Enzyme and Microbial Technology 47 (2010) 4451

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Enzyme and Microbial Technology

journal homepage: www.elsevier.com/locate/emt

Improved bio-catalytic conversion by novel immobilization process using cryogel

beads to increase solvent production
Anuj Tripathi a,b , Haider Sami a,b , Seema R. Jain b , Maria Viloria-Cols b , Natalia Zhuravleva b ,
Gran Nilsson b , Hans Jungvid b , Ashok Kumar a,b,
a
b

Department of Biological Sciences and Bioengineering and Centre for Environmental Sciences and Engineering, Indian Institute of Technology Kanpur, 208016 Kanpur, India
Protista Biotechnology AB, Protista, Kvarngatan 2, P.O. Box 86, 26722 Bjuv, Sweden

a r t i c l e

i n f o

Article history:
Received 10 February 2010
Received in revised form 19 March 2010
Accepted 22 March 2010
Keywords:
Cryogel beads
Immobilization
Butanol production
Agarose-alginate matrix
Fermentation

a b s t r a c t
The use of immobilization matrix in bio-processing is a promising approach to immobilize a catalytic
strain for production of biomolecules. In this study, a novel immobilization system of agarose-alginate
cryogel was developed in the format of beads and characterized to facilitate the effective cell immobilization followed by enhanced solvent production compared to other immobilization matrix. Cryogel beads
showed macroporous internal architecture and nano-range grooves on outer periphery. Study suggests
that these grooves facilitate the convective medium transport throughout the cryogel beads in order to
eliminate the substrate and product inhibition and also prevent cell leakage. The immobilization study
was carried out on a typical anaerobic system of Clostridium acetobutylicum ATCC 824 for butanol production. The experiment was carried out in three different sets (A, B and C) with varying medium and
substrate concentration. The adsorption of cells on agarose-alginate cryogel beads produced 11.79 g/l
of butanol and 21.64 g/l total ABE (acetone, butanol and ethanol), while entrapment of cells on agarosealginate cryogel beads showed high glucose consumption, high butanol and total ABE production that was
92.16%, 14.47 g/l and 27.80 g/l, respectively, which was much higher than the control and other matrices.
2010 Elsevier Inc. All rights reserved.

1. Introduction
Arrays of man-made chemicals in the environment have led
an ever-increasing pressure on industrial developments leading
to the tremendous deterioration in environmental quality. Biocatalytic conversion for the production of useful chemicals is the best
way to produce particular substance at industrial scale. Though, the
use of biomass as the raw material for production of some important solvents like n-butanol, acetone, ethanol etc, is still appealing
and amending environmentally. In contrast, the synthetic processes have replaced fermentation for commercial production in
the early 1960s due to several reasons, of having low productivity, low solvent yield and substantially high recovery cost by
distillation process [1,2]. Since then, solvent fermentation could
not compete economically with the chemical processes. However,
in recent years research has progressed in an attempt to make
the solvent fermentation not only environmentally favourable but
also economically competitive. In the pharmaceutical and biotech-

Corresponding author at: Department of Biological Sciences and Bioengineering,

Indian Institute of Technology Kanpur, 208016 Kanpur, India. Tel.: +91 512 2594051;
fax: +91 512 2594010.
E-mail address: ashokkum@iitk.ac.in (A. Kumar).
0141-0229/$ see front matter 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.enzmictec.2010.03.009

nology industries, fermentation is an important step of upstream

processing. The fermentation process includes large-scale cultivation of microbes or other single cell type, occurring either in aerobic
or anaerobic conditions. The industrially important biotechnology
processes are generally utilizing microorganism and their application in the fermentation medium during the process. Such classical
fermentations undergo several constrains like, nutritional limitations, low cell density, solvent toxicity and batch-mode operations
with high down times [3,4]. It has been well recognized that
the concentration of microbial cells is prime important during
the downstream process to achieve higher volumetric productivity from the fermented medium. Designing of advance bioreactor
and its continuous operation with controlled parameters is an
important area of research and requires great focus indeed. Many
experimental ventures have been carried out on small scale and
scaled up but there had been problems of cell leakage through the
running fermenter. Therefore, the future research should focus on
development of executable microbiological processes with immobilized cells and also perform broad research to gure out some
of the engineering problems like scale up and diffusion limitations
with high cell density.
The cell concentration inside the bioreactor can be increased
by cell immobilization technology. Adsorption and entrapment
are two main techniques which have been extensively examined

