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Anaerobe 11 (2005) 217–224

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Ecology/environmental microbiology

Immobilization of anaerobic bacteria on rubberized-coir forpsychrophilic digestion of night soil

Ram Kumar Dhaked, Karna Venkat Ramana1, Arvind Tomar, Chandrakant Waghmare,Dev Vrat Kamboj, Lokendra Singh�

Biotechnology Division, Defence Research and Development Establishment, Gwalior, MP 474 002, India

Received 11 August 2004; accepted 5 January 2005

Available online 9 April 2005

Abstract

Low-ambient temperatures, o30 1C, are known to cause drastic reduction in the efficiency of anaerobic biodigesters due to low-

growth rate of the constituent bacterial consortium. Immobilization of anaerobic bacteria has been attempted in the biodigester

operating at 10 1C. Various matrices were screened and evaluated for the immobilization of bacteria in digesters. Anaerobic

digestion of night soil was carried out with hydraulic retention time in the range of 9–18 days. Among the tested matrices,

rubberized-coir was found to be the most useful at 10 1C with optimum hydraulic retention time of 15 days. Optimum amount of

coir was found as 25 g/L of the working volume of biodigesters. Immobilization of bacteria on the coir was observed by scanning

electron microscopy and fluorescent microscopy. The study indicates that rubberized coir can be utilized to increase biodegradation

of night soil at higher organic loading. Another advantage of using this matrix is that it is renewable and easily available in

comparison to other synthetic polymeric matrices.

r 2005 Elsevier Ltd. All rights reserved.

Keywords: Anaerobic-digestion; Biomethanation; Night soil; Rubberized-coir; Immobilization

1. Introduction

Stringent governmental regulation combined withpublic awareness necessitated the treatment of domesticand industrial wastes in eco-friendly manner. Thedisposal of night soil (NS) at high-altitudes inhabitedareas of Himalayas, India, where no proper disposalmethod is in practice, is a burning problem. EvenAntarctica, which is known for its pristine environment,is not an exception in this regard. Un-decomposed NS isresponsible for epidemic outbreaks, aesthetic nuisanceand organic pollution. Anaerobic biodegradation hasbeen preferable as it offers complete stabilization of NS.

e front matter r 2005 Elsevier Ltd. All rights reserved.

aerobe.2005.01.003

ing author. Tel.: +91751 2233489;

41148.

ess: lst2397@rediffmail.com (L. Singh).

ress: Microbiology Discipline, Defence Food Research

ysore 570 011, India.

Prevailing subzero temperature (as low as �40 1C)completely hampers the activity of microbes responsiblefor biodegradation of organic waste and hilly terrainlimits the volume of digesters for transportation andinstallation. Moreover, non-availability of conventionalenergy sources restricts the operation of complicatedtreatment system and maintenance of the digesterstemperature. Anaerobic digestion has the advantage interms of cost, low energy requirement because agitationis avoidable and temperature maintenance in lowtemperature areas is easy due to close system. Further,the process generates inflammable biogas.

Anaerobic disposal of NS has been sparsely studiedwith reference to sub-ambient temperatures, 5–25 1C[1,2]. At temperatures o30 1C drastic reduction in theefficiency of digester performance has widely beennoticed which is mainly attributed to the reducedgrowth rate of the anaerobic bacterial population [3].As a consequence, the digesters operating at low

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inlet

gas

outlet

13 cm

15 cm

5 cm

Fig. 1. Schematic overview of the biodigester.

R.K. Dhaked et al. / Anaerobe 11 (2005) 217–224218

temperatures require high hydraulic retention time(HRT) [4].

A wide variety of immobilized cell processes havebeen investigated, such as adsorption (or attachment),covalent binding, crosslinking, encapsulation and en-trapment. The adsorption method has been used for thetraditional biological treatment processes. There is ascope to increase the efficiency of biodegradation byusing suitable immobilization matrix which will not onlyincrease the methanogenic and other microbial popula-tion in the biodigester but may also resist the wash outdue to short HRT [5] as well as organic over loadsouring [6]. Immobilization will also minimize thedeleterious effects arising from toxicants, low pH andtemperature of the biodigesters and cells offers thepossibility of continuous operation and higher stability.Moreover, immobilization may reduce the volume of thebiodigesters due to retention of higher bacterial mass.

