Enzyme and Microbial Technology 47 (2010) 44–51

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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 ,
Göran 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 figure 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

The cryogel beads were repeatedly washed using PBS (pH 7. All cultures were kept anaerobic by purging O2 free nitrogen gas through 0. All the salts of P2 medium were autoclaved separately in the serum bottle.2. Sweden. acetobutylicum ATCC 824.e. We are aiming to generate an efficient 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. sodium chloride 0. While checking the production of solvents. In addition.4. the work will also focus to optimize the process parameters to improve the n-butanol productivity from immobilized clostridial cells. N-hydroxysuccinimide (NHS) was bought from Spectrochem (Mumbai. The beads were then further used for immobilization experiments.2. These support matrices were processed into 2–3 mm in size from their raw sources except polymeric support matrices i.1. through LGC Promochem. 2. Selection and processing of support matrices for immobilization The support matrices for cell immobilization are an efficient approach to increase the productivity of end-product. The beads were incubated in paraffin oil at subzero temperature i. In our study. The growth was monitored spectrophotometrically at 560 nm. EDC followed by NHS was added and mixed by vortexing. Boras.A. Sterile alginate solution was put in plastic syringe and slowly dropped into 2% of sterile CaCl2 solution using fine needle. The previous study suggested that different cell immobilization support matrices like clay brick. −20 ◦ C for 16 h.4. ammonium acetate 2. clostridial nutrient broth (Fluka) (containing meat extract 10 g/l. Materials and methods 2. Immobilized cell systems facilitate to maintain high cell densities in the system. 2. magnesium sulphate 0. On the other hand. which were used without any further purification. (St. When the temperature of polymer solution comes down to 45 ◦ C.e.1 g/l and p-aminobenzoic acid 0.1. using homogeneous or heterogeneous monomers/polymer solution mixture.2 g/l. Previous studies have shown that lots of efforts have been done for screening potential support matrix to improve the immobilization efficiency. potassium hydrogen phosphate 0. yeast extract 3 g/l. These beads were incubated overnight in the same solution to provide strength. hydrogel beads and fibrous supports etc. manganese sulphate 0.71) was purchased from Sigma Chemical Co. FW-191. Tripathi et al.001 g/l. SEM micrograph allows direct measurement of porosity.75%) was added in the completely dissolved 5 ml of hot agarose solution (6%) and mixed by vortexing.5 g/l. 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.75%) was prepared in 50 ml plastic tube using deionized water as a solvent. acetobutylicum cells (ATCC 824) were obtained from the American Type Culture Collection (ATCC).01 g/l. 2. sporulated cells (from the glycerol stock) were activated by heat shock at 80 ◦ C for 10 min. thin sheets. 2. For cell immobilization experiment. The immobilization systems are also stable at high dilution rates with little cell washout and provide simplicity of operation. iron sulphate 0. acetobutylicum is a gram positive rod shaped bacteria which grow in strict anaerobic conditions. Mixture of agarose-alginate was incubated for 5–10 min at 60 ◦ C and then the heterogeneous solution was kept for cooling at room temperature. biotin 0. USA). potassium dihydrogen phosphate 0.9].5 g/l. Other advantages are that the fermentor configuration can be relatively simple and immobilization material can often be reused. sodium chloride 5 g/l. MO.5 g/l. we have chosen the following support material such as coconut fibres. This active cell culture was used for immobilization and bio-catalytic conversion experiments. India). C. These gels can be produced from various types of chemical molecules (monomers) or polymeric precursors which can be both synthetic and natural.4) for overnight under gentle magnetic stirring to remove paraffin oil completely. Here we are emphasizing the generation of novel polymeric scaffold based cell immobilization approach for solvent production without cell leakage problem through an efficient transport of solvent occurring within cryogel beads. final pH 6. 