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Enzyme and Microbial Technology 47 (2010) 44–51

Contents lists available at ScienceDirect

Enzyme and Microbial Technology

journa l homepage: www.e lsev ier .com/ locate /emt

mproved bio-catalytic conversion by novel immobilization process using cryogeleads to increase solvent production

nuj Tripathia,b, Haider Samia,b, Seema R. Jainb, Maria Viloria-Colsb, Natalia Zhuravlevab,öran Nilssonb, Hans Jungvidb, Ashok Kumara,b,∗

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

r t i c l e i n f o

rticle history:eceived 10 February 2010eceived in revised form 19 March 2010ccepted 22 March 2010

eywords:ryogel beads

mmobilization

a b s t r a c t

The use of immobilization matrix in bio-processing is a promising approach to immobilize a catalyticstrain for production of biomolecules. In this study, a novel immobilization system of agarose-alginatecryogel was developed in the format of beads and characterized to facilitate the effective cell immobiliza-tion followed by enhanced solvent production compared to other immobilization matrix. Cryogel beadsshowed macroporous internal architecture and nano-range grooves on outer periphery. Study suggeststhat these grooves facilitate the convective medium transport throughout the cryogel beads in order toeliminate the substrate and product inhibition and also prevent cell leakage. The immobilization studywas carried out on a typical anaerobic system of Clostridium acetobutylicum ATCC 824 for butanol pro-

utanol production

garose-alginate matrixermentation

duction. The experiment was carried out in three different sets (A, B and C) with varying medium andsubstrate concentration. The adsorption of cells on agarose-alginate cryogel beads produced 11.79 g/lof butanol and 21.64 g/l total ABE (acetone, butanol and ethanol), while entrapment of cells on agarose-alginate cryogel beads showed high glucose consumption, high butanol and total ABE production that was92.16%, 14.47 g/l and 27.80 g/l, respectively, which was much higher than the control and other matrices.

. Introduction

Arrays of man-made chemicals in the environment have ledn ever-increasing pressure on industrial developments leadingo the tremendous deterioration in environmental quality. Biocat-lytic conversion for the production of useful chemicals is the bestay to produce particular substance at industrial scale. Though, these of biomass as the raw material for production of some impor-ant solvents like n-butanol, acetone, ethanol etc, is still appealingnd amending environmentally. In contrast, the synthetic pro-esses have replaced fermentation for commercial production inhe early 1960s due to several reasons, of having low produc-ivity, low solvent yield and substantially high recovery cost byistillation process [1,2]. Since then, solvent fermentation could

ot compete economically with the chemical processes. However,

n recent years research has progressed in an attempt to makehe solvent fermentation not only environmentally favourable butlso economically competitive. In the pharmaceutical and biotech-

∗ Corresponding author at: Department of Biological Sciences and Bioengineering,ndian Institute of Technology Kanpur, 208016 Kanpur, India. Tel.: +91 512 2594051;ax: +91 512 2594010.

E-mail address: ashokkum@iitk.ac.in (A. Kumar).

141-0229/$ – see front matter © 2010 Elsevier Inc. All rights reserved.oi:10.1016/j.enzmictec.2010.03.009

© 2010 Elsevier Inc. All rights reserved.

nology industries, fermentation is an important step of upstreamprocessing. The fermentation process includes large-scale cultiva-tion of microbes or other single cell type, occurring either in aerobicor anaerobic conditions. The industrially important biotechnologyprocesses are generally utilizing microorganism and their applica-tion in the fermentation medium during the process. Such classicalfermentations undergo several constrains like, nutritional limita-tions, low cell density, solvent toxicity and batch-mode operationswith high down times [3,4]. It has been well recognized thatthe concentration of microbial cells is prime important duringthe downstream process to achieve higher volumetric productiv-ity from the fermented medium. Designing of advance bioreactorand its continuous operation with controlled parameters is animportant area of research and requires great focus indeed. Manyexperimental ventures have been carried out on small scale andscaled up but there had been problems of cell leakage through therunning fermenter. Therefore, the future research should focus ondevelopment of executable microbiological processes with immo-bilized cells and also perform broad research to figure out some

of the engineering problems like scale up and diffusion limitationswith high cell density.

