors increasing DNA methylation level
(mannitol, salicylic acid, jasmonates) or mobilizing the
activity of the whole genome (infection by non-virulent
microorganisms) takes place. The comparative analysis
of flax, in which the lycopene cyclase gene was induced
by a vector method (GMO) and non-vector method
(alterGMO), revealed the two time higher effectiveness of
the latter. Beside from the evidently higher effectiveness,
the most important is, that the alterGMO method allow
to generate the favorably altered plants, whose cultivation
make the plant producer independent from the complicated and unstable procedure of obtaining agreement on
their release into environment.
L8.3
RNAi in cereal functional genomics
Waclaw Orczyk1, Anna Nadolska-Orczyk2, Sebastian Gasparis2,
Wojciech Zalewski2, Yuliya Yanushevska1
Dept. of Genetic Engineering, Acclimatization
and Plant Breeding Institute Natl Research Institute,
Radzikow, 05-870 Blonie, Poland; 2Dept. of Functional
Genomics, Acclimatization and Plant Breeding Institute Natl
Research Institute, Radzikow, 05-870 Blonie, Poland
1
One of the most important biological achievements of

last two decades of XXc. was discovery of novel class of
RNA molecules and discovery of the process RNA interference (RNAi) where the molecules act as regulators. The impact of the discovery on basic and applied
biology (recognized by Nobel Prize awarded in 2006) is
enormous and is still growing.
Experimental gene silencing and silencing-based functional gene analysis is one of the possibilities opened
by the knowledge. Depending on the experimental design the silencing can be specific to a particular gene or
agroup of genes. It is particularly important in allopolyploids (such as wheat or triticale) where a group of homologous genes exist and their functional analysis might
require this level of specificity.
We have adapted and we have been currently using this
strategy to study function of the genes selected based
on their putative involvement in conferring important
agricultural traits in cereals [1, 2, 3]. We tested this approach by silencing expression of genes responsible for
grain hardness: Pina / Pinb and Sina / Sinb in wheat and
triticale respectively. Both Pina / Pinb genes are located
in hardness locus in D genome of allohexaploid wheat
(AABBDD). Wheat plants transformed with silencing
cassettes showed 8091% reduction of Pina / Pinb transcript level compared with control, what was followed by
significant reduction in PINA and PINB proteins in seed
endosperm. Observed in the plants increase of kernel
hardness, up to the level found in hard grain T. durum
[4], was consistent with the earlier knowledge of the gene
function based on mutant analysis. Sina / Sinb genes are
believed to be Pin orthologs located in R genome in allohexaploid triticale (AABBRR). Transgenic triticale (with
Pina silencing constructs) led to silencing of Sina / Sinb
ortologs and to lowering SINA /SINB proteins. The unexpected and surprising in the light of the previous results
was grain hardness. It remained unchanged in transgenic
plants. The results clearly confirmed the expected role of
Pina / Pinb genes in endosperm texture of wheat kernels
and contradicted the anticipated role of the genes and the
proteins in triticale seeds (unpublished).
HvCKX and TaCKX, existing in various species as agene
family, represent another group of tested genes. They
encode cytokinin oxidase an enzyme responsible for
catabolism of cytokinin. Expression pattern of the genes
(as well as the activity of the enzyme) is organ and developmental dependent but detailed functions remain un-
Eurobiotech 2013
known. We found that HvCKX1 and HvCKX2 were specifically induced in developing barley spikes. Silencing
of the genes in barley strongly correlated with increased
productivity shown as higher number and higher mass of
kernels. The trait was inherited in the next generations.
The results confirm the hypothesis that organ specific
expression of the CKX genes influences traits related to
plant productivity [3].
Comparison of two different transformation methods
(Agrobacterium-based and particle bombardment) revealed that the experimental design might have a big
impact in final results. Introduction of the same silencing cassette via Agrobacterium and by particle bombardment gave clearly different results. In our case depression
of the CKX transcript level was observed only in Agro
transformed plants. Particle delivery of the silencing
construct led to inconsistent and self-contradictory results [5].
Current project, and unpublished results, is focused on
identification and functional analysis of putative barley
ortholog of brassinosteroid regulators OsGSK1 in rice
and BIN2 / AtSK2 in Arabidopsis. Depressing transcript
level of the tested gene to 0.09 0.22 of the transcript in
control plants revealed the clear correlation with elevated
salt tolerance of the seedlings. The structure of the gene
and biological function of the gene will presented and
discussed.
Acknowledgements
The research was financed by grants: 620/N-COST/09/2010/0
(AN-O), UMO-2011/03/B/NZ9/01383 (AN-O), N302 013 31/1517
(AN-O), 718/N-COST/2010/0 (WO), UMO-2011/01/B/NZ9/02387
(WO).
References
[1] Gasparis S., Bregier C., Orczyk W., Nadolska-Orczyk A. (2008)
Agrobacterium-mediated transformation of oat (Avena sativa L.)
cultivars via immature embryo and leaf explants. Plant Cell Rep, 27:
17211729.
[2] Binka A., Orczyk W., Nadolska-Orczyk A. (2012) The Agrobacteriummediated transformation of common wheat (Triticum aestivum L.) and
triticale (x Triticosecale Wittmack): role of the binary vector systems and
selection cassettes. J Appl Genet, 53: 18.
[3] Zalewski W., Galuszka P., Gasparis S., Orczyk W., Nadolska-Orczyk
A. (2010) Silencing of the HvCKX1 gene decreases the cytokinin oxidase/
dehydrogenase level in barley and leads to higher plant productivity.
J Exp Botany, 61: 18391851.
[4] Gasparis S., Orczyk W., Zalewski W., Nadolska-Orczyk A. (2011)
The RNA-mediated silencing of one of the Pin genes in allohexaploid
wheat simultaneously decreases the expression of the other, and
increases grain hardness. J Exp Botany, 62: 40254036.
[5] Zalewski W., Orczyk W., Gasparis S., Nadolska-Orczyk A. (2012)
HvCKX2 gene silencing by biolistic or Agrobacterium-mediated
transformation in barley leads to different phenotypes. BMC Plant
Biology, 12: 206.
127
L8.4
Expression of three diadinoxanthin
de-epoxidase genes of Pheodacylum
tricornutum in Escherichia coli Origami
b strain
Monika Olchawa-Pajor1, Monika Bojko1, Wojciech Strzaka2,
Paulina Kuczyska1, Dariusz Latowski1, Kazimierz Strzaka1
Poland; 2Department of Plant Biotechnology, Faculty
of Biochemistry, Biophysics and Biotechnology,
Jagiellonian University, Gronostajowa 7, 30-387 Crakow, Poland
1
All photosynthetic organisms develop photoprotective

mechanisms with carotenoids playing a fundamental role
in the dissipation of excess light energy. Photoprotection
is connected with de-epoxidized forms of xanthophyll
pigments which are formed by enzymatic removal of epoxy groups under high light conditions. These reactions
occur in the processes commonly known as the xanthophyll cycles, the most common among them being the
violaxanthin and diadinoxanthin cycles. In violaxanthin
cycle, violaxanthin is converted to zeaxanthin via antheraxanthin whereas in diadinoxanthin cycle epoxy group
is removed from diadinoxanthin and diatoxanthin is created. This conversion takes place e.g. in diatoms with involvement of the enzyme diadinoxanthin de-epoxidase.
Previously, only one gene of this de-epoxidase, designed
DDE, was postulated. Today in one of the diatoms, Phaeodactylum tricornutum (CCAP 1055/1 strain which all
genomes are sequenced) three forms of de-epoxidases
were identified but only one of them corresponded to
VDE. This form of DDE is also called VDE, its gene is
marked as PtVDE and might be involved in the conventional xantophyll cycle. While two VDE-like de-epoxidases (designated as PtVDL1 and PtVDL2) may be more
specialized in the chromist-specific diadinoxanthin cycle.
The purpose of our research was to obtain the PtVDE, PtVDL1 and PtVDL2 genes of Phaeodactylum tricornutum
(UTEX 646 strain) and constructing expression vectors
pET-15b/VDE subsequently used for production of these
enzymes with polyhistidine tag.
Total RNA was prepared from 5 day old culture of Ph.
tricornutum using GeneJET Plant RNA Purification kit.
cDNA was synthesized using M-Mul V Reverse transcriptase and Random primer. Primer pairs and cDNA
were used as a template for amplification of PtVDE, PtVDL1 and PtVDL2. Sense primer contained the initiation Met, codons of amino acid from the mature protein
N terminus and Nde I site. Antisense primers contained
a stop codon, codons from C terminus and BamH I site.
PCR products were visualized and confirmed by agarose
gel electrophoresis, then digested and inserted to digested
by Nde I and BamH I pet-15b vector using ligation kit.
Plasmid was named pet-15b/PtVDE, pet-15b/PtVDL1
and pet-15b/PtVDL2.
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The sequences of PtVDE, PtVDL1 and PtVDL2 were

compared with sequences of these gene of Ph. tricornutum CCAP 1055/1 strain.
Pet-15b/PtVDE, and pet-15b/PtVDL2 plasmids (similar
sequences to the respective Ph. tricornutum gene) were
used to transform E. coli Origami b and BL21 strain cells.
After IPTG induction, the presence of VDE and VDL2
proteins was confirmed by western-Blot analysis. The stable expression of enzymes was observed in both tested
E. coli strains (Origami b, BL21). Obtained VDE and
VDL2 with molecular weight about 49 kDa and 60 kDa
respectively, exhibited ability to de-epoxidise violaxanthin.
L8.5
The role of ubiquitins in cucumber
(C. sativus L.) flower morphogenesis
Magdalena Pawekowicz, Cezary Kowalczuk, Pawe Osipowski,
Micha Wojcieszek, Grzegorz Kojder, Katarzyna Kosik,
Zbigniew Przybecki
Department of Plant Genetics, Breeding & Biotechnology,
Faculty of Horticulture and Landscape Architecture,
Warsaw University of Life Sciences- SGGW,
Nowoursynowska 166 St., 02-776, Warsaw, Poland
Sex determination and flower morphogenesis are a very

broad and complex processes controlled at many levels.
We isolated four clones from cucumber transcriptomes
connected with ubiquitination processes and checked if
the expression of the genes corresponding to those clones
is different among vegetative and generative tissues (leaf,
shoot apex, flower buds 12 mm) in monoecious and
gynoecious cucumber lines. Therefore, to determine to
the extent the role of sequences in flower morphogenesis
a comprehensive analysis was performed using computational studies to acquire insight into characteristic features in genes structure, upstream regulatory elements
and protein motifs. We conclude that the cucumber
generative organs have different sensitivity to plant hormones due to distinct signal transduction mediated by
ubiquitins in male and female organs in floral buds and
shoot apex. Ubiquitins could be correlated with alternative way of hormones signal transduction in flowers of
opposite sex, taking part in inhibition of unwanted generative organs causing the development of unisex flower.
Acknowledgements
This research was supported by grants from the Polish Ministry of
Science and Higher Education (MNiSW) N302 3633 33 and National
Science Center 2011/01/B/NZ2/01631.
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129
Posters
P8.2
P8.1
Efficient Agrobacterium rhizogenes-mediated

transformation of haploid and diploid
sugar beet (Beta vulgaris L.) explants
Comparative analysis of Gal4

and split-ubiquitin-based yeast twohybrid systems for Arabidopsis thaliana
Proliferating Cell Nuclear Antigen studies
Agata Jakubowska, Olga Sztatelman, Wojciech Strzaka
Biophysics and Biotechnology, Jagiellonian University,
Gronostajowa 7, 30-378 Crakow, Poland
The PCNA (proliferating cell nuclear antigen) protein

is wellconserved across Eukaryota both in structure
and function. It plays an essential role in DNA replication, DNA repair and cell cycle regulation. Human and
yeast PCNA has been shown to form complexes with
many different nuclear proteins. Multiprotein complexes are increasingly recognized as the molecular basis of
a substantial amount of biological processes. Mapping
of protein interaction networks in such complexes is of
prime importance. It allows to characterize the physiological functions of the component proteins and usually
results in better understanding of multifactorial-dependent processes. One of the most common methods of
studying protein-protein interactions in vivo is the yeast
two hybrid (Y2H) system. Since its first introduction by
Fields in 1989 various modifications have appeared. Two
features determine the Y2H system as the first choice for
interaction studies: simplicity and high-throughput capacity, which enables screening for interactions on a genome-wide scale.
The aim of this study was to determine which Y2H system is potentially better for screening proteins interacting with Arabidopsis thaliana PCNA1 and PCNA2. We
compared two commercially available Y2H systems: the
classic one based on the reconstitution of the Gal4 transcription factor (Invitrogen) and a split-ubiquitin system
based on the reconstitution of ubiquitin (MoBiTech),
both adapted to Gateway technology. Since A. thaliana
PCNA1 and PCNA2 were shown to form homo- and
hetero-trimeric ring structures, PCNA1/1, PCNA1/2,
PCNA2/2 and PCNA2/1 interactions were studied to test
the selected Y2H systems. The investigated interactions
could only be detected with the split-ubiquitin Y2H system. This observation suggests that this system might be
more effective in screening for proteins interacting with
plant PCNA.
Magdalena Klimek-Chodacka, Iwona Zapaa, Rafal Baraski

Unit of Genetics, Plant Breeding and Seed Science, Institute
of Plant Biology and Biotechnology, Faculty of Horticulture,
Hairy root cultures obtained after Agrobacterium rhizogenes-mediated genetic transformation can serve as model
system for studying plant metabolism and physiology or
can be utilized for the production of secondary metabolites. So far sugar beet hairy roots were used mainly for
studying resistance to BNYVV, Heterodera schachtii or
Tetanops myopaeformis, but no efficient protocol of hairy
root production has been publically released. Usually explants of diploid or tetraploid donor sugar beet plants were
used for transformation however, the use of haploids could
be more desirable. Diploidization of genetically modified
haploid hairy roots could result in fast development of tissue that is homozygous at the transgene locus.
Here we report an efficient system for sugar beet genetic
transformation leading to the development of hairy roots.
The study included two A. rhizogenes strains (A4T and
LBA1334) carrying a binary vector pBIN-m-gfp5-ER or
pCAMBIA1301 possessing gfp and uidA reporter genes, respectively. Petiole and midrib explants of two haploid and
two diploid sugar beet genotypes were inoculated in five
treatment combinations including time of explants exposure
to ultrasound, application of bacteria suspension before or
after sonication and time of bacteria-explant co-culture.
The formation of hairy roots was observed as early as two
weeks after inoculation at the site of tissue wounding.
Hairy roots appeared on 0% to 36% explants depending on the treatment combination. The highest frequency was achieved when explants of a haploid genotype
No. 169 were sonicated for 15 s in the A4T pCAMBIA1301
inoculum of OD600 = 0.5 followed by a 3-day co-culture.
Using the same treatment combination the explants of
the remaining genotypes developed hairy roots with the
frequency ranging from 10% to 30%.
The T-DNA presence in hairy roots was confirmed by PCR
and the copy number of transgenes was assessed by Southern hybridization and Real-Time PCR and determined
as one to several copies depending on the transformation
event. Expression of the transgenes in hairy roots was confirmed visually i.e., beta-glucuronidase activity was verified by histochemical staining and the synthesis of green
fluorescent protein by its fluorescence in UV light. 54% of
the used hairy root cultures showed transgene expression.
The results indicate that the developed protocol allows production of 42 hairy roots per one hundred explants.
Acknowledgements
This work was supported by the Polish Ministry of Agriculture and
Rural Development (Decision No. HOR hn-801-10/13).
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Eurobiotech 2013
P8.3
DesC desaturase of cyanobacterium
Synechococcus vulcanus expression
in canola plants does not improve the low
positive temperature growth
Mariia Slyvets1, 2, Liudmyla Sakhno1, Yuri Sheludko1
Institute of Cell Biology and Genetic Engineering NAS
of Ukraine; 2National Technical University of Ukraine
Kyiv Polytechnic Institute
1
One of the plant adaptation mechanisms to cold is the increase in the unsaturation of fatty acid residues in cellular
membranes, sustaining the required membrane fluidity.
The important role in this process is attributed to fatty
acid desaturases, catalyzing the transformation of a single
bond between carbon atoms in acyl chains (CC) into the
double bond (C=C).
Earlier we have obtained spring canola (Brassica napus L.,
cv Obreey) lines bearing hybrid gene of acyl-lipid 9-desaturase (des) from cyanobacterium Synechococcus vulcanus translationally fused with termostable lichenase
from Clostridium thermocellum (licBM3) reporter gene
in their nuclear genome using Agrobacterium tumefaciens-mediated leaf disk transformation. Desaturase gene
expression was estimate qualitatively (lichenase plate
test) and quantitatively (photometry) on the basis of activity of licBM3 gene product as a part of hybrid protein.
Gas-chromatographic data revealed that cyanobacterium
DesC expression did not lead to qualitative changes in
canola leaf fatty acid composition but resulted in increase
in the lipid content (up 48%) and in the quantitative alteration in the fatty acid composition. Trienoic (16:3 and
18:3) fatty acid content was increased up 33% and saturated fatty acid content (16:0) was decreased up 16%.
These alterations allows suppose that desC canola lines
might be more resistant to chilling damage in comparison with control ones.
The present work was aimed at investigation of transgenic line growth under low positive temperature (+4C).
During experiments control and des expressing canola
lines (primary transformants 18, 18 and plants of first
generation Bn18/6, Bn18/25) were analysed after propagation by cutting under aseptic conditions. Plants were
grown during four weeks in termal room (16/8 photoperiod, +23) in Sigma 25150 (Sigmawave) tubes
containing 15 ml agar solidified MS medium without
hormones. Then they were transferred to chamber (16/8
photoperiod, +4) for 5 days. After growth under low
temperature plants were returned under initial conditions for four weeks. Stress effect on plant growth was
evaluated using measurement of fresh weight, total soluble protein content and superoxide dismutase activity.
We documented that desC canola plants did not differ
significantly from control under all tested temperature
regimes. It seems trienoic fatty acid content increase in
our canola due to DesC expression was not sufficient for
cold resistance improved. Perhaps, desC activity was not

effective in cytosol. Earlier it was reported that tobacco
plants expressing native desC gene from S.vulcanus had
enchanced chilling resistance though information about
chloroplast targeting of introduced gene was absent. We
plan to modify our genetic construction by inclusion of
chloroplast signal peptide to obtain canola with cold resistance improvement.
Acknowledgements
The work was supported with the grant of National Academy of Science
of Ukraine 0110U006062.
Eurobiotech 2013
131
P8.4
P8.5
A method of epoxides determination

in bacterial cells
Comparison of expression and activity

of violaxanthin and diadinoxanthin
de-epoxidases in Escherichia coli Origami b
strain
Paulina Kuczynska1, Sylwia Leskiewicz1, 2, Wojciech Strzalka3,

Monika Olchawa-Pajor1, Monika Bojko1, Dariusz Latowski1,
Kazimierz Strzalka1
Jagiellonian University; 2Faculty of Chemistry,
Jagiellonian University; 3Department of Plant Biotechnology,
Jagiellonian University
1
Organic compounds containing a ternary cyclic complex

consisting of an oxygen linked to two carbon atoms are
named epoxides. They arise from oxidative metabolism
of compounds derived from the cell and the environment
via chemical and enzymatic processes and have high
chemical reactivity, genotoxicity, cytotoxicity. Our studies deal with determination of epoxides in bacterial cells
overexpressing zeaxanthin epoxidase (ZEP). This enzyme
catalyzes conversion of zeaxanthin (an epoxy-free xanthophyll) into violaxanthin (di-epoxide xanthophyll) in
the violaxanthin cycle. It has been assumed that the lack
of ZEP substrate may cause nonspecific reactions leading to production of toxic epoxides. We noticed a correlation between ZEP expression in Escherichia coli and
increase in amount of epoxides. A spectrofluorometric
method has been used for the determination of total epoxides according to Sano and Takitani (1985). As a result
of standardizing the method the best conditions to carry out the experiment using bacteria cells were worked
out. Two steps reaction was performed; the first with or
without sodium sulfide and the second with taurine and
o-phthalaldehyde reagents. The studies were conducted
on E. coli strain Origami B (DE3) transformed with Arabidopsis thaliana ZEP open reading frame cloned into
pDEST17 vector. Increase in amount of epoxides was
observed in transformed cells during the time of growth.
Moreover, it was also correlated with lower growth rate of
bacteria what was probably caused by toxic nonspecific
products of epoxidation. Higher epoxides content during
the time of ZEP overexpression induction shows that this
enzyme is responsible for the epoxide formation. This
study shows that described method can be used for determination of total epoxides in cells thus it might be useful
for studies on proteins engaged in epoxide formation or
degradation.
Monika Olchawa-Pajor1, Monika Bojko1, Wojciech Strzaka2,

Paulina Kuczyska1, Dariusz Latowski1, Kazimierz Strzaka1
Poland; 2Department of Plant Biotechnology,
Jagiellonian University, Gronostajowa 7, 30-387 Crakow, Poland
1
Photoprotection in the most of photoautotrophs is

strongly connected with de-epoxidized forms of xanthophyll pigments. These compounds are formed in the
processes commonly known as xanthophyll cycles. The
most widespread in the nature is the violaxanthin cycle,
in which epoxy groups are removed from violaxanthin by
enzyme violaxantin deepoxidase (VDE) forming transiently antheraxanthin and then zeaxanthin as the final
product. In another type of xanthophyll cycle, under
high light conditions monoepoxide, diadinoxanthin, is
de-epoxidised into diatoxanthin. This conversion takes
place e.g. in diatoms with involvement of the enzyme diadinoxanthin de-epoxidase and this xanthophyll cycle is
called diadinoxanthin cycle. Previously, only one gene of
this de-epoxidase, designed DDE, was postulated. Since
2008 it has been known that in one of the diatoms, Phaeodactylum tricornutum three forms of de-epoxidase are
present. One of them corresponds to known VDE occuring in higher plants. This form of DDE is also called
VDE and its gene is marked as PtVDE. The purpose of
our research was to obtain the VDE gene from Arabidopsis thaliana (WT Columbia strain) (AtVDE) and
VDE gene of Phaeodactylum tricornutum (CCAP 1055/1
strain) (PtVDE) and constructing expression vectors
pET-15b/VDE subsequently used for production of these
enzymes with polyhistidine tag. Finally we compared
the effects of one step purification and activity of the enzymes.
Total DNA prepared from 5 day old culture of Ph. tricornutum was used as a template for PtVDE and primer
pairs. Sense primer contained the initiation Met, codons
of amino acid from the mature protein N terminus and
Nde I site. Antisense primers contained a stop codon, codons from C terminus and BamH I site. PCR products
were visualized and confirmed by agarose gel electrophoresis, then digested and inserted to digested by Nde I and
BamH I pet-15b vector using ligation kit. Plasmid was
named pet-15b/PtVDE.
The wild type AtVDE gene uses rare codons with ahigh
frequency. Moreover, it contains several negatively
cis-acting motifs which might hamper expression in prokaryota. Therefore, AtVDE gene was obtained by chemical synthesis adapting the codons from A. thaliana to the
132
Eurobiotech 2013
codons bias of Escherichia coli gene. During the optimization process the following cis-acting sequence motifs
were omitted: internal TATA-boxes, chi-sites and ribosomal entry sites; AT-rich or GC-rich sequence stretches; repeat sequences and RNA secondary structures. The
optimized gene was inserted to pet-15b vector using ligation kit. Purified plasmid containing AtVDE named
pet-15b/AtVDE was received.
Both plasmids were used to transform E. coli Origami b
strain cells. PtVDE and AtVDE effective expression after
IPTG induction were evidenced by SDS-Page electrophoresis and Western-Blot methods. VDE proteins were purified using TALON super flow Metal Affinity resin. The
stable expression in E. coli (Origami b strain) provided
enzymes with molecular weight about 42 kDa for VDE
from A. thaliana and 49 kDa for VDE from Ph. tricornutum. Both obtained enzymes were active relative to Vx
although AtVDE exhibited higher activity than PtVDE.
P8.6
Identification and analysys of WUSCHEL
(WUS/WOX) and CLAVATA genes
in cucumber (Cucumis sativus L.)
Micha Wojcieszek1, Magdalena Pawekowicz1, Marcin Olszak1,
Agata Jdrzejuk2, Zbigniew Przybecki1, Wojciech Burza1
Department of Plant Genetics, Breeding & Biotechnology,
Faculty of Horticulture and Landscape Architecture,
Warsaw University of Life Sciences- SGGW,
Nowoursynowska 166 St., 02-776, Warsaw, Poland;
2
Department of Ornamental Plants, Faculty of Horticulture
and Landscape Architecture, Warsaw University of Life
Sciences- SGGW, Nowoursynowska 166 St., 02-776, Warsaw,
Poland
1
The genes WUSCHEL (WUS), WUSCHEL-RELATED