2.3. Growth conditions and maintenance Clostridial cells are endospore forming cells. Louis. l-cysteine hydrochloride 0. high diffusivity and high mechanical strength [10]. sodium acetate 3 g/l. are able to improve reactor productivity [5–7]. The alginate solution took a shape of bead when it merged in CaCl2 . Initially.4. The activated spore culture (2 ml) was inoculated in 60 ml sterile clostridial nutrient medium (CNB) and grown at 37 ◦ C under anaerobic conditions. 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. thus providing unique chemistry to tailor them for specific applications. Bacterial strain and medium selection The strain used for immobilization purpose was C. P2 medium (containing glucose 60 g/l. dried in oven at 60 ◦ C and finally sterilized by autoclaving at 121 ◦ C for 15 min. It is one of the best studied solventogenic clostridia strain which has been widely 45 studied from the physiological and bioengineering points of view.5 g/l and agar 0. For maintaining the growth of clostridial cells. cryogelation technology has emerged as a potential approach to generate three-dimensional (3D) macroporous polymeric support matrix called cryogel.e. The non-polymeric matrices were further washed thoroughly with water. starch 1 g/l. agarose (low EEO. The slightly warm polymer solution took a round shape when dropped in to moderately frozen paraffin oil. immobilized cells were cultured in production medium i. disc shaped. Other important aspect has been that these macroporous matrices can be synthesized in different formats like monoliths. Cryogels have been used as a carrier for various biomedical and bioengineering applications [8.2 ␮m filters. In contrast. Agarose (low EEO. alginate hydrogel beads and agarose-alginate cryogel beads for immobilization of clostridia. After 44 h. The air dried cryogel samples were coated with gold using a sputter coater (Vacuum .1 g/l).8 ± 0. gelling temperature 38–40 ◦ 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. Addition of vitamins was done after filter sterilization under sterile conditions.5. d(+) glucose 5 g/l. 20 ml of cell culture medium was transferred in to 500 ml of fresh CNB medium and was incubated for 22 h. Low viscosity alginate solution (3. coal (burned). After incubation. To this was added sterile glucose solution to make a total concentration of 60 g/l of glucose. All the solutions were purged with O2 free N2 before autoclaving. This novel immobilization approach of the bacterial cells on the specially designed cryogel beads can be an efficient approach for industrial applications.2 g/l. C. average diameter of pores and strut thickness by image analyzing software. etc. To achieve this main goal. These approaches have potential possibility for improvement of solvent fermentation. beads were taken out from the paraffin oil. 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.01 g/l. 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. 2. beads. thiamin 0. 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 paraffin liquid oil. Materials Low viscosity alginic acid sodium salt (from brown algae). Morphological analysis of immobilization support matrices The morphology of synthesized agarose-alginate cryogel beads was analyzed by scanning electron microscopy (SEM). thus offering a unique combination of high interconnected porosity. N-(3dimethylaminopropyl)-N -ethylcarbodiimide hydrochloride (EDC. Then the beads were washed with autoclaved distilled water three times for 15 min each. Thus owing to these properties these cryogels have suitable chemistry and its porosity can be altered as per application. Cryogels are special type of hydrogels which are synthesized at subzero temperatures and have supermacroporous structure with interconnected pores. peptone 5 g/l. The ratio of agarose to alginate was 2:1. Switzerland). In addition. Synthesis of alginate hydrogel beads Alginate solution (2%) was prepared in deionized water and then autoclaved the solution. / Enzyme and Microbial Technology 47 (2010) 44–51 for the use of cell immobilization with support matrix.01 g/l. Other chemicals used were of analytical grade. clay bricks. gelling temperature ∼38 to 40 ◦ C) was purchased from Sisco Research Laboratories (Mumbai. which improve reaction rates and provide high productivity.2 at 25 ◦ C) was used in concentration of 33 g/l. alginate hydrogel beads and agarose-alginate cryogel beads. India). Then 4 ml of stock solution of alginate (3. The cells were cultured in clostridial nutrient medium purchased from Fluka (Buchs. 2.