The cell concentration inside the bioreactor can be increasedby “cell immobilization technology”. Adsorption and entrapmentare two main techniques which have been extensively examined

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or the use of cell immobilization with support matrix. Immobi-ized cell systems facilitate to maintain high cell densities in theystem, which improve reaction rates and provide high produc-ivity. The immobilization systems are also stable at high dilutionates with little cell washout and provide simplicity of operation.ther advantages are that the fermentor configuration can be rel-tively simple and immobilization material can often be reused.n addition, immobilization systems serve to enhance the sol-ent productivity due to one of the major advantage that involveshe dissociation of immobilized whole-cell growth from cellularynthesis of favoured compounds. The previous study suggestedhat different cell immobilization support matrices like clay brick,ydrogel beads and fibrous supports etc. are able to improve reac-or productivity [5–7]. These approaches have potential possibilityor improvement of solvent fermentation.

In contrast, cryogelation technology has emerged as a potentialpproach to generate three-dimensional (3D) macroporous poly-eric support matrix called cryogel, using homogeneous or het-

rogeneous monomers/polymer solution mixture. Cryogels haveeen used as a carrier for various biomedical and bioengineeringpplications [8,9]. Cryogels are special type of hydrogels which areynthesized at subzero temperatures and have supermacroporoustructure with interconnected pores, thus offering a unique com-ination of high interconnected porosity, high diffusivity and highechanical strength [10]. These gels can be produced from various

ypes of chemical molecules (monomers) or polymeric precursorshich can be both synthetic and natural, thus providing unique

hemistry to tailor them for specific applications. Other importantspect has been that these macroporous matrices can be synthe-ized in different formats like monoliths, disc shaped, thin sheets,eads, etc. Thus owing to these properties these cryogels have suit-ble chemistry and its porosity can be altered as per application.

We are aiming to generate an efficient and suitable novel sup-ort matrix for immobilizing the clostridial cells using cryogelationethod and then this developed process can be used on indus-

rial scale for the production of improved n-butanol. To achievehis main goal, the objective is to carry out studies on immobi-ization of the clostridial strain (Clostridium acetobutylicum ATCC24) as a model cell line on different supports matrix includingryogel to reveal its potentiality in comparison with other sup-ort matrices. In addition, the work will also focus to optimizehe process parameters to improve the n-butanol productivity frommmobilized clostridial cells. Here we are emphasizing the genera-ion of novel polymeric scaffold based cell immobilization approachor solvent production without cell leakage problem through anfficient transport of solvent occurring within cryogel beads. Thisovel immobilization approach of the bacterial cells on the speciallyesigned cryogel beads can be an efficient approach for industrialpplications.

. Materials and methods

.1. Materials

Low viscosity alginic acid sodium salt (from brown algae), N-(3-imethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC; FW-191.71)as purchased from Sigma Chemical Co. (St. Louis, MO, USA). Agarose (low EEO,

elling temperature ∼38 to 40 ◦C) was purchased from Sisco Research LaboratoriesMumbai, India). N-hydroxysuccinimide (NHS) was bought from SpectrochemMumbai, India). For cell immobilization experiment, C. acetobutylicum cells (ATCC24) were obtained from the American Type Culture Collection (ATCC), throughGC Promochem, Boras, Sweden. The cells were cultured in clostridial nutrientedium purchased from Fluka (Buchs, Switzerland). Other chemicals used were of

nalytical grade, which were used without any further purification.