A. Tripathi et al. / Enzyme and Microbial Technology 47 (2010) 4451

for the use of cell immobilization with support matrix. Immobilized cell systems facilitate to maintain high cell densities in the
system, which improve reaction rates and provide high productivity. The immobilization systems are also stable at high dilution
rates with little cell washout and provide simplicity of operation.
Other advantages are that the fermentor conguration can be relatively simple and immobilization material can often be reused.
In addition, immobilization systems serve to enhance the solvent productivity due to one of the major advantage that involves
the dissociation of immobilized whole-cell growth from cellular
synthesis of favoured compounds. The previous study suggested
that different cell immobilization support matrices like clay brick,
hydrogel beads and brous supports etc. are able to improve reactor productivity [57]. These approaches have potential possibility
for improvement of solvent fermentation.
In contrast, cryogelation technology has emerged as a potential
approach to generate three-dimensional (3D) macroporous polymeric support matrix called cryogel, using homogeneous or heterogeneous monomers/polymer solution mixture. Cryogels have
been used as a carrier for various biomedical and bioengineering
applications [8,9]. Cryogels are special type of hydrogels which are
synthesized at subzero temperatures and have supermacroporous
structure with interconnected pores, thus offering a unique combination of high interconnected porosity, high diffusivity and high
mechanical strength [10]. These gels can be produced from various
types of chemical molecules (monomers) or polymeric precursors
which can be both synthetic and natural, thus providing unique
chemistry to tailor them for specic applications. Other important
aspect has been that these macroporous matrices can be synthesized in different formats like monoliths, disc shaped, thin sheets,
beads, etc. Thus owing to these properties these cryogels have suitable chemistry and its porosity can be altered as per application.
We are aiming to generate an efcient and suitable novel support matrix for immobilizing the clostridial cells using cryogelation
method and then this developed process can be used on industrial scale for the production of improved n-butanol. To achieve
this main goal, the objective is to carry out studies on immobilization of the clostridial strain (Clostridium acetobutylicum ATCC
824) as a model cell line on different supports matrix including
cryogel to reveal its potentiality in comparison with other support matrices. In addition, the work will also focus to optimize
the process parameters to improve the n-butanol productivity from
immobilized clostridial cells. Here we are emphasizing the generation of novel polymeric scaffold based cell immobilization approach
for solvent production without cell leakage problem through an
efcient transport of solvent occurring within cryogel beads. This
novel immobilization approach of the bacterial cells on the specially
designed cryogel beads can be an efcient approach for industrial
applications.
2. Materials and methods
2.1. Materials
Low viscosity alginic acid sodium salt (from brown algae), N-(3dimethylaminopropyl)-N -ethylcarbodiimide hydrochloride (EDC; FW-191.71)
was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Agarose (low EEO,
gelling temperature 38 to 40 C) was purchased from Sisco Research Laboratories
(Mumbai, India). N-hydroxysuccinimide (NHS) was bought from Spectrochem
(Mumbai, India). For cell immobilization experiment, C. acetobutylicum cells (ATCC
824) were obtained from the American Type Culture Collection (ATCC), through
LGC Promochem, Boras, Sweden. The cells were cultured in clostridial nutrient
medium purchased from Fluka (Buchs, Switzerland). Other chemicals used were of
analytical grade, which were used without any further purication.
2.2. Bacterial strain and medium selection
The strain used for immobilization purpose was C. acetobutylicum ATCC 824. It
is one of the best studied solventogenic clostridia strain which has been widely

45

studied from the physiological and bioengineering points of view. C. acetobutylicum