45 ␮m syringe filter into autosampler vials. potential support matrix among all the other immobilization support matrices used. Results and discussion 2. Two types of medium as mentioned above i.1. where the used beads for immobilization were of 2–3 mm in size. SEM examinations were made on a FEI Quanta 200 at high vacuum at 20 kV with spot size 3. clostridial cells were entrapped within the cryogel beads during its synthesis. The alginate Fig. 3. which had no immobilization support.8 mm) equipped with a Biorad Micro-Guard cartridge (30 mm × 4. attachment to a support matrix or entrapment within the matrix. Column temperature was adjusted to 30 ◦ C in a column oven. Analysis of glucose consumption Cell growth and active bio-conversion of glucose by clostridial cells were examined at pre-defined time intervals in batch culture. The mobile phase was 0. Sample preparation and morphological analysis The immobilization support matrices were prepared into 2–3 mm size from their respective raw material (Fig. 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. The naturally attached cells exhibit better growth than the cells immobilized on support matrices. 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. It however. For immobilization of cells on agarose-alginate cryogel beads. (C) agarose-alginate cryogel beads and (D) clay bricks. In the first approach.15 mM sulphuric acid which was filtered through a 0. butanol.9. intermediate product butyrate and other end product i. ethanol and acetone were observed in the batch tests.e. In general.5 mm. 3. the profile of glucose degradation as well as production of biomass. (B) coal (burned). where the 4 ml of immobilization support was saturated in 15 ml of CNB (set 1). India).6. The fermentation broth samples were centrifuged in closed micro-centrifuge tubes whch clarified the samples and then filtered through 0. The active cell culture was centrifuged at 6000 rpm for 20 min. The sample (20 ␮l) volume was injected onto the column to analyse the production of butanol.46 A. two approaches were employed.e. where the cells were allowed to grow as well as adhere on the support matrix surface. The fermentation was done in 40 ml crimp top glass vials. Several research groups developed several approaches for whole-cell immobilization. Different sized cryogel beads are shown in (C). Mode of immobilization on support matrices 2. Each set of experiment was designed with all the support matrices (as mentioned above) along with one control. The material in the moulds shows the processed material further used for immobilization. In second approach. In the third set. As for immobilized cells. 1. Batch fermentation Three different modes of experiments were setup in batch mode to check the effect of different support matrices in solvent production. two broad type of methods have been used to immobilize microorganism i. These results further supported in the identification 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. 1).7 ml/min through a Biorad Aminex HPX87H column (300 mm × 7. The synthesized beads were washed with sterile water purged with nitrogen. / Enzyme and Microbial Technology 47 (2010) 44–51 Tech. 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.45 ␮m filter. will depend upon whether the artificial environment provides favourable or unfavourable conditions to cells.8. 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. Bangalore. The flow rate was adjusted to 0. The digital images of immobilization support matrices: (A) coconut fibres.7. 2.6 mm).e. The active cell culture was used to load on the support matrix merged in fresh medium. CNB (first set) and P2 (second set) were separately used with the support matrices for immobilization and production analysis. These vials were then incubated at 37 ◦ C for solvent production and were monitored up to 141 h of fermentation process. . Analysis of solvent production by HPLC The immobilization of clostridial cells on non-polymeric samples was done using adsorption method.5 ml of active bacterial culture. 2. cells were adhered on already synthesized cryogel beads by physical adsorption similar method used for other immobilization support matrices. P2 (set 2) or CNB to P2 (set 3) medium and then each vial was inoculated with 1. Tripathi et al.

The use of agarose with alginate provided substantial stiffness to the beads. which might be attractive for solvent flow and can support cell adherence. The selected and morphologically examined matrices were further used to establish batch fermentation system for butanol production. liquid paraffin oil was used which helped to provide the smoothness to the outer surface of cryogel beads. However. Alginate has shown potential property as soft polymeric material. the cryogel beads were prepared at subzero temperature using EDC-NHS. During the maturation of cryogel beads at subzero temperature. 3). 4 ml of support matrices. . 2. Tripathi et al. The use of NHS to improve the performance of EDC crosslinking is well documented in the literature [14. 15 ml of medium and 1. 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. 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. 2B) has shown small nano-range grooves (Fig. An ideal immobilization support matrix should be non-toxic. the incubation system i. In the previous studies.12] or the use of EDC may involve in the crosslinking within the carboxyl group rich alginate chains [13]. 2A and C. the diameter of grooves was recorded in nano-range. 2D) investigated by scanning electron microscopy (SEM). where the polymer surface charges attract the cells and help in their adherence. In contrast. The scanning electron microscopic (SEM) images showing external and internal morphology of agarose-alginate cryogel beads as shown in (A). 