HOMEOBOX (WOX) and CLAVATA (CLV) play key
roles in many life processes in plants. In Arabidopsis the
homeobox gene WUS encodes a homeodomain transcription factor that has been shown to be a regulator of
apool of pluripotent stem cells in the shoot apical meristem (SAM). In SAM, an essential mechanism for meristem organization and maintenance is a regulatory feedback loop between WUSCHEL (WUS) and CLAVATA
(CLV), which functions in a non-cell-autonomous manner. This intercellular signaling network coordinates the
development of the organization center, organ boundaries and distant organs. In root apical meristem (RAM)
gene WOX5 can be considered analogous to WUS that
acts to maintain stem cells of the shoot apex. The studies
of other authors showed that induced overexpression of
the PGA6 gene (identical to WUS) caused high-frequency somatic embryo formation in all tissues and organs
tested, without any external plant hormones.
In our research we performed broad in silico analysis of
WUS and CLV genes from C. sativus genomes Polish B10 and Chinese 9930. Upstream regions examination, protein function determination and collation with
A.thaliana analogues were the main procedures utilized
in this work. The results obtained, especially for line B10
are the first step to a better understanding and control
of the plant morphogenesis processes under in vitro and
field conditions. This outcome will be useful for our investigations concerning plant pluripotent and totipotent
induced stem cells.
Acknowledgements
This research was partially supported by grants from the Polish Ministry
of Science and Higher Education (MNiSW) N302 3633 33 and
National Science Center 2011/01/B/NZ2/01631.
Eurobiotech 2013
P8.7
P8.8
Protoplasts of Lathyrus sp. optimization

of isolation procedure
Processing of plant tissue bearing

S-HBsAg for an oral vaccine against
hepatitis B
Barbara Piwowarczyk1, Wojciech Rybiski2

Faculty of Horticulture, University of Agriculture in Crakow,
Poland; 2Institute of Plant Genetics, Polish Academy
of Sciences, Poznan, Poland
1
Interspecific hybridization between various species of genus Lathyrus is difficult. Conventional methods of crossing have been successful only in several instances. This
obstructs obtaining of hybrids and introduction of new
desirable features to crops. Application of biotechnological methods seems to be the only solution of this obstacle.
Currently, among the available biotechnology techniques,
protoplast fusion is often used for obtaining somatic hybrids. Besides, protoplasts are excellent explants for genetic transformation due to the absence of barrier that
is cell wall. Prerequisite to use these methods is development of efficient procedures of protoplasts isolation,
their cultivation and plant regeneration. Lathyrus species
belong to large-seeded Fabaceae plants that are very sensitive to in vitro manipulations and very recalcitrant to
regeneration.
In the study, protoplasts were isolated from mesophyll of
two Lathyrus species: L. tingitanus and L. cicera. Leaves,
originating from either 3 week-old ex vitro seedlings or
2 month-old plants growing in the field conditions, were
used as explants. Different enzymatic mixtures (composed of Cellulase, Pectinase, Macerozyme and osmotic
agent (sorbitol) in different concentration) as well as various time of tissue incubation were investigated. Effectiveness of applied isolation conditions was evaluated on the
basis of the number of protoplasts per 1 g plant tissue,
protoplasts viability and the rate of cell wall digestion.
These studies will determine the optimal conditions for
efficient isolation of viable protoplasts that will be a contributive research to perform the fusion between protoplasts of Lathyrus species in the future.
Acknowledgements
This work was supported by the Ministry of Science and Higher
Education of Republic of Poland (BM 4541).
133
Marcin Czy1, Tomasz Pniewski1, Jzef Kapusta2,

Radosaw Dembczyski3, Roman Marecik3,
Andrzej Pucienniczak2
Institute of Plant Genetics, Polish Academy of Sciences,
Poznan, Poland; 2Institute of Biotechnology and Antibiotics,
Warszawa, Poland; 3Poznan Life Science University, Poland
1
Potential plant-derived oral vaccine against Hepatitis

B Virus must have a formula which would enable efficacious and convenient administration under defined
regime in addition to long-term stability of borne antigen, here the small surface antigen (S-HBsAg) formed
into highly immunogenic Virus-Like Particles (VLPs).
Freeze-drying was a chosen technique that could allow to
fulfil above-mentioned requirements.
The aim of this project was to investigate and optimise
the processing of previously obtained transgenic lettuce
plants expressing S-HBsAg to prepare an oral vaccine
formula. Conditions of lyophilisation and storage of obtained preparation were investigated regarding the stability of the antigen.
Several physical freeze-drying parameters were tested.
That included freezing method of an initial plant material, in-process shelf temperatures and primary and secondary drying times. Established conditions were used in
the second stage of research on effect of added lyoprotectants. A number of substances and their concentrations
were studied, in addition to parameters of tissue saturation. Subsequently, antigen preservation after prolonged
storage was evaluated. Freeze-dried and powdered plant
tissue was stored at various temperatures, with optional
presence of silica gel desiccator and neutral nitrogen atmosphere. Impact of lyoprotectants, applied process and
storage parameters were assessed.
Based on obtained results the most effective profile of
freeze-drying process, and the most potent lyoprotectant
were determined. Under the selected conditions 80% of
VLPs-assembled S-HBsAg was retained after freeze-drying. Lyophilised tissue was successfully used for mouse
oral immunisation and also could be converted into
a formula for possible human vaccination. However,
long-term storage of that semi-product was successful
only at lowered temperatures. Therefore, unrestricted
long-term storage of lyophilised formula still requires
some elaboration.
So far obtained results indicate that it is possible to efficiently preserve S-HBsAg in immunogenic VLP form
in lyophilised stored plant tissue, providing rationale
for further research on formulation of plant-derived anti-HBV oral vaccine.
Acknowledgements
This work was supported by grant No. N N302 157837 from Polish
Ministry of Science and Higher Education.
Animal biotechnology in biomedicine

Lectures
L9.1
Stress preconditioning of cells
for improved output in cloning, IVF
and stem cell research
Csaba Pribenszky
Szent Istvan University Budapest, Hungary
In vitro procedures with gametes and embryos, including

their culture, manipulation or storage, has always been
hampered by their inherent sensitivity to environmental
changes and external interventions. Consequently, most
of the strategies to increase success rates in embryo production, cryopreservation, cloning and other procedures
focus on reducing the inevitable damage caused by the
manipulation, and, whenever it is applicable, imitating
the natural physiology. In contrast to this principle, the
efficiency of various procedures could be improved by
preconditioning gametes and embryos with precisely adjusted and applied sublethal stress treatment (reviewed in
Callesen 2010, Pribenszky et al. 2010a, 2012). Precisely
adjusted stress preconditioning in the form of hydrostatic
pressure (HP) (at 600 times the physiological value) has
been demonstrated to improve the cryosurvival of blastocysts in a mouse model (Pribenszky et al. 2005). Advantageous effect on porcine, bovine and ovine embryos,
oocytes and spermatozoa, embryonic stem cells and human chord blood in combination with different in vitro
techniques such as slow freezing, vitrification, in vitro
fertilization and somatic cell nuclear transfer has also
been documented (reviewed in Pribenszky et al. 2010a,
Pribenszky & Vajta 2010). Studies showed, that DNA and
membrane integrity has also been improved together with
the reduced number of apoptotic cells and nuclei. Hydrostatic pressure treatment of porcine oocytes prior to vitrification resulted in significantly increased blastocyst rates
and higher cell numbers after warming, parthenogenetic
activation and in vitro culture (Pribenszky et al. 2008a,
b, Du et al. 2008, Lin et al. 2010). These results were confirmed in studies on activated human oocytes, where discarded in vitro matured or non-fertilized oocytes showed
increased survival and cleavage rates following HP treatment (Pribenszky et al. 2010b, Matyas et al. 2010).
Despite these observations of a biological effect of the
HP technique, a fundamental understanding of its mechanism of action at the molecular level is still far from
being complete. Different groups have shown that the
HP-increased cryotolerance of embryos or spermatozoa
is concomitant with the altered expression of growth arrest-, oxidative stress-, cell pluripotency-, apoptosis- and
lipid metabolism-related genes (Bock et al. 2010, Siqueira Filho et al. 2011, Bogliolo et al. 2011). Huang et al.
2009a showed HP related changes in the proteome of
treated porcine spermatozoa that was detected after the
HP treatment, but remained throughout the cryopreservation procedure and after thawing as well, and might be
associated with improved survival and fertilizing capacity
resulting in higher pregnancy rate and litter size upon insemination. Studies indicate that HP treatment results in
significant changes of gene expression and highlight the
need for a comprehensive transcriptome analysis in mammalian cells. Recently, global gene expression profiling of
HP-treated porcine oocytes, and the parthenogenetically
activated or cloned embryos developed from these oocytes, identified several HP-responsive genes.
Preconditioning gametes and embryos with a predefined,
controlled sublethal stress (hydrostatic pressure) seems to
enhance significantly cell survival and function after various in vitro procedures such as cryopreservation, in vitro
culture or cloning.
References
Bogliolo L., Ariu F., Leoni G., Uccheddu S. & Bebbere D. (2011) High
hydrostatic pressure treatment improves the quality of in vitro-produced ovine blastocysts. Reproduction, Fertility, and Development 23,
809817.
Callesen H. (2010) Challenge testing of gametes to enhance their viability. Reproduction, Fertility, and Development 22, 4046.
Du Y., Pribenszky C., Molnar M., Zhang X., Yang H., Kuwayama M.,
Pedersen A.M., Villemoes K., Bolund L. & Vajta G. (2008) High hydrostatic pressure: a new way to improve in vitro developmental competence of porcine matured oocytes after vitrification. Reproduction 135,
1317.
Huang S.Y., Pribenszky C., Kuo Y.H., Teng S.H., Chen Y.H., Chung M.T.
& Chiu Y.F. (2009a) Hydrostatic pressure pre-treatment affects the protein profile of boar sperm before and after freezing-thawing. Animal
Reproduction Science 112, 136149.
Lin L., Luo Y., Srensen P., Prtorius H., Vajta G., Callesen H., Pribenszky C., Bolund L. & Kristensen T.N. (2013) Effects of high hydrostatic
pressure on genomic expression profiling of porcine parthenogenetic activated and cloned embryos. Reproduction, Fertility, and Development
[in press] (http://dx.doi.org/10.1071/RD13037).
Lin L., Pribenszky C., Molnar M., Kragh P.M., Du Y., Zhang X., Yang H.,
Bolund L., Callesen H., Machaty Z. et al. (2010) High hydrostatic pressure treatment of porcine oocytes induces parthenogenetic activation. Cellular Reprogramming 12, 475480.
Matyas S., Pribenszky C., Kovacs P., Rajczy K., Molnar K., Losonczi E.,
Molnar M., Kaali S.G. & Vajta G. (2010) Preconditioning oocytes by
sublethal hydrostatic pressure stress in order to improve cryosurvival
animal models and human application. Proceedings of The 1st International Congress on Controversies in Cryopreservation of Stem Cells,
Reproductive Cells, Tissue and Organs Valencia, Spain.
Pribenszky C. & Vajta G. (2010) Cells under pressure: how sublethal
hydrostatic pressure stress treatment increases gametes and embryos
performance? Reproduction, Fertility, and Development 23, 4855.
Pribenszky C., Du Y., Molnar M. & Vajta G. (2008b) Sublethal stress
on porcine oocytes enhances the efficacy of ART procedures. Human
Reproduction 23, 161.
Eurobiotech 2013
Pribenszky C., Du Y., Molnar M., Harnos A. & Vajta G. (2008a) Increased stress tolerance of matured pig oocytes after high hydrostatic
pressure treatment. Animal Reproduction Science 106, 200207.
Pribenszky C., Lin L., Du Y., Losonczi E., Dinnyes A. & Vajta G. (2012)
Controlled stress improves oocyte performance-cell preconditioning in
assisted reproduction. Reproduction in Domestic Animals 47, 197206.
Pribenszky C., Matyas S., Losonczi E., Stanca C., Bock I. & Vajta G.
(2010b) Stress for stress tolerance: improving cell survival by sublethal
stress treatment of eggs before vitrification pilot study. Fertility and
Sterility 9, S32.
Pribenszky C., Molnar M., Cseh S. & Solti L. (2005) Improving postthaw survival of cryopreserved mouse blastocysts by hydrostatic pressure challenge. Animal Reproduction Science 87, 143150.
Pribenszky C., Vajta G., Molnar M., Du Y., Lin L., Bolund L. & Yovich
J. (2010a) Stress for stress tolerance? A fundamentally new approach in
mammalian embryology. Biology of Reproduction 83, 690697.
Siqueira Filho E., Caixeta E.S., Pribenszky C., Molnar M., Horvath A.,
Harnos A., Franco M.M. & Rumpf R. (2011) Vitrification of bovine blastocysts pretreated with sublethal hydrostatic pressure stress: evaluation
of post-thaw in vitro development and gene expression. Reproduction,
Fertility, and Development 23, 585590.
135
L9.2
Large animal models in biomedical
research
Marlena Szalata1, 2, Daniel Lipiski1, 2, Joanna Zeyland1,
Magdalena Boksa1, Hanna Przystaowska1,
Bartosz Grzekowiak1, 3, Karol Tunio1, 3,
Marzena Skrzypczak-Zieliska2, Ryszard Somski1, 2, 3
Department of Biochemistry and Biotechnology,
Poznan University of Life Science, Poland; 2Institute
of Human Genetics, Polish Academy of Sciences, Poznan,
Poland; 3NanoBioMedical Centre (NBMC) at Adam Mickiewicz
University in Poznan, Poland
1
Large animal models has become a common expression

in biomedicine and refers to livestock (pigs, goats, sheep,
cattle, horses, donkeys), cats, dogs, non-human primates
and sometimes rabbits. Large animal models are not as
widely used as mice, flies, or nematodes, often require
different research infrastructure than small rodents and
higher funding. Historically, large animals have made
many contributions to greater understanding of human
health.
Large animal research opens up the possibility to take
frequent biopsies and samples, non-invasive monitoring
by ultrasound without having to sacrifice the animal. The
similarity in organ size in some animal models allows for
better models of surgery, hemodynamics and possible future xenotransplantation. Large animals can be used for
pharming to produce large quantities of useful proteins
in milk of transgenic cows, pigs, sheep or goats. Using
large animal models in biomedical research may also
benefits in advantages for animal agriculture.
We are currently involved in generating and analyzing
genetically modified pig models for xenotransplantation
and using of transgenic rabbits and goats as bioreactors.
Another field of our research covers the assessment of
closely related pigs.
Acknowledgements
The studies were financially supported by the projects N R12 0036
06/2009, N R13 0075 06/2009 and UDA/POIG.01.04.00-24-002/11-00.
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Eurobiotech 2013
L9.3
L9.4
Risk of viral infections

in xenotransplantation and prevention
strategies
Novel strategies in the somatic cell cloning

of mammals
Urszula Mazurek, Joanna Gola
National Research Institute of Animal Production,

Department of Biotechnology of Animal Reproduction, Balice
near Crakow, Poland
Department of Molecular Biology, Medical University of Silesia,

1 Narcyzow St., 41-100 Sosnowiec, Poland
The shortage of organs and other tissues for use in human

transplantation procedures is increasingly being supplied
with material taken from pigs. Among several types of virus found in pigs which might be transmitted to humans
as a result of xenotransplantation, one of the greatest
problems is presented by porcine endogenous retroviruses. Of particular concern in this respect are porcine endogenous retroviruses (PERVs). Three subtypes of PERV
have been identified, of which both PERV-A and PERV-B
have the ability to infect human cells in vitro. While the
third subtype, PERV-C, does not show this ability, recombinant PERV-A/C forms have demonstrated infectivity in
human cell culture. PERVs have been shown to be able
to infect human cells in vitro. However, this phenomenon has not been confirmed in vivo. In view of the risk
presented by these observations, the International Xenotransplantation Association recently recommended four
strategies to prevent transmission of PERVs during xenotransplantation. Screening xenotransplant recipients
for PERV transmission can be done in a number of ways.
Those include detection of PERV DNA, RNA, reverse
transcriptase (RT) activity assessment and proteins as
well as anti-PERV antibodies evaluation. In summary, detection of PERVs in xenotransplantations should be carried out with the necessary caution at all stages to eliminate the possibility of transmission of PERV infection.
Marcin Samiec, Maria Skrzyszowska
Nuclear transfer (NT)-derived offspring have already

been obtained not only in livestock species or infertile
crossbreds/bastards (cattle, sheep, goats, Chinese swamp
buffalos, rabbits, horses and mules), but also in laboratory animals (mice, rats), companion animals (cats,
dogs, domestic ferrets, horses), as well as unthreatened
or endangered species living in the wild (gaur, mouflon,
red deer, dromedary camel, African wild cats, sand cats,
Euro-Asian gray wolves) and even extinct species (bucardo mountain wild goat - Pyrenean ibex; a subspecies of
Spanish/Iberian ibex). The propagation of mammalian
specients by somatic cell nuclear transfer (SCNT) has
important economical implications in biotechnology and
biomedicine as so far has been demonstrated by generation of transgenic cloned animals with either the ability
to produce valuable recombinant human proteins in their
body fluids (milk, urine, seminal plasma, blood) or the resistance to interspecies transmissible and heritable diseases. Pig embryo engineering (transgenesis combined with
somatic cell cloning) is also a particularly important research field of assisted reproductive technologies (ARTs)
due to increasing role of the porcine tissues and organs
in xenotransplantology or creation of animal bioreactors
synthesizing and secreting biopharmaceuticals. In spite
of tremendous improvement in somatic cell cloning efficiency of mammals, high early-, mid- and late-gestation
mortality rates of nuclear-transferred (NT) embryos/foetuses, cumulative occurrence of stillbirth, increased incidence rates of both sudden perinatal death and serious
neonatal morbidity as well as numerous malformations
(i.e., various congenital histo- and anatomopathological
abnormalities) of cloned newborn offspring still often
appear. Therefore, the effectiveness of SCNT technology
in mammals remains unsatisfactory. Generally, the main
cause of low somatic cell cloning efficiency in different
mammalian species, with many severe developmental
anomalies (anatomo-histological disorders in foetal and
extrafoetal/placental tissues, immune dysfuntions) leading to high pregnancy losses and neonatal deaths, may be
an incomplete epigenomically-conditioned reprogramming of transcriptional activity for donor cell-descended genes. The results of somatic cell cloning-associated
experiments indicate that NT embryos of farm livestock
species (pigs, rabbits, horses, sheep, goats, cattle) and
laboratory animal species (mice, rats) rarely reach full
pre- and/or postimplantation development, especially in
relation to the number of reconstructed oocytes and/or
to the number of embryos suitable for surgical transfer
(reimplantation) into the reproductive tracts of surrogate
Eurobiotech 2013
recipient females. That is why, the attempts to increase the

efficacy of SCNT in mammals have been made by many
investigators lately. For these purposes, they have developed the original and interesting methods or innovative
solutions in this research field of genomic/genetic embryo engineering. Their efforts have been aimed at evolving, adopting and optimizing the new systems for preparation of nuclear donor cells (somatic cells) and nuclear
recipient cells (oocytes) at different stages prior to and
throughout the SCNT. These latter involve among others:
1) epigenomic transformation of cultured donor somatic cells and/or 2) in vitro maturing recipient oocytes as
well as 3) activated nuclear-transferred oocytes using either non-specific histone deacetylase (HDAC) inhibitors
(e.g., trichostatin A, scriptaid, valproic acid, oxamflatin,
sodium butyrate) or non-specific DNA methyltransferease (DNMT) inhibitors (e.g., 5-aza-2-deoxycytidine,
zebularine, S-adenosylhomocysteine). Moreover, these
novel approaches involve the generation of NT-derived
embryos applying such inventive systems of artificial
stimulation for developmental program of reconstructed
oocytes as: 1) pseudophysiological transcomplementary
activation mediated by zygote-descended cytoplasts (i.e.,
zygoplasts) and 2) sequential physicochemical activation
based on combination of various agents (electric pulses,
calcium ionophore antibiotics, protein synthesis blockers, selective and non-selective inhibitors of cyclin-dependent protein kinases; CDKs). Other approaches encompass the production of cloned embryos with the use
of completely new sources of either: 1) nuclear recipient
cells (zygotes, single blastomeres of two cell-stage embryos) or 2) nuclear donor cells [bone marrow- and peripheral blood-derived multipotent mesenchymal stem cells
(MSCs), induced pluripotent stem cells (iPSCs)]. All the
above-mentioned strategies are applied to facilitate and
accelerate the epigenomic reprogrammability for gene
expression of donor cell nuclei that had been transplanted into cytoplasmic microenvironment of recipient cells
and subsequently underwent the dedifferentiating and
re-establishing the epigenetically dependent status of
their transcriptional activity during the pre- and/or postimplantation development of cloned embryos. On the
one hand, a better understanding of the molecular mechanisms underlying epigenetic transcriptional reprogramming of the donor nuclear genome can contribute to improvements in the efficiency of this process. On the other
hand, it can give rise to enhancement of the developmental potential of NT embryos. This will be helpful in solving the problems resulting from mammalian somatic cell
cloning and may open up new possibilities for common
utilization of this technology in human biomedicine.
Acknowledgements
This study was supported by the Polish Ministry of Science and Higher
Education as the statutory activity No. 02-1.00.1, which is realized from
2012 to 2014.
137
L9.5
Retroviral restriction factor gene
expression in normal human dermal
fibroblasts after porcine endogenous
retrovirus infection
Magorzata Kimsa, Magdalena Kimsa, Barbara Strzaka-Mrozik,
Joanna Gola, Jolanta Adamska, Urszula Mazurek
Narcyzow 1, 41-200 Sosnowiec, Poland
Xenotransplantation can offer a potential solution for the

shortage of allogeneic human organs, tissues and cells.
However, possibility of pathogen interspecies transmission from xenografts into humans should also be considered. In pigs, one of the greatest problems is presented by
porcine endogenous retroviruses (PERVs). Replication of
the retroviruses can depend on the balance between cellular cofactors and host antiviral restriction factors, e.g.
tripartite motif (TRIM) protein family, apolipoprotein B
mRNA-editing catalytic polypeptides (APOBEC), bone
marrow stromal cell antigen 2 (BST-2, tetherin) or zinc
finger antiviral protein (ZAP). Many studies have shown
that these factors may play an important role in safety of
xenotransplantation, but it is still unclear whether PERVs
may be inhibited by these factors.
The present study focused on retroviral restriction factor
gene expression in normal human dermal fibroblasts after porcine endogenous retroviruses infection.
The PERV infectivity was analyzed using a co-culture system of normal human dermal fibroblasts (NHDFs) and
porcine kidney epithelial cells (PK15 cell line). Total RNA
was extracted from cells using TRIzol reagent. Detection
of the copy number of PERV A, PERV B DNA and PERV
A, PERV B RNA was performed using real-time Q-PCR
and QRT-PCR. The expression of retroviral restriction
factor genes was compared between PERV-infected and
uninfected NHDF cells using commercially available oligonucleotide microarrays HG-U133A 2.0. Validation of
microarray data was performed by QRT-PCR.
PERV A DNA was detected in NHDF cells after co-cultures with PK15 cell line, whereas PERV B DNA was not
found. In turn, both PERV A and PERV B RNA were observed in NHDF cells. Typing of differentiation genes was
performed in a panel of selected 95 retroviral restriction
factor transcripts for 58 genes. The up-regulated transcripts were recorded for three genes: TRIM1, TRIM16,
TRIM48.
Our results suggest that the TRIM family may play an important role in innate immunity to PERV infection.
Acknowledgements
This study was supported by the grant No. KNW-2-031/D/3/N from
Medical University of Silesia, Katowice, Poland.
138
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Posters
P9.2
P9.1
The influence of porcine endogenous

retrovirus infection on the expression
of toll-like receptor genes in human dermal
fibroblasts
Infection of Human Cell Lines with

porcine endogenous retroviruses
Sabina Gaka, Dagna Sotysik, Daria Matczyska,
Daniel Sypniewski, Tomasz Loch, Ewa Nowak, Ilona Bednarek
Department of Biotechnology and Genetic Engineering, Medical
University of Silesia in Katowice, Narcyzow 1,
An origin of most human transplants is allotransplatation.

