INTRODUCTION

Microalgae:
are a very important component of the aquatic ecosystem;
have the ability to fix CO
2
while capturing solar energy with efficiency 10 to 50
times greater than that of terrestrial plants
higher biomass production compared to energy crops (Wang et al., 2008).

Chlorella:
a unicellular, non-motile green microalga
found both in fresh and marine water.
cells are solitary, very small (2-12 m) and spherical, globular or ellipsoidal in
shape (Sharma, 1986).

Nanoparticles:
loosely defined as manufactured materials that are smaller than 100 nanometer in
at least one dimension,
Have been of scientific interest for several decades, but are now being used in a
wide range of commercial applications (Kulacki and Cardinale, 2012).


1

Introduction Contd

Titanium dioxide (TiO
2
) nanoparticles
An example of manufactured nanosized materials that are already widely used
(Hartmann et al., 2010).
Also known as titanium (IV) oxide or Titania
Is the naturally occurring oxide of titanium, and titanium is the ninth most
abundant element in the world, it is five times less abundant than iron but 100
times more abundant than copper (IARC, 2010).
Generally titanium dioxide is sourced from Ilmenite ore, rutile and anatase, which
are mined from deposits located throughout the world. Ilmenite ore is the widest
spread of titanium dioxide bearing ore in the world. Rutile (TiO
2
) and Ilmenite
(FeTiO
3
) are commonly found as accessory minerals in plutonic and metamorphic
rocks but occur also as detrital minerals in beach sands.
Titanium dioxide (TiO
2
) is primarily used as a pigment because of its brightness,
high refractive index, and resistance to discolouration (Diebold, 2003).
The global production of titanium dioxide for all uses is in the millions of tons
per year. In the cosmetics industry the use of nanosized particles.



2

Introduction Contd
Microalgae require nutrient for their growth

Nitrogen is an important element for algae growth, it is essential in protein synthesis
and in pigment construction.

Phosphorus is also an important nutrient element.
Many important molecules within a cell contain phosphorus. Adenosine
Triphosphate (ATP) is an example of a molecule that uses phosphorus.
As a component of nucleic acids governing protein synthesis and of the adenosine
phosphate transformations that power intracellular transport, phosphorus is an
essential requirement of living, functional plankters.

Nitrogen and phosphorus are the primary nutrients of concern in relation to water
quality issues because they can stimulate primary productivity. (Dodds, 2002).

Nitrogen and phosphorus are often the primary limiting nutrients for aquatic algal
production (Lv, 2011) because they are frequently in short supply relative to cellular
growth requirements.



3
Introduction Contd


Accrual of algal biomass, and thus overall ecosystem productivity, may be controlled
by the type and intensity of nutrient limitation (Dodds et al., 2002)

The term oxidative stress refers to the situation of serious imbalance between
production of reactive species and antioxidant defense.

According to Sies, 1991, it is a disturbance in the prooxidantantioxidant balance in
favour of the former, leading to potential damage.

Antioxidant defense mechanisms include enzymes that catalyze reactions of ROS
scavenging, such as catalase, ascorbate peroxidase, glutathione peroxidase and
superoxide dismutase (Pinto et al., 2003; Mallick, 2004), lipophylic compounds and
free radical scavengers like carotenes and a-tocopherol, reducers like ascorbate and
reduced glutathione (GSH) (Buchanan et al., 2000; Mallick, 2004).


4
Introduction Contd
Oxidative stress also leads to cell death, which occurs by two mechanisms; necrosis
and apoptosis (Halliwell, 2001).

During evolution, living organisms have adapted to the presence of natural
nanoparticles in the environment. For synthetic nanoparticles, however, it is recognized
that their potential harmful properties on ecosystems have to be evaluated (Handy et
al., 2008; Nowack, 2009).


5
STATEMENT OF THE RESEARCH PROBLEM

The potential harmful properties of synthetic nanoparticles on ecosystems have to be
evaluated (Handy et al., 2008; Nowack, 2009).

The increasing presence of nanoparticles in many products has made it all the more likely
that they will also be released into the aquatic environment.

The recent advances in nanotechnology and the corresponding increase in the use of
nanomaterials in products in every sector of society have resulted in uncertainties regarding
environmental impacts (Klaine et al, 2008).

Scientists are concerned for the effects of the synthetic nanoparticles on biological systems
because of their novel appearance in the environment and the lack of protective mechanisms
in the course of biological evolution in living organisms (Valavanidis and Vlachogianni,
2010).