is a gram positive rod shaped bacteria which grow in strict anaerobic conditions.
For maintaining the growth of clostridial cells, clostridial nutrient broth (Fluka)
(containing meat extract 10 g/l, peptone 5 g/l, yeast extract 3 g/l, d(+) glucose 5 g/l,
starch 1 g/l, sodium chloride 5 g/l, sodium acetate 3 g/l, l-cysteine hydrochloride
0.5 g/l and agar 0.5 g/l; nal pH 6.8 0.2 at 25 C) was used in concentration of 33 g/l.
While checking the production of solvents, immobilized cells were cultured in production medium i.e. P2 medium (containing glucose 60 g/l, magnesium sulphate
0.2 g/l, sodium chloride 0.01 g/l, manganese sulphate 0.01 g/l, iron sulphate 0.01 g/l,
potassium dihydrogen phosphate 0.5 g/l, potassium hydrogen phosphate 0.5 g/l,
ammonium acetate 2.2 g/l, biotin 0.001 g/l, thiamin 0.1 g/l and p-aminobenzoic acid
0.1 g/l). All the salts of P2 medium were autoclaved separately in the serum bottle. To
this was added sterile glucose solution to make a total concentration of 60 g/l of glucose. Addition of vitamins was done after lter sterilization under sterile conditions.
All the solutions were purged with O2 free N2 before autoclaving.
2.3. Growth conditions and maintenance
Clostridial cells are endospore forming cells. Initially, sporulated cells (from the
glycerol stock) were activated by heat shock at 80 C for 10 min. The activated spore
culture (2 ml) was inoculated in 60 ml sterile clostridial nutrient medium (CNB) and
grown at 37 C under anaerobic conditions. The growth was monitored spectrophotometrically at 560 nm. After 44 h, 20 ml of cell culture medium was transferred in
to 500 ml of fresh CNB medium and was incubated for 22 h. This active cell culture
was used for immobilization and bio-catalytic conversion experiments. All cultures
were kept anaerobic by purging O2 free nitrogen gas through 0.2 m lters.
2.4. Selection and processing of support matrices for immobilization
The support matrices for cell immobilization are an efcient approach to
increase the productivity of end-product. Previous studies have shown that lots
of efforts have been done for screening potential support matrix to improve the
immobilization efciency. In our study, we have chosen the following support
material such as coconut bres, coal (burned), clay bricks, alginate hydrogel beads
and agarose-alginate cryogel beads for immobilization of clostridia. These support
matrices were processed into 23 mm in size from their raw sources except polymeric support matrices i.e. alginate hydrogel beads and agarose-alginate cryogel
beads. The non-polymeric matrices were further washed thoroughly with water,
dried in oven at 60 C and nally sterilized by autoclaving at 121 C for 15 min.
2.4.1. Synthesis of alginate hydrogel beads
Alginate solution (2%) was prepared in deionized water and then autoclaved the
solution. Sterile alginate solution was put in plastic syringe and slowly dropped into
2% of sterile CaCl2 solution using ne needle. The alginate solution took a shape
of bead when it merged in CaCl2 . These beads were incubated overnight in the
same solution to provide strength. Then the beads were washed with autoclaved
distilled water three times for 15 min each. The beads were then further used for
immobilization experiments.
2.4.2. Synthesis of agarose-alginate cryogel beads
Agarose-alginate
cryogel
beads
were
synthesized
using
N-(3hydrochloride
(EDC)
with
dimethylaminopropyl)-N -ethylcarbodiimide
N-hydroxysuccinimide (NHS) for chemical crosslinking. Low viscosity alginate solution (3.75%) was prepared in 50 ml plastic tube using deionized water
as a solvent. On the other hand, agarose (low EEO; gelling temperature 3840 C)
solutions (6%) was prepared in deionized water by putting the agarose containing
plastic tube in the boiling water bath for 30 min or until the solution become
transparent. Then 4 ml of stock solution of alginate (3.75%) was added in the
completely dissolved 5 ml of hot agarose solution (6%) and mixed by vortexing. The
ratio of agarose to alginate was 2:1. Mixture of agarose-alginate was incubated for
510 min at 60 C and then the heterogeneous solution was kept for cooling at room
temperature. When the temperature of polymer solution comes down to 45 C, EDC
followed by NHS was added and mixed by vortexing. The solution was transferred
in disposable polyethylene syringe (internal tip diameter was varied for synthesis
of different sized beads) and dropped in to moderately frozen light viscous parafn
liquid oil. The slightly warm polymer solution took a round shape when dropped
in to moderately frozen parafn oil. The beads were incubated in parafn oil at
subzero temperature i.e. 20 C for 16 h. After incubation, beads were taken out
from the parafn oil. The cryogel beads were repeatedly washed using PBS (pH
7.4) for overnight under gentle magnetic stirring to remove parafn oil completely.
The agarose-alginate cryogel beads were air dried for morphological studies while
cell immobilization was done on ethanol sterilized (overnight incubation in 70%
ethanol) cryogel beads.
2.5. Morphological analysis of immobilization support matrices
The morphology of synthesized agarose-alginate cryogel beads was analyzed by
scanning electron microscopy (SEM). SEM micrograph allows direct measurement of
porosity, average diameter of pores and strut thickness by image analyzing software.
The air dried cryogel samples were coated with gold using a sputter coater (Vacuum

46

A. Tripathi et al. / Enzyme and Microbial Technology 47 (2010) 4451

Tech, Bangalore, India). SEM examinations were made on a FEI Quanta 200 at high
vacuum at 20 kV with spot size 3.5 mm.

potential support matrix among all the other immobilization support matrices
used.