Allografts are limited by graft donor deficiency. Under
those circumstances xenotransplantation, being interspecies transplantation, may serve as an alternative solution.
A domestic swine has been identified as the most suitable
donor animal. However all pigs carry a viral particles called
porcine endogenous retroviruses (PERVs). Gene sequences of PERVs have been integrated into the genomes of all
pig. It is well known that retroviral infection may involve
significant changes in the human genes which are important for the tumor growth and progression. Therefore, its
really important to investigate, whether in vitro human cell
lines are prone to PERVs infection.
The aim of the study was to investigate the long-term effects of PERVs infection on human cells and estimation
of PERVs integration, stability in human genome and
replication of new virions.
Three different cell lines were used in our experiments:
HeLa cells [ATCC nr: CCL-2], (human cervical cancer
line); the human embryonic kidney cell line HEK 293
[ATCC: CRL-1573] and the porcine kidney cell line
PK-15 [ATCC: CCL33 ], producing porcine endogenous
retroviruses. All cells were maintained under standard
condition (37C, 5% CO2), in a medium containing 10%
FBS. The culture supernatant of the PK-15 cells was filtrated to remove cellular debris, subsequently was used as
the inoculum to infect two other human cell lines. After
exposure to PERVs, after each of cultures passage, during
about 6 weeks, DNA and RNA were extracted, respectively: from cells and from supernatant. Presence of PERVs
integrated in cells human genome was verified by PCR
and virus replication was confirmed in cellssupernatant
by RealTime RT-PCR with primers complementary to
four different PERVs genes (env A, env B, gag, pol).
Our results showed positive infection of both human cell
lines the sequences of PERVs genes were detected after
cells infection, however, successive loss of PERV genes
took place. Moreover, ability to produce and release active PERV particles was confirmed only in human embryonic kidney cell line. We assumed, that efficient PERV
infection of human cells is possible in vitro, but is not stable. Moreover, porcine endogenous retrovirus infects but
does not replicate in HeLa cancer human cell line.
Acknowledgements
This work was supported by research grants of Polish Ministry of
Sciences No. N R12 0036 06/7/2009; DOP-D/138/09.
Magdalena Kimsa1, Sawomir Dudek2, Magorzata Kimsa1,

Barbara Strzaka-Mrozik1, Joanna Gola1,
Celina Kruszniewska-Rajs1, Jolanta Adamska1,
Urszula Mazurek1
Department of Molecular Biology, Medical University
of Silesia, Narcyzow 1, 41-200 Sosnowiec, Poland;
2
Department of Pharmacognosy and Phytochemistry, Medical
University of Silesia, Jagiellonska 4, 41-200 Sosnowiec, Poland
1
The therapeutic usage of porcine material in xenotransplantation is not confirmed to be completely safe
because of the presence of various pathogens such as
porcine endogenous retroviruses (PERVs). Numerous
studies have demonstrated that PERVs are transmitted
to different human cell lines but it is still necessary to
conduct research to explain the interaction between
PERVs and human cells and to search for diagnostic
markers of infection.
Toll like receptors (TLRs) are specific immunologic
markers. The understanding of the molecular details
of effector immune responses and signal transduction
activated by TLRs may provide guidelines for the development of novel diagnostic and therapeutic strategies in
the aspect of PERV infection during xenotransplantation.
The present study focuses on the identification the
effect of porcine endogenous retroviruses on human
dermal fibroblasts by investigating the changes of the
aspect of the expression of TLR and TLR-dependent
genes.
The PERV infectivity was analyzed in the co-culture system. Normal human dermal fibroblasts (NHDF cell line)
were co-cultured for 5 days with normal porcine kidney
epithelial cells (PK15 cell line). Genomic DNA was isolated from harvested cells using the salting out extraction
method. Total RNA was extracted from cells using TRIzol
reagent. The infectivity of PERVs was determined using
real-time Q-PCR and QRT-PCR assay. The analysis of
the expression profile of TLR and TLR-dependent genes
was performed using HG-U133A 2.0 oligonucleotide microarrays.
The copy number of PERV A and PERV B RNA in NHDF
cells (637.30363.0; 77.0058.0, respectively) were revealed after co-culture. However, only the copy number
of PERV A DNA was observed (10.147.6). The study
indicated that after PERV infection, NHDF cells showed
a statistically significant increase of expression of TLR3
(p = 0.008, FC = 1.46). Among TLR3-dependent genes,
45 genes were differentially regulated with a fold change
above 1.1 and pOf these 45 genes, 22 were up-regulated,
23 were down-regulated and 6 were found to be regulated
by more than 1.5-fold.
Eurobiotech 2013
These findings contribute to the clarification of molecular

mechanisms involved in the interactions between porcine endogenous retroviruses and Toll like receptors of
human cells. The expression changes in TLR-dependent
genes may lead to further insights into their role in the
PERV infection during xenotransplantation.
Acknowledgements
This study was supported by the project No. KNW-2-032/D/3/N from
139
P9.3
Cyclosporine A and expression of genes
associated with cell cycle in normal human
dermal fibroblasts cultured in vitro
Grzegorz Hibner1, Adam Wilczok1, Tomasz Janikowski2,
Urszula Mazurek2
Department of Biopharmacy, Medical University of Silesia,
Narcyzow 1, 41-200 Sosnowiec, Poland; 2Department
of Molecular Biology, Medical University of Silesia, Narcyzow 1,
1
The cell cycle or cell division cycle is a series of events that

occur in eukaryotic cell, leading to its division. Generally, these events can be divided into two, usually not very
long, periods: interphase and division phase M. During
interphase the cell grows, accumulating nutrients needed for cytokinesis and division of its genetic material
(karyokinesis). Within the M phase the cell divides into
two separate ones. Regulation of cell cycle contains the
key elements, including the detection of DNA lesions and
genetic material repair and various restriction points to
prevent uncontrolled cell division. Molecular processes
that control the cell cycle are ordered and oriented, which
means that each process occurs in a sequential manner,
and it is impossible to reverse the cycle. The object of
our study was to assess the impact of cyclosporine A
a peptide with immunosuppressive activity widely used
in transplant patients - on expression of genes associated
with the cell cycle in normal human dermal fibroblasts
(NHDF).
Using oligonucleotide microarray technique HGU133A
2.0 (Affymetrix) we compared transcriptional activity of genes associated with the cell cycle in NHDF cells
exposed to cyclosporine A (C = 100 ng/ml; t = 8 h) in
relation to control cells. GeneSpring GX fluorescence
signals analysis of 7845 probes (T test, asymptotic p-value computation with Benjamini-Hochberg correction),
which represented the expression of 5726 genes selected
from the NetAffx Analysis Center database, demonstrated the inhibited expression of 16 genes, including ASPM,
BUB1B, CDC45, CDK1, FOS, RRM2, and TOP2A (p4.0).
Understanding the molecular patterns, which characterize the impact of cyclosporine A on the cell cycle is
important for determining the drug action mechanisms
and resistance formation pathways. Such knowledge also
enables the design of new, more effective drugs and increases the effectiveness and safety of post-transplant patients treatment.
Keywords: cyclosporine, cell cycle, NHDF, oligonucleotide microarray.
Acknowledgements
This study was supported by the grant No. KNW-1-047/D/1/0 from
140
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P9.4
P9.5
Expression of wnt/-catenin signalling

pathway target genes in endometrial
cancer
Changes in the transcriptome of genes

related to TNF induced pathways PERV
infected cells
Tomasz Janikowski1, Grzegorz Cwynar2, Agnieszka Jda2,

Grzegorz Hibner3, Joanna Orchel1, Andrzej Witek2,
Urszula Mazurek1
Joanna Gola, Barbara Strzaka-Mrozik, Magdalena Kimsa,

Magorzata Kimsa, Jolanta Adamska, Celina Kruszniewska-Rajs, Urszula Mazurek
Department of Molecular Biology, Medical University

of Silesia, Narcyzow 1, 41-200 Sosnowiec, Poland;
2
Department and Clinic of Obstetric and Gynecology,
Medical University of Silesia, Medykow 16 St.,
40-752 Katowice; 3Department of Biopharmacy, Medical
University of Silesia, Narcyzow 1, 41-200 Sosnowiec

1 Narcyzow St., 41-200 Sosnowiec, Poland
The wnt/-catenin signalling pathway regulates multiple

key cell processes. The influence of -catenin on cell metabolism, differentiation and death is well known. Its stabilization is regulated by a multiple protein complex consisting of two enzymes: GSK3, CK1 and scaffold proteins:
APC, Axin. When this complex is ineffective it result in
uncontrollable accumulation of -catenin in the cell and
its entering to the nucleus as well as activating transcription factor TCF/LEF. This aberration can lead to cancer
genesis by stimulating angiogenesis, progression and metastasis. The crucial role of -catenin is well documented
in various cancer types, but its role in endometrial cancer
progression is still not fully explained.
The analyses was made on endometrial cancer samples
collected from patients during surgery and afterwards
histopathologically classified accordingly to WHO standards. From all samples total RNA was extracted using
TRIZOL reagent. Next was performed HGU 133A microarrays (Affymetrix) analysis.
There were obtained 22283 ID mRNA from which were
selected 823 associated with wnt/-catenin signalling
pathway and for the statistical analysis GeneSpring GX
was employed. Our results suggest that in endometrial
cancer wnt signalling is down-regulated, but biologically significant genes in proliferation and angiogenesis are
up-regulated by other pathways.
Background. The interaction between PERVs and human cells in inflammatory conditions is still unclear. The
key factors of the immune response in the presence of infectious agents are many cytokines, including tumour necrosis factor (TNF). TNF acts through receptors TNFR1
and TNFR2, inducing MAPK, NFkB and caspase signalling cascades.
Aim. The aim of this work was to assess the changes in
the transcriptome of genes related to TNF signal transduction pathways infected with PERVs with and without
lipopolysaccharide stimulation.
Methods. Human fibroblasts (NHDF) were co-cultured
with normal epithelial porcine kidney cells (PK15 cell
line) in the presence of lipopolysaccharide (LPS). Total
RNA was extracted with the use of phenol-chlorophorm
method. The expression profile of genes related to the
TNF signal transduction pathways was appointed with
the use of oligonucleotide microarrays HG-U133A 2.0
(Affymetrix). Data analysis was performed with the use
of GeneSpring 12.0 platform (Agilent Technologies).
Genes were considered differentiating when p0,001 and
FC3,0 (fold change).
Results. In PERV infected fibroblasts in the presence of lipopolysaccharide six genes were differentiating: MAP3K7, MAP3K8, MAP4K3, BIRC5 (survivin),
MEF2C and TANK, comparing to control. The only
downregulated gene was BIRC5 survivin (FC = 4.09).
Conclusion. PERV infection and the presence of inflammatory factor lead to significant changes in the mRNA
level of survivin and MAPK genes.
Eurobiotech 2013
P9.6
Abundance of blastocysts developed
in vitro from porcine cloned embryos
reconstructed with cell nuclei of adult bone
marrow-descended mesenchymal stem
cells
Marcin Samiec, Jolanta Opiela, Jurij Koseniuk
Department of Biotechnology of Animal Reproduction,
Balice near Crakow, Poland
The present study was carried out to explore the effect

of adult bone marrow-derived mesenchymal stem cells
(ABM-MSCs) that were used as nuclear donor cells on
the in vitro developmental potential of cloned pig embryos. In the somatic cell nuclear transfer (SCNT), oocytes that had reached the metaphase II stage following
in vitro meiotic maturation (IVM) provided the source
of nuclear recipient cells. The cumulus-oocyte complexes (COCs) were matured in vitro for 20 to 22 h in TC 199
medium enriched with 10% foetal bovine serum (FBS),
10% porcine follicular fluid (pFF), 5 ng mL1 recombinant human basic fibroblast growth factor (rh-bFGF),
10 ng mL1 recombinant human epidermal growth factor (rhEGF), 0.6 mM L-cysteine, 1 mM dibutyryl cyclic
adenosine monophosphate (db-cAMP), 0.1 IU mL1
human menopausal gonadotropin (hMG) and 5 mIU
mL1 porcine follicle-stimulating hormone (pFSH). The
COCs were subsequently cultured for a further 22 to
24 h in the fresh IVM medium depleted of db-cAMP,
hMG and pFSH. To form the ooplast-nuclear donor
cell complexes, the previously enucleated oocytes were
subjected to microinjection of either fully confluent/
trypsinised ABM-MSCs (Group I) or foetal fibroblast
cells (FFCs; Group II) under their zonae pellucidae. The
ooplasts were then underwent simultaneous fusion and
electrical activation (SF-EA). The electroactivated nuclear-ooplasmic hybrids (clonal cybrids) were exposed
to 5 g mL-1 cytochalasin B (CB) for 2 h, followed by
in vitro culture to morula and blastocyst stages in 0.4%
bovine serum albumin (BSA)- and 10% FBS-supplemented NCSU-23 medium for 6 to 7 days. A total of 293
and 234 enucleated oocytes that were electrically fused
with either ABM-MSC nuclei or FFC nuclei were simultaneously activated in Groups I and II, respectively. In
Groups I and II, 275/293 (93.9%) and 208/234 (88.9%)
oocytes were successfully fused/activated and selected
for in vitro culture, respectively (P 0.05; 2 test). Out
of 275 and 208 cultured SCNT-derived embryos allotted into Groups I and II, 262 (95.3%) and 164 (78.8%)
were able to divide, respectively (P2 test). The percentages of embryos that completed their development to the
morula and blastocyst stages were 216/275 (78.5%) and
125/275 (45.5%) or 123/208 (59.1%) and 67/208 (32.2%)
in Groups I or II, respectively (P2 test). In conclusion,
porcine cloned embryos reconstituted with ABM-MSC
141
nuclei displayed significantly higher frequencies of both

cleavage divisions and morula/blastocyst formation as
compared to those reconstituted with FFC nuclei.
Acknowledgements
The research project was funded by the Polish National Science Centre
resources allocated on the basis of decision number DEC-2011/03/D/
NZ9/05537.
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Eurobiotech 2013
P9.7
Scriptaid-induced epigenetic modulation
of adult dermal fibroblast cells before their
use for somatic cell nuclear transfer
in pigs
Marcin Samiec, Maria Skrzyszowska
National Research Institute of Animal Production, Department
of Biotechnology of Animal Reproduction, Balice near Crakow,
Poland
The current study was conducted to examine the in vitro

developmental competences of porcine cloned embryos reconstructed with oocytes receiving the cell nuclei
of adult cutaneous fibroblast cells that had been epigenetically transformed by treatment with new-generation
non-specific inhibitor of histone deacetylases, known
as scriptaid. Cumulus-oocyte complexes (COCs) were
matured in vitro for 20 h in Tissue Culture Medium 199
(TCM 199) enriched with 1 mM L1 dibutyryl cyclic adenosine monophosphate (db-cAMP), 10 IU mL1 equine
chorionic gonadotropin (eCG), 10 IU mL1 human
chorionic gonadotropin (hCG), 10% porcine follicular
fluid (pFF), 10 ng mL1 recombinant human epidermal
growth factor (rhEGF), 5 ng mL1 recombinant human
basic fibroblast growth factor (rh-bFGF) and 0.6 mM
L1 L-cysteine. Afterwards, the COCs were cultured for
an additional 22 to 24 h in the db-cAMP- and eCG+hCG-deprived medium. Prior to use for somatic cell cloning, the permanent fibroblast cell lines (between passages
1 and 3) that had been established from the primary cultures derived from ear skin biopsies of a prepubertal gilt
were exposed to 350 nM L1 scriptaid (6-(1,3-dioxo-1H,
3H-benzo[de]isoquinolin-2-yl)-hexanoic acid hydroxyamide) during 24-h serum starvation. Reconstruction of
enucleated oocytes was accomplished by their electrofusion with epigenetically modulated fibroblast cells. Fusion of ooplast-somatic cell couplets was triggered using
two consecutive DC pulses of 1.2 kV cm1 for 60 s. The
same electric pulses that evoked the fusion of ooplast-nuclear donor cell complexes were simultaneously applied
to initiate the activation of reconstituted oocytes (clonal
cybrids). Immediately after electrofusion/electroactivation, clonal cybrids were incubated in North Carolina
State University-23 (NCSU-23) medium supplemented
with 5 g mL-1 cytochalasin B for 1 to 2 h, followed by in
vitro culture up to morula and blastocyst stages in NCSU23/BSA/FBS medium for 144 to 168 h. The rates of dividing embryos (99/131; 75.6%A), morulae (73/131; 55.7%C)
and blastocysts (38/131; 29.0%C) that originated from nuclear-transferred oocytes reconstituted with adult dermal
fibroblast cells undergoing scriptaid treatment were significantly higher than in the scriptaid-unexposed group
(68/116; 58.6%B, 52/116; 44.8%D and 21/116; 18.1%D, respectively) [A,B PC,D P2 test]. Altogether, the improvements
in not only cleavage activity of cloned pig embryos, but
also their morula/blastocyst yields seem to arise from en-
hanced abilities for promotion of faithful and complete

epigenetic reprogramming of scriptaid-treated adult cutaneous fibroblast cell nuclei in a cytoplasm of reconstituted oocytes.
Acknowledgements
This study was supported by the Polish Ministry of Science and Higher
Education as the statutory activity No. 02-1.00.1, which is realized from
2012 to 2014.
Eurobiotech 2013
P9.8
P9.9
The effects of the alloferon and its

analogues on the immune responses
and heart beating in Tenebrio molitor beetle
Can we use the beetle heart as a model

of cardiac ageing?
Arkadiusz Urbaski1, Elbieta Czarniewska2,

Szymon Chowaski2, Jan Lubawy2, Mariola Kuczer3,
Edward Baraniak1, Grzegorz Rosiski2
Department of Systematical Zoology, Faculty of Biology,
Adam Mickiewicz University, Poznan, Poland; 2Department
of Animal Physiology and Development, Faculty of Biology,
Adam Mickiewicz University, Poznan, Poland; 3Faculty
of Chemistry, University of Wroclaw, Wroclaw, Poland
143
Karolina Walkowiak, Monika Szymczak,