6
STATEMENT OF THE RESEARCH PROBLEM CONTD

The fate and toxicity of engineered nanoparticles in natural waters therefore need to be
studied as the situation is complicated by the existence of a very broad spectrum of
engineered nanoparticles, differing in their chemical, physical and morphological properties
(Behra et al., 2009).

But precisely these properties need to be taken into account in efforts to understand how
nanoparticles act on algae, since they influence the bioavailability of particles and the
mechanisms of toxicity (Behra et al., 2009).

Mechanisms of nanomaterial toxicity include cellular damage due to oxidative stress,
physical damage to the cell surface, dissolution at the cell surface, and impacts via
bioaccumulation. Bioaccumulation via the food chain is also possible. (Batley and
McLaughlin, 2010)


7
STATEMENT OF THE RESEARCH PROBLEM CONTD

According to available literature, impacts of nanoparticles on nutrient availability in aquatic
ecosystems have not been assessed in detail yet. The effect of titanium dioxide nanoparticle
on the nutrient uptake by Chlorella vulgaris needs to be studied.

However, according to Chakraborty, 2009, since nanopaticles interact with ions and diverse
other components, it is very likely that they also interact with nutrients essential for aquatic
organisms especially if these nutrients are present only in very low concentrations, such as
for microelements.

8
JUSTIFICATION

While they may seem insignificant compared to rainforests or other accumulated biomass
on land, algae contribute approximately half of the global primary production as well as
atmospheric oxygen (Aruoja, 2011).

Algae play an important role in the aquatic ecosystem, not only producing biomass that
forms the basic nourishment for food webs, but also contributing to the self-purification of
polluted water.

Due to their importance, algal growth response to existing and potential new environmental
threats has to be clarified.

Changes in the structure and productivity of the algal community may induce direct
structural changes in the rest of the ecosystem and/or indirectly affect the ecosystem by
affecting water quality (Nyholm and Peterson, 1997).

Alga is one of normally used model organism for the toxicity examination of toxicants and
nanoparticles as well, (Ji et al, 2011)


9
JUSTIFICATION CONTD

Chlorella vulgaris is distributed widely in freshwater and seawater and has a short growth
cycle and this makes it ideal for aquatic eco-toxicity studies and it can be used to directly
observe toxicity at the cellular level (Wong et al., 1997).

Bioavailability and toxicity of nanoparticles are largely unknown (Biswas and Wu, 2005)

Due to the increased use, titanium dioxide nanoparticles will inevitably reach the aquatic
environment, where they have so far been traced from urban applications into receiving
waters of urban runoff (Kaegi et al., 2008)

Engineered nanoparticles might be released along the lifecycle of consumer products
during their production, use, and disposal.

Algae and crustaceans were also the most sensitive environmentally relevant species for
synthetic nanoparticles (Kahru and Dubourguier, 2010)
10
AIM OF THE STUDY

The aim of this is to study the effect of titanium dioxide nanoparticles and nutrient
concentrations on the growth, biomass production, biochemical composition and
physiological response of Chlorella vulgaris.

11
OBJECTIVES OF THE STUDY
To determine the effect of titanium dioxide nanoparticles at different nutrient concentrations
on the growth and biomass production of Chlorella vulgaris.

To determine the effect of titanium dioxide nanoparticles as a function of nutrient
concentrations on the biochemical composition of Chlorella vulgaris.

To determine the effect of titanium dioxide nanoparticles and nutrient concentrations on the
antioxidant response of Chlorella vulgaris.

12
HYPOTHESES
There is no significant effect of titanium dioxide nanoparticles and nutrient concentrations
treatment on the growth and biomass of Chlorella vulgaris.

There is no significant effect of titanium dioxide nanoparticles and nutrient concentrations
treatment on the biochemical composition of Chlorella vulgaris.

There is no significant effect of titanium dioxide nanoparticles and nutrient concentrations
treatment on the antioxidant response of Chlorella vulgaris.

13
MATERIALS AND METHODS

ALGAL CULTURE SPECIES
The microalgae Chlorella vulgaris strain was obtained from the freshwater microalgae
culture collection of the University of Texas, USA.

CULTURE MEDIA
The algal species will be cultured in the OECD medium (2011).

CULTURE CONDITIONS:
Culture will be maintained at 23 2
o
C under continuous lighting at an intensity of
120mol m
-2
s
-1
with white fluorescent lamps.
Algae suspension will be periodically shaken by manual means daily, to prevent clumping
(Wei et al., 2010).