2.6. Mode of immobilization on support matrices

2.9. Analysis of solvent production by HPLC

The immobilization of clostridial cells on non-polymeric samples was done using

adsorption method. The active cell culture was used to load on the support matrix
merged in fresh medium, where the cells were allowed to grow as well as adhere on
the support matrix surface. For immobilization of cells on agarose-alginate cryogel
beads, two approaches were employed. In the rst approach, cells were adhered on
already synthesized cryogel beads by physical adsorption similar method used for
other immobilization support matrices. In second approach, clostridial cells were
entrapped within the cryogel beads during its synthesis. The active cell culture was
centrifuged at 6000 rpm for 20 min. The cell mass (pellet) was mixed in agarosealginate solution at 45 C. The crosslinkers were added and then followed the same
procedure mentioned for synthesis of cryogel beads. The synthesized beads were
washed with sterile water purged with nitrogen.

Samples were taken out using syringe at different time points from the each
set of batch cultures and examined for the concentrations of different solvents produced. The mobile phase was 0.15 mM sulphuric acid which was ltered through a
0.45 m lter. The ow rate was adjusted to 0.7 ml/min through a Biorad Aminex
HPX87H column (300 mm 7.8 mm) equipped with a Biorad Micro-Guard cartridge
(30 mm 4.6 mm). Column temperature was adjusted to 30 C in a column oven.
The fermentation broth samples were centrifuged in closed micro-centrifuge tubes
whch claried the samples and then ltered through 0.45 m syringe lter into
autosampler vials. The sample (20 l) volume was injected onto the column to
analyse the production of butanol.

3. Results and discussion

2.7. Batch fermentation

Three different modes of experiments were setup in batch mode to check the
effect of different support matrices in solvent production. Two types of medium as
mentioned above i.e. CNB (rst set) and P2 (second set) were separately used with
the support matrices for immobilization and production analysis. In the third set,
the cells were initially grown and immobilized in CNB media for 48 h and then the
medium was changed to P2 medium for production of butanol. Each set of experiment was designed with all the support matrices (as mentioned above) along with
one control, which had no immobilization support. The fermentation was done in
40 ml crimp top glass vials, where the 4 ml of immobilization support was saturated
in 15 ml of CNB (set 1), P2 (set 2) or CNB to P2 (set 3) medium and then each vial was
inoculated with 1.5 ml of active bacterial culture. These vials were then incubated
at 37 C for solvent production and were monitored up to 141 h of fermentation
process.
2.8. Analysis of glucose consumption
Cell growth and active bio-conversion of glucose by clostridial cells were
examined at pre-dened time intervals in batch culture. In general, the prole
of glucose degradation as well as production of biomass, butanol, intermediate
product butyrate and other end product i.e. ethanol and acetone were observed
in the batch tests. These results further supported in the identication of most

The immobilization of microbial cells in biological processes

can occur either as a natural process or in the course of providing
external support matrix. The naturally attached cells exhibit better
growth than the cells immobilized on support matrices. It however, will depend upon whether the articial environment provides
favourable or unfavourable conditions to cells. Several research
groups developed several approaches for whole-cell immobilization. As for immobilized cells, two broad type of methods have
been used to immobilize microorganism i.e. attachment to a support matrix or entrapment within the matrix. Here we utilized both
techniques and tried to develop a novel approach for cell immobilization to increase the end-product concentration in the solvent
and also minimize or overcome the cell leakage problem.
3.1. Sample preparation and morphological analysis
The immobilization support matrices were prepared into
23 mm size from their respective raw material (Fig. 1). The alginate

Fig. 1. The digital images of immobilization support matrices: (A) coconut bres, (B) coal (burned), (C) agarose-alginate cryogel beads and (D) clay bricks. The material in
the moulds shows the processed material further used for immobilization. Different sized cryogel beads are shown in (C), where the used beads for immobilization were of
23 mm in size.