Joanna Pacholska-Bogalska, Grzegorz Rosiski
Department of Animal Physiology and Developmental Biology,
Faculty of Biology, Adam Mickiewicz University
Alloferon for the first time was isolated from the blowfly
Calliphora vicina larvae. This compound was characterized by a strong antibacterial activity which protected
blow fly larvae against adverse environmental conditions.
In the last few years, increased an interest in using alloferon in biomedicine. This is caused by discovery of antitumor activities and inhibition of Human herpesvirus,
Papillomaviruses and Coxsackieviruses replication by
alloferon. Furthermore, this peptide stimulated cytotoxic
activity of natural killer cells, haemocytes apoptosis and
interferon synthesis. For many biological activities, the
most important is discovering the active structures which
will affect the immune and circulatory system.
In presented research were used alloferon and its 10
structural analogues, which were characterized by the
highest proapoptotic activities. The significant differences
in the actions of alloferon analogues on cellular and humoral responses were observed, but only two analogues
decreased both immune responses. These compounds
had structural modification in position 1 where native
amino acid histidine was changed on synthetic aromatic amino acid Phe(p-Cl) or Phe(p-OMe). Both structural
modifications in position 1 probably increase proteolytic
stability of alloferon analogues. In addition, long-term
immunomodulation effect may suggest a long half-life of
these peptides in the haemolymph of T. molitor. Alloferon and its analogues didnt affect the heart beat frequency. This result indicates a high specify activity of these
compounds in T. molitor. Presented results enhance the
knowledge about relationship between bioactivity and
structure of alloferon. The discovery of alloferon active
core is necessary for the design and synthesis of peptidomimetics, which can be used for pest control and new
antitumor therapy.
Over the past several years insects have gained interest as model organisms used in studies of fundamental
physiological processes. Some beneficial features, such as
short life cycle, large number of offspring and low costs
of rearing, but also genetic simplicity and less complex
metabolism make insects promising alternative to mammalian models in studying mechanisms of many human
diseases.
The aim of this study was to analyze the relationships in
the heart performance during ageing between beetle Tenebrio molitor L. and humans.
In vitro bioassays of a semi-isolated myocardium and
video microscopic techniques combined with dynamic
image analysis were used to trace mechanic and hemodynamic parameters of ageing beetle heart. We detected
that parameters, such as ejection fraction, stroke volume
and cardiac output of myocardium of ageing beetles undergo changes similar to the decline of cardiac function
in elderly people with heart failure.
Our results suggest that beetle myocardium seems to be
an appropriate candidate as a model to study cardiac performance decline with age and disease and can be used
e.g. in pharmacological or sport medicine researches.
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P.9.10
Identification of 77 novel SNPs within
entire intron A of the pregnancy-associated
glycoprotein 2-like gene subfamily
(PAG2-L) in cross-breed pigs*
Martyna Bieniek, Grzegorz Panasiewicz, Aleksandra Zamojska,
Bozena Szafranska
Department of Animal Physiology, Faculty of Biology
and Biotechnology, University of Warmia and Mazury in Olsztyn,
10-719 Olsztyn-Kortowo, Poland
Pregnancy-associated glycoprotein gene family (PAGs)

encodes multiple chorionic polypeptide precursors that
start expression in trophoblast cells during peri-implantation (Szafranska et al. 2006), the period of the
highest embryonic mortality in many eutherians, including the domestic pig (Cetartiodactyla). Previous
analysis of genomic DNA (gDNA) revealed conserved
structure of PAG genes (approx. 9.5 kbp), consisting of
5-flanking promoter sequence, 9 exons and 8 introns
(A-H), within identified bovine PAG1 and PAG2 (Xie et
al. 1995; Telugu et al. 2009) and porcine PAG2 pPAG2
(Szafranska et al. 2001), so far. Previously in the pig
genome, there were identified all 9 exonic and only 3
short intronic (C, G and H) sequences of the pPAG2like (pPAG2-L) gene subfamily, however 5 still missing
long introns (A, B, D, E & F) were identified more recently and their sequences deposited in the GenBank
database (Bieniek et al. 2013).
The aim of this study was to identify single nucleotide
polymorphism (SNP) within 1093 bp novel sequence of
intron A of the pPAG2-L gene subfamily, which was deposited in the GenBank under accession no. KF471015
(Bieniek et al. 2013). Frequency (px) of homozygous and
heterozygous genotypes (alleles) was calculated for each
SNP. The gDNA templates were isolated from leukocytes
of cross-breed pigs (N = 30) and then amplified parallel to positive control templates of Bacterial Artificial
Chromosome clones: CH242-60C13 and CH242-294016
(BACPAC Resources, USA; http://bacpac.chori.org), containing the pPAG3 and pPAG6 or pPAG3 gene sequences,
respectively. Amplicons were produced by PCR with the
use of sensATG/Ex2as or IntrAseSr/IntrBasdo2 primers
(~1050, 1150, 1250 bp or ~654 bp, respectively). Electrophoreticaly separated pPAG2-L amplicons were cut out
from agarose gel, purified and then sequenced in both
sense and antisense directions (3130 Genetic Analyzer,
Applied Biosystems). Automatically generated sequences were verified qualitatively on the chromatograms with
the use of Finch TV program (Geospiza, Inc., USA) and
SNP variations were encoded according to IUPAC (International Union of Pure and Applied Chemistry). All
sequences were compared by DNASIS v.3.0 software (Hitachi Software Engineering Co. Ltd., Japan).
Among 77 novel SNPs (iA.8iA.1042; intron A numbering), 59 SNPs were identified within the 5-donor
flanking region of intron A (iA.8iA.449), including one
insertion (iA.157/158ins c, px = 0.37), 32 transitions, 24

transversions and 2 SNPs, in which occurred both transitions and transversions (iA.118 / iA.167). Within the
3-acceptor flanking region (iA.558iA.1042), 18 SNPs
were identified, including 13 transitions and 5 transversions. Amongst all 77 SNPs, 70 SNPs were identified as
dominant homozygous genotype (px = 0.570.96), 6 SNPs
as dominant heterozygous genotype (px = 0.5290.913)
and 1 SNP (iA.92a>g) was equivalently frequent for homozygous and heterozygous genotypes (px = 0.5). This
is the first study that describes identification of 77 novel SNPs within the intronic A sequence of the pPAG2-L
gene subfamily. Our data extend the current knowledge
about domestic pig genome and polymorphism of the
pPAG multigene family in cross-breed pigs.
Acknowledgements
Support MNiSW/NN311/066237.
Eurobiotech 2013
P9.11
Identification of 5 novel SNPs within intron
E & F of porcine Pregnancy-Associated
Glycoprotein 2-like subfamily (pPAG2-L)
in two hybrid lines*
Grzegorz Panasiewicz1, Aleksandra Zamojska1,
Martyna Bieniek1, Roman Jdryczko2, Bozena Szafranska1
Department of Animal Physiology, Faculty of Biology
and Biotechnology, University of Warmia and Mazury in Olsztyn,
10-719 Olsztyn-Kortowo; 2Veterinary Diagnostic Laboratory,
11-036 Gietrzwald
1
Multiple PAG gene family (PAGs) codes large group of

secretory products identified in some Eutherians (95
cloned cDNAs; and 15 predicted gDNAs). So far, some
PAG cDNAs have been already patented (USA), mainly
for the Ruminantia or Carnivora taxa. However, structural exon-intron organizations of 3 genes have been identified only: bovine PAG-1 & -2 (Xie et al. 1995; Telugu
et al. 2009); and pPAG2 (Szafranska et al. 2001). Lately,
still missing 5 intron sequences (A, B, D, E & F) of the
pPAG2-L subfamily have been identified in cross-breed
pigs (Bieniek et al. 2013).
The aim of this study was to identify SNPs, mainly within
intron E (between exon 5 & 6) and the largest intron F
(between exon 6 & 7), amongst organizational structure
of eight introns between nine exons of the pPAG2-L subfamily (Szafranska et al. 2001), in two swine hybrid lines
(Hirschmann HM; and Naima Per Ar Lan PAL). All
gDNA templates of pigs (N = 13) were isolated from leukocytes of the HM (n=7) or PAL (n=6), then subjected for
modified PCR with the use of primer pair (IntrEsensd6
and IntrFasGap), amplifying internal region (1483 bp) of
the pPAG2-L genes encompassing (288 bp of 3-flanking
acceptor end of intron E, 117 bp of entire exon 6, and
1078 bp of 5-flanking donor of intron F; GenBank Acc.
Nos.: KF527576/927, U41424/117 and KF537535/1455
bp, respectively). Obtained amplicons of the hybrid
pigs, were examined parallel to commercial BAC clone
CH242-60C13, as positive control template. Separated
pPAG2-L amplicons were visualized by UV, cut out from
agarose gels, purified, labeled (Big Dye Terminator v.3.1)
and sequenced in both sense and antisense directions
(3130 Genetic Analyzer, Applied Biosystems). Amplicon
sequences were in silico analyzed (Megablast or Blastn,
NCBI) for homology discovery, due to the entire intron
E & F sequences of the pPAG2-L (GenBank). Finally, frequency (px) of homozygous or heterozygous genotypes
was calculated for each SNP.
Within intron E, among 8 SNPs previously found in
cross-breed pigs (CBP), presently we identified only 2
SNPs (E numbering; IU code): transitions iE.867g>a (R)
and iE.872g>a (R) in HM, while iE.867R in PAL only.
Three iE.867R genotypes (gg/ga/aa) were identified in
both hybrid lines, similarly as in CBP, with dominant
heterozygous (ga) genotype (HM px = 0.71 and PAL
px = 0.5). The second SNP (iE.872R), was identified only
145
in HM hybrids with two genotypes only (ga/px = 0.43 and

dominant gg/px = 0.57), in contrast to three (gg/ga/additional gt) in CBP. Within the entire exon 6 sequence,
SNPs were not identified in both porcine hybrid lines, in
contrast to 4 SNPs previously found in CBP.
Within intron F, among 6 identified SNPs, 5 were novel in
hybrids (F numbering; IU code); and only one transition
iF.831c>t (Y) was identical, among 17 SNPs in CBP. This
single SNP was dominant genotype (cc) in PAL (px = 0.6)
and in HM (px = 0.7), in contrast to two genotypes
(tt/tc) in CBP. The first novel transition iF.492t>c (Y),
with dominant heterozygous (tc) genotype was identified
in PAL (px = 0.8) and HM (px = 1). Second SNP, transversion iF.493t>g/a (K/W), with genotype gg (px = 0.2)
and dominant ga (px = 0.8) was identified in PAL; while
only genotype ga (px = 1) in HM. Third, transversion
iF.515a>c (M) was with three genotypes in PAL (aa/px = 0.4;
ac/px = 0.4; cc/px = 0.2), and dominant (ac) in HM only.
The last two SNPs, transition iF.516a>g (R) and transversion iF.970g>t (K), with heterozygous genotypes
(ag/px = 0.2 and gt/px = 0.2) were identified in PAL only.
This is the first study describing initial discovery of novel SNPs within intron E & F of the pPAG2-L gene subfamily, presently identified in porcine hybrid lines. Our
preliminary results provide novel data concerning polymorphism of the pPAG multigene family, and increase
current knowledge extending domestic pig genome data.
Further study will allow to define some correlations
between SNP-genotypes with important reproductive
QTLs, then use as pre-selection markers of piglets for
their economic growth required for breeding of the best
progenitors among hybrid lines.
Acknowledgements
Supported by NN311/066237.
146
Eurobiotech 2013
P9.12
Novel glycoprotein family within human
placenta detected with polyclonals
against recombinant or native porcine
Pregnancy-Associated Glycoproteins*
Marta Majewska1, aleksandra Zamojska2,
Grzegorz Panasiewicz2, Martyna Bieniek2,
Zbigniew aganowski3, Bozena Szafranska2
Department of Human Physiology, Faculty of Medical
Sciences, University of Warmia and Mazury in Olsztyn,
10-082 Olsztyn; 2Department of Animal Physiology,
Faculty of Biology and Biotechnology, University of Warmia and
Mazury in Olsztyn, 10-719 Olsztyn-Kortowo; 3The Womans
Health Clinic, 10-684 Olsztyn, Poland
1
Multiple PAG family (PAGs) has been previously identified in various domestic and wild species (Szafranska
et al. 2006). So far, we detected the PAGs in the pig (Majewska et al. 2005, 2006), the European bison (Majewska
et al. 2008), and three Camelidae species: the alpaca, C.
bactrianus and C. dromedarius (Majewska et al. 2009,
2011, 2013). The PAGs belong to aspartic proteinase
superfamily (AP), which includes different proteolytic and lysosomal enzymes (i.e. pepsins or cathepsins).
All AP members possess a two-bilobe structure with a
cleft capable for short peptide binding. As a role of the
PAGs is still unclear, it is however involved in proper
embryo-maternal interaction and placenta development
(Szafranska et al. 2007; Panasiewicz et al. 2007). Thus
abnormal PAG expression reflects placental disorders.
Therefore, the PAGs are routinely used as prenatal diagnostic markers for various pregnancy tests (similarly to
hCG-tests), based on the PAG concentration in peripheral maternal blood or milk of ruminant species (US
Patents; USA).
The aim of this study was to identify PAG-like expression
within hemochorial human placenta by heterologous detection with the use of two different rabbit polyclonals
raised against recombinant porcine PAG2 antigen (anti-RpPAG2) or polyvalent polyclonals raised against native pPAG-like (pPAG-L). Late placental tissues of three
healthy women were collected after caesarean section and
immediately stored at 70oC. The PAG-like immunoreactivity was examined by immunohistochemistry (IHC)
and Western blotting. Total proteins were extracted from
homogenized placental explants and concentrated by
ultrafiltration (MWCO >10 kDa). Extracted placental
proteins were directly used for Western dot-blotting or
separated by SDS-PAGE. Proteins were semi-dry transferred onto nitrocellulose membranes and used for
Western. Primary rabbit polyvalent anti-pPAG-L (1:300)
or anti-RpPAG2 (1:50) polyclonals, characterised previously (Szafranska et al. 2002), were used to identify
molecular mass of human PAG-like proteins. The immuno-complexes were detected with mouse anti-rabbit IgG
monoclonals (1:100 000)conjugated with alkaline phosphatase and then visualized with BCIP/NBT substrates.
Cryosectioned human placental tissues were fixed, dehydrated and used for heterologous dual fluorescent IHC
(htdF-IHC) with the same polyclonals as for Western
blotting. The PAG immuno-complexes within human
placental sections were visualized with secondary goat
anti-rabbit immunoglobulins (1:1000)conjugated with
Alexa 488 dye (495ext/519em nm), as the visualizing
fluorochrome (green). Finally placental sections were
counterstained with propidium iodide (red) to visualize
nuclei.
This is the first study identifying cellular localization of
the PAGs in human placental proteome. The human PAG
family (hPAGs) expression was found to be restricted to
various trophectoderm (chorionic epithelium) cells only.
The separated human placental proteins weakly reacted
with polyvalent anti-pPAG-L polyclonals, but strongly
with anti-recombinant pPAG2 polyclonals. Western blotting revealed dominant approx. 60 kDa hPAG isoform.
The cross-reactivity of the anti-RpPAG2 polyclonals with
the hPAGs revealed structural epitope similarities of the
PAG isoforms in human placenta and other eutherian
species.
Acknowledgements
Supported by UWM (WBiB#528-0206-806 and WNM#1501.801).
Eurobiotech 2013
P9.13
Antibacterial peptides from WPC
hydrolisate
Paulina Worsztynowicz, Dagmara Leniak, Wodzimierz Grajek
Poznan University of Life Sciences
Introduction. The bioactive peptides derived from food

proteins show antibacterial activity. Great interest among
researchers antimicrobial peptides has milk protein. The
reason for this is the fact that milk contains antimicrobial
proteins: lactoperoxidase and lactoferrin but the hydrolysis of whey protein could increase their spectrum antimicrobial effects.
It has been shown that the antimicrobial peptides have
several advantages: quick kill the bacterial cells, and exhibit a broad spectrum of activity, including some resistant nosocomial pathogens [Rizzello et al. 2005]. Include
demonstrated that peptides generated during proteolysis
of lactoferrin have activity against both Gram (+) and
Gram () example: Escherichia, Helicobacter, Listeria,
Salmonella, Staphylococcus, yeast and filamentous fungi
[Korhonen and Pihlanto. 2006].
AIM. The aim of this study was to investigate the antibacterial activity of the whey protein hydrolyzate obtained in
the process of enzymatic hydrolysis carried out with the
participation of protease isolated from lactic acid bacteria.
Materials and methods. Materials. A commercial whey
protein concentrate (WPC80) from dairy cows was obtained from Spomlek. Lactococcus lactis purchased from
Poznan University of Life Sciences, Department of Biotechnology and Food Microbiology.
Enzyme production. In the isolation of bacterial proteases was used modification the method of Requena et al.
(1993) with a slight modification. The resulting supernatants were the source of proteases.
Preparation of WPC hydrolysate. WPC was dissolved in
0,1M Tris-HCl, pH 8,0 at a concentration of 4% (w/v)
and heated in 65C for 30 min. The enzymatic hydrolysis
was carried out at 40C with an enzyme to substrate ratio
(E/S) of 5:1. Samples were collected at different hydrolysis
times (0 h, 8 h, 16 h, 22 h, 28 h, 40 h) and immediately
heated in 100C water bath for 5 min to inactivate the
protease and stop the hydrolysis reaction. After the hydrolysate solution was centrifuged at 4500x g for10 min
in 4C to remove non-hydrolysed protein. The supernatant was collected and lyophilised.
Determination of antibacterial effects. The antibacterial
effect of WPC hydrolysate against E. coli, L. monocytogenes and S. enteritidis was examined according to the
method of Nonnecke and Smith (1984) with a slight
modification. Briefly, 0.5 ml working inoculums of bacterial cultures ware added to 40 mL nutrient broths and
incubated in oscillation incubator for 12 h at 37C. The
cultures ware then diluted with physiological saline to
afinal concentration of approximately 1x106 cfu/ml and
147
used for the antibacterial assay. The growth medium consisted of (w/v) 1% peptone, 0.5% yeas extract and 0.5%
sodium chloride. Each tube was filled with 4 ml growth
medium, 0.5 ml bacterial inoculum and 0.5 ml WPC hydrolysate (10 mg/ml). WPC hydrolysate was dissolved
in physiological saline which was sterilised by filtration
through a0.22 mm membrane. Control assays contained
all components except WPC hydrolysate. The mixtures
were incubated at for 37C 12 h. Viable bacterial counts
of E. coli were obtained using the plate count method
after being cultured on nutrient agar. Plates were incubated at 37C for 24 h. The antibacterial effect of WPC
hydrolysate and its fraction was defined as antibacterial
rate again E. coli, L. monocytogenes and S. enteritidis was
calculated by the following equation: Antibacterial rated
(%) = (Nc Ns)/Nc x 100 where Nc and Ns are the bacteria numbers of the control group and the sample group,
respectively.
SEM analysis. Scanning electron microscopy (SEM) was
used to examine the ultrastructural changes in bacteria
induced by antimicrobial peptides.
Results. It has been shown that the studied strain
L. lactis produced three kinds of proteases: cell wall-associated proteinase, intracellular and extracellular proteinase. The antimicrobial potential of WPC hydrolyzed on
E. coli, L. monocytogenes and S. enteritidis were determined. While hydrolysates for 8 h, 16 h, 22 h and
28h did not show antibacterial activity, whey proteins
hydrolyzed for 40 h by cell wall-associated proteinase,
intracellular and extracellular proteinase exhibited antibacterial activity.
Animal biotechnology in agriculture

Lectures
L10.1
Leptin and leptin antagonists in research
and biotechnology
Arieh Gertler
Institute of Biochemistry, Food Science and Nutrition,
The Hebrew University of Jerusalem, Rehovot, 76100, Israel
Superactive leptin antagonists (D23L/L39A/D40A/F41A,

mutants) of mouse, human, rat or ovine leptins were developed in our lab, expressed in E. coli, refolded and purified to homogeneity as monomeric proteins. Pegylation
of leptin antagonists resulted in a potent and effective
long-acting reagents suitable for in vivo studies. In the
present report we summarize the possible use of leptin
antagonists as (a) research reagents in the study of bone
development, autoimmune diseases, metabolic syndrome
and T2DM with particular emphasis on creation of a novel, fast and reversible model of metabolic syndrome and
T2DM in mice and (b) possible leptin blockers in various
human pathologies such as uremic cachexia, inflammatory and autoimmune diseases, and cancer. In conclusion,
recognition and mutagenesis of D23L of previously developed leptin antagonists (L39A/D40A/F41A) enabled
construction of novel compounds that induce potent and
reversible central and peripheral leptin deficiency. The
possible use of leptin in agriculture was explored using
small weight new born piglets comparable to human babies with intra-uterine growth restriction (IUGR) as it is
well established that low birth weight resulting from intrauterine growth retardation (IUGR) is a risk factor for
further development of metabolic diseases. It was found
that (1) IUGR piglets present altered postnatal growth
and increased adiposity; (2) IUGR piglets exhibit abnormal hypothalamic distribution of leptin receptors that
may be linked to further disturbance in food-intake behavior; and (3) postnatal leptin administration can partially reverse the IUGR phenotype by correcting growth
rate, body composition, and development of several organs involved in metabolic regulation. Early postnatal
6-days leptin treatment induced also an increase in body
weight and the relative weights of the liver, spleen, pancreas, kidneys, and small intestine without any changes
in triglycerides, glucose and cholesterol levels. This study
highlights a new role for leptin in general developmental
processes and may provide new insight into IUGR pathology and show that leptin supplementation can partially
reverse the IUGR phenotype. Long-term follow-up study
is required to conclude whether short-term post-natal

treatment of IUGR piglets may be beneficial to agriculture. Furthermore as pig appears to reproduce nearly all
of the phenotypic pathological consequences of human
IUGR and is likely to be more relevant than rodents in
studies of neonatal development in humans
Eurobiotech 2013
L10.2
Development of non-antibiotic treatment
to prevent animal digestive tract from
bacterial infections
Stefan Kwiatkowski1, Karl Dawson1, Maciej Gryszel2
1
2
Alltech Biotechnology Center USA, Alltech Inc.;

Faculty of Chemistry Warsaw University of Technology
Virulent strains of Escherichia coli and Salmonella cause

serious infections of human and animal digestive and urinary tracts. These bacteria are present in feces that can
contaminate water sources or feed if not proper housing
of animals is in place. Antibiotics have been used as feed
additives to prevent infections. Their vide use in food
production caused bacterial resistance to antibiotics and
their use was banned. Therefore new, effective, non antibiotic feed additives that will not promote bacterial resistance are needed.
The infection starts from bacterial cell adherence to epithelial cells lining of animal or human digestive tract.
E.coli uses type 1 pili which have mannose binding site
(FimH protein) located at its tip. The FimH protein forms
extended complex with -D-mannose reach glycosyl
groups of the epithelial cells proteins. The complex between FimH protein and Man--(13)-(Man--(16)Man--(14)-GlcNAc--(14)-GlcNAc have been crystallized and its 3D structure showed that only three: Man-(13)-Man--(14)-GlcNAc out of five monosaccharides are involved in complex interactions [1].
Aqueous solutions of -D-Mannose and methylmannoside can partially inhibit infections [2] but only at
very high concentrations (2.5%w/v). Alltech products:
BioMos and Actigen which are reach in mannose poliand oligosaccharides are also able to bind to these bacteria [3] but they are not as effective as antibiotics.
Man--(13)-Man--(14)-GlcNAc
trisaccharide,
which has the highest, known inhibitory activity [2] is not
commercially available. Published, multi step syntheses
of this compound are able to supply only minute quantities of it, and thus the product has never been proved
to work in animals challenged with bacterial infections.
Our goal is to develop feasible method for a large scale
production of this material and use it in animal trials. We
assume that irreversible binding of bacteria by this water
soluble trisaccharide will prevent digestive tract colonization without causing bacterial resistance.
Two monosaccharides: -D-mannose (from yeast cell
wall) and 2-acetamido-2-deoxy-D-glucose (from lobster
shells) are used as starting materials. They are converted into Man--(13)-Man-1-OH heptaacetate and GlcNAc-1,3,6-triacetate in simple and high yield transformations. Condensation of the mannose disaccharide using
trichloroacetonitrille with the GlcNAc derivative yields
fully protected with acetate groups: Man--(13)-Man-(14)-GlcNAc trisaccharide, which is than transesterified (with methanol) to yield the desired trisaccharide.
References
[1] Wellens A. et al., PLOS one 3(4):e2040 (2008).
[2] Neeser J-R. et al., Infection and Immunity 52(2): 433.
[3] Parks C.W. et al., Poultry Science 84(12):196773.
149
150
Eurobiotech 2013
L10.3
L10.4
ESC technology in farm animals current

state of knowledge and prospects
for the future
Animals as a tool for metabolic processes

modeling
Zofia Madej
University of Life Sciences Poznan, Department of Genetics
and Animal Breeding
The establishment of stable embryonic stem cell lines