In the present study, we have evaluated natural andsynthetic matrices viz; coconut coir, polystyrene pads,woolen cloth, glass wool (GW), polyurethane foam(PUF) and wooden scrap fibres (WSF) for immobiliza-tion. Immobilized microorganisms were evaluated forbiodegradation of night soil and observed underscanning electron and fluorescent microscope.

2. Materials and methods

2.1. Matrices

Glass wool was obtained form Hi-Media Labora-tories Limited, Mumbai (India). Rubberized-coir (RC,the fibres coated with natural rubber), PUF andpolystyrene sheets were selected from packing material.Rope coir, polished coir (PC), raw coir and WSF wereobtained commercially. All the matrices were soaked intap water for 3–4 h before use.

2.2. Screening of matrices for NS digestion

Biodigesters of 2L capacity (working volume, Fig. 1)were charged initially with low temperature adaptedbacterial consortium obtained from biodigester operat-ing with NS at 10 1C. The pH of the inoculum was 7.24and TS and VS contents were 2.93% and 2.19%,respectively. Immobilization matrices (equivalent to300 mL of water displaced) were added to the biodige-sters separately (in duplicates). The digesters were keptat 10 1C for 15 days to allow the microbial film todevelop on the matrices. There, after semi-continuousfermentation (daily batch fed digestion) of NSwas carried out by feeding with 1:1 (wet wt/vol)diluted NS. The initial values of influent were pH7.170.2; TS 9.370.11%, VS 8.6170.09% and COD585767813 mg O2/L. Feeding was done at every 24 h

interval at 15 days HRT. Biodigesters without matrixwas run at 25 days HRT in parallel and served ascontrol. (It was observed that biodigesters fail at HRTsof lower than 25 days at 10 1C [13]. It is mainly due tolower number of methanogens and their reduced activityat low temperatures. Hence, control biodigesters wereoperated at 25 days HRT throughout this study).

2.3. Evaluation and optimization of amount of coir

For the purpose coir material of different forms wereselected with the following properties: (1) rubberizedcoir or packing coir fibres covered with polymericmaterial or latex. The material is mainly used to providesafety during transportation of equipments; (2) Ropecoir is the one that has been made into a rope; (3) rawcoir is the crude form of outer coat of the coconut; and(4) the polished coir contained smooth surface fibres.Consequent to the finding that RC is superior for thepurpose, it was added to 2L digesters in variousamounts i.e., 25/50/75/100 g (in duplicates), and thedigestion was carried out at 10 1C with 15 days HRT.Control was run at 25 days HRT.

2.4. Enrichment of VFA utilizing bacteria

Low temperature adapted microbial consortium(600 mL) was taken in 1 L anaerobic bottles. Bottleswere flushed with N2:H2:CO2 (8.5:1.0:0.5) gases andincubated for 5 days at 10 1C for stabilization of culture.Subsequently, acetate, propionate and butyrate wereadded individually up to 4000 ppm and incubation wascontinued. At 15 days intervals, VFA were monitoredand bottles were recharged with individual VFA so thattheir concentrations were maintained at the level of4000 ppm. This cycle of enrichment was repeated fivetimes. The enriched cultures were subsequently used for

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0

0.5

1

1.5

2

2.5

3

1 2 3 4 5 6 7 8

Time (week)

Bio

gas

(L

)

controlrubberized coirPUFpolystyreneWSFGlass woolwoolen cloth

9

Fig. 2. Biogas production in presence of different matrices.

R.K. Dhaked et al. / Anaerobe 11 (2005) 217–224 219

biodegradation studies. Each of these enriched cultureswas used as inoculum in immobilization study and NSwas fed at 15 days HRT in 2 L biodigesters in thepresence of RC after 10 days of stabilization at 10 1C.

2.5. Analysis

Total biogas was measured by water displacementmethod. Methane contents and volatile fatty acids(VFAs )were determined by Shimadzu gas chromato-graph (GC 9A) equipped with a flame ionizationdetector (FID) and free fatty acid phase (FFAP)column. For methane estimation, nitrogen flow was50 mL/min and column and injector temperatures were90 and 110 1C, respectively. VFAs concentrations weredetermined after acidification with phosphoric acid andether extraction of samples. The conditions used forVFAs estimations were nitrogen flow, 50 mL/min;column temperature, 120 1C and injector temperature,140 1C. Total solids, volatile solids and chemical oxygen(COD) demand were estimated as per standard methods[7]. Biogas volume, VFAs, pH of the slurry and methanecontents of biogas were measured at weekly intervals.The results reported are the average values of duplicates.