A. Tripathi et al. / Enzyme and Microbial Technology 47 (2010) 4451

hydrogel beads and agarose-alginate cryogel beads (Fig. 1C) were

also prepared in the same size, which were further used to explore
the potentiality of different matrices for cell immobilization. The
alginate hydrogel beads were prepared by physical crosslinking of
alginate polymer chains into CaCl2 solution at room temperature
which turned into a less porous gel beads.
In contrast, the cryogel beads were prepared at subzero temperature using EDC-NHS. The crosslinking of agarose and alginate
using EDC is not specically studied, but the previous studies suggest that EDC mediates acid anhydride formation between two
carboxyl groups of alginate and eventually the resultant acid anhydride may readily react with a hydroxyl group of agarose to form an
ester bond [11,12] or the use of EDC may involve in the crosslinking within the carboxyl group rich alginate chains [13]. The use of
NHS to improve the performance of EDC crosslinking is well documented in the literature [14,15]. In addition, agarose is a well
known polymer, which can also physically self-gelate and make
a gel. The use of agarose with alginate provided substantial stiffness to the beads, while the alginate has high tendency to bind
the bacterial cells due to charged surface property. The cryogelation process generated large pores within the cryogel beads as
shown in Fig. 2A and C, while the outer surface of cryogel beads
(Fig. 2B) has shown small nano-range grooves (Fig. 2D) investigated by scanning electron microscopy (SEM). The outer surface
was matured in such a manner to work as a closed compact system
for cell immobilization. During the maturation of cryogel beads at
subzero temperature, the incubation system i.e. liquid parafn oil
was used which helped to provide the smoothness to the outer
surface of cryogel beads. The presence of grooves on bead surface helps in the transport of solvent in between the inner side to

47

outer side of cryogel beads. However, the diameter of grooves was

recorded in nano-range, which prevents the leakage of microbes
from inside to outside of bead (in case of entrapment). These beads
were mechanically stable and very spongy, which helped to prevent
breakage of the beads as cell growth occurs inside. Other matrices
were also examined by SEM, where coconut bres based matrix
showed entangled brous network, while coal (burned) and clay
brick showed non-uniform rough surface containing some small
pouches on the surface.
An ideal immobilization support matrix should be non-toxic,
highly porous and can provide large surface area for cell attachment. Among all the used matrices, only the cryogel beads showed
highly porous and interconnected internal network, which might
be attractive for solvent ow and can support cell adherence. In
the previous studies, agarose and alginate has been most common
polymers used for whole cell immobilization. Alginate has shown
potential property as soft polymeric material, where the polymer
surface charges attract the cells and help in their adherence. These
features support agarose-alginate cryogel beads that can be used
for cell immobilization applications.
The selected and morphologically examined matrices were
further used to establish batch fermentation system for butanol
production. Before starting a real experiment, the mass of the
immobilization support matrices along with the medium and
inoculum was optimized i.e., 4 ml of support matrices, 15 ml of
medium and 1.5 ml of inoculum, which covered approximately 50%
volume of the 40 ml glass bottle (Fig. 3). As the system was anaerobic, so the sufcient volume was required to maintain conditions
like pressure, for long time run of batch fermentation in 40 ml glass
bottle. This optimized volume was utilized for further studies.

Fig. 2. The scanning electron microscopic (SEM) images showing external and internal morphology of agarose-alginate cryogel beads as shown in (A). (B) and (C) are the
images taken at high magnication. Image (D) shows nano-range grooves present on the outer surface of the cryogel beads.

48

A. Tripathi et al. / Enzyme and Microbial Technology 47 (2010) 4451

3.3. Estimation of glucose consumption and cell behaviour

Fig. 3. The digital image shows the physical appearance of immobilization support
matrices setup in 40 ml crimp top glass bottles, which were further utilized for the
production of butanol in batch fermentation. In the gure, coconut bres (A), coal
(burned) (B), clay brick (C), alginate hydrogel beads (D), agarose-alginate cryogel
beads (E) and control (without support matrix) (F) were volumetrically optimized
along with the medium and inoculum in volume ratio.