(ESC) from farm animals creates a potent resource for
both agricultural and biomedical research. ESC technology could greatly improve the efficiency of transgenic animal production. The benefits would be both agricultural
(animals resistant to certain diseases or producing therapeutic proteins) and medical (creating animal models of
human diseases or testing the efficiency of gene therapy).
However, despite many years of intensive studies, true
ESC lines have been derived only from mouse (Evans,
1981), rat (Buehr, 2008) and human (Thomson, 1998) embryos. In case of bovine and other animal species, such as
pigs, sheep, goats, rabbits, horses, hamsters, minks, dogs
and cats only ESC-like cell lines have been obtained. This
is mainly due to the limited knowledge about the signalling pathways and species specific factors responsible for
maintaining pluripotency in these species. Existing cell
lines show several major deficiencies raging from short
life in culture to the lack of controlled pluripotency. It is
now evident that although there are some shared mechanism regulating pluripotency and self-renewal in the
ESC, mammalian species differ in morphology, surface
marker and gene expression patterns. Presently scientific efforts concentrate on (1) establishing the optimal developmental stage (timing) to isolate primary embryonic
cell lines, (2) finding the appropriate markers that would
enable the identification of true ESC and (3) identifying
the right set of conditions to propagate and sustain these
lines in culture.
Acknowledgements
Research supported by: Foundation for Polish Science (FNP)
Programme Homing HOM/2009/B and The Ministry of Science and
Higher Education research grant: MNiSzW:4429/B/P01/2010/39.
Ewa Oco, Krystyna Pierzchaa-Koziec, Joanna Zubel-ojek,

Anna Latacz
University of Agriculture in Crakow, Department of Animal
Physiology and Endocrinology, al. Mickiewicza 24/28,
Animal models represent important tools for investigating the physiological and pathological mechanisms of
metabolic processes. The main reason of using animal
models for regulation of metabolism is that the results
can be extrapolated of the humans. Traditionally, animal models can be grouped into the following categories: spontaneous models; genetically modified models;
induced or experimental models and negative models
[3]. Continuous development of genomics, proteomics,
biotechnology and bioinformatics changed trends in applying of animal models. For instance, transgenic technique allows to study the role of specific gene products
involved in the physiological regulation, development or
pathogenesis [5].
Most of the available models are based on rodents because
of their small size, short generation interval, easy availability and economic considerations [1]. Unfortunately,
rodents models differ from human metabolic processes
in many aspects. For example, normal mouse lipoprotein
profiles have essentially atheroprotective HDL, whereas
human lipoprotein profiles contain originally atherogenic low-density lipoproteins (LDL) [7]. For modelling the
metabolic disorder, obese mice combined with dyslipidemia, hypertension and/or elevated glucose levels has
been determined. In recent years, large number of new
genetically modified animal models including transgenic,
generalized knock-out and tissue-specific knockout mice
or rat have been engineered for the study of metabolic
processes and disorders (e.g. mice IRS2-KO, AG4KO,
MG4KO, AMG4KO, IR/IRS-1) [2]. Unfortunately, none
rodents model can exactly mimic all aspects of human
metabolism [7].
The selection of an animal model is difficult and might
lead to misinterpretation of data or even to the wrong
conclusions. Therefore, non-rodent models of metabolic
diseases are urgently needed as a valuable supplement to
rodents for improving an extrapolation of study results.
The pig has many similarities in structure and function to
humans, including size, feeding patterns, digestive physiology, dietary habits, propensity to obesity and social
behaviours [6]. Additionally, pig cardiovascular anatomy and physiology, in combination with the porcine response to atherogenic diets, have made them a universally standard model for the study of atherosclerosis. Their
gastrointestinal anatomy has some significant differences from that of humans; however, the physiology of the
digestive processes has made them a valuable model for
studying disorders. Therefore, the Gottingen, Yucatan,
Eurobiotech 2013
and Ossabaw pigs breeds have been used widely for investigations of adipose tissue activities, glucose and lipids
metabolism, obesity or cardiovascular diseases [4].
Undoubtedly, there are some limitations like expensiveness, practical difficulties and ethical considerations associated with the use of large animal species (pigs, dogs,
monkey). However, in order to better understanding the
regulation of metabolism mechanisms; discover new targets for the treatment of metabolic diseases in humans,
these models are required [5].
Acknowledgements
Supported by DS 3243/KFiEZ/13.
References
[1] Artinamo A., Castro M. (2009) Experimental rat models to study the
metabolic syndrome. Br. J. Nutr. 102(9): 124653.
[2] Gilliam L.A., Neufer P.D. (2012) Transgenic mouse models resistant
to diet-induced metabolic disease: is energy balance the key? Pharmacol.
Exp. Ther. 342(3): 6316.
[3] McMurray F., Moir L., Cox R.D. (2012) From mice to humans. Curr.
Diab. Rep. 12(6): 6518.
[4] Neeb Z.P., Edwards J.M., Allosh M., Long X., Mokelke E.A., Sturek
M. (2010) Metabolic syndrome and coronary artery disease in Ossabaw
compared with Yucatan swine. Comp. Med. 60(4): 30015.
[5] Speakman J., Hambly C., Mitchell S., Krol E. (2008) The contribution
of animal models to the study of obesity. Lab. Anim. 42(4): 41332.
[6] Spurlock M.E., Gabler N.K. (2008) The development of porcine
models of obesity and metabolic syndrome. J. Nutr. 138(2): 397402.
[7] Tschop M., Heiman M.L. (2001) Rodent obesity models: an
overview. Exp. Clin. Endocrinol. Diab. 109(6): 30719.
151
Posters
P10.1
Cytogenetic mapping of the HSPB genes
in the domestic Bovids
Barbara Danielak-Czech, Katarzyna Kruczek,
Anna Kozubska-Sobocika, Barbara Rejduch, Agnieszka Bk
Department of Animal Cytogenetics and Molecular Genetics,
National Research Institute of Animal Production, Krakowska 1,
32-083 Balice near Crakow, Poland
The stress response involving up-regulation of small

heat shock proteins is an important mechanism to deal
with harmful conditions such as hyperthermia, hypoxia
or oxidative stress. The sHSPs expressed in the nervous
system exhibits a clear neuroprotective potential, whereas the mutations of genes encoding these proteins lead
to cell dysfunction associated with myopathies, motor
neuropathies and neurodegenerative disorders. The aim
of this study was the cytogenetic mapping of five HSPB
genes encoding small heat shock proteins involved in
neurodegenerative processes in the domestic Bovids: cattle, sheep and goat. Clones, containing sequences of the
HspB1, HspB2, HspB5, HspB6 and HspB8 genes, were derived from the CHORI-240 Bovine BAC library (http://
bacpac.chori.org/librariesphp) and used as probes which
facilitated the successful assignment of these genes to the
bovine (BTA), ovine (OAR) and caprine (CHI) chromosomes by FISH technique. Because it was not possible
to select separate BAC clones for the HspB2 and HspB5
genes (due to their close proximity in the mammalian genome), thus the same clone containing sequences
of both these genes was used. In this study we present
localizations of the above mentioned five HSPB genes,
respectively, in BTA and CHI genome regions: 25q22,
15q14-q21, 18q24 and 17q24-q25 as well as OAR 24q22,
15q21-q21, 14q24 and 17q24-q25 corresponding regions.
The HspB2 and HspB5, two small heat shock genes located adjacently in the vertebrate genome, were mapped
to the same chromosome band 15q14-q21. Similarly in
humans, these two related genes, composed of two and
three exons, respectively, are located in HSA11q22-q23
genome region, arranged in a head-to-head manner (with
an intergenic sequence about 1 kb) and transcribed in the
opposite direction. All the localizations presented in this
report were in agreement with their physical positions in
the human genome, based on the human-cattle comparative chromosome-painting map (ZOO-FISH). In order to
better understand the function of the HSPB gene family
in livestock mammals, further research should be undertaken, especially in animals exhibiting a neuropathic and
neurodegenerative disorders.
Acknowledgements
Research work financed from National Science Centre funds, authors
project no. N N311 082540.
152
Eurobiotech 2013
P10.2
P10.3
Chromosomal assignment of the small

heat shock proteins genes in the pig
genome
Comparative mapping of UCP2 and UCP3

genes in the pig genome
Barbara Danielak-Czech, Katarzyna Kruczek,

Anna Kozubska-Sobociska, Barbara Rejduch, Agnieszka Bk
Anna Kozubska-Sobociska, Agnieszka Bk,

Barbara Danielak-Czech, Barbara Rejduch,
Magorzata Miszczak


The small heat shock proteins (sHSPs) exert housekeeping role in normal cell metabolism as well as protective
functions under stress conditions such as exposure to
elevated temperature or toxic environmental and metabolic products. Developmentally regulated expression
of sHSPs is required for differentiation of nervous cells
whereas the mutations of genes encoding these proteins
are responsible for desmin-related myopathies or neurodegenerative and autoimmune diseases. The aim of this
study was the chromosomal localization of five sHSPs
genes involved in neurodegenerative processes in the
pig genome. Clones, containing sequences of the HspB1,
HspB2, HspB5, HspB6 and HspB8 genes, were obtained
from the CHORI-242 Porcine BAC library (http://bacpac.
chori.org/librariesphp). The clones, selected based on information about BAC-end sequences (BES) (http://www.
sanger.ac.uk/Projects/S_scrofa/BES.shtml), were used
as probes in the FISH experiments. It was not possible
to select separate BAC clones for the HspB2 and HspB5
genes due to their close proximity in the pig genome, thus
the same clone containing sequences of both these genes
was used. The genes were assigned to the following pig
chromosome (SSC) regions: HspB1 (SSC3p15), HspB2
(SSC9p21), HspB5 (SSC9p21), HspB6 (SSC6q12) and
HspB8 (SSC14q21). The HspB2 and HspB5 loci, clustered
at the distance of above 0.6 kb in the pig genome (http://
www.ncbi.nlm.nih.gov/gene/), were identified in the same
SSC9p21 chromosome band. Similarly in humans, these
two related genes are located in HSA11q22-q23 genome
region and arranged in a head-to-head manner with an
intergenic sequence of less than 1 kb, raising a possibility
of shared regulatory elements for their expression. The
human HspB2 and HspB5 genes (composed of two and
three exons, respectively) are transcribed in the opposite
direction and expressed in most cells under physiological
conditions. However, recent studies point out that they
both are dispensable for cardiac function and maintenance of myocardial integrity as well as suggest their important neuronal function under stress conditions. The
experiments involving genomic context and structure of
porcine sHSPs genes provide a compelling rationale of future biomedical studies to define the complete interaction
of small heat shock proteins in cardiac and neuronal cells
under both basal and stress conditions.
Uncoupling proteins 2 and 3 (UCP2 and UCP3) belong to

a family of mitochondrial transmembrane carriers, which
participate in processes of thermoregulation and energy
metabolism. UCP2 gene is expressed in almost all mammalian tissues, which suggests that it plays a functional
role in global energy metabolism in the body, whereas
UCP3 gene is predominantly expressed in mammalian
skeletal muscle and brown adipose tissue. In humans
UCP2 and UCP3 genes form a cluster localized in the distal q13-14 segment of chromosome 11. Studies of UCP2
and UCP3 genes on the level of nucleotide sequences revealed the relatively high conservation among different
species. Comparative analyses of pig and human UCP2
genes showed 86% identical nucleotide sequences, so genetic conservation of these genes made it possible to use
human molecular probes in interspecies in situ hybridizations to ascertain physical localization of UCP2 and UCP3
genes on pig chromosomes. For this purpose, cross-species in situ hybridizations (FISH) with human, commercial, fluorescent labelled probe (R110 probe, Q-BIOgene
UK) specific for human genome region HSA11q13-14
containing loci of UCP2 and UCP3 genes were carried out
on pig metaphase chromosomes. Fluorescence signals
obtained were observed in Axio Imager.D2 (Zeiss) fluorescence microscope equipped with Axio Vision computer-assisted image analysis system Axio Vision. In an
effect of ZOO-FISH technique red fluorescence signals in
SSC9p21-24 pig chromosome region were obtained,
which enabled to assign localization of UCP2 and UCP3
genes on physical pig genome map. Cytogenetic localization of UCP2 and UCP3 genes on pig chromosomes
confirmed results of previous physical mapping with application of somatic cells hybrid (RH) panels as well as genetic mapping on the basis of linkage analysis. Cross-species in situ hybridizations carried out confirmed conserved nature of the linkage group containing UCP2 and
UCP3 genes as well as homology of HSA11q13-14 and
SSC9p21-24 chromosome regions in which these genes
were localized in humans and pigs. The results obtained
may contribute precise pointing out of evolutional rearrangements concerning chromosomes complied in comparative mapping.
Acknowledgements
Research work financed from National Science Centre funds, authors
project No. N N311 082540.
Acknowledgements
This study was conducted as part of NRIAP statutory activity, projects
No. 04-2.01.1, 04-6.03.1.
Eurobiotech 2013
P10.4
P10.5
Visfatin as a bioindicator of endothelial

cells dysfunction
An universal method for the quantitative

determination of cattle and horse
components contaminating raw and
processed pork meat
Ewa Oco, Krystyna Pierzchaa-Koziec, Joanna Zubel-ojek,

Anna Latacz
University of Agriculture in Crakow, Department of Animal
Physiology and Endocrinology, al. Mickiewicza 24/28,
Visfatin is a protein with several suggested functions: cytokine, enzyme as well as adipokine. This adipokine, also
known as extracellular pre-B-cell colony-enhancing factor (PBEF) and nicotinamide phosphoribosyltransferase
(Nampt), is peptide whose circulating levels are enhanced
during metabolic disorders [1]. The study has demonstrated that visfatin may exert direct destructive actions
on the cardiovascular system, including cell proliferation,
monocyte/macrophage activation, vascular inflammation and remodeling, all of them leading to the development of atherosclerosis [2].
Thus, the aim of the study was to determine the synthesis and secretion of visfatin from endothelial cells in response to hyperglycemia. Additionally, the adipokine was
estimated as an indicator of endothelium dysfunction.
Human aortic endothelial cells (HAEC) were cultured in
a standard medium (M 199) supplemented with LSGS
kit (Invitrogen). The experiment was performed two
times on fourth passage of HAEC culture. The cells were
incubated for 24 hours with glucose in concentrations
22.2mmol/l. After completing the experiment, total RNA
was isolated from cells using Ambion Cells-to-CT Kits
(Life Technologies). Reverse transcription was undertaken using High Capacity Reverse Transcription Kit (Life
Technologies). Quantitative PCR analysis was performed
using StepOnePlus Real-Time PCR System (Life Technologies) with the Universal Master Mix and TaqMan
chemistry (Life Technologies). Visfatin concentration
was measured using a commercial ELISA kit (BioVendor
R&D), according to the manufacturers protocol.
The activity of aortic endothelial cell negatively changed
in response to hyperglycemia. The cells proliferation was
decreased by 36,9% compare to control group (p
Acknowledgements
Supported by DS 3243/KFiEZ/13.
References
[1] Fukuhara A., Matsuda M., Nishizawa M. (2005) Visfatin: a protein
secreted by visceral fat that mimics the effects of insulin. Science.
307(5708): 426430.
[2] Stam F., van Guldener C., Schalkwijk C.G. (2003) Impaired renal
function is associated with markers of endothelial dysfunction and
increased inflammatory activity. Nephrology Dialysis Transplantation
18(5): 892898.
153
Magorzata Natonek-Winiewska, Piotr Krzycin,

Katarzyna Kruczek
National Research Institute of Animal Production, Balice,
Poland
Species identification of pork meat adulterated with beef

and horse meat is an important factor in checking the
reliability of food product information provided by the
producer. Samples of pork adulterated with 0.1% to 80%
beef were analysed. DNA was isolated using the Ax Food
kit (A&A Biotechnology). This method allows for quantitative determination of identified species using standard
curve and TaqMan MGB probes by real-time PCR. A test
that uses short amplicons (69 bp) in the gene encoding
ATPase6 was developed to identify bovine components.
The primer and probe sequences were as follows:
F/Bov(ATPase6)
5-CTGTGAGCAGGAGCCGTAATT
R/Bov(ATPase6)
5-TGGTAAGAAATGGGCAAGTGATG
P/Bov(ATPase6)
5-Vic ATTCCGCAATAAAAC
The primers and the probe that detect horse components
were taken from TANABE et al. (2007), Biosci. Biotechnol. Biochem., 71; 3131-3135. A 75-bp cytB fragment of
horse mtDNA was amplified. The limit of quantitation
(LOQ) for the beef and horse component in pork meat
is 0.2%.
The small size of the amplicon used in the above analyses
is useful for detecting species composition of processed
food products with degraded DNA. For this reason, the
method is applied to analyse adulterations in both raw
and processed meat.
154
Eurobiotech 2013
P10.6
P10.7
A real-time quantitative PCR detection

method for beef, pork, chicken and mutton
components in foods
New polymorphisms in regulatory regions

of CAPN3 gene in broiler chickens
Magorzata Natonek-Winiewska, Piotr Krzycin,

Katarzyna Kruczek
National Research Institute of Animal Production, Balice,
Poland
In order to determine species composition of food products for humans as well as pet food, a method for rapid and sensitive identification of their components was
developed. Species specific primers and TaqMan probes
were designed on mitochondrial DNA: ATPase6 for cattle, cox1 for sheep and pigs, and 16SrRNA for chickens.
In the case of chicken and sheep identification, the proposed primers amplify the DNA of the species for which
they were designed. When identifying cattle and pigs,
across-reaction was found between these species during
the 35th reaction cycle. However, this did not hamper the
analysis of the results because during this cycle a reaction
takes place for the identified product at a level of 0.05%
and at 100% for the second species.
The limit of quantitation for chickens and sheep has not
been determined, but it is lower than 0.1%. The limit of
quantitation for cattle and pigs, due to the cross-reaction,
is 0.1%.
The developed method is suitable for species identification of raw meat, processed meat and components of
meat-and-bone meals subjected to thermobaric treatment at a temperature of 133C and pressure of 3 bar for
20 minutes.
Katarzyna Pirkowska2, Joanna Doktor3, Katarzyna Potowicz3,

Katarzyna Ropka-Molik2
Laboratory of Genomics, National Research Institute of Animal
Production, Krakowska 1, 32-083 Balice, Poland; 2Department
of Animal Genetics and Breeding, National Research Institute
of Animal Production, Krakowska 1, 32-083 Balice, Poland
1
The calpains are the enzymes belong to calcium-dependent, non-lysosomal cysteine proteases expressed in
mammals and many other organisms. The CAPN3 gene
encodes a major intracellular protease, which is muscle
specific. In chickens it is localized on chromosome 5. The
calpains have a high capacity degradation of cytoskeletal
and muscle fibers proteins. Therefore they play an important role in fusion of mioblasts, proliferation, growing
and migration of cells. The CAPN3 has been chosen as
candidate gene associated with meat quality in chickens.
Consequently the aim of our study was to identified new
polymorphisms in regulatory region of CAPN3 gene and
examine their impact on CAPN3 transcript abundance in
breast muscles. In experiment used broilers of two genetic lines: fast and slow-growing. As a screening method
the High Resolution Melting (HRM) was used. The polymorphisms were identified with on Beckman Coulter sequencer based on the Sanger method. The CAPN3 gene
expression was estimated on Real Time 7500 Applied Biosystems using succinate dehydrogenase complex; subunit
A (SDHA) and 60S ribosomal protein L4 (RPL4) genes
as endogenous control. Statistical and linkage analyses
were performed using SAS Enterprise. Four new polymorphisms were found. One in promoter region c. 450
G > A and three in 3ÙTR (c. 176* C>T, c. 137*_147* del.,
c. 144* G > C) region. One of them in 3ÙTR region was
deletion of 11 nucleotides (CAGCCCTGCTT). New polymorphisms were identified by used restriction enzymes
ScrFI, BslI, AcuI, HpyHV, respectively. The frequency of
polymorphisms found in 3ÙTR region was similar in
both lines. Nevertheless, in fast growing line appeared
more genotypes than in slow growing according to c.
C > T 176* and c. G > C 144* polymorphisms. Moreover,
both broiler lines were in Hardy-Weinberg disequilibrium of c. 450 G > A polymorphism, although this polymorphism does not change any transcription binding
site. Analysis of effect of new polymorphisms on CAPN3
gene expression showed, that in fast growing lines the
chickens with GG genotype according to c. 450 G > A
polymorphism characterized with the highest CAPN3
gene expression. Other polymorphisms in 3ÙTR region
seem not effect on CAPN3 gene expression.
Acknowledgements
This work was supported from National Research Institute of Animal
Production statutory activity, research project No. 01-4.02.1
Eurobiotech 2013
P10.8
Different culture media affect growth rate
and morphology of porcine bone
marrow-derived mesenchymal stromal
cells- the preliminary results of media
screening suitable for both porcine MSCs
and bovine embryo in vitro culture
Jolanta Opiela, Micha Bochenek, Joanna Romanek,
Zdzisaw Smorg
Department of Biotechnology of Animal Reproduction, National
Research Institute of Animal Production, 32-083 Balice near
Crakow, Poland
The presented results are the part of the experiment which

aims to study the co-culture of bovine embryos with porcine mesenchymal stem cells. We assume that the stem,
multipotencial nature of the MSCs used for co-culture
systems would significantly improve the bovine embryo
in vitro development. Therefore, as a result the significant
enhancement of in vitro embryo production efficiency
is expected. So far, the in vitro culture of bovine embryos along with MSC as a feeder layer was applied in two
media (MENEZO B2 + 2,5% fetal calf serum (FCS) and
SOF + 10% estrus calf serum (ECS), however the embryo development was blocked at the morula stage. The
quality of MSC was noticeably decreased which indicated
the suboptimal culture conditions. This prompted us to
explore for the medium suitable for both porcine MSCs
and bovine embryo in vitro culture.
MSCs were isolated from porcine bone marrow
and expanded in DMEM supplemented with 10%
FCS. Selected MSC surface markers (CD31, CD34,
CD90) were analyzed by flow cytometry to confirm
their mesenchymal characteristics. Then, positively
verified MSCs were cultured in nine established cell
culture media: TCM 199 + 10% FCS, NCSU-23 + 10%
ECS, TCM 199 + 2,5% FCS, NCSU-23 + 2,5% FCS,
MENEZO B2 + 10% FCS and MENEZO B2 + 2,5% FCS,
SOF + 10% ECS, G2 + 2,5% FCS, DMEM/F12 + 2,5%
FCS. The MSC were cultured in two systems: in a dish
with 3 ml of medium and the 4-well dish in 0.5 ml of
medium under mineral oil. MSC morphology and
proliferation rate were observed on Days 3 and 7 of
in vitro culture in all of analyzed media. The cells
in each analysed media were given a score from 1
to 5 subjectively where 1 indicates poor or low and 5
indicates excellent and high.
The flow cytometry of MSC markers proved their mesenchymal origin. The positive expression of the CD90
was observed in 99.8% of analyzed cells and the negative
expression of CD31 and CD34 antigens was observed in
3.8% of analyzed cells. Cell morphology and proliferation
rate varied importantly between the media. The best cell
morphological quality and proliferation rate were observed in TCM 199 and NCSU-23. Interestingly, the least
suitable media for MSC culture turned out to be Menezo
155
B2 and SOF, which are the media routinely used for bovine embryo culture.
The choice of MSCs expansion medium can have a significant influence on both growth and cell morphology
which is of fundamental importance for their implementation in other biotechnological procedures like e.g.
embryo in vitro culture. The in vitro co-culture system of
embryos with MSCs requires further research.
Acknowledgements
This research was supported by the Statutory Activity of National
Research Institute of Animal Production 02-4.03.1 and by the Polish
National Science Centre resources allocated on the basis of decision
number DEC-2011/03/D/NZ9/05537.
156
Eurobiotech 2013
P10.9
P10.10
The impact of long-term in vitro culture

and differentiation of porcine
mesenchymal stem cells (pMSC)
on the cell cycle distribution and nuclear
DNA profile
RNA-seq analysis of differentially

expressed genes in muscle tissue
of Pietrain and Puawska pigs
Jolanta Opiela, Marcin Samiec, Micha Bochenek,