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. so the sufficient volume was required to maintain conditions like pressure. These beads were mechanically stable and very spongy. which can also physically self-gelate and make a gel. which were further used to explore the potentiality of different matrices for cell immobilization. where coconut fibres based matrix showed entangled fibrous network.e. / Enzyme and Microbial Technology 47 (2010) 44–51 hydrogel beads and agarose-alginate cryogel beads (Fig. Other matrices were also examined by SEM. Image (D) shows nano-range grooves present on the outer surface of the cryogel beads. only the cryogel beads showed highly porous and interconnected internal network. which covered approximately 50% volume of the 40 ml glass bottle (Fig. which helped to prevent breakage of the beads as cell growth occurs inside. while coal (burned) and clay brick showed non-uniform rough surface containing some small pouches on the surface.5 ml of inoculum.A.. while the outer surface of cryogel beads (Fig. 1C) were also prepared in the same size. Before starting a real experiment. As the system was anaerobic. The crosslinking of agarose and alginate using EDC is not specifically studied. the mass of the immobilization support matrices along with the medium and inoculum was optimized i. In addition. Among all the used matrices. 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. which prevents the leakage of microbes from inside to outside of bead (in case of entrapment). agarose is a well known polymer. These features support agarose-alginate cryogel beads that can be used for cell immobilization applications. agarose and alginate has been most common polymers used for whole cell immobilization.e. This optimized volume was utilized for further studies. for long time run of batch fermentation in 40 ml glass bottle. (B) and (C) are the images taken at high magnification. The outer surface was matured in such a manner to work as a closed compact system for cell immobilization. highly porous and can provide large surface area for cell attachment. Fig.15].

48 A. Estimation of glucose consumption and cell behaviour Fig. 4E). The initial glucose concentration was 5 g/l in CNB medium. Apart from that. the entrapped cells needed some extra time to get activated before utilizing substrates. the glucose consumption was also found to be higher than the control bottle. 4. 4C) due to shrinkage of surface during the dehydration process. the percent glucose consumption was 97. the clay bricks matrix containing bottle showed highest glucose utilization (Table 1) up to the end of batch fermentation i. clay brick (C). In set B. we first increased the cell population by growing cells in CNB medium and simultaneously immobilizing them on the support matrices up to 72 h. while cells were entrapped in agarose-alginate cryogel beads as shows in (F). 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.3. 3. In set C. In control bottle. d-glucose was used as a sugar substrate in the medium. While in other support matrices. In set A. While the alginate hydrogel beads showed less cells adherence (Fig. 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. coal. The cells were adhered on coconut fibres (A). was slower as compared to the other matrices. coal (burned) (D) and agarose-alginate cryogel beads (E). alginate hydrogel beads (D). with the production medium (P2). 3.04% in all the support matrices at 141 h of batch fermentation. Scanning electron microscopic images of different support matrices showing cell immobilization. The rate of glucose consumption in cell entrapped agarose-alginate cryogel beads (E-AAC). 141 h. no support matrix was used. agarose-alginate cryogel beads Fig. 4E) or in entrapped mode (Fig. The solvent producing clostridia metabolize pentose sugars by way of the pentose phosphate pathway [17–20]. These results show efficient cell immobilization property of agarose-alginate cryogel beads. 4D. Once the cells were immobilized. coal (burned) (B). coconut fibres. while P2 medium is a production medium with high substrate concentration. the CNB medium was replaced with P2 medium for solvent production. C. Tripathi et al. . The digital image shows the physical appearance of immobilization support matrices setup in 40 ml crimp top glass bottles. In this study. alginate hydrogel beads (C). which lead to the detachment of cells. the initial glucose concentration was 60 g/l. Among all the support matrices. agarose-alginate cryogel beads (E) and control (without support matrix) (F) were volumetrically optimized along with the medium and inoculum in volume ratio.58 ± 2.2. The uniform distribution of cells in the entire porous cryogel bead was observed during SEM analysis at different places of the sample. Morphological analysis of immobilized support matrices The immobilization efficiency and cell behaviour on the selected matrices was examined using scanning electron microscopy (Fig. 4A and B). clay bricks (B). 4). clay bricks. Unlike adhered cells. / Enzyme and Microbial Technology 47 (2010) 44–51 3. there was no further utilization of glucose after 72 h of growth in the different support matrices i. the glucose consumption was analysed from the step where the medium was changed from CNB to P2 medium.e. The utilization of glucose by clostridial cells was analyzed at various time intervals. The CNB medium was generally used for growth and proliferation of clostridial cells. The results obtained from HPLC analysis of the samples revealed that. acetobutylicum is capable of utilizing all the prevalent sugars (pentose and hexose) present in the medium [16]. the cryogel beads showed high number of cell immobilization either in adsorbed condition (Fig.e. In this set. In the figure. coconut fibres (A). which were further utilized for the production of butanol in batch fermentation. The coconut fibres and clay bricks showed affable cell adherence due to the rough surface of matrices (Fig.