.2. Bacterial strain and medium selection

The strain used for immobilization purpose was C. acetobutylicum ATCC 824. Its one of the best studied solventogenic clostridia strain which has been widely

ial Technology 47 (2010) 44–51 45

studied from the physiological and bioengineering points of view. C. acetobutylicumis 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 hydrochloride0.5 g/l and agar 0.5 g/l; final 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 pro-duction medium i.e. P2 medium (containing glucose 60 g/l, magnesium sulphate0.2 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 acid0.1 g/l). All the salts of P2 medium were autoclaved separately in the serum bottle. Tothis was added sterile glucose solution to make a total concentration of 60 g/l of glu-cose. Addition of vitamins was done after filter 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 theglycerol stock) were activated by heat shock at 80 ◦C for 10 min. The activated sporeculture (2 ml) was inoculated in 60 ml sterile clostridial nutrient medium (CNB) andgrown at 37 ◦C under anaerobic conditions. The growth was monitored spectropho-tometrically at 560 nm. After 44 h, 20 ml of cell culture medium was transferred into 500 ml of fresh CNB medium and was incubated for 22 h. This active cell culturewas used for immobilization and bio-catalytic conversion experiments. All cultureswere kept anaerobic by purging O2 free nitrogen gas through 0.2 �m filters.

2.4. Selection and processing of support matrices for immobilization

The support matrices for cell immobilization are an efficient approach toincrease the productivity of end-product. Previous studies have shown that lotsof efforts have been done for screening potential support matrix to improve theimmobilization efficiency. In our study, we have chosen the following supportmaterial such as coconut fibres, coal (burned), clay bricks, alginate hydrogel beadsand agarose-alginate cryogel beads for immobilization of clostridia. These supportmatrices were processed into 2–3 mm in size from their raw sources except poly-meric support matrices i.e. alginate hydrogel beads and agarose-alginate cryogelbeads. The non-polymeric matrices were further washed thoroughly with water,dried in oven at 60 ◦C and finally sterilized by autoclaving at 121 ◦C for 15 min.

2.4.1. Synthesis of alginate hydrogel beadsAlginate solution (2%) was prepared in deionized water and then autoclaved the

solution. Sterile alginate solution was put in plastic syringe and slowly dropped into2% of sterile CaCl2 solution using fine needle. The alginate solution took a shapeof bead when it merged in CaCl2. These beads were incubated overnight in thesame solution to provide strength. Then the beads were washed with autoclaveddistilled water three times for 15 min each. The beads were then further used forimmobilization experiments.

2.4.2. Synthesis of agarose-alginate cryogel beadsAgarose-alginate cryogel beads were synthesized using N-(3-

dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) withN-hydroxysuccinimide (NHS) for chemical crosslinking. Low viscosity algi-nate solution (3.75%) was prepared in 50 ml plastic tube using deionized wateras a solvent. On the other hand, agarose (low EEO; gelling temperature 38–40 ◦C)solutions (6%) was prepared in deionized water by putting the agarose containingplastic tube in the boiling water bath for ∼30 min or until the solution becometransparent. Then 4 ml of stock solution of alginate (3.75%) was added in thecompletely dissolved 5 ml of hot agarose solution (6%) and mixed by vortexing. Theratio of agarose to alginate was 2:1. Mixture of agarose-alginate was incubated for5–10 min at 60 ◦C and then the heterogeneous solution was kept for cooling at roomtemperature. When the temperature of polymer solution comes down to 45 ◦C, EDCfollowed by NHS was added and mixed by vortexing. The solution was transferredin disposable polyethylene syringe (internal tip diameter was varied for synthesisof different sized beads) and dropped in to moderately frozen light viscous paraffinliquid oil. The slightly warm polymer solution took a round shape when droppedin to moderately frozen paraffin oil. The beads were incubated in paraffin oil atsubzero temperature i.e. −20 ◦C for 16 h. After incubation, beads were taken outfrom the paraffin oil. The cryogel beads were repeatedly washed using PBS (pH7.4) for overnight under gentle magnetic stirring to remove paraffin oil completely.The agarose-alginate cryogel beads were air dried for morphological studies whilecell 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 byscanning electron microscopy (SEM). SEM micrograph allows direct measurement ofporosity, 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