Joanna Romanek

The aim of experiment was to determine the impact of

long-term in vitro culture and differentiation of porcine
mesenchymal stem cells (pMSC) on the cell cycle distribution and nuclear DNA profile. This determination
could be helpful to confirm or exclude the suitability of
physico-chemical culture conditions for the purposes of
both the maintenance of an undifferentiated state and to
promote differentiation in pMSCs.
Flow cytometry was applied to analyse the cell cycle and
occurrence of aneuploidy/diploidy. The 2 test was used
to compare the total rates of G0/G1-, S-, and G2/M-phase
cell fractions with diploid and aneuploid DNA and the
DNA index ratios between three experimental groups of
pMSCs.
Five weeks of in vitro culture under differentiating conditions resulted in a considerable reduction of DNA stability and a remarkable increase in the rate of cells exhibiting
an aneuploid DNA stemline; however, a similar dependence was not found in the non-differentiated MSCs.
To sum up, we propose cytokinetic diagnostics using flow
cytometry as an objective and useful method for screening the tumour-forming capacity and malignancy potential of both in vitro long-term cultured MSCs and MSCs
subjected to ectopic differentiation.
Acknowledgements
Research Institute of Animal Production 02-4.03.1.
Katarzyna Ropka-Molik1, Kacper ukowski2, Robert Eckert2,

Artur Gurgul1, Katarzyna Pirkowska1
Laboratory of Genomics, National Research Institute of Animal
Production, Krakowska 1, 32-083 Balice, Poland; 2Department
of Animal Genetics and Breeding, National Research Institute
of Animal Production, Krakowska 1, 32-083 Balice, Poland
Next Generation Sequencing RNA-seq technology is a powerful tool which creates new possibilities in
whole-transcriptome analysis. The RNA analysis using
high-throughput sequencing method allows to measure
gene expression on a genome-wide scale. In our study,
RNA-seq method was applied to analyze global changes
in transcriptome form the muscle tissue (m. semimembranosus) in two pig breeds (Pietrain 5 pigs, Puawska
7pigs). The total RNA wasisolated using TRI-Reagent.
The quantity and quality of RNA were evaluated with the
NanoDrop 2000 and by 2% agarose gel electrophoresis.
cDNA libraries were prepared by using the TruSeq RNA
Sample Preparation Kit v2 (Illumina) according to standard protocol. Sequencing by synthesis of the libraries
was performed on a HiScanSQ System with 50 single-end
cycles using TruSeq SBS Kit v 3 HS chemistry (Illumina).
Using two different approaches by using DESeq, egdeR
freewares and considered the most restrictive criteria
we identified 184 differentially expressed genes between
Pietrain and Puawska pigs. In both breeds, the most
abundant were: transcripts encoding ribosomal proteins,
genes associated with metabolic processes, which play
akey role in respiratory electron transport chain in mitochondrion, intercellular or cation transport (NDUFS6,
SYPL2, CHCHD3, ATP5A1, DLD).
In Puawska pigs we indicated up-regulation of several
genes that play a variety of roles crucial for metabolic
processes metabolism of polysaccharides an carbohydrates (CHID1, HS3ST5, MFSD10), amino acid and protein metabolic processes (NUBP2, ALDH16A1, OTUB1).
Furthermore, when compare to Pietrain pigs, the significantly higher expression was obtained for genes encoding proteins associated with cell adhesion and cell cycle
(EMILIN1, EMILIN3, GADD45G, H1FX). In Pietrain
breed, only 37 genes were over-expressed and majority
of them play an important role in cell developmental processes i.a. regulation of apoptosis, mitosis, cell-cell signaling and adhesion (SGPP1, HTRA2,SLITRK2, MAFB,
ACTL6B), as well as in the muscle contraction and cation/anion transport (GLRB, SLC9A7). The different expression profile of selected genes obtained for two pig
breeds may be the result of long-term selection to improve the meat content in carcasses conducted especially
in Pietrain breed.
Acknowledgements
This study was supported by the National Research Institute of Animal
Production statutory activity, Research Project No. 01-5.03.1.
Eurobiotech 2013
P10.11
P10.12
An increase of piglets survivability after

biolactin per os administration
Association of calpastatin gene

polymorphisms and meat quality traits
in pig
Barbara Gajda1, Barbara Szczniak-Fabiaczyk1,

Katarzyna Poniedziaek-Kempny1, Izabela Mandryk1,
Florian Ryszka2, Barbara Doliska2, Lucyna Leszczyska2,
Zdzisaw Smorg1
32-083 Balice near Crakow, Poland; 2Pharmaceutical Research
and Production Plant (FZNP)
1
Prolactin (PRL) is a protein hormone synthesized in and

secreted predominantly by lactotroph cells of the anterior pituitary gland. It has been found to stimulate the
immune system in animals as well. Because prolactin secreted in milk by the mothers body is often insufficient,
the administration of exogenous prolactin may significantly contribute to improving the survivability of piglets.
The objective of this study was to determine the effect of
0.1mg PRL per kg of body weight of piglets administered
per os to newborn piglets.
Prolactin (Biolactin, Biochefa, Poland) was obtained
from freeze-dried pig hypophyses. An experimental
group (63 litters/757 piglets) received per os 0.1 mg PRL
per kg of body weight and a control group (30 litters/358
piglets) 1.0 mL of 0.9% physiological saline solution. The
number of piglets born alive at birth and on 21, 28 and
70 days of age was monitored. Based on this the mortality of piglets in the experimental and control groups was
estimated.
At birth the average litter size was 12.0 and 11.9 piglets
in experimental (after per os Biolactin administration)
and control groups respectively. At 21 and 28 days (weaning) after farrowing the number of piglets, the average litters were 10.8 and 10.7 piglets in experimental and 10.5
and 10.3 piglets in control litters respectively. An average
of 10.6 and 10.0 piglets were left from experimental and
control litters respectively at 70 days of age. The reduction of mortality in the experimental group (9.9%, 11.1%
and 12.0%) in comparison with the control group (11.7%,
14.0% and 15.9%) during the first three, four and ten
weeks, respectively was observed.
In conclusion, the results demonstrated an increase in the
survivability of piglets that received Biolactin.
Acknowledgements
Supported by grant No. NR12-0057-10 of NCBiR, Poland.
157
Anna Bereta2, Katarzyna Ropka-Molik1, Mirosaw Tyra2,

Marian Rycki2
Laboratory of Genomics National Research Institute of Animal
Production National Research Institute of Animal Production,
Krakowska 1, 32-083 Balice, Poland; 2Department of Animal
Genetics and Breeding National Research Institute of Animal
Production National Research Institute of Animal Production,
Krakowska 1, 32-083 Balice, Poland
1
Calpastatin is an endogenous inhibitor specific for the

calpains (calcium-dependent cysteine protease). Due to
the regulation of proteolysis processes in cells, the activity of calpastatin is related with several meat quality traits
such as tenderness, drip loss, water holding capacity,
conductivity or color. Calpastatin is also associated with
the rate of degradation of muscle structural proteins post
mortem via controlling of calpain activity. Furthermore,
the calpastatin protein is thought to influence the expression levels of genes encoding structural or regulatory proteins. Based on above function, calpastatin gene (CAST)
is considered to be a candidate gene for the meat quality
in many domestic animals including pig.
The aim of the study was to determine the effect of CAST
gene polymorphisms on selected meat quality traits in
different pig breed used as maternal component: Polish
Landrace (PL)(n = 157) and Polish Large White (PLW)
(n = 224), paternal component Pietrain (n = 94), Duroc
(n = 93) and in the Puawska, which is included in conservative breeds (n = 110). Animals were maintained in
the Pig Station of the National Research Institute of Animal Production in Pawowice according SKURTH procedure. All animals were genotyped for three polymorphisms using PCR-RFLP method (CAST/HinfI; CAST/
HpaII; CAST/RasI) according to Ernest et al. (1998).
The results obtained showed that two among 3 analyzed
polymorphisms were significantly associated with meat
pH and color. Due to the CAST/HinfI and CAST/HpaII
polymorphisms, homozygotes BB and DD (respectively)
were characterized by the highest pH measured 45 minutes and 24 hours after slaughter in both ham and loin.
The significant differences in red and yellow color intense
of meat between CAST/HinfI polymorphism were also
observed. The BB genotype had the highest red intensity
(a*) and the lowest yellow intensity (b*). Moreover, the FF
genotype (CAST/RsaI) significantly affects water-holding
capacity (PCAST gene polymorphisms can be potential
use as a marker for meat quality traits in pig.
Acknowledgements
The study was supported by the Polish Ministry of Science and Higher
Education (project No. NN311349139).
References
Ernst C.W., Robic A., Yerle M., Wang L., Rothschild M.F. (1998)
Mapping of calpastatin and three microsatellites to porcine chromosome
2q2.1q2.4. Animal Genetics, 29, 212215.
158
Eurobiotech 2013
P10.13
P10.14
Genetic characterization of Hucul horse

populations based on microsatellite
polymorphism
The impact of varied values of High

Hydrostatic Pressure applied on porcine
Mesenchymal Stem Cells on their
survival rate and early apoptosis after
cryopreservation
Agnieszka Fornal, Anna Radko, Agata Piestrzyska-Kajtoch

of Cytogenetics and Molecular Genetics of Animals, Balice near
Crakow, Poland
Short tandem repeat (STR) loci, i.e. microsatellites are

aclass of genetic markers commonly used for population
studies and parentage control. This study determined the
usefulness of microsatellite markers recommended by International Society for Animal Genetics (ISAG) for identification and pedigree analysis in horses based on an example of Hucul horses populations (Equus caballus). The
set of 17th STRs was tested (AHT4, AHT5, ASB2, HMS2,
HMS3, HMS6, HMS7, HTG10, HTG4, HTG6, HTG7,
VHL20, ASB17, ASB23, CA425, HMS1, LEX3) for three
Hucul horses populations. 216 individuals were genotyped and mean number of alleles per locus was estimated as 6.039. The low diversity between each population
was observed. Means of observed (Ho) and expected (He)
heterozygosity calculated for all populations were 0.735
and 0.669, respectively. Ho was similar in all populations
analyzed (respectively: 0.692; 0.625; 0.689). The average
polymorphism information content (PIC) for seventeen
microsatellite markers indicates the usefulness of this set
of markers for Hucul horses parentage testing.
Joanna Romanek, Jolanta Opiela, Zdzisaw Smorg

Department of Biotechnology of Animal Reproduction National
Research Institute of Animal Production, Krakowska 1,
32-083 Balice, Poland
High Hydrostatic Pressure (HHP) by stimulating the defensive reaction to stress increases cells resistance to another adverse factor. Cells after HHP treatment overexpress resistance proteins, therefore they can quicker adapt
to next adverse factor like cryopreservation. As a result,
a higher survival rates after cryopreservation can be obtained. As a beneficial effect of HHP on sperm, oocytes
and embryos cryotolerance is already documented, we
assume that the HHP treatment may also have a positive
effect on the Mesenchymal Stem Cells (MSCs) quality after cryopreservation.
The aim of this study was to examine the influence of
varied HHP values on porcine MSCs to increase survival
rate and quality after cryopreservation. The survival rate
and phosphatidylserine (PS) exposure in external surface
of the cells were analyzed to indicate the most optimal
HHP value for further experiments. In normal cells PS
is located on the cytosolic side of cell membranes but
during the early stages of apoptosis PS becomes exposed
on the surface of the cell. Annexin V has the highest affinity for PS, therefore it is used as a probe to detect cells
that have expressed PS.
The MSCs were isolated from bone morrow. Aspirate was
diluted with PBS and subjected to centrifugation with
Ficoll. The mononuclear cells were isolated from the interface, washed twice in PBS and resuspended in DMEM
supplemented with 10% FCS. The MSCs were cultured for
about 2 weeks until 80% confluence was reached. Then,
MSCs were detached with 0.25% trypsin/EDTA, centrifuged at 300 g and aspirated into 0.5 ml straws with M199
Hepes modification supplemented with 2% FCS. Such
prepared straws were subjected to the five different HHP
treatments (20 MPa, 30 MPa, 40 MPa, 50 MPa, 60MPa)
for 1 h in 24C. After HHP treatment, MSCs were suspended in 10% DMSO and stored in liquid nitrogen for
at least 24 h. Thawed MSCs were used for survival rate
and early apoptosis detection by trypan blue staining and
Annexin V-Biotin Apoptosis Detection Kit, respectively.
Regarding MSCs viability, the high significant difference
(P < 0.001) was noted between MSCs subjected to HHP
ranging for 30 MPa to 60 MPa and control MSCs. The
significant difference (P < 0.05) was noted between MSC
subjected to 20 MPa HHP and control. No significant difference was observed in PS exposure in any of analyzed
experimental groups.
Eurobiotech 2013
According to the presented result, HHP increases MSC

survival rate regardless the applied HHP value. Moreover,
HHP does not have any negative impact on cells quality
as measured by early apoptotic marker PS. The further
experiments will be performed to indicate the most optimal HHP treatment for increasing the MSCs tolerance
after cryopreservation.
Acknowledgements
Research Institute of Animal Production 02-4.03.1 and by the Polish
National Science Centre resources allocated on the basis of decision
number DEC-2011/03/D/NZ9/05537.
159
P10.15
Analysis of the changes in the expression
profile of liver transcriptome by RNA-seq
in pigs fed with various diet supplements
Maria Oczkowicz1, Katarzyna Ropka-Molik1,
Magorzata witkiewicz2, Kacper ukowski3, Artur Gurgul1
National Research Institute of Animal Production, Laboratory
of Genomics; 2National Research Institute of Animal Production,
Department of Animal Nutrition And Feed Science;
3
of Animal Breeding and Genetics
1
RNA sequencing is a new, cutting edge technology which

allows for quantification of transcripts, identification of
a new SNPs and a new splicing variants. In our study we
aimed to compare the liver transcriptome of pigs fed with
different diet supplements. All animals were kept in the
same housing conditions and fed with standard feed mixture according to their nutritional requirements. There
were three experimental groups, differing with type of
used fat: beef tallow (n = 4), coconut oil (n = 4) and rapeseed oil (n = 4) and one group with addition of rapeseed
fat but no addition of DGGS (Dried Destilled Grains
with Soluble) in contrast to the other groups. When the
animals reached the weight of 118 Kg were slaughtered.
Samples of liver were collected and frozen in a liquid nitrogen immediately. Next, RNA was isolated from the
samples using Trizol reagent. Quality and concentration
of RNA were evaluated using Nanodrop 2000 and by
2% gel electrophoresis. cDNA library was prepared with
400 ng of good quality RNA and TruSeq RNA Sample
Preparation Kit v2 (Illumina). Clustering was performed
on cBot (Illumina) and Sequencing by Synthesis was performed on a Hi Scan SQ System with 50 single-end cycles
using TruSeqSBS Kit v 3 HS chemistry (Illumina). After
data analysis, one sample was excluded from the experiment because of low quality of mapping. Transcrpits expression differences were analyzed with Cuffdiff freeware.
In total, 40 genes differed in the expression level when all
experimental groups were compared. The highest number of differentially expressed genes (n = 15) were present between animals supplemented with coconut oil and
pigs without DGGS in the feed stuff. Some of the identified genes are responsible for lipid transport (ABCD3,
APOA4), metabolism and cholesterol metabolic processes (HMGCS1). These genes will be further analyzed by
Real-Time PCR.
Acknowledgements
This work was supported by National Research institute of Animal
Production.
160
Eurobiotech 2013
P10.16
Regulation of Tenebrio molitor heart
beating by monoamines and tropane
alkaloids
Szymon Chowaski, Jan Lubawy, Arkadiusz Urbaski,
Marta Spochacz, Grzegorz Smykalla, Grzegorz Rosiski
Department of Animal Physiology and Development,
Faculty of Biology, Adam Mickiewicz University in Poznan
Insects generally possess pumping structures and various

diaphragms to ensure that haemolymph flows throughout the haemocel. The primary pump for moving haemolymph is a dorsal vessel also named as heart. The myocardium in all insects is spontaneously active. This type of
heart is termed myogenic because the electrical activity
underlying contractions a rises in the myocardium itself.
This is in contrast to a neurogenic heart present in, for example, crustaceans. The heart is innervated by paired lateral nerves from segmental ventral ganglia or by cardiac
nerve running along the heart and coming out from the
brain. Furthermore, the heart contractility is regulated in
neurohormonal way by neurotransmitters and neurohormone peptides for example by proctolin and crustacean
cardioactive peptide.
Monoamines function as a neurotransmters, neuromodulators and neurohormones in nervous system both in
vertebrates as in invertebrates. In case of vertebrate, the
catecholamines (epinephrine, dopamine and serotonin)
have the main role in nervous system regulation and in
case of invertebrates it is trace amines (octopamine and
tyramine). In insects, this compounds influent on many
different physiological processes. They modulate central
nervous system activity, control contractility activity and
metabolism of muscles.
Tropane alkaloids, atropine and scopolamine, are an antagonist of cholinergic muscarinic receptors (mAChRs).
In vertebrates, acting of atropine and scopolamine on
mAChRs leads to increased heart rate. These receptors
are also found in insects. The available data suggest that
they are involved in behaviour related to locomotion,
chewing and pharyngeal movements and the behaviour
associated with singing and also in regulation of wings
and antennae movements. Furthermore, activation of
muscarinic receptors may affect the changes in gene expression in neurons as well as regulation of neuro-endocrinic processes. However, there is not information, how
agonists or antagonists of mAChR affect insect myocardium. In case of vertebrates, atropine and scopolamine
cause an increase of heart rate and abolition bradycardia
and asystoles.
The aim of study was to determine the influence of
tropane alkaloids and monoamines on the myocardial
activity in the beetle Tenebrio molitor. The results
showed that compounds tested have diversed effect
on the myocardium contractile activity in T. molitor.
Tropane alkaloids did not cause changes in the action
of semi-isolated heart in the whole concentration range
(1010103 M). But the in vivo experiment carried out on

1-day old pupae T. molitor showed, that these compounds
lead to decrease in heart rate what was observed as
negative chronotropic effect both in ortodromic and
antidromic phase of contractility between 4th and 10th
hour after application of scopolamine and atropine.
Epinephrine did not cause changes in the heart contractility
both in vivo and in vitro experiments. Dopamine had an
opposed effect. In vitro it leads to a slight increase of heart
rate (only at highest tested concentration 103 M) but in
vivo it acts as a myoinhibitor. Tyramine and octopamine
both in vivo and in vitro caused negative chronotropic
effect but only between 6th and 10th hour after application.
Obtained data may suggest indirectly participation of
scopolamine and atropine in regulation of heart beating.
There is probable that these compounds affect neuro-secretory structures, which are involved in the myocardium
activity regulation. No changes in the heart action under
influence of tropane alkaloids can be a result of lack of
mAChRs in the heart or presence of receptors insensitive on these antagonists. Trace amines (octopamine and
tyramine) influenced on myocardium both in vivo and
in vitro what indicaste, that these compounds take part
directly in regulation of myocardium activity.
Eurobiotech 2013
161
P10.17
P10.18
Genetic polymorphisms of lep and lepr

genes in relation with production
and reproduction traits in cattle
Prp4 kinase is required for proper

segregation of chromosomes during
meiosis in Schizosaccharomyces pombe
Anna Trakovick, Nina Moravkov, Radovan Kasarda
Miroslava Pogajov1, ubo ipk2, Anna Trakovick1
Slovak University of Agriculture in Nitra, Department of Animal

Genetics and Breeding Biology, Tr. A. Hlinku 2, 949 76 Nitra,
The Slovak Republic
Leptin and leptin receptor genes are considered as markers of production traits in dairy or beef cattle. The aim
of this study was to verify the associations of polymorphisms in bovine LEP and LEPR genes with production
and reproduction traits in Slovak Spotted and Pinzgau
cows. Evaluated were long life production: milk, protein, and fat yield and reproduction traits: age at first
calving, calving interval, days open, and insemination interval. In total 296 blood samples of Slovak Spotted and
85 hair roots samples of Pinzgau cows were analyzed. In
order to detect LEP/Sau3AI (BTA 4, inron 2) and LEPR/
T945M (BTA 3, exon 20) genotypes PCR-RFLP method
was used. In Slovak Spotted and Pinzgau cows were the
allele frequencies 0.838/0.162 and 0.694/0.306 for A and
B LEP variants, and 0.954/0.046 and 0.912/0.088 for C
and T LEPR variants, respectively. For testing the associations between SNPs LEP/Sau3AI and LEPR/T945M
and evaluated traits the General Linear Model (GLM)
procedure in SAS Software was used. Statistical analysis
showed that the SNP LEP/Sau3AI significant affected
milk, protein and fat yield (P
Chromosome segregation during meiosis is a complex

process which leads to production of four haploid gametes from two precursor cells. Reversible phosphorylation of proteins plays a crucial role in this process. The
Schizosaccharomyces pombe Prp4 is an essential serine/
threonine kinase which belongs to the Clk/Sty family.
To study the role of Prp4 in meiosis, we analysed chromosome segregation in a strain carrying analog-sensitive
allele of Prp4 (prp4-as2). Our data show, that Prp4 plays
important role in chromosome segregation during meiosis, as revealed by enhanced missegregation of chromosomes in prp4-as2 mutant cells.
Acknowledgements
This work was supported by the Slovak Research and Development
Agency under the contract No. APVV-0636-11.
Slovak University of Agriculture, Tr. A. Hlinku 2, 94976 Nitra,

Slovakia; 2Max F. Perutz Laboratories, Dr. Bohr-Gasse 9,
A-1030 Vienna, Austria
Acknowledgements
Agency under the Contract No. APVV-0636-11.
162
Eurobiotech 2013
P10.19
P10.20
Genetic diversity in populations of Slovak