this was nearly the same concentration .7 26.04 39. The graph shows different parameter of cell entrapped agarose-alginate cryogel beads (E-AAC) in batch fermentation of set C i.0 0.8 3. It might also be possible that the rest of the cells which adhered on to support matrices were dead due to medium shock i.4 5.802 1. 6. the butanol concentration range was not very different in all the bottles which ranged from 0.8 2.58 11. from CNB to P2 medium.31 1. Glucose substrate consumption and other physiological parameters were monitored.201 g/l. Fig. 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. alginate hydrogel beads and cryogel beads (A-AAC) showed approximate same amount of butanol.e A-AAC) and alginate hydrogel beads as well control (Fig.5 3.8 5.29 19.71 g/l. clostridium nutrient broth (CNB) followed by production (P2) medium. good cell growth was observed in the CNB medium and the medium was turned into translucent because of high cell growth in suspension. 5.84 4.92 79.661 0. B and C) with varying medium and substrate concentrations.6 5.38 Different support matrices were examined upto 141 h for butanol. (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 fibres Coal Clay brick Control 2. Support type O..05). Support type O.9 4.616 3. The high butanol production was found in case of clay bricks i. (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 fibres Coal Clay brick Control 3.5 4.57 1.05). 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.90 1. The glucose concentration in the CNB medium was low. initially. 5.21 40. while other support matrices showed less efficiency than above mentioned matrices but still higher than the control.47 1.5 4. In E-AAC bottle. butyric acid and total ABE production. also the comparative changes in optical density and pH of the medium with time was analysed (Fig. 5 and Table 2).437 1. 11. But in case of E-AAC.64 10.2 4. The efficiency of agarose-alginate cryogel beads over other support matrices in the batch fermenter for producing butanol and total ABE (acetone.275 g/l (Fig.80 10.5 4.e. (in case of adhered cells i.66 20. The graph shows percent glucose consumption by clostridial cell in the presence of different support matrices in set C i. The experiments were conducted in triplicates (P < 0. the cells were safely entrapped inside the cryogel beads as there was no cell loss and also it prevented cells from medium shock. The CNB medium supports cell growth and can be used for developing an active biofilm on a support matrix by growing cells over a period of time.863 0.56 44.6 1.34 0.0 1.24 12. 7) with an ABE concentration of 0. 8). 3.e. in cell entrapped agarose-alginate cryogel beads (E-AAC). While in case of cryogel beads (A-AAC).5 76.13 92. Analysis of butanol production by HPLC Fig.9 4.65 3. However.317 0. the continuous and efficient glucose consumption was observed as shown in Fig. in all the bottles except E-AAC bottle.618 ± 0.79 5. the cells were entrapped and grew and proliferated within the porous cryogel beads. butyric acid and total ABE production.5 4.4.e.41 0.654 0. / Enzyme and Microbial Technology 47 (2010) 44–51 49 Table 1 Batch fermentation process in production (P2) medium i.922 0.6 2.02 3. The control batch fermentation experiment was run in three different sets (set A.10 20.e.37 9. The coal. At the end of fermentation of set A.94 82.737 1. set B.48 0.A. so the solvent production was limited.706 1.5 4.83 23. 6).e 13.D.218 3.071 1.0 4.5 4.097 to 0.79 g/l butanol production was achieved.95 90.737 0.5 5.71 5. clostridium nutrient broth (CNB) followed by production (P2) medium.59 13.1 2.D. Apart from that. Tripathi et al.423 21.711 4. set C.869 27. Table 2 Batch fermentation process in clostridium nutrient medium (CNB) followed by production (P2) medium i.8 4.87 24. Glucose substrate consumption and other physiological parameters were also monitored.e.59 14. and the outer surface prevented or decreased cell leakage.34 0.92 89.4 2.06 20. The experiments were conducted in triplicates (P < 0. In set B.1 0. the cells were directly grown in P2 medium with 60 g/l glucose concentration (Fig.56 13.7 11.457 Different support matrices were examined after 96 h for butanol.378 0.9 1. butanol and ethanol) was studied at various time intervals.68 26.