2.6. Microbial counts

Proteolytic, lipolytic, cellulolytic and amylolyticbacteria were enumerated by inoculating a fixed amountof the diluted slurry into casein, tributyrin, carboxy-methyl cellulose (CMC) and starch containing agar [4],respectively. The media plates were incubated inanaerobic jars for 3 days at 10 1C under N2, H2, andCO2 at a ratio of 8.5:1.0:0.5. Methanogenic bacteriawere enumerated by the most probable number (MPN)method by growing them in the media described byBalch et al. [8]. Serum vials containing 18 mL of liquidmedium, prepared under anaerobic conditions (N2, H2

and CO2 in the ratio of 8.5:1.0:0.5), were used formethanogenic counts. Ten fold serial dilutions of thewell-mixed samples were prepared in the dilutionmedium containing NaH2PO4 � 2H2O, 2.964; Na2HPO4,11.49; Cystine HCl, 0.025; Sodium sulphide, 0.025 andresazurin, 0.001 g/L. Two millilitre of each dilution wasinoculated in a set of five vials under aseptic conditions.The bottles along with control were incubated at 37 1Cfor 15 days. Gas from the headspace was analysed forpresence of methane on a gas chromatograph. Metha-nogenic MPN was computed on the basis of bottleshowing positive test using standard table.

2.7. Scanning electron and fluorescent microscopy

Scanning electron microscopy of matrices RC and PCwith immobilized microorganisms was carried out using15-nm thick gold layer using a JFC-1100 sputter-coating

unit (Japan). The specimens were observed at 5 and10 kV with a JSM-840 JEOL scanning electron micro-scope (Japan). The matrices (RC and PC) were alsoobserved before keeping in the digester, for comparison.The auto-fluorescence of methanogens was ascertainedafter eluting the adsorbed bacteria using 0.05 Mphosphate buffer with gentle shaking. Similarly thebacteria present in the slurry in free form were alsoobserved under fluorescent microscope (Leica Micro-scope, Germany).

3. Results

3.1. Screening of matrices

During preliminary screening of various matrices in2 L biodigesters at 15 days HRT and 10 1C, the highestbiogas production was observed after 6 weeks ofoperation in case of coconut coir. The volume of biogasproduced was considerably low in case of other matriceseven lower than control (Fig. 2). Biogas production withwoolen cloth was the least and about half than thecontrol. After 6 weeks of operation (steady stateconditions) VFAs content in the control digester wasin the range of 6700–6960 ppm and has been much lowerthan the test biodigesters (Table 1). The amount of VFAproduced in coir immobilized biodigesters were approxi-mately 10 000 ppm where as increased VFAs levels wererecorded in slurry of biodigesters with other matrices.The pH of the slurries of stabilized control and coircontaining biodigesters was in the range of 6.9–7.3. ThepH of other biodigesters was comparatively lower andwas found in between 6.2 and 6.8. The methane contentof biogas was 69% in coir containing biodigesters,which was almost similar to control (68%). Howeverbiogas contained low amount of methane (45–53%) inbiodigesters with other matrices (data not shown).

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Table 1

Total volatile fatty acids (VFAs) concentration (ppm) in the presence of various matrices

Week PUF Polystyrene sheets Coir Woolen cloth Glass wool WSF Control

1 3201 3009 3450 2847 3519 3104 3313

2 5551 6022 5677 4269 4897 5385 5300

3 7640 8958 6989 10220 8915 9028 5713

4 8340 10421 7243 12058 10741 11286 6292

5 9559 12607 7546 11100 11286 11654 6807

6 14757 16455 9189 15765 11982 12500 6700

7 14900 16976 10126 16068 12821 14689 6740

8 15120 17187 10960 16068 14915 14558 6960

0

0.5

1

1.5

2

2.5

3

Time (week)

Bio

gas

(L

)

controlrubberized coirrope coirraw coirpolished coir

1 2 3 4 5 6 7 8

Fig. 3. Biogas production by various types of coir.

0

0.5

1

1.5

2

2.5

3

Time (week)

Bio

gas

(L

)

control

25 g

50 g

75 g

100 g

1 2 3 4 5 6 7 8

Fig. 4. Effect of coir amount on biogas production.