Spotted cattle based on snps analyses
Analysis of Slovak Spotted breed for

bovine beta casein A1 variant as risk factor
for human health
Nina Moravkov, Anna Trakovick, Alica Navrtilov

Slovak University of Agriculture in Nitra, Department of Animal
Genetics and Breeding Biology, Tr. A. Hlinku 2, 949 76 Nitra,
The Slovak Republic
Martina Miluchov, Michal Gbor, Anna Trakovick
The aim of this study was identification of SNPs in leptin

(LEP), leptin receptor (LEPR) and growth hormone (GH)
genes in order to analyze genetic diversity of Slovak Spotted cattle. The total numbers of blood samples were taken from 353 Slovak Spotted cows originating from four
farms. Genomic DNA was isolated by phenol-chloroform
extraction method and analyzed by PCR-RFLP method.
After digestion with restriction enzymes were detected in whole population of cows alleles with frequency:
LEP/Sau3AI A 0.83 and B 0.17 (0.0141); LEPR/BseGI C
0.96 and T 0.04 (0.0076) and GH/AluI L 0.69 and V 0.31
(0.0173). Based on the observed vs. expected genotypes
frequencies populations across loci were in Hardy-Weinberg equilibrium (P > 0.05). Predominant for SNP LEP/
Sau3AI was AA genotype (0.69), for SNP LEPR/T945M
CC genotype (0.92), and for SNP GH/AluI was dominant
LV genotype (0.47). The observed heterozygosity of SNPs
across populations was also transferred to the low or median polymorphic information content 0.25 (He 0.28),
0.07 (He 0.08) and 0.33 (He 0.42) for LEP, LEPR and GH
genes, respectively. Within genetic variability estimating
negative values of fixation indexes FIS (0.09 0.05) and
FIT (0.07 0.03) indicating heterozygote excess were
observed. The value of FST indexes (0.018-0.023) shows
very low levels of genetic differentiation in allele frequencies of loci among evaluated subpopulations. The low values of genetic distances (0.00180.0159) indicated high
genetic relatedness among animals in subpopulations
probably caused of common ancestry used in breeding
program of farms.
The goal of work was identification A1 variant of bovine

beta casein which involves ischemic heart disease and
diabetes mellitus in human. The digestion of A1beta casein can result in the production of the bioactive beta
casomorphin-7 (BCM-7), this is not the case with A2.
This bioactive peptide has been linked to physiological
traits that may elicit effects on components of the vascular and immune systems. The material involved 111 Slovak Spotted breed. Bovine genomic DNA was extracted
from whole blood by using commercial kit and used in
order to estimate beta casein genotypes by means of
PCR-RFLP method. The PCR products were digested
with DdeI restriction enzyme. In the population included
in the study were detected all three genotypes, homozygote genotype A1A1 (14 animals), heterozygote genotype
A1A2 (37 animals) and homozygote genotype A2A2
(60 animals). In the total population of cattle homozygotes
A2A2 0.5405 were the most frequent, while homozygotes A1A1 0.1261 were the least frequent ones. This
suggests a superiority of allele A2 (0.7072) which does
not produce BCM-7 and thus is safe for human consumption. The expected homozygosity for gene CSN2 is
in the population stated a slight increase in homozygosity
(0.5858). This caused a slight decrease in the level of possible variability realization (41.80%), which corresponds
to the level of polymorphic locus (1.7071).
Acknowledgements
Agency under the contract No. APVV-0636-11.
Slovak University of Agriculture in Nitra, Department of Genetics

and Breeding Biology, Tr. A Hlinku, 2, 949 76 Nitra, Slovakia
Acknowledgements
This work has been supported by the grants: The Slovak Research
and Development Agency under the contract No. LPP-0220-09 and
No. APVV-0636-11.
Eurobiotech 2013
163
P10.21
P10. 22
Analysis of CAPN1 and CAST gene

polymorphisms in native dual purpose
breeds of cattle in Slovakia
Allatostatins as potential bioinsecticides?
Michal Gbor, Martina Miluchov, Anna Trakovick
Slovak University of Agriculture in Nitra, Department of Genetics

and Breeding Biology, Tr. A. Hlinku 2, 949 76 Nitra, Slovakia
The aim of this work was to optimize fast, reliable and efficient modification of the PCR methods for identification
of polymorphism for candidate genes calpain 1 (CAPN1)
and calpastatin (CAST) and to analyze the genetic structure in population of 145 animals of Slovak Spotted
breed and 137 animals of Slovak Pinzgau breed. Genetic
structure for the selected breeds was evaluaeted by using
three markers of CAPN1 gene (CAPN316, CAPN4685,
CAPN4751) and three markers of CAST gene (CAST-T1,
CAST-UoG, CAST-WSU). Genomic DNA was isolated
from whole blood, sperm and hairs by using commercial
column kit. The study of CAPN1 and CAST gene polymorphisms was done by using molecular-genetics methods such as PCR-RFLP, ARM-PCR and HRMA. Genetic
analysis confirmed a higher frequency of preferred alleles
(C; C; A; C) and genotypes (CC; CC; AA; CC) of markers CAPN316, CAPN4751, CAST-T1 and CAST-UoG associated with meat tenderness, in Slovak Pinzgau breed
unlike Slovak Spotted breed, which was characterized by
the lowest frequence of preferred alleles and genotypes
of these markers. For marker CAPN4685 associated with
ahigher lean share in valuable cuts was detected the prevalence of favourable allele T and genotype TT in Slovak
Pinzgau breed too. Our primary results suggest, that the
Slovak Pinzgau breed has favourable genetic base for improvement of quality meat such as tenderness in compare
with the Slovak Spotted breed, which has markedly prevalency of favourable allele C for marker CAST-WSU which
is associated with longevity and reproduction traits.
Acknowledgements
This work has been supported by the projects APVV No. LPP-0220-09
and APVV-0636-11.
Jan Lubawy1, Arkadiusz Urbaski2, Szymon Chowaski1,

Ewelina Paluch3, Mariola Kuczer4, Grzegorz Rosiski1
Department of Animal Physiology and Development,
Adam Mickiewicz University in Poznan; 2Department
of Systematic Zoology, Adam Mickiewicz University in Poznan;
3
Department of Plant Physiology, Adam Mickiewicz University
in Poznan; 4Faculty of Chemistry, University of Wroclaw
Neurohormones synthesized by neuro-endocrine system

are importnt factors regulating processes of growth and
development, reproduction and behavior in insects. The
quick development of analytical techniques has lead to
discovery of more than 30 families of peptide neurohormones. In recent years research is conducted on usage of
these compounds in agriculture and medicine. There are
being designed, on the basis of naturally occurring neurohormones, new biostable pseudopetides and peptidomimetics which can be used as environmentally friendly
pesticides. One of vast group of insect neurohormones
are allatostatins, which show pleiotropic activities influencing a number of physiological processes in insects,
such as inhibition of the synthesis of juvenile hormone
in corpora allata of insects, regulation of visceral muscle
contraction, synthesis and release of vitellogenins from
fat body or synthesis of digestive enzymes. Such features
made them interested for scientists and peptide analogs
based on them are now tested on fighting with mosquitoes or aphids.
In this paper we, present the effects of synthetic allatostatin Grybi-AS B1 (GWQDLNGGWa) on haemocytes
morphology, phenoloxidase (PO) activity and oviduct
contractions of Tenebrio moitor beetle, a synanthrope
which is a pest of cereal. The studies show that activity of
PO has increased after application of 500 pM and 50 nM
of peptide. Simultaneously the peptide caused morphological changes in cytoskeleton structure of haemocytes
of the beetle. Bioassays with oviduct have shown biomodal myotropic activity of Grybi-AS B1, at lower concentrations (1012109 M)it increases endogenic muscle
contractions and at the higher concentrations (106 and
105 M) it nhibits contractions of oviduct.
164
Eurobiotech 2013
P10.23
Relationships between type traits
and functional productive life in Slovak
Holstein Cows
Eva Strapkov, Peter Strapk, Juraj Candrk
Slovak University of Agriculture in Nitra
The relationships between type traits and functional productive life were analyzed in 60208 Slovak Holstein cows
first calved from 1996 to 2010. Functional productive
life was defined as the number of days from first calving
to death, culling or censoring. All cows were scored for
conformation during the first lactation. Weibull models
were fitted to analyze the data. The strongest relationship
between estimated breeding values for survival and type
traits were found for rear legs side view, rump width, angularity, fore udder attachment, dairy strenght, body condition and udder depth. Analyses were done one at atime
for each of those 7 type traits. Based on analyses was
found that cows with shallower udder, moderate fore udder attachment, marked angularity, correct rear legs side
wiev or narrower rump achieved longer productive life.
Evaluation of body condition confirmed that very thin or
very fat cows reached higher involuntary risk of culling.
Acknowledgements
Financial support was provided by the project APVV-0636-11 and
KEGA 027SPU-4/2012.
Legislation for biotechnologists

Lectures
L11.2
L11.1
Genetically modified soybean with altered

fatty acid profile and direct consumer
benefits
Recent developments in gene and stem cell

patenting in Europe
Rainer Friedrich, Elisabeth Greiner
df-mp Patents Trademarks Designs
The presentation will introduce you to requirements and

limitations regarding the protection of your biotechnological inventions in Europe.
Andrew Tommey
DuPont Pioneer, Avenue des Arts 44, 1040 Brussels, Belgium
Commodity soybean oil has been widely used in the food

industry for many years and is often hydrogenated by
oil processors in order to increase its stability and
shelf life. Whilst this results in a functionally stable and
longer lasting partially hydrogenated soybean oil, the hydrogenation process also leads to an increase in trans fats
in the final product and this can impact on cholesterol
levels thereby increasing the risk of heart disease. These
issues have been addressed through genetic modification
of the fatty acid profile in soybean (GM soybean event
DP-35423-1) and the resultant oil from the high oleic
soybeans is zero trans fat providing direct benefits to the
final consumer, in addition to benefits for the farmer and
food industry. Food producers have continued to test
this product and interest is high due to the improved
oil profile of these soybeans allowing food companies to
bring enhanced, healthier products to the market.
This GM soybean product has now been approved by regulatory authorities for either cultivation and/or import,
food and feed uses in all major parts of the world with the
exception of the European Union. The EU risk assessment
and regulatory approval processes are exceedingly stringent and timelines are lengthy given the quantity and
quality of safety data requested as well as the time taken
for review by the EU authorities. However, the European
Food Safety Authority (EFSA) is now close to completing
its risk assessment and Member States in the EU should
soon be voting as to the formal approval of this healthy
high oleic soybean product.
166
Eurobiotech 2013
L11.3
L11.4
Genetically modified plants

in Polish agriculture
Biotechnology regulation and society,

Anno Domini 2013
Sawomir Sowa, Janusz Zimny
Tomasz Twardowski
Plant Breeding and Acclimatization Institute National Research

Institute, Biotechnology and Cytogenetic Department,
GMO Controlling Laboratory
Institute of Bioorganic Chemistry PAN, Poznan, Poland
Green biotechnology plays an important role in bioeconomy strategies in both developed and developing
countries. According to the Strategy Europe 2020 bioeconomy is a key element for smart and green growth
in Europe. Now it is obvious that Europe needs new
approaches to production and consumption in order to
meet global environmental and economical challenges.
In 2012 genetically modified plants were cultivated in 28
countries worldwide and its role in food/feed production
is increasing steadily in the last years. On the other hand
Europe is very reluctant in adoption of GM plants but
remains big GM feed importer. Poland is one of the few
EU countries where GM plants have been cultivated in
the past years on commercial scale. In South of Poland
where infestation with the European corn borer (Ostrinia nubilalis) pose severe damages to maize production
Polish farmers used to grow authorized for cultivation in
EU genetically modified maize varieties. As from 28th of
January 2013 regulation of Polish Council of Ministers
prohibited the use of MON810 maize seeds and Amflora
potato the only GM events approved for cultivation in
EU. Poland along with Austria, France, Hungary become
one of the European countries where GM plants are not
being cultivated. Wide adoption of genetically modified
plants in European and Polish agriculture could be important not only to ensure food security, reduce dependence on non-renewable resources but also to create jobs
and maintain competitiveness on the world market. Currently GM plant varieties are not available to Polish farmers but recent advancements in innovative plant breeding
techniques brings new insight to application of genetic
engineering in Polish and European agriculture.
It is too late to close the door on GM products: biomaterials, bioenergy, biopharmaceuticals as well as food and
feed. The political economy of biotechnology policies
depends on consumers. In the United Europe the consumers (= the voters) said no to the GM farming giants in
several states, however that didnt stop millions of tones
of GM soya and corn entering the European food chain.
Legislation including IPR, innovative technology, public
acceptance and many more factors are critical for the further development of biotechnology.
Eurobiotech 2013
L11.4
Posters
European patents for human embryonic

stem cells
P11.1
Ewa Waszkowska
Conjoint duty for biotechnologists
Patent Office of the Republic of Poland
Sebastian Kwiatkowski1, 2, 3
167
University of Warsaw; 2Faculty of Biology; 3Students Scientific

Association of Genetics and Epigenetics UW
1
Human embryonic stem cells hold great promise for advancing human health. However, patents for the use of
human embryonic stem cells have been the subjects of
extensive public discussion and are regarded as extremely
problematical. The ethical and legal problems are associated.
In October 2011, The Court of Justice of the European Union issued its decision in Brstle v Greenpeace
(C-34/10), which relates to the patentability of technology based on the use of human embryonic stem cells.
The European court ruled that German scientists could
not patent a technique based on human embryonic stem
cells because it involved the destruction of something
capable of commencing the process of development of
ahuman being in other words a human embryo.
The definition of a human embryo used by the court included types of artificially created embryos covering
these that are not capable of developing into a fetus.
Is this definition too broad?
The initial aim of patents is to promote innovation and
reward scientific efforts.
The ban on patenting human stem cell research could
harm its future development in Europe?
First of all, when we are talking about biotechnology as

interdisciplinary science which could solve global famine
problem, we have to emphasize the importance of GMOs
together with the attitude towards public opinion this
technology and, finally, legislation which concerns all colours of biotechnology [1].
Everybody knows how controversial all kind of genetic
modifications of: bacteria, plants and animals could be.
All genetic modifications, including genes coding Bt toxins and EPSP synthase, are not acceptable by public opinion because of the minimal knowledge about biology and
biotechnology. The different cause is simply the fear as
aresult of the unknown. Other reason of that state is lack
of trust in science and scientists [2].
To conclude, fear of unknown and lack of trust are the
reasons of unfavorable legislation in several countries
in European Union (e.g. in Poland). On the other hand
there are countries (e.g. Great Britain or Czech Republic)
in which attitude towards public opinion and legislation
are more favorable for biotechnology [3].
In my opinion one of the most important duties for all
biotechnologists is taking care of improvement opinion
about biotechnology. Especially nowadays, when some
organisations are showing scientists (above all biotechnologists) as corrupt and danger for society.
References
[1] Twardowski T. (ed.) (2012) Aspekty Spoeczne i Prawne Biotechnologii. Polska Akademia Nauk.
[2] Aerni P. (2013) Resistance to agricultural biotechnology: The
importance of distinguishing between weak and strong public attitudes.
Biotechnology Journal. doi: 10.1002/biot.201300188.
[3] USDA Foreign Agricultural Service (2013) Agricultural Biotechnology Annual. Global Agricultural Information Network.
Induced pluripotent stem cells: a future of biomedicine
Lectures
L1.2
L1.1
There and back again: short history

of reprogramming. From a somatic cell
to an inducible pluripotent cell
Hypoxia and oxidative stress

in reprogramming and differentiation
Alicja Jozkowicz
Department of Medical Biotechnology, Faculty of Biochemistry,
Biophysics, and Biotechnology, Jagiellonian University,
Gronostajowa 7, 30-387 Crakow
Human induced pluripotent stem cells (hiPS) are generated by epigenetic reprogramming of somatic cells through
the exogenous expression of four transcription factors
(Oct4, Klf4, Sox2, c-Myc). These cells has the all characteristic features of human embryonic stem cells (hES)
as self-renewal and ability to differentiate to cells of all
three germ layers. Growing body of published data clearly
shows that that epigenetic mechanisms are involved in the
process of reprogramming of somatic cells into pluripotent state. TRIM28/KAP1 protein is one of the epigenetic regulators that regulate heterochromatin formation
through histone modifications and DNA methylation.
Over 500 unique zinc finger proteins containing KRAB
domain recruit TRIM28/KAP1 protein to genomic DNA
in sequence-specific fashion thus leading to transcriptional repression of neighboring genes. Here, we showed
that TRIM28/KAP1 protein controls self-renewal of hES
and hiPS cells. Stable knockdown of TRIM28/KAP1 gene
expression in both hES and hiPS cells achieved using lentiviral vectors carrying specific shRNAs resulted in progressive loss of pluripotent phenotype as confirmed by
analysis of surface embryonic markers expression using
immunofluorescence and FACS analyses. Moreover, we
have documented progressive loss of expression of selected embryonic transcription factors in hES and hiPS cells
depleted of TRIM28/KAP1 exkpression. Our ongoing experiments are focused on detailed analysis of the molecular mechanisms that are involved in the observed phenotype and may shed new light on epigenetic mechanisms
that preserve self renewal of hES and hiPS cells.
Maria Anna Ciemeryc

Department of Cytology, Faculty of Biology,
University of Warsaw, Miecznikowa 1, 02-096 Warsaw
Embryonic stem cells (ESCs), derived from prelimplantation embryos at the blastocyst stage, are unique in unlimited self-renew ability and pluripotency allowing their
differentiation into any cell type. Until the beginning of
XXI century ESCs were considered as the only cell lines
which pluripotency was proved both in vitro and in vivo.
In 2006 first paper describing reprogramming of fibroblasts into sa called induced pluripotent cells (iPSCs) was
published. The basis of this spectacular achievement done
by Yamanaka and his collaborators was establised in 50s
and 60s of the XX century. The short history of the cellular
reprogramming and pluripotent stem cells research that
led to the Nobel Prize for Yamanaka and Gurdon will be
a topic of this talk.
Eurobiotech 2013
169
L1.3
L1.4
Epigenetic mechanisms of human induced

pluripotent stem cells (iPS)
The importance of peeing earnest: urine

derived cells as model systems in biology
and biomedicine
Maciej Wiznerowicz1, 2
Gene Therapy Laboratory, Department of Cancer Immunology,
Poznan University of Medical Sciences, Chair of Medical
Biotechnology, Poland; 2Gene Therapy Laboratory,
Department of Cancer Immunology, Greater Poland Cancer
Centre, Poland
1
Induced pluripotent stem cells (iPS) cells are generated by dedifferentiation of adult cells through the forced
expression of few embryonic transcription factors. The
reprogramming process involves ordered and global
epigenetic alterations including histone modifications
and DNA methylation that progressively silence expression of lineage-specific genes whilst enabling transcription of embryonic factors genes. We explored the role
of KRAB-containing zinc finger proteins (KRAB-ZFPs)
and their cofactor TRIM28/KAP1 during the reprogramming process. The results of our work strongly suggest
that knockdown of TRIM28/KAP1 expression facilitate
de-differentiation of human primary fibroblasts towards
the iPS cells. In the other hand the inhibition of TRIM28/
KAP1 function in established human pluripotent cells results in progressive loss of their self-renewal potential in
contrast the the wild-type human iPS cells. Additionally,
our parallel lines of research clearly demonstrated that
binding of TRIM28/KAP1 through KRAB-containing
transcriptional repressors results in specific methylation
of the cellular promoters. Finally, we have revealed that
epigenetic factors recruited by TRIM28/KAP1 controls
retrotransposition events during the reprogramming of
human somatic cells to pluripotency. Taken together, our
results demonstrated novel role for TRIM28/KAP1 protein in the reprogramming process and self-renewal of
human iPS cell as well as their involvement in modeling
landscape of genomic DNA methylation during that process. In-depth understanding of epigenetic mechanisms
involved in the cellular reprogramming and self-renewal of human iPS cells will have implications for basic research and may pave ways to novel therapies.
Ting Zhou3, Christina Benda3, Sarah Dunzinger2,

Matthias Wieser2, Heinz Redl5, Johannes Grillari1,
Miguel A Esteban3, Regina Grillari2
Evercyte GmbH, Vienna, Austria; 2Department of Biotechnology,
BOKU Vienna, Austria 3Key Laboratory of Regenerative
Medicine, Chinese Academy of Sciences, Guangzhou, China;
4
Guangdong Stem Cell and Regenerative Medicine Research
Centre, University of Hong Kong, China; 5Ludwig Boltzmann
Institute for Clinical and Experimental Traumatology, Vienna,
Austria, 6Austrian Cluster for Tissue Regeneration, Vienna,
Austria
1
Human cell cultures are of ever increasing importance in

many scientific areas ranging from basic biology, biotechnology to medicine. In all these fields cells find application as model systems, products and producers. Especially their use as model systems to understand cellular behaviour or pathogenic conditions relies on the availability
of sufficient quantities of cells.
We have therefore established several cell lines that closely resemble their primary counterparts in key characteristics. Among these cells range endothelial cells, adult
stem cells and renal proximal tubular epithelial cells
(RPTECs). During these studies, we realized that RPTEC
like cells are exfoliated into urine. These cells can be
converted into iPS cells with high efficiency, and can be
re-differentiated into neuronal cells, hepatocyte-like cells
and cardiomyocytes.
As these cells are derived from a non-invasive source that
is not exposed to DNA damaging UV radiation like skin,
we propose urine to become the preferred source for generating iPSCs in many instances. The ease of this method may facilitate the standardization of iPSC technology,
will boost the generation of cell based disease model systems and is also an advance in the direction of clinical use
of iPSCs.
170
Eurobiotech 2013
L1.5
L1.6
Stem cells in drug development

and toxicity testing: implementation
of emerging technologies
Use of cell reprogramming technologies

for neurobiological research
Leonora Buzanska
International Institute of Molecular and Cell Biology
Stem Cell Bioengineering Laboratory, NeuroRepair Department