Titanium dioxide treatment
Dry Titanium (IV) oxide-anatase nano-powder will be purchased from Sigma-Aldrich, USA;
(CAS number 637254), particle size <25nm.


14
MATERIALS AND METHODS CONTD
Preparation of nanoparticles dispersion
Titanium dioxide nanoparticles obtained will be used to produce suspensions in
OECD algal medium 201 (OECD, 2011).

Titanium dioxide nanoparticle stock solution will be prepared by suspending
titanium dioxide nanoparticles in OECD medium in a concentration of 5000mgL
-1
(Chen et
al., 2012).
This suspension will be kept at 5
o
c in the dark and will be sonicated for 10 minutes
prior to preparation of the test suspensions.

Nanoparticles Treatment

When the algal cultures are at their exponential growth phase, 35 mL (Wei et al.,
2010) algal suspension will be distributed into 500 mL Polycarbonate Erlenmeyer flask.
Then 5 mL (Wei et al., 2010) of various concentrations of titanium dioxide nanoparticles will
be added to the algal suspensions in the Erlenmeyer flasks. Treatment will be replicated
thrice and will be for 96hrs.

Selection of these concentrations will be based on 96hrs EC50 of n-Tio
2
that will
be obtained in preliminary experiment on determining the acute toxicity.

15
MATERIALS AND METHODS CONTD

Nutrient source
Nitrogen will be provided as NH
4
Cl at 1.110
-3
M (control), 2.910
-6
M, 1.110
-5
M,
which represent limiting and environmentally replete nitrogen concentrations in aquatic
environments (Reynolds, 2006).

At each nitrogen concentration, n-TiO
2
will be added at the chosen n-TiO
2
concentrations after the LC50 determination, while the control will have no n-TiO
2.


Phosphorus will be provided as KH
2
PO
4
at 0.02mg/l, 0.06mg/l, 0.2mg/l, which
represents phosphorus concentrations at different aquatic trophic levels (Mainstone, 2002).
At each phosphorus concentration, n-TiO
2
will be added at the chosen n-TiO
2
concentrations after the LC50 determination, while the control will have no n-Tio
2



16
T
0
T
1
T
2
T
3
N
0
N
0
T
0
N
0
T
1
N
O
T
2
N
O
T
3
N- N-T
0
N-T
1
N-T
2
N-T
3
N+ N+T
0
N+T
1
N+T
2
N+T
3
T
0
T
1
T
2
T
3

P
0
P
0
T
0
P
0
T
1
P
O
T
2
P
O
T
3
P- P-T
0
P-T
1
P-T
2
P-T
3
P+ P+T
0
P+T
1
P+T
2
P+T
3
T- Titanium dioxide nanoparticles, N- Nitrate, P- Phosphate

MATERIALS AND METHODS CONTD

Combinations of titanium dioxide with nitrate and phosphate




17
MATERIALS AND METHODS CONTD

Biomass Determination
Spectrophotometry at optical density at 500 nm (Liu et al., 2008)

Dry Weight Measurement
A Whatman's GF/C filter paper (5 cm diameter) will be dried for 2 h at 60C, cooled in a
desiccator and weighed to the nearest mg. These filter papers will be used to filter 20 mL of
the samples and then the filter papers will be dried again for 2 h at 60C, cooled in a
desiccator and weighed.
Dry weight will be calculated in mg L
1
DW as the difference between the initial dry weight
of the filter paper without microalgal biomass and the final weight of the filter paper with the
retained microalgal biomass.

Density (cells mL
-1
)
The algal cell culture density will be monitored by spectrophotometry at 400 to 700 nm .
0.5ml of algal cells will be taken daily and cell quantity will be counted with a Neubauer
haemocytometer (cells/ml) under a phase contrast Zeiss microscope will be used to
determine cell density with the aid of an improved Neubauer haemocytometer.
You have to buy this as the former one we have was destroyed.