3.2. Morphological analysis of immobilized support matrices

The immobilization efciency and cell behaviour on the selected
matrices was examined using scanning electron microscopy
(Fig. 4). The coconut bres and clay bricks showed affable cell
adherence due to the rough surface of matrices (Fig. 4A and B).
While the alginate hydrogel beads showed less cells adherence
(Fig. 4C) due to shrinkage of surface during the dehydration process, which lead to the detachment of cells. The burned coal as a
substrate for cell adherence showed number of cells and spores on
its smooth surface and pouches as shown in Fig. 4D. Apart from
that, the cryogel beads showed high number of cell immobilization
either in adsorbed condition (Fig. 4E) or in entrapped mode (Fig. 4E).
The uniform distribution of cells in the entire porous cryogel bead
was observed during SEM analysis at different places of the sample. These results show efcient cell immobilization property of
agarose-alginate cryogel beads.

C. acetobutylicum is capable of utilizing all the prevalent sugars

(pentose and hexose) present in the medium [16]. In this study,
d-glucose was used as a sugar substrate in the medium. The solvent producing clostridia metabolize pentose sugars by way of the
pentose phosphate pathway [1720]. The utilization of glucose by
clostridial cells was analyzed at various time intervals. The initial glucose concentration was 5 g/l in CNB medium. In set A, the
percent glucose consumption was 97.58 2.04% in all the support
matrices at 141 h of batch fermentation. The rate of glucose consumption in cell entrapped agarose-alginate cryogel beads (E-AAC),
was slower as compared to the other matrices. Unlike adhered cells,
the entrapped cells needed some extra time to get activated before
utilizing substrates. In set B, with the production medium (P2), the
initial glucose concentration was 60 g/l. The growing cells on all
the support matrices showed continuous utilization of glucose and
the samples were analysed at different time intervals up to 141 h
of fermentation process. Among all the support matrices, the clay
bricks matrix containing bottle showed highest glucose utilization
(Table 1) up to the end of batch fermentation i.e. 141 h. While in
other support matrices, the glucose consumption was also found
to be higher than the control bottle. In control bottle, no support
matrix was used.
In set C, the glucose consumption was analysed from the step
where the medium was changed from CNB to P2 medium. The
CNB medium was generally used for growth and proliferation of
clostridial cells, while P2 medium is a production medium with
high substrate concentration. In this set, we rst increased the cell
population by growing cells in CNB medium and simultaneously
immobilizing them on the support matrices up to 72 h. Once the
cells were immobilized, the CNB medium was replaced with P2
medium for solvent production. The results obtained from HPLC
analysis of the samples revealed that, there was no further utilization of glucose after 72 h of growth in the different support matrices
i.e. coconut bres, coal, clay bricks, agarose-alginate cryogel beads

Fig. 4. Scanning electron microscopic images of different support matrices showing cell immobilization. The cells were adhered on coconut bres (A), clay bricks (B), alginate
hydrogel beads (C), coal (burned) (D) and agarose-alginate cryogel beads (E), while cells were entrapped in agarose-alginate cryogel beads as shows in (F).

A. Tripathi et al. / Enzyme and Microbial Technology 47 (2010) 4451

49

Table 1
Batch fermentation process in production (P2) medium i.e. set B.
Support type

O.D. (at 560 nm)

pH

Glucose consumption (%)

Butanol (g/l)

Butyric acid (g/l)

ABE (g/l)

Cryogel bead-adsorbed
Cryogel bead-entrapment
Alginate hydrogel bead
Coconut bres
Coal
Clay brick
Control

2.1
0.5
3.8
3.1
2.6
2.9
1.9

4.5
4.5
4.5
4.5
4.5
4.5
4.5

76.56
44.92
89.94
82.92
79.95
90.21
40.80

10.79
5.24
12.7
11.58
11.59
13.71
5.378

0.706
1.317
0.863
0.737
0.922
0.437
1.423

21.64
10.83
23.06
20.29
19.87
24.37
9.38

Different support matrices were examined upto 141 h for butanol, butyric acid and total ABE production. Glucose substrate consumption and other physiological parameters
were also monitored. The experiments were conducted in triplicates (P < 0.05).