Mossakowski Medical Research Centre, 02-106 Warsaw,
Poland
Developing reliable human-based in vitro systems to

study drug toxicity and their mode of action is a major
challenge for establishing new and safe therapies. The
emerging technologies involved in the creation of such
systems combine advancement in stem cell research with
bioengineering of the stem cell niche. This allows creating microscale biomimetic microenvironments on high
content/throughput platforms to test response of cells
representing relevant human disease models with specific
genetic background.
The generation of disease and patient-specific cell lines
become possible during the last few years due to development of induced pluripotent stem cells (iPSC) technology. iPSCs can be obtained from any tissue of the body
by ectopicly delivered transgenes of specific sets of transcription factors, thus circumventing both ethical and
immunological limitations implicated by the use of human embryonic stem cells. The iPSC-based disease models tested on microengineered drug discovery platforms
have been already shown to be useful in filling the gap
between animal testing and clinical trials.
The important role of human embryonic and somatic
tissue-derived stem cell models for drug discovery and
toxicity testing, especially in the aspect of developmental biology, was widely proven during the last decade and
such examples, including the results of our group, will be
presented. However the iPSC technology provides a giant advancement step for pharmacology and regenerative
medicine applications, enabling personalized testing, diagnosis and treatment.
This lecture will provide the state of the art on the development of human-based in vitro systems with stem cells
and micro/nano engineered cell-growth platforms, used
to test a variety of compounds and possibly to correct the
disease-related phenotypes.
Acknowledgements
Supported by: MSHE grant No. N N302597838 and status funds of
Mossakowski Medical Research Centre.
Jacek Jaworski1, Ewa Liszewska
Reprogramming of somatic cells made possible to study

in vitro inaccessible human cells, such as different types
of neurons. Almost immediate consequence of the emergence of this technology was the development of a number of cellular models of the nervous system diseases.
They are used both to explore the cellular mechanisms
of these diseases and for the development of new pharmacological strategies. Reprogrammed cells are also
apotential alternative to embryonic stem cells for transplantation. During my talk I will introduce methods used
for cellular reprogramming for neurobiological research
(e.g. reprogramming to iPS vs. direct reprogramming to
neurons). I will present the most important achievements
in the use of cell reprogramming technology in modeling
of nervous system diseases such as spinal muscular atrophy, Rett Syndrome, schizophrenia and neurodegenerative diseases. I will also illustrate how reprogrammed cells
can be used for potential therapy of spinal cord injuries.
At the same time, I will point out the limitations of the
methodology and the expected directions of its development. Finally I will present preliminary results of my
group in establishing cellular models of Tuberous Sclerosis, a developmental disease caused by mutation in genes
encoding either hamartin or tuberin and manifested by
epilepsy and autism.
Acknowledgements
Authors express their gratitude to Prof. K. Kotulska and S. Jozwiak
for sharing skin biopsies. This work is financed by ERA-NET
NEURON/06/2011 AMRePACELL (co-financed by NCBiR) to JJ and
Homing-Plus (HOMING PLUS/2012-5/6) of Polish Science Foundation
to EL.
Eurobiotech 2013
Posters
P1.2
P1.1
Differentiation of iPS toward

endothelial cells
Role of heme oxygenase-1 in generation

and differentiation of iPS cells
Jacek Stepniewski, Tomasz Pacholczak, Alicja Jozkowicz,
Jozef Dulak
Department of Medical Biotechnology, Faculty of Biochemistry,
Biophysics, and Biotechnology, Jagiellonian University,
Gronostajowa 7, 30-387 Crakow
Reprogramming of somatic cells which leads to the

generation of induced pluripotent stem cells (iPSCs) is
a complex process requiring many modifications at the
level of gene expression profile, metabolism and chromatin structure. However, the initial response to the viral
vectors delivering reprogramming transcription factors
(Oct4, Sox2, Klf4 and c-Myc) involves the elevation in
production of reactive oxygen species (ROS). This may
activate Nrf2 transcription factor master regulator of
response to oxidative stress and heme oxygenase-1
(HO-1), one of its target genes. T
h e aim of our study was
to evaluate the role of these two cytoprotective proteins
in generation and differentiation of iPS cells. It appeared
that the lack of either Nrf2 or HO-1 decreased the efficiency of reprogramming. Accordingly, induction of their
expression or activity resulted in higher number of generated iPS colonies. We also observed that the level of HO-1
was elevated in the later stages of reprogramming. Interestingly, stimulation of murine fibroblasts with valproic
acid, which was previously demonstrated to increase the
reprogramming outcome, resulted in the lower HO-1 level in the cells. Accordingly, this compound decreased the
reprogramming efficiency in our experiments. Obtained
iPS cells were further cultured and characterized. Interestingly, we observed that lack of Nrf2 can increase the
number of contracting bodies in spontaneously differentiating cells. These results indicate that Nrf2-pathway play
a role in the reprogramming process and in differentiation of iPSC.
171
Neli Kachamakova-Trojanowska1, Michael Beilharz3,

Karolina Bukowska-Strakova1, Jacek Stpniewski 3,
Antonia Chmura-Skirliska2, Jozef Dulak3, Alicja Jozkowicz3
Laboratory of Molecular Pharmacology of Endothelium;
Laboratory of EPR Spectroscopy, JCET; 3Department
of Medical Biotechnology, Faculty of Biochemistry, Biophysics,
and Biotechnology, Jagiellonian University, Gronostajowa 7,
30-387 Crakow
1
2
Induced pluripotent stem (iPS) cells can be a very

useful model for studies of disease-specific endothelial
dysfunctions, especially those dependent on genetic
background of patients. Our aim was to elaborate
a method for differentiation of iPS toward endothelial
cells.
Tail-tip fibroblasts were isolated from the wild type
C57Bl6 mice and then used for generation of iPS cells
by delivery of four reprogramming factors (Sox2, Klf4,
c-Myc and Oct4) with a single lentiviral vector. Pluripotency of resulting cells was confirmed by staining for
SSEA-1 or NANOG and by teratoma formation. Before
starting the differentiation, two distinct cell populations
were present in the cultures, defined by expression of
either CD31 or CD34. They shifted with time towards
a CD34+CD31- phenotype with a rising percentage of
CD34+Tie-2+ double positive cells. However, almost no
CD31 or CD105 could be detected. Tie-2 and KDR were
also found up-regulated at RNA level. Nevertheless, no
KDR positive cells could be observed by flow cytometry.
Importantly, immunocytochemical staining revealed,
that the cultured cells were positive for activated endothelial nitric oxide synthase (eNOS, phosphorylated at
Ser1177) and positive for von Willebrand factor. Moreover, eNOS functionality was confirmed by detection of
nitric oxide production. Direct angiogenic capacity of the
generated endothelial cells was tested by matrigel tube
formation assay and by outgrowth of capillaries from
spheres embedded in collagen gel. No tube formation
was observed after 24 h or later time-points on matrigel,
unless the iPS-derived cells were cultured together with
mature endothelial cells. Also the sphere formation was
delayed in comparison to endothelial cells, as the process took 6 days, instead of 1-2 days. However, after embedding of the spheres in collagen the pronounced outgrowth of capillary network was observed, confirming
the endothelial activity.
To sum up, murine iPS cells can be differentiated into
endothelial-like cells, with some immunophenotype features and angiogenic activities typical for endothelium.
They resemble the phalanx cells (like in stable arteries),
more than tip or stalk cells.
172
Eurobiotech 2013
P1.3
P1.4
Application of epigenetic switch

for generation of human IPS cells
Dynamics of retroelements activity during

reprogramming of human fibroblast
to induced pluripotent stem cells (iPSc)
Joanna Wrblewska, Katarzyna Kulcenty,

Urszula Oleksiewicz1, Wiktoria Suchorska1, 2, Andersen Jannik4,
Giulio Draetta4, Gustavo Mostoslavsky, Maciej Wiznerowicz,
Biotechnology, Poland; 2Gene Therapy Laboratory, Department
ofCancer Immunology, Greater Poland Cancer Centre, Poland;
Boston University School of Medicine, Dept. of Medicine,
Boston, MA, USA; 4Institute for Applied Cancer Science,
University of Texas MD Anderson Cancer Center, Houston, TX,
USA
1
Human induced pluripotent stem cells (IPS) are generated by reprogramming of somatic cells through enforced
expression of embryonic transcription factors. However,
clinical applications require that expression of introduced
transgenes must be permanently switched off in the IPS
cells and obtained differentiated progenies. Here, we took
advantage of epigenetic switch that relies on reversible
binding of tTRKRAB transrepressor to tetO element,
which results in tight transcriptional repression of proximal promoter through heterochromatin formation. In
order to apply this system for reprogramming, the tetO
element was inserted into pSTEMCCA lentiviral vector
carrying OCT4, SOX2, KLF4 and cMYC under control
of EF-1alpha promoter. Transduction of human skin fibroblasts with obtained pSTEMCCA-tetO allowed for
expression of reprogramming factors and thus efficient
generation of human iPS clones. Obtained clones were
picked and further cultured until establishing stable IPS
cell lines. Then cells were then transduced with lentiviral
vector pLV-HK carrying tTRKRAB, in order to switch off
reprogramming transgene expression.
Tight repression of introduced transgenes in all human
IPS clones was analyzed by RT-PCR and confirmed
full functionality of our system. Obtained IPS cell lines
showed no abnormalities in karyotypes. Pluripotent phenotype of IPS cells was revealed by analysis of endogenous embryonic genes expression using RT-PCR and
immunofluorescence staining. Analyzed cells were also
able to form embryonic bodies in vitro and teratomas in
immunocompromised mice, which proved their ability
to differentiate into cells derived from three germ layers. tTRKRAB-mediated epigenetic repression persisted
through prolonged culture of obtained IPS cell lines. Importantly, expression of introduced transgenes remained
undetectable after differentiation into embryonic bodies.
In order to confirm molecular homogeneity of obtained
IPS cell lines, high throughput molecular profiling including RNA-Seq and global DNA methylation analysis
are currently being performed.
Our results confirm that our epigenetic switch effectively
prohibits re-expression of embryonic transgenes in human IPS cells and their differentiated progenies paving
the way for their applications in various fields of regenerative medicine, disease modeling and drug discovery.
Katarzyna Tomczak1, 2, Urszula Oleksiewicz2,

Postgraduate School of Molecular Medicine, Medical University
of Warsaw, Warsaw; 2Gene Therapy Laboratory, Department
of Cancer Immunology, Poznan University of Medical Sciences,
Chair of Medical Biotechnology, Poland; 3Gene Therapy
Laboratory, Department of Cancer Immunology, Greater Poland
Cancer Centre, Poland
1
Retroelements constitute around 90% of all transposable

elements in the human genome. These endogenous parasite sequences are able to move through the genome from
one location to another contributing to genome diversity
and disease, including cancer. The expression of mobile
elements is regulated by epigenetic mechanisms including histone modifications and DNA methylation. Intensive epigenetic remodelling of the chromatin structure is
important process occurring during cellular reprogramming and formation of induced pluripotent stem cells
(iPS) through forced expression of selected transcription
factors. Here, we investigated the dynamics of retroelement activity during generation of induced pluripotent
stem cells (iPS). Our research including molecular and
biochemical analysis confirmed that cellular reprogramming process induces activation of retroelements (such
as LINE-1) in human cells. In order to obtain insight into
epigenetic mechanisms of the observed phenomena we
analysed the role of chosen protein in regulation of retroelements activity during formation of human iPS cells.
Our ongoing experiments are aimed towards profiling of
dynamics of retroelements activity during reprogramming of human cells depleted of selected epigenetic factors gene expression. Our project will contribute to better
understanding of the molecular mechanisms that control
retroelements during cellular reprogramming and are involved in host defence against these genomic parasites.
Eurobiotech 2013
P1.5
P1.6
KAP1 protein is involved in reprogramming

of human induced pluripotent stem cells
(iPS)
TRIM28/KAP1 triggers DNA methylation

of cellular promoters during
reprogramming of human fibroblasts
to induced pluripotent cells
Katarzyna Kulcenty1, Urszula Oleksiewicz1,

of Cancer Immunology, Greater Poland Cancer Centre, Poland
1
Induced pluripotent stem cells (iPSC) are derived from

human somatic cells through ectopic expression of few
embryonic transcription factors. They have two unique
properties-self-renewal and pluripotency. Their potential
raises the possibility to develop variety of novel clinical
applications in patient specific regenerative medicine.
Recent studies of human iPS cells implicate, that epigenetic mechanisms are involved in the process of reprogramming of somatic cells into pluripotent state. Here
we probe, that KAP1 protein together with transcription
repressors carrying KRAB domain regulate expression
of specific genes through histone modification and DNA
methylation.
First in our experiments, we achieved efficient knockdown of KAP1 in human fibroblast using lentiviral vectors carrying KAP1 specific shRNA. Then, our KAP1
knockdown cells together with wild type were reprogrammed using lentiviral vector carrying cDNA for four
transcription factors: Oct4, Sox2, Klf4 and c-Myc. Pluripotent phenotype of emerging iPS cells lines was revealed
by analysis of cell surface protein markers by immunofluorescence and flow cytometry analysis. The effect of
KAP1 knockdown on the reprogramming process was
revealed by growth kinetics of the emerging iPS colonies.
Our results show, that KAP1 knockdown accelerates the
emergence of iPS colonies at early stages of reprogramming process. At this early stage, KAP1 knockout cells
show higher expression of epithelial markers as well as
markers characteristic for pluripotent state (alkaline
phosphatase, SSEA4, Tra-1-81, Nanog). Our results implicate, that epigenetic mechanisms controlled by KAP1
protein are involved in the reprogramming process.
173
Marta Gadych1, Urszula Oleksiewicz1, Maciej Wiznerowicz1, 2

1
Induced pluripotent stem cells (iPSCs) are generated by

dedifferentiation of adult cells through the forced expression of few embryonic transcription factors. The
reprogramming involves ordered and global epigenetic
alterations including histone modifications and DNA
methylation that progressively silence expression of lineage-specific genes whilst enabling transcription of embryonic factors. TRIM28/KAP1 protein is potent epigenetic modulator that induce heterochromatin formation
when recruited to DNA in sequence-specific fashion
by zinc finger proteins containing KRAB domain thus
leading to transcriptional repression of neighboring
genes through histone modifications and HP1 recruitment. Exclusively during early embryonic development,
TRIM28/KAP1-mediated epigenetic repression leads to
DNA methylation that is largely restricted to repetitive
elements thus protecting the genomes stability. Here, we
have shown that trigger DNA methylation of cellular promoters during the reprogramming of human primary fibroblasts to iPS cells. Conditional repression of PGK promoter was achieved by chimeric transrepressor (tetRKRAB) containing KRAB domain fused with DNA binding
domain of tetracycline repressor (tetR) that binds to tetO
element from E. coli tetracycline operator in doxycycline
(dox)-controllable manner. The tetRKRAB transgene and
tetO-PGK-GFP expression cassette were stably integrated
into genome of human primary fibroblasts using lentiviral
vectors. Next, the engineered cells were reprogrammed to
pluripotency by forced expression of Oct4, Klf4, Sox2 and
Myc genes, either in presence (dox+) or absence (dox)
of doxycycline. Phenotype of obtained iPS cell lines was
confirmeed by immunoflurescence analysis of expression of selected embryonic markers (NANOG, SSEA4,
TRA1-60 and TRA 1-81). Bisulfite sequencing of PKG
promoter that was subjected to tetKRAB-mediated epigenetic repression during reprogramming in the absence
of dox revealed high level of CpG methylation (98,6%).
In contrast, PGK promoter in human iPS cells obtained
from the engineered fibroblasts in the presence of dox
that sequestrated tetRKRAB from the tetO-PGK-GFP
cassette remained largely unmethylated (1,42,8%). Our
results strongly suggest that transcriptional repressors
containing KRAB domain may be involved in changing
landscape of genomic DNA methylation during cellular
reprogramming.
174
Eurobiotech 2013
P1.7
P1.8
Role of TRIM28/KAP1 protein in

reprogramming of mouse fibroblasts
to induced pluripotent stem cells (miPSCs)
Differentiation of induced pluripotent

stem cells (iPSC) into functional neurons
Marta Klimczak1, Anna Misiewicz1, Andrzej Mackiewicz1, 2,

1
Mouse induced pluripotent stem cells (miPSCs) can be

generated mouse somatic cells by forced over-expression
of embryonic transcription factors that most commonly
involve: Oct4, Sox2, Klf4 and c-Myc. miPSCs are highly
similar to embryonic stem cells (ESCs) with reference to
proliferation and differentiation capacity. Therefore, they
provide a promising source of pluripotent cells for not
only basic stem cell biology but also clinical cell-based
therapies and disease modeling. The major goal of this
study is to determine the role of KAP1 protein in reprograming and self-renewal of miPSCs. TRIM28/Kap1 is a
cofactor of KRAB-containing zinc finger proteins (KRAB-ZFPs). These are known as the transcriptional repressors which act by formation of heterochromatin through
histone modifications, HP1 binding and DNA methylation. In the first step, TRIM28/Kap1 expression in primary mouse embryonic fibroblasts (MEF) was silenced
by a mean of lentiviral vectors carrying short hairpin
RNA (shRNA) specific for TRIM28/Kap1. Next, the cells
were reprogrammed to miPSC using lentiviral vector
carrying Oct4, Sox2, Klf4 and c-Myc transgenes. Immunofluorescence analysis of embryonic markers revealed
that TRIM28/Kap1 knockdown results in accelerated reprogramming of mouse fibroblasts to iPS cells. Our preliminary results suggests that repressive chromatin state
imposed by TRIM28/Kap1 prohibits the reprogramming
thus helps to maintain identity of differentiated cells.
Sylwia Mazurek1, Joanna Wrblewska1, Maciej Wiznerowicz1, 2

1
Efficient differentiation of pluripotent stem cells (iPS)

to neural stem cells (NSC), and further to neurons, astrocytes and oligodendrocytes, offers various opportunities including: regenerative medicine, neurological
diseases modelling and neurotoxicity drug testing. iPS
cells are generated by reprogramming of somatic cells
to pluripotency through forced expression of embryonic
transcription factors and serve as potentially unlimited
source of various cell types that can be used in regenerative medicine. In order to induce neuronal differentiation from human iPSC, we used Gibco protocol with
Neurobasal Medium and Gibco Neural Induction Supplement. First neural rosettes appeared after 6 days of
differentiation. Immunofluorescent staining of obtained
cells confirmed presence of neuronal marker (Nestin)
and absence of pluripotency markers (Oct4, NANOG).
The NSC differentiation was confirmed by expression
analysis of selected markers for: pluripotent cells (SOX2)
and neural stem cells (PAX6, SOX1, Nestin). NSC differentiation into neurons, astrocytes and oligodendrocytes
was performed with medium consisting of Neurobasal
Medium, B27 Supplement and L-Glutamine for 14 days,
additionally, during first 7 days of differentiation medium was supplemented with brain-derived neurotrophic
factor (BDNF), glial cell line-derived neurotrophic factor
(GDNF), insulin-like growth factor (IGF), ascorbic acid,
dibutyryl-cAMP. Immunocytochemical staining (Nestin,
Tuj) confirmed differentiation into neurons. The results
of our studies can be applied for generation of functional human neural cells from patient-specific iPS cells and
pave the way for regeneration of central nervous system
injury in future.
Eurobiotech 2013
P1.9
TRIM28/KAP1 protein controls self-renewal
of human induced pluripotent stem cells
Wojciech Barczak1, Katarzyna Kulcenty1, Maciej Wiznerowicz1, 2
1
Human induced pluripotent stem cells (hiPS) are generated by epigenetic reprogramming of somatic cells through
the exogenous expression of four transcription factors
(Oct4, Klf4, Sox2, c-Myc). These cells has the all characteristic features of human embryonic stem cells (hES)
as self-renewal and ability to differentiate to cells of all
three germ layers. Growing body of published data clearly
shows that that epigenetic mechanisms are involved in the
process of reprogramming of somatic cells into pluripotent state. TRIM28/KAP1 protein is one of the epigenetic regulators that regulate heterochromatin formation
through histone modifications and DNA methylation.
Over 500 unique zinc finger proteins containing KRAB
domain recruit TRIM28/KAP1 protein to genomic DNA
in sequence-specific fashion thus leading to transcriptional repression of neighboring genes. Here, we showed
that TRIM28/KAP1 protein controls self-renewal of hES
and hiPS cells. Stable knockdown of TRIM28/KAP1 gene
expression in both hES and hiPS cells achieved using lentiviral vectors carrying specific shRNAs resulted in progressive loss of pluripotent phenotype as confirmed by
analysis of surface embryonic markers expression using
immunofluorescence and FACS analyses. Moreover, we
have documented progressive loss of expression of selected embryonic transcription factors in hES and hiPS
cells depleted of TRIM28/KAP1 exkpression. Our ongoing experiments are focused on detailed analysis of the
molecular mechanisms that are involved in the observed
phenotype and may shed new light on epigenetic mechanisms that preserve self renewal of hES and hiPS cells.
175

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Contents

Nanotechnologies in industrial processes.............................................................................. 3

Development of renewable energy in biotechnology........................................................... 13

Bioplastics and biobased polymers......................................................................................... 26

Plant molecular breeding......................................................................................................... 60

Plant genetic engineering......................................................................................................... 125

Animal biotechnology in biomedicine................................................................................... 134

Animal biotechnology in agriculture...................................................................................... 148

Legislation for biotechnologists............................................................................................... 165

Induced pluripotent stem cells: a future of biomedicine...................................................... 168

Business Session Life Science Open Space

Preface by the Organizing Committee

On behalf of the Congress Organizing Committee

Synthetic Plant Biotechnology Tools

Oxidative Stress Signal Transduction

Neal Stewart, Jr.

Frank Van Breusegem

Department of Plant Sciences, University of Tennessee,

VIB Department of Plant Systems Biology, UGent Technologie

Synthetic biology tools have the ability to greatly increase

Different biotic and abiotic stresses adversely affect plant

Teaching from the Plant Kingdome;

Luuk A.M. van der Wielen

Almost all of the modern infrastructure of our civilization

Delft University of Technology, The Netherlands, Industrial

Nanotechnologies in industrial processes

Use of titanium dioxide for purification,

The use of biofiltration in the rendering plants

The study evaluated the effectiveness of air biofiltration of

Agata Markowska-Szczupak1, Beata Tryba1,

Titanium dioxide is commonly used as a white pigment

angle, thus gives increased self-cleaning capacity and

The human body is exposed to microorganisms such as

Phosphorus cycle possibilies of its

Nanoemulsion as a base to controlled

Katarzyna Gorazda, Zbigniew Wzorek, Anna K. Nowak,

Magorzata Jaworska1, Elbieta Sikora1,

Crakow University of Technology, Warszawska 24 St.,

The recovery of phosphorus from waste can take place

Faculty of Chemical Engineering and Technology,

Forskolin is one of labdane diterpenoids. It is isolated

Studies on the formation of O/W nanoemulsions, by low-energy emulsification

Preparation of Ag nanoparticles using

Present study reports a green chemistry approach for

Magorzata Jaworska1, Elbieta Sikora1, Micha Zielina2,

Influence of process parameters on

Nanometals as tool in fight against plant

Elwira Laso, Elbieta Sikora, Jan Ogonowski

Nanostructured lipid carriers (NLC) are stabile colloidal

Agnieszka Sobczak-Kupiec1, Dagmara Malina1,

In recent years, a great interest in nanotechnology has

pared materials provide good electrical properties. The

Characterization and properties of silver

Silver nanoparticles as an effective

Agnieszka Sobczak-Kupiec1, Katarzyna Bialik-Ws2,

Nowadays nanomaterials and nanotechnology are very

Jolanta Pulit1, Marcin Banach1, Renata Szczygowska2,

Nanosilver is one of the most thoroughly investigated

Thus, according to the varying degrees of effectiveness

The development of nanotechnologies and the related

Development of renewable energy in biotechnology

tion or knock out of ATG13 gene involved in autophagy

Phytic acid (myo-inositol hexa-phosphoric acid, IP6) is