18
MATERIALS AND METHODS CONTD

TOTAL CHLOROPHYLL DETREMINATION
At the end of the experiment (96h), 10mL of algae cells suspension will be collected and
centrifuged at 4000 rpm for 10min. The supernatant will be removed and 5mL of 80% (V/V)
acetone will be added, mixed, and kept in the dark for 24hrs. The extracts will then be
centrifuged at 4000 rpm for 10 min and the supernatant will be analyzed for optical density
(Li et al., 2005). Absorbance will be measured at663, 645, 440 nm light wavelength using a
UV-VIS Spectrophotometer (B. Bran scientific and instrument company, England).The effect
of toxic agents on the photosynthesis of algae could be measured by using pigments such as
chlorophyll and carotenoids.
Chlorophyll a, b and carotenoid will be extracted using acetone. 5mL of the algal sample will
be filtered through a cellulose acetate filter paper, which will be placed in a 5mL plastic
centrifuge tube containing 3ml of acetone. The container will be wrapped with foil paper and
the extract will then be refrigerated at -20
o
C for 3hrs. After refrigerating, the supernatants
will be removed. Chlorophyll a, chlorophyll b and carotenoid quantification will be done
using absorbance measured at 470, 653 and 666 nm with a UV-VIS Spectrophotometer
(B.bran scientific and instrument company, England). Pigment concentrations will be
determined using the following equations provided by Nmeth (1998) and Wellburn (1994):
C
a
(mg/L) =17.12A
666
- 8.86A
653

C
b
(mg/L) = 32.23A
653
- 14.55A
666

C
T
(mg/L) = 2.57A
666
+ 23.6A
653

C
K
(mg/L) =1000A
470
2.86(15.65 A
666
- 7.34 A
653
) -129.2(27.05A
653
-11.21A
666
)/221.
Where C
a
is the content of chlorophyll-a (mg/L), C
b
the content of chlorophyll-b, C
T
the
content of total chlorophyll, and C
K
is the content of carotenoids.

19
MATERIALS AND METHODS CONTD

BIOCHEMICAL COMPOSITION DETERMINATION
Total proteins
Determination of the total protein content of the microagal cells will follow the procedure of
Bradford (1976) with bovine serum albumin (BSA) as standard. These values will be used to
plot the total protein standard calibration curve.
Total carbohydrates
Carbohydrates analysis will be performed according to the modified phenol-sulfuric acid
technique (Liu, et. al., 1973) using glucose as standard. Carbohydrate concentrations will be
obtained from a calibration curve of glucose with concentrations from 10g mL
-1
to 150g
mL.
Total lipids
10 mL of algal suspension will be harvested by centrifugation at different time points, and
the pellet will be washed with distilled water and then extracted with chloroform-methanol
(2:1 v/v) using the method described by Bligh and Dyer (1959). The extraction will be
carried out until a blue residue is left. The extract will be evaporated to dryness at 37
o
C and
then weighed.
Fatty acid composition????? (You can carry this out at NARICT)





20
MATERIALS AND METHODS CONTD

Antioxidants Enzyme determination
The enzymatic antioxidants that will be analysed include superoxide dismutase, catalase,
peroxidase and glutathione S-transferase.

Assay of Superoxide Dismutase (SOD)
Misra and Fridovich (1972) with some modifications.

Assay of catalase
Luck, (1974) with some modifications.

Assay of Peroxidase
Reddy et al., (1995) with some modifications.

21
MATERIALS AND METHODS CONTD

Assay of Glutathione S-Transferase
The assay of glutathione S-transferase activity will be performed using the method of Habig
et al., (1979) with some modification. Glutathione S-transferase conjugates GSH with CDNB
and the extent of conjugation is used as a measure of enzyme activity from the proportionate
change in the absorption at 340 nm.

CHEMICAL ANALYSES
Nitrate analysis
Nitrates concentrations in culture media will be determined according to the American Public
Health Association: APHA (1998) using the Phenol-disulphonic acid method.
Phosphates analysis
Phosphates concentrations in culture media will be determined according to the American
Public Health Association: APHA (1998) using the stannous chloride method.



22
MATERIALS AND METHODS CONTD

STATISTICAL ANALYSES
Growth inhibition tests of the algae will be performed in triplicates; the EC
50
values
(titanium dioxide concentration required to cause a 50% reduction in growth) will be
computed using EPA Probit analysis program.

SPSS version 20 Release 20.0.0 (IBM 2011)

Significant differences among the different treatments of algal samples will be determined
using Analysis of Variance (ANOVA) and where significant Duncans Multiple Range Test
will be used to separate the means.

STATISCA version 10 (Stat Soft. Inc. (2011)) program will be used for data analysis.

Significant correlations between the growth, biomass production, biochemical composition
and physiological responses of the microalgal with the treatment conditions will be
determined using Principal Component Analysis (PCA).

23