R.K. Dhaked et al. / Anaerobe 11 (2005) 217–224220

Since immobilization on coconut coir resulted in highamount of biogas production, its different forms, i.e.rubberized coir, polished coir, rope coir and raw coirwere evaluated for their performance at 10 1C. Thebiogas produced in different types of coir is shown inFig. 3. In all biodigesters, biogas production reached toa maximum after 3 weeks of operation and subsequentlygot stabilized. Based on biogas production rubberizedcoir was found superior for immobilization than othermatrices. It was followed by raw and rope coirs.Polished coir with smooth surfaced fibrils was leastsuitable for immobilization purpose and biogas wasproduced was comparable to control operating at 25days HRT. The total VFAs concentrations were inbetween 6295 and 6663 ppm and the pH was in the rangeof 6.9–7.3 at the end of the experiment.

3.2. Immobilization on rubberized coir

The effect of quantity of RC on biogas productionhas been shown in Fig. 4. Optimum amount of RC forimmobilization was 25 g/L of biodigester volume, which

produced higher volume of biogas. Biodigesters with12.5 g/L of RC yielded lower amount of biogas followedby 37.5 and 50 g/L. RC biodigesters were operated at9–18 days HRTs and results are shown in Table 2. Thepattern of biogas production, methane content, pH andVFAs with the time indicates that biodigester operatingat 9 and 12 days HRT tend to sour. There wascontinuous decrease in amount of biogas and methanecontent with the time. Biodigesters operating at 15 and18 days HRTs (as well as control) got stabilized by 75days of operation and maximum biogas was produced at15 days HRT with 25 g/L of RC used as immobilizationmatrix. At the end of experiment, total solids (TS),volatile solids (VS) and chemical oxygen demand (COD)were reduced from 9.83% to 2.70%, 8.61% to 2.02%and 58 576 to 15 870 mg O2/L, respectively, at 15 daysHRT (Table 3). The pH was in the range of 6.9–7.3 andVFAs were 4593–5711 ppm. The biodigesters operatingat 9 and 12 days HRTs contained higher TS, VS, CODand accumulated VFAs.

Proteolytic, lipolytic, amylolytic and cellulolyticbacteria per gram of matrix on the rubberized coir were

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Table 2

Effect of HRT on biogas and methane content under immobilized condition

Days after HRT (days)

9 days 12 days 15 days 18 days Control

Gas (ml) CH4 (%) Gas (ml) CH4 (%) Gas (ml) CH4 (%) Gas (ml) CH4 (%) Gas (ml) CH4 (%)

15 1540 50 1470 53 1410 57 1540 52 1080 57

30 1630 (3170) 65 1650 (3120) 62 1590 (3000) 68 1670 (3210) 61 1510 (2590) 60

45 1425 (4595) 68 1375 (4495) 69 1493 (4493) 62 1328 (4538) 70 1223 (3813) 63

60 1313 (5908) 67 1238 (5733) 70 1378 (5871) 65 1260 (5798) 73 758 (4571) 59

75 621 (6529) 55 761 (6494) 68 1032 (6903) 72 1104 (7866) 71 461 (5032) 68

90 364 (6893) 49 498 (6992) 61 900 (7803) 68 964 (7866) 68 364 (5396) 66

105 279 (7172) 47 354 (7364) 57 861 (8664) 67 836 (8702) 70 357 (5753) 70

120 189 (7361) 36 300 (7646) 52 839 (9503) 69 893 (9595) 72 393 (6146) 69

Figures in parentheses indicate the cumulative biogas.

Table 3

Effect of HRT on TS, VS and COD

HRT (days) pH Total VFA (ppm) TS (%) VS (%) COD (mgO2/l)

9 5.4 9113 5.93 4.29 31740

12 6.1 7834 4.46 3.87 25960

15 7.2 5614 2.70 2.02 15870

18 7.3 5143 2.54 1.89 11950

Control 7.2 6788 3.18 2.36 21620

Initial values 7.3 Not detected 9.83 8.61 58576

Fig. 5. Scanning electron micrograph of rubberized coir (� 2500

magnification).

Fig. 6. Scanning electron micrograph of polished coir (� 2500

magnification).