Table 2
Batch fermentation process in clostridium nutrient medium (CNB) followed by production (P2) medium i.e., set C.
Support type

O.D. (at 560 nm)

pH

Glucose consumption (%)

Butanol (g/l)

Butyric acid (g/l)

ABE (g/l)

Cryogel bead-adsorbed
Cryogel bead-entrapment
Alginate hydrogel bead
Coconut bres
Coal
Clay brick
Control

3.0
1.4
5.4
2.8
2.6
1.0
0.8

5.0
4.8
4.6
5.2
4.9
4.5
5.7

26.13
92.04
39.10
20.68
26.66
20.56
13.31

1.59
14.47
1.34
0.34
0.41
0.90
1.218

3.654
0.711
4.616
3.84
4.02
3.65
3.737

1.869
27.802
1.661
0.48
0.57
1.071
1.457

Different support matrices were examined after 96 h for butanol, butyric acid and total ABE production. Glucose substrate consumption and other physiological parameters
were monitored. The experiments were conducted in triplicates (P < 0.05).

(in case of adhered cells i.e A-AAC) and alginate hydrogel beads
as well control (Fig. 5 and Table 2). However, in cell entrapped
agarose-alginate cryogel beads (E-AAC), the continuous and efcient glucose consumption was observed as shown in Fig. 5, also
the comparative changes in optical density and pH of the medium
with time was analysed (Fig. 6). The low glucose utilization might
be because of ineffective cell adherence which occurred on to support matrices and the most of the bacterial cells come out with the
CNB medium when changed to P2. It might also be possible that
the rest of the cells which adhered on to support matrices were
dead due to medium shock i.e. from CNB to P2 medium. But in case
of E-AAC, the cells were safely entrapped inside the cryogel beads
as there was no cell loss and also it prevented cells from medium
shock. Apart from that, initially, good cell growth was observed
in the CNB medium and the medium was turned into translucent
because of high cell growth in suspension, in all the bottles except
E-AAC bottle. In E-AAC bottle, the cells were entrapped and grew
and proliferated within the porous cryogel beads, and the outer surface prevented or decreased cell leakage. These results may suggest
that the growth of cells and cellular activity for solvent production
was found higher in E-AAC as compared to other support matrices.

3.4. Analysis of butanol production by HPLC

Fig. 5. The graph shows percent glucose consumption by clostridial cell in the presence of different support matrices in set C i.e. clostridium nutrient broth (CNB)
followed by production (P2) medium.

Fig. 6. The graph shows different parameter of cell entrapped agarose-alginate cryogel beads (E-AAC) in batch fermentation of set C i.e. clostridium nutrient broth (CNB)
followed by production (P2) medium.

The efciency of agarose-alginate cryogel beads over other support matrices in the batch fermenter for producing butanol and
total ABE (acetone, butanol and ethanol) was studied at various
time intervals. The control batch fermentation experiment was run
in three different sets (set A, B and C) with varying medium and
substrate concentrations. At the end of fermentation of set A, the
butanol concentration range was not very different in all the bottles
which ranged from 0.097 to 0.275 g/l (Fig. 7) with an ABE concentration of 0.618 0.201 g/l. The coal, alginate hydrogel beads
and cryogel beads (A-AAC) showed approximate same amount of
butanol, while other support matrices showed less efciency than
above mentioned matrices but still higher than the control. The
glucose concentration in the CNB medium was low, so the solvent
production was limited. The CNB medium supports cell growth and
can be used for developing an active biolm on a support matrix by
growing cells over a period of time. In set B, the cells were directly
grown in P2 medium with 60 g/l glucose concentration (Fig. 8).
The high butanol production was found in case of clay bricks i.e
13.71 g/l. While in case of cryogel beads (A-AAC), 11.79 g/l butanol
production was achieved, this was nearly the same concentration

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A. Tripathi et al. / Enzyme and Microbial Technology 47 (2010) 4451

Fig. 7. The graph shows pattern of butanol production using different support
structure in clostridium nutrient broth (CNB) (set A). The experiment was done
in triplicate (P < 0.05).