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. This study also suggests that the cell entrapment in cryogel beads can significantly increase the solvent production by maintaining high cell mass and preventing cell leakage. entrapment in cryogel beads showed high solvent productivity compared to adsorption process. where many classical problems like sporulation.64 g/l in cryogel beads (A-AAC) (Table 1). In set B. respectively (Table 2). agarose-alginate cryogel beads were screened and compared with other well known potential support matrices. Further. 7. The cells were initially grown on different support matrices in clostridium nutrient broth (CNB) up to 72 h (set C). 9). In set C. The experiment was done in triplicate (P < 0. The graph shows pattern of butanol production using different support structure in clostridium nutrient broth (CNB) (set A).05). Set C explains the importance of growth of entrapped cells followed by production of solvent. Entrapped cells utilized substrate from the medium with convective medium transport through nano-range grooves present on outer surface of Fig. The medium change caused some loss in cell number. The graph shows butanol production kinetics by clostridial cells immobilized on different support matrices in production (P2) medium (set B). the entrapped cells in cryogel beads (E-AAC) were active after some lag time as shown in Fig. So. The experiment was done in triplicate (P < 0. the exposure of low glucose concentration medium to high glucose concentration medium might cause some shock to cells. which can further actively work in fermentation. E-AAC cryogel beads showed efficient supports for cell growth and butanol production. the alginate polymer has well known property to enhance the cellular attachment on the matrix because of positive charge. These unique and novel properties of cryogel beads suggest its potential use in bio-catalytic conversion process to enhance the solvent productivity. In that case the cells were entrapped inside the cryogel beads and were safe from the cell loss problem. The cells entrapped inside the cryogel beads can be reused in next fermentation process. In contrast. In conclusion immobilization of cells for the use in continuous or batch culture has several advantages which make the reactor configuration relatively simple and the support structure can often be reused. pH inhibition. 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. 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. which had some lag period to get metabolically active and then start growing and utilizing the substrate for solvent production.38 g/l butanol and 9. So.37 g/l. The study was carried out on a typical anaerobic system of clostridial cells. After medium change. These findings suggest that the cryogel beads as a support adsorbent can be a good matrix for bio-catalytic conversion process. the cells were grown in CNB medium up to 72 h and then medium was changed to P2 medium (Fig. of the maximum butanol production in clay bricks.05).38 g/l ABE production. / Enzyme and Microbial Technology 47 (2010) 44–51 Fig. which suggest poor performance of all support matrices.50 A. On the other hand. the set C was set up as per the above mentioned approach. Other supports also showed good butanol production (Fig. where the maximum production was 24. In case of E-AAC cryogel beads. 8. 9. if the entrapped cells will be initially exposed to growth medium for a certain time period. can help to produce high butanol concentration as compared to other support matrices (as results obtained in set C). In batch mode. 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.80 g/l. it may metabolically activate cells. 8). 9). found in clay bricks and 21. The cryogel beads showed good amount of butanol production when used as an adsorption structure. Perhaps. In contrast. the butanol and total ABE production was 14. The graph shows pattern of butanol production using different support matrices in production (P2) medium. The exposure of cells to medium is not direct and which may also help to prevent the cells from any type of shocks. 8 and Table 1). cryogel beads. The control bottle showed 5. The experiment was done in triplicate (P < 0. . 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. substrate concentration and solventogenic inhibition etc. except EAAC cryogel beads. All the batch fermentation bottles containing different support matrices showed insignificant production of solvent except E-AAC cryogel beads (Fig.47 g/l and 27. The ABE productivity was also found in the same order.05). which makes matrix more efficient for active biofilm genesis. this behaviour within cryogel beads was because of entrapped cells. reduces the butanol productivity. the estimation of butanol and total ABE production at different time intervals was done. Fig. In this study. Tripathi et al. if the cryogel beads can be used as a support matrix (where cells are entrapped inside the beads).

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