R.K. Dhaked et al. / Anaerobe 11 (2005) 217–224 221

2.0� 107, 1.73� 108, 1.54� 108 and 5.6� 105, respec-tively. However, the corresponding counts in RCdigester slurry in suspended form were 1.2� 106,1.32� 105, 1.19� 104 and 1.6� 102/ml. Similarly, themethanogenic population on RC was 2.5� 107/g whereas methanogens in the slurry and in control biodigesterswere 1.2� 105 and 2.0� 105/ml, respectively.

The SEM of RC and polished coir fibres prior toimmobilization displayed remarkable variation in theirsurface characteristics. The RC fibres had uniformsmooth surface with dispersed pores, on the contrarypolished coir fibres had grooves and scaly structure(Figs. 5 and 6). After reaching the steady state

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conditions, the surface of RC fibres displayed a uniformlayer of coccus type of bacteria (Fig. 7). However, thebacterial mat was not uniform in case of PC and wasrather discontinuous. In contrast, the PC showed toadhere with lesser number of rod and coccus shaped of

Fig. 7. Scanning electron micrograph of rubberized coir with

immobilized bacteria (� 2500 magnification).

Fig. 8. Scanning electron micrograph of polished coir with immobi-

lized bacteria (� 2500 magnification).

Table 4

Biodegradation of night soil with VFA utilizing cultures at 10 1C (HRT-15d

Parameters Acetate Acetate enriched Propionate

Biogas (ml/week) 164 295 131

Methane content (%) 65.1 70.0 62.6

Total VFA (ppm) 4863 4060 4685

pH 6.8 6.9 6.9

bacteria on its surface (Fig. 8). Under fluorescentmicroscope both rods and cocci shaped bacteria werefound to produce auto-fluorescence indicating metha-nogenic population.

3.3. Immobilization of VFA utilizing consortium

In order to increase VFA degradation, the microbialpopulation utilizing particular VFA was enriched at10 1C. Production of biogas and methane content wereobserved for 12 weeks and the average values are shownin Table 4. Presence of coir stimulated biogas produc-tion in all the cases indicating immobilization ofbacterial consortium, which was more pronounced withpropionate utilizing inoculum and 2.1 fold increase inbiogas production was recorded. Methane contentwas between 62% and 71%, which increased in thepresence of coir by 1–5%. This increase has beennoticeable in acetate and propionate utilizing consor-tium and remained marginal by immobilization ofbutyrate degrading inoculum. Total VFA and pHrecorded at the end of 12 weeks indicated stablefermentation with VFA levels ranging from 3738 to6931 ppm and pH from 6.8 to 7.1.

4. Discussion and conclusions

High methanogenic biomass is essential to enhancethe performance of anaerobic biodigesters. Currently,digesters operational at lower HRT are based on theimmobilization of bacterial populations over matricessuch as glass beads, wooden pieces, clay, polyurethanefoam and many other porous and fibrous materials[9,12]. Immobilization of bacterial biomass in thedigester as biofilm or bacterial aggregates allowsefficient interspecies mass transfer with discriminationbetween hydrolytic retention time (HRT) and meansolids retention time (MSRT) [10]. Beside this it helps inthe adaptation of microbial communities to low-temperatures as their residence time increases in thebiodigester [5,11].

In the present study, we have used various matrices,which are widely available, and to the best of ourknowledge this is the first study where coconut coir was

ays)

Propionate enriched Butyrate Butyrate enriched Control

277 197 298 116

66.1 70.0 71.1 63.4

3738 4759 3963 5037

7.0 6.9 7.1 6.9

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evaluated for the immobilization of anaerobic bacteria.Rubberized coir is commonly used as packing materialand cheaply available than other matrices like polyester,coal, dextran, alginate etc. It was observed and reportedthat biodigesters fail at HRTs of lower than 25 days at10 1C [13] as low temperature reduced population andactivity of methanogens. Hence, control biodigesterswere operated at 25 days HRT.

During preliminary screening we found that biogasproduction was maximum in RC containing biodigesters(Fig. 2). Immobilization of bacteria on RC producedapproximately two-fold higher biogas as compared toother matrices. RC immobilized biodigesters yieldedhigher amount of biogas (37.5mL/g) as compared tocontrol (31.2 mL/g). The yield has been poor with othermatrices. The analysis of VFAs in different biodigestersindicated that with increased organic loading, higherlevels of VFAs accumulated than the control (Table 1).Higher VFAs might have adversely affected themethanogenic population leading to lower biogas yield.However, the RC immobilized biodigester containedlarge numbers of methanogens leading to higher biogasyield (Fig. 7).