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

.6. Mode of immobilization on support matrices

The immobilization of clostridial cells on non-polymeric samples was done usingdsorption method. The active cell culture was used to load on the support matrixerged in fresh medium, where the cells were allowed to grow as well as adhere on

he support matrix surface. For immobilization of cells on agarose-alginate cryogeleads, two approaches were employed. In the first approach, cells were adhered onlready synthesized cryogel beads by physical adsorption similar method used forther immobilization support matrices. In second approach, clostridial cells werentrapped within the cryogel beads during its synthesis. The active cell culture wasentrifuged at 6000 rpm for 20 min. The cell mass (pellet) was mixed in agarose-lginate solution at 45 ◦C. The crosslinkers were added and then followed the samerocedure mentioned for synthesis of cryogel beads. The synthesized beads wereashed with sterile water purged with nitrogen.

.7. Batch fermentation

Three different modes of experiments were setup in batch mode to check theffect of different support matrices in solvent production. Two types of medium asentioned above i.e. CNB (first set) and P2 (second set) were separately used with

he support matrices for immobilization and production analysis. In the third set,he cells were initially grown and immobilized in CNB media for 48 h and then the

edium was changed to P2 medium for production of butanol. Each set of experi-ent was designed with all the support matrices (as mentioned above) along with

ne control, which had no immobilization support. The fermentation was done in0 ml crimp top glass vials, where the 4 ml of immobilization support was saturated

n 15 ml of CNB (set 1), P2 (set 2) or CNB to P2 (set 3) medium and then each vial wasnoculated with 1.5 ml of active bacterial culture. These vials were then incubatedt 37 ◦C for solvent production and were monitored up to 141 h of fermentationrocess.

.8. Analysis of glucose consumption

Cell growth and active bio-conversion of glucose by clostridial cells werexamined at pre-defined time intervals in batch culture. In general, the profilef glucose degradation as well as production of biomass, butanol, intermediateroduct butyrate and other end product i.e. ethanol and acetone were observed

n the batch tests. These results further supported in the identification of most

ig. 1. The digital images of immobilization support matrices: (A) coconut fibres, (B) coahe moulds shows the processed material further used for immobilization. Different sized–3 mm in size.

al Technology 47 (2010) 44–51

potential support matrix among all the other immobilization support matricesused.

2.9. Analysis of solvent production by HPLC

Samples were taken out using syringe at different time points from the eachset of batch cultures and examined for the concentrations of different solvents pro-duced. The mobile phase was 0.15 mM sulphuric acid which was filtered through a0.45 �m filter. The flow rate was adjusted to 0.7 ml/min through a Biorad AminexHPX87H 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 tubeswhch clarified the samples and then filtered through 0.45 �m syringe filter intoautosampler vials. The sample (20 �l) volume was injected onto the column toanalyse the production of butanol.

3. Results and discussion

The immobilization of microbial cells in biological processescan occur either as a natural process or in the course of providingexternal support matrix. The naturally attached cells exhibit bettergrowth than the cells immobilized on support matrices. It how-ever, will depend upon whether the artificial environment providesfavourable or unfavourable conditions to cells. Several researchgroups developed several approaches for whole-cell immobiliza-tion. As for immobilized cells, two broad type of methods havebeen used to immobilize microorganism i.e. attachment to a sup-port matrix or entrapment within the matrix. Here we utilized bothtechniques and tried to develop a novel approach for cell immobi-lization to increase the end-product concentration in the solventand also minimize or overcome the cell leakage problem.

3.1. Sample preparation and morphological analysis

The immobilization support matrices were prepared into2–3 mm size from their respective raw material (Fig. 1). The alginate

l (burned), (C) agarose-alginate cryogel beads and (D) clay bricks. The material incryogel beads are shown in (C), where the used beads for immobilization were of

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ydrogel beads and agarose-alginate cryogel beads (Fig. 1C) werelso prepared in the same size, which were further used to explorehe potentiality of different matrices for cell immobilization. Thelginate hydrogel beads were prepared by physical crosslinking oflginate polymer chains into CaCl2 solution at room temperaturehich turned into a less porous gel beads.