of the maximum butanol production in clay bricks. Other supports

also showed good butanol production (Fig. 8 and Table 1). The ABE
productivity was also found in the same order, where the maximum production was 24.37 g/l, found in clay bricks and 21.64 g/l in
cryogel beads (A-AAC) (Table 1). The control bottle showed 5.38 g/l
butanol and 9.38 g/l ABE production. The E-AAC cryogel beads did
not show butanol and ABE production up to 60 h of batch fermentation and after that a sudden increase in the butanol and ABE
concentration was observed (Fig. 8). Perhaps, this behaviour within
cryogel beads was because of entrapped cells, which had some lag
period to get metabolically active and then start growing and utilizing the substrate for solvent production. These ndings suggest
that the cryogel beads as a support adsorbent can be a good matrix
for bio-catalytic conversion process. In contrast, the alginate polymer has well known property to enhance the cellular attachment
on the matrix because of positive charge, which makes matrix more
efcient for active biolm genesis.
In set C, the cells were grown in CNB medium up to 72 h and
then medium was changed to P2 medium (Fig. 9). After medium
change, the estimation of butanol and total ABE production at different time intervals was done. All the batch fermentation bottles
containing different support matrices showed insignicant production of solvent except E-AAC cryogel beads (Fig. 9). E-AAC cryogel
beads showed efcient supports for cell growth and butanol production. The medium change caused some loss in cell number.
Further, the exposure of low glucose concentration medium to high
glucose concentration medium might cause some shock to cells,
which suggest poor performance of all support matrices, except EAAC cryogel beads. In that case the cells were entrapped inside the
cryogel beads and were safe from the cell loss problem. Entrapped
cells utilized substrate from the medium with convective medium
transport through nano-range grooves present on outer surface of

Fig. 8. The graph shows butanol production kinetics by clostridial cells immobilized
on different support matrices in production (P2) medium (set B). The experiment
was done in triplicate (P < 0.05).

Fig. 9. The graph shows pattern of butanol production using different support matrices in production (P2) medium. The cells were initially grown on different support
matrices in clostridium nutrient broth (CNB) up to 72 h (set C). The experiment was
done in triplicate (P < 0.05).

cryogel beads. The exposure of cells to medium is not direct and

which may also help to prevent the cells from any type of shocks. In
case of E-AAC cryogel beads, the butanol and total ABE production
was 14.47 g/l and 27.80 g/l, respectively (Table 2).
Set C explains the importance of growth of entrapped cells followed by production of solvent. In set B, the entrapped cells in
cryogel beads (E-AAC) were active after some lag time as shown
in Fig. 8 and then started producing solvent after 60 h of batch fermentation process. The lag time required for cells may be because
the process of entrapment may cause polymer shielding on the
entrapped cells within the cryogel beads. So, if the entrapped
cells will be initially exposed to growth medium for a certain
time period, it may metabolically activate cells, which can further actively work in fermentation. In contrast, the set C was
set up as per the above mentioned approach, where the initial
incubation in growth medium (CNB medium) helped cells to get
active and further the active high cell mass produced high concentration of butanol as shown in set C. These results suggest that
agarose-alginate cryogel beads were shown potentiality as a useful immobilization support matrix for either adsorbing the cells on
surface or entrapping the cells within beads.
In conclusion immobilization of cells for the use in continuous or
batch culture has several advantages which make the reactor conguration relatively simple and the support structure can often be
reused. In this study, agarose-alginate cryogel beads were screened
and compared with other well known potential support matrices. The study was carried out on a typical anaerobic system of
clostridial cells, where many classical problems like sporulation,
pH inhibition, substrate concentration and solventogenic inhibition
etc. reduces the butanol productivity. The cryogel beads showed
good amount of butanol production when used as an adsorption
structure. On the other hand, entrapment in cryogel beads showed
high solvent productivity compared to adsorption process. This
study also suggests that the cell entrapment in cryogel beads can
signicantly increase the solvent production by maintaining high
cell mass and preventing cell leakage. In batch mode, the major
factor of solventogenic inhibition causes decrease in productivity
and in such case continuous system is generally preferred which
reduces the solventogenic inhibition and increases the productivity. So, if the cryogel beads can be used as a support matrix (where
cells are entrapped inside the beads), can help to produce high
butanol concentration as compared to other support matrices (as
results obtained in set C). The cells entrapped inside the cryogel
beads can be reused in next fermentation process. These unique
and novel properties of cryogel beads suggest its potential use in
bio-catalytic conversion process to enhance the solvent productivity.

A. Tripathi et al. / Enzyme and Microbial Technology 47 (2010) 4451

Acknowledgements
Authors would like to acknowledge the support received from
Department of Biotechnology (DBT), Ministry of Science and Technology, Govt. of India and Protista AB, Bjuv, Sweden. AT and HS
acknowledges the nancial support received from Protista AB, Sweden for working in Sweden. AT also acknowledges CSIR for granting
Sr. Research Fellowship (SRF).
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