In our attempts to evaluate different types of coirnone of the other types displayed activity equivalent toRC. The performance of rope/raw coir was relativelypoor than RC but better than PC (Fig. 3). SEM revealedporous structure of the RC and smooth surface of PC(Figs. 5 and 6). The immobilized bacteria were seen inlarge numbers on the surface of RC whereas discontin-uous bacterial mat with much lower number of bacteriawere observed on the PC (Figs. 7 and 8). Higherbiodegradation in presence of RC is because of betterimmobilization of desired bacteria that is attributed toits physiochemical nature and porous structure. Porousmaterials have been the choice of immobilizationmatrices as it provides much large surface area per unitvolume of matrix [12].

It was observed that amount of RC is crucial forbiodegradation of night soil. Twenty-five grams of coir/litre volume of biodigester was found to be the mostappropriate (Fig. 4). The lower biogas with less amountof RC is justified due to lower numbers of immobilizedbacteria at this juncture it is difficult to explain lowerbiogas production with higher amount of coir, However,it may be because of lower free space available for thebiodegradation reaction.

Different group of hydrolytic bacteria were found toadhere on RC and the population varied from 5.6� 105

to 1.73� 108/g. Methanogenic population which isconsidered to be the most crucial for biomethanationat psychrophilic temperatures also immobilized in goodnumbers and count 2.5� 107/g.

Using the immobilization process in the present study,HRT was considerably reduced from 25 to 15 days at10 1C, hence biodigesters volume can also be expected to

reduce. Lower volume biodigesters will be usefulbecause of the low cost and ease in transportation inhilly terrain. The better performance of biodigesterswith immobilization matrix is mainly attributed tohigher methanogenic and other hydrolytic bacterialpopulation, which is less susceptible to wash out as wellas to physico-chemical fluctuations. Total bacterialpopulation in immobilized biodigester was 100 timesmore in comparison to the control. Based on thepresent data be proposed that the surface character-istics (hydrophilic/hydrophobic) of the matrices playvital role in addition to the porous nature of thematrix. The study also emphasizes the significanceof the SEM and fluorescent microscopic observa-tions to follow the course of bacterial attachment tomatrix surface.

The immobilization efficiency of RC was alsoobserved with enriched VFA utilizing inoculum. In-creased biogas and methane content with immobilizedacetate, propionate and butyrate utilizing cultures alsoindicates increased methanogenic and other desiredbacterial population. Acetate, propionate and butyrateare either directly utilized by methanogens or inassociation with their syntrophic partners. Propionatehas been reported as most toxic fatty acid amongdifferent VFAs and our experience shows its accumula-tion at low temperature biomethanation [13,14]. Theimmobilized propionate degrading bacteria can be usedto reduce the probability of biodigester souring becauseof accumulated propionate at psychrophilic tempera-tures.

Immobilization of anaerobic bacterial biomass hasbecome an important aspect for process intensificationand to protect the bacteria from environmental andchemical perturbations. It enhances the residence timeand simultaneously adaptation of the bacterial commu-nities. The study indicates the utility of RC for NSdigestion at 10 1C in biodigesters operating with higherorganic load. The material is widely available at lowercost and has good stability (more than 1 year) at 10 1C.The matrix is relatively non-settling in the biodigesterslurry and can provide a large surface area for micro-bial attachment. The surface characteristics of theabiotic matrix such as hydrophilic and hydrophobicproperties probably play a vital role in the immobiliza-tion process. Further experiments are required todemonstrate the effect on the stability and performanceof biodigester at fluctuating pH and temperatures.The matrix can also be useful in organic over-load-ing conditions, as short periods of hydraulic overloadingare difficult to avoid in digesters operating at transitfacilities of the field and other locations where numberof users are not fixed. Furthermore, attemptswill be made to utilize the matrix for methanogenicdegradation of various organic wastes at psychrophilictemperatures.

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Acknowledgments

The authors are thankful to Director, DRDE,Gwalior, for their support and encouragement. We aregrateful to Dr R.S. Chauhan, Electron MicroscopyDivision, who kindly helped in electron microscopy

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