In contrast, the cryogel beads were prepared at subzero tem-erature using EDC-NHS. The crosslinking of agarose and alginatesing EDC is not specifically studied, but the previous studies sug-est that EDC mediates acid anhydride formation between twoarboxyl groups of alginate and eventually the resultant acid anhy-ride may readily react with a hydroxyl group of agarose to form anster bond [11,12] or the use of EDC may involve in the crosslink-ng within the carboxyl group rich alginate chains [13]. The use ofHS to improve the performance of EDC crosslinking is well doc-mented in the literature [14,15]. In addition, agarose is a wellnown polymer, which can also physically self-gelate and makegel. The use of agarose with alginate provided substantial stiff-ess to the beads, while the alginate has high tendency to bindhe bacterial cells due to charged surface property. The cryoge-ation process generated large pores within the cryogel beads ashown in Fig. 2A and C, while the outer surface of cryogel beadsFig. 2B) has shown small nano-range grooves (Fig. 2D) investi-ated by scanning electron microscopy (SEM). The outer surfaceas matured in such a manner to work as a closed compact system

or cell immobilization. During the maturation of cryogel beads atubzero temperature, the incubation system i.e. liquid paraffin oilas used which helped to provide the smoothness to the outer

urface of cryogel beads. The presence of grooves on bead sur-ace helps in the transport of solvent in between the inner side to

ig. 2. The scanning electron microscopic (SEM) images showing external and internal mmages taken at high magnification. Image (D) shows nano-range grooves present on the

ial Technology 47 (2010) 44–51 47

outer side of cryogel beads. However, the diameter of grooves wasrecorded in nano-range, which prevents the leakage of microbesfrom inside to outside of bead (in case of entrapment). These beadswere mechanically stable and very spongy, which helped to preventbreakage of the beads as cell growth occurs inside. Other matriceswere also examined by SEM, where coconut fibres based matrixshowed entangled fibrous network, while coal (burned) and claybrick showed non-uniform rough surface containing some smallpouches on the surface.

An ideal immobilization support matrix should be non-toxic,highly porous and can provide large surface area for cell attach-ment. Among all the used matrices, only the cryogel beads showedhighly porous and interconnected internal network, which mightbe attractive for solvent flow and can support cell adherence. Inthe previous studies, agarose and alginate has been most commonpolymers used for whole cell immobilization. Alginate has shownpotential property as soft polymeric material, where the polymersurface charges attract the cells and help in their adherence. Thesefeatures support agarose-alginate cryogel beads that can be usedfor cell immobilization applications.

The selected and morphologically examined matrices werefurther used to establish batch fermentation system for butanolproduction. Before starting a real experiment, the mass of theimmobilization support matrices along with the medium andinoculum 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 anaer-obic, so the sufficient volume was required to maintain conditionslike pressure, for long time run of batch fermentation in 40 ml glassbottle. This optimized volume was utilized for further studies.

orphology of agarose-alginate cryogel beads as shown in (A). (B) and (C) are theouter surface of the cryogel beads.

48 A. Tripathi et al. / Enzyme and Microbi

Fig. 3. The digital image shows the physical appearance of immobilization supportmatrices setup in 40 ml crimp top glass bottles, which were further utilized for theproduction of butanol in batch fermentation. In the figure, coconut fibres (A), coal(burned) (B), clay brick (C), alginate hydrogel beads (D), agarose-alginate cryogelba

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medium for solvent production. The results obtained from HPLC

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eads (E) and control (without support matrix) (F) were volumetrically optimizedlong with the medium and inoculum in volume ratio.

.2. Morphological analysis of immobilized support matrices

The immobilization efficiency and cell behaviour on the selectedatrices was examined using scanning electron microscopy

Fig. 4). The coconut fibres and clay bricks showed affable celldherence due to the rough surface of matrices (Fig. 4A and B).hile the alginate hydrogel beads showed less cells adherence

Fig. 4C) due to shrinkage of surface during the dehydration pro-ess, which lead to the detachment of cells. The burned coal as aubstrate for cell adherence showed number of cells and spores onts smooth surface and pouches as shown in Fig. 4D. Apart fromhat, the cryogel beads showed high number of cell immobilizationither in adsorbed condition (Fig. 4E) or in entrapped mode (Fig. 4E).he uniform distribution of cells in the entire porous cryogel bead

as observed during SEM analysis at different places of the sam-le. These results show efficient cell immobilization property ofgarose-alginate cryogel beads.

ig. 4. Scanning electron microscopic images of different support matrices showing cell imydrogel beads (C), coal (burned) (D) and agarose-alginate cryogel beads (E), while cells w

al Technology 47 (2010) 44–51

3.3. Estimation of glucose consumption and cell behaviour

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 sol-vent producing clostridia metabolize pentose sugars by way of thepentose phosphate pathway [17–20]. The utilization of glucose byclostridial cells was analyzed at various time intervals. The ini-tial glucose concentration was 5 g/l in CNB medium. In set A, thepercent glucose consumption was 97.58 ± 2.04% in all the supportmatrices at 141 h of batch fermentation. The rate of glucose con-sumption 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 beforeutilizing substrates. In set B, with the production medium (P2), theinitial glucose concentration was 60 g/l. The growing cells on allthe support matrices showed continuous utilization of glucose andthe samples were analysed at different time intervals up to 141 hof fermentation process. Among all the support matrices, the claybricks matrix containing bottle showed highest glucose utilization(Table 1) up to the end of batch fermentation i.e. 141 h. While inother support matrices, the glucose consumption was also foundto be higher than the control bottle. In control bottle, no supportmatrix was used.

In set C, the glucose consumption was analysed from the stepwhere the medium was changed from CNB to P2 medium. TheCNB medium was generally used for growth and proliferation ofclostridial cells, while P2 medium is a production medium withhigh substrate concentration. In this set, we first increased the cellpopulation by growing cells in CNB medium and simultaneouslyimmobilizing them on the support matrices up to 72 h. Once thecells were immobilized, the CNB medium was replaced with P2

analysis of the samples revealed that, there was no further utiliza-tion of glucose after 72 h of growth in the different support matricesi.e. coconut fibres, coal, clay bricks, agarose-alginate cryogel beads

mobilization. The cells were adhered on coconut fibres (A), clay bricks (B), alginateere entrapped in agarose-alginate cryogel beads as shows in (F).

A. Tripathi et al. / Enzyme and Microbial Technology 47 (2010) 44–51 49

Table 1Batch 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 2.1 4.5 76.56 10.79 0.706 21.64Cryogel bead-entrapment 0.5 4.5 44.92 5.24 1.317 10.83Alginate hydrogel bead 3.8 4.5 89.94 12.7 0.863 23.06Coconut fibres 3.1 4.5 82.92 11.58 0.737 20.29Coal 2.6 4.5 79.95 11.59 0.922 19.87Clay brick 2.9 4.5 90.21 13.71 0.437 24.37Control 1.9 4.5 40.80 5.378 1.423 9.38

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

Table 2Batch 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 3.0 5.0 26.13 1.59 3.654 1.869Cryogel bead-entrapment 1.4 4.8 92.04 14.47 0.711 27.802Alginate hydrogel bead 5.4 4.6 39.10 1.34 4.616 1.661Coconut fibres 2.8 5.2 20.68 0.34 3.84 0.48Coal 2.6 4.9 26.66 0.41 4.02 0.57Clay brick 1.0 4.5 20.56 0.90 3.65 1.071

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ifferent support matrices were examined after 96 h for butanol, butyric acid and tere monitored. The experiments were conducted in triplicates (P < 0.05).

in case of adhered cells i.e A-AAC) and alginate hydrogel beadss well control (Fig. 5 and Table 2). However, in cell entrappedgarose-alginate cryogel beads (E-AAC), the continuous and effi-ient glucose consumption was observed as shown in Fig. 5, alsohe comparative changes in optical density and pH of the mediumith time was analysed (Fig. 6). The low glucose utilization might

e because of ineffective cell adherence which occurred on to sup-ort matrices and the most of the bacterial cells come out with theNB medium when changed to P2. It might also be possible thathe rest of the cells which adhered on to support matrices wereead due to medium shock i.e. from CNB to P2 medium. But in casef E-AAC, the cells were safely entrapped inside the cryogel beadss there was no cell loss and also it prevented cells from mediumhock. Apart from that, initially, good cell growth was observedn the CNB medium and the medium was turned into translucentecause of high cell growth in suspension, in all the bottles except-AAC bottle. In E-AAC bottle, the cells were entrapped and grewnd proliferated within the porous cryogel beads, and the outer sur-

ace prevented or decreased cell leakage. These results may suggesthat the growth of cells and cellular activity for solvent productionas found higher in E-AAC as compared to other support matri-

es.

ig. 5. The graph shows percent glucose consumption by clostridial cell in the pres-nce of different support matrices in set C i.e. clostridium nutrient broth (CNB)ollowed by production (P2) medium.

1.218 3.737 1.457

BE production. Glucose substrate consumption and other physiological parameters

3.4. Analysis of butanol production by HPLC

The efficiency of agarose-alginate cryogel beads over other sup-port matrices in the batch fermenter for producing butanol andtotal ABE (acetone, butanol and ethanol) was studied at varioustime intervals. The control batch fermentation experiment was runin three different sets (set A, B and C) with varying medium andsubstrate concentrations. At the end of fermentation of set A, thebutanol concentration range was not very different in all the bottleswhich ranged from 0.097 to 0.275 g/l (Fig. 7) with an ABE con-centration of 0.618 ± 0.201 g/l. The coal, alginate hydrogel beadsand cryogel beads (A-AAC) showed approximate same amount ofbutanol, while other support matrices showed less efficiency thanabove mentioned matrices but still higher than the control. Theglucose concentration in the CNB medium was low, so the solventproduction was limited. The CNB medium supports cell growth andcan be used for developing an active biofilm on a support matrix bygrowing 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.e13.71 g/l. While in case of cryogel beads (A-AAC), 11.79 g/l butanolproduction was achieved, this was nearly the same concentration

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

50 A. Tripathi et al. / Enzyme and Microbial Technology 47 (2010) 44–51

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ig. 7. The graph shows pattern of butanol production using different supporttructure in clostridium nutrient broth (CNB) (set A). The experiment was donen triplicate (P < 0.05).

f the maximum butanol production in clay bricks. Other supportslso showed good butanol production (Fig. 8 and Table 1). The ABEroductivity was also found in the same order, where the maxi-um production was 24.37 g/l, found in clay bricks and 21.64 g/l in

ryogel beads (A-AAC) (Table 1). The control bottle showed 5.38 g/lutanol and 9.38 g/l ABE production. The E-AAC cryogel beads didot show butanol and ABE production up to 60 h of batch fermen-ation and after that a sudden increase in the butanol and ABEoncentration was observed (Fig. 8). Perhaps, this behaviour withinryogel beads was because of entrapped cells, which had some lageriod to get metabolically active and then start growing and uti-

izing the substrate for solvent production. These findings suggesthat the cryogel beads as a support adsorbent can be a good matrixor bio-catalytic conversion process. In contrast, the alginate poly-

er has well known property to enhance the cellular attachmentn the matrix because of positive charge, which makes matrix morefficient for active biofilm genesis.

In set C, the cells were grown in CNB medium up to 72 h andhen medium was changed to P2 medium (Fig. 9). After mediumhange, the estimation of butanol and total ABE production at dif-erent time intervals was done. All the batch fermentation bottlesontaining different support matrices showed insignificant produc-ion of solvent except E-AAC cryogel beads (Fig. 9). E-AAC cryogeleads showed efficient supports for cell growth and butanol pro-uction. The medium change caused some loss in cell number.urther, the exposure of low glucose concentration medium to highlucose concentration medium might cause some shock to cells,

hich suggest poor performance of all support matrices, except E-AC cryogel beads. In that case the cells were entrapped inside theryogel beads and were safe from the cell loss problem. Entrappedells utilized substrate from the medium with convective mediumransport through nano-range grooves present on outer surface of

ig. 8. The graph shows butanol production kinetics by clostridial cells immobilizedn different support matrices in production (P2) medium (set B). The experimentas done in triplicate (P < 0.05).

ces in production (P2) medium. The cells were initially grown on different supportmatrices in clostridium nutrient broth (CNB) up to 72 h (set C). The experiment wasdone in triplicate (P < 0.05).

cryogel beads. The exposure of cells to medium is not direct andwhich may also help to prevent the cells from any type of shocks. Incase of E-AAC cryogel beads, the butanol and total ABE productionwas 14.47 g/l and 27.80 g/l, respectively (Table 2).

Set C explains the importance of growth of entrapped cells fol-lowed by production of solvent. In set B, the entrapped cells incryogel beads (E-AAC) were active after some lag time as shownin Fig. 8 and then started producing solvent after 60 h of batch fer-mentation process. The lag time required for cells may be becausethe process of entrapment may cause polymer shielding on theentrapped cells within the cryogel beads. So, if the entrappedcells will be initially exposed to growth medium for a certaintime period, it may metabolically activate cells, which can fur-ther actively work in fermentation. In contrast, the set C wasset up as per the above mentioned approach, where the initialincubation in growth medium (CNB medium) helped cells to getactive and further the active high cell mass produced high con-centration of butanol as shown in set C. These results suggest thatagarose-alginate cryogel beads were shown potentiality as a use-ful immobilization support matrix for either adsorbing the cells onsurface or entrapping the cells within beads.

In conclusion immobilization of cells for the use in continuous orbatch culture has several advantages which make the reactor con-figuration relatively simple and the support structure can often bereused. In this study, agarose-alginate cryogel beads were screenedand compared with other well known potential support matri-ces. The study was carried out on a typical anaerobic system ofclostridial cells, where many classical problems like sporulation,pH inhibition, substrate concentration and solventogenic inhibitionetc. reduces the butanol productivity. The cryogel beads showedgood amount of butanol production when used as an adsorptionstructure. On the other hand, entrapment in cryogel beads showedhigh solvent productivity compared to adsorption process. Thisstudy also suggests that the cell entrapment in cryogel beads cansignificantly increase the solvent production by maintaining highcell mass and preventing cell leakage. In batch mode, the majorfactor of solventogenic inhibition causes decrease in productivityand in such case continuous system is generally preferred whichreduces the solventogenic inhibition and increases the productiv-ity. So, if the cryogel beads can be used as a support matrix (wherecells are entrapped inside the beads), can help to produce highbutanol concentration as compared to other support matrices (asresults obtained in set C). The cells entrapped inside the cryogel

beads can be reused in next fermentation process. These uniqueand novel properties of cryogel beads suggest its potential use inbio-catalytic conversion process to enhance the solvent productiv-ity.

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cknowledgements

Authors would like to acknowledge the support received fromepartment of Biotechnology (DBT), Ministry of Science and Tech-ology, Govt. of India and Protista AB, Bjuv, Sweden. AT and HScknowledges the financial support received from Protista AB, Swe-en for working in Sweden. AT also acknowledges CSIR for grantingr. Research Fellowship (SRF).

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