ARTICLE IN PRESS

Anaerobe 11 (2005) 217–224

www.elsevier.com/locate/anaerobe

Ecology/environmental microbiology

Immobilization of anaerobic bacteria on rubberized-coir for

psychrophilic 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 Prevailing subzero temperature (as low as 40 1C)

Stringent governmental regulation combined with
public awareness necessitated the treatment of domestic
and industrial wastes in eco-friendly manner. The
disposal of night soil (NS) at high-altitudes inhabited
areas of Himalayas, India, where no proper disposal
method is in practice, is a burning problem. Even
Antarctica, which is known for its pristine environment,
is not an exception in this regard. Un-decomposed NS is
responsible for epidemic outbreaks, aesthetic nuisance
and organic pollution. Anaerobic biodegradation has
been preferable as it offers complete stabilization of NS.
Corresponding author. Tel.: +91 751 2233489;
fax: +91 751 2341148.
E-mail address: lst2397@rediffmail.com (L. Singh).
1
Present Address: Microbiology Discipline, Defence Food Research
Laboratory, Mysore 570 011, India.
completely hampers the activity of microbes responsible
for biodegradation of organic waste and hilly terrain
limits the volume of digesters for transportation and
installation. Moreover, non-availability of conventional
energy sources restricts the operation of complicated
treatment system and maintenance of the digesters
temperature. Anaerobic digestion has the advantage in
terms of cost, low energy requirement because agitation
is avoidable and temperature maintenance in low
temperature areas is easy due to close system. Further,
the process generates inflammable biogas.
Anaerobic disposal of NS has been sparsely studied
with reference to sub-ambient temperatures, 5–25 1C
[1,2]. At temperatures o30 1C drastic reduction in the
efficiency of digester performance has widely been
noticed which is mainly attributed to the reduced
growth rate of the anaerobic bacterial population [3].
As a consequence, the digesters operating at low

1075-9964/$ - see front matter r 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.anaerobe.2005.01.003
 ARTICLE IN PRESS
218 R.K. Dhaked et al. / Anaerobe 11 (2005) 217–224
temperatures require high hydraulic retention time
(HRT) [4].
A wide variety of immobilized cell processes have
been investigated, such as adsorption (or attachment),
covalent binding, crosslinking, encapsulation and en-
trapment. The adsorption method has been used for the
traditional biological treatment processes. There is a
scope to increase the efficiency of biodegradation by
using suitable immobilization matrix which will not only
increase the methanogenic and other microbial popula-
tion in the biodigester but may also resist the wash out
due to short HRT [5] as well as organic over load
souring [6]. Immobilization will also minimize the
deleterious effects arising from toxicants, low pH and
temperature of the biodigesters and cells offers the
possibility of continuous operation and higher stability.
Moreover, immobilization may reduce the volume of the
biodigesters due to retention of higher bacterial mass.
In the present study, we have evaluated natural and
synthetic 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 for
biodegradation of night soil and observed under
scanning 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 and
polystyrene sheets were selected from packing material.
Rope coir, polished coir (PC), raw coir and WSF were
obtained commercially. All the matrices were soaked in
tap water for 3–4 h before use.
2.2. Screening of matrices for NS digestion
Biodigesters of 2 L capacity (working volume, Fig. 1)
were charged initially with low temperature adapted
bacterial consortium obtained from biodigester operat-
ing with NS at 10 1C. The pH of the inoculum was 7.24
and TS and VS contents were 2.93% and 2.19%,
respectively. Immobilization matrices (equivalent to
300 mL of water displaced) were added to the biodige-
sters separately (in duplicates). The digesters were kept
at 10 1C for 15 days to allow the microbial film to
develop on the matrices. There, after semi-continuous
fermentation (daily batch fed digestion) of NS
was carried out by feeding with 1:1 (wet wt/vol)
diluted NS. The initial values of influent were pH
7.170.2; TS 9.370.11%, VS 8.6170.09% and COD
585767813 mg O2/L. Feeding was done at every 24 h
inlet
gas
5 cm
outlet
15 cm
13 cm
Fig. 1. Schematic overview of the biodigester.
interval at 15 days HRT. Biodigesters without matrix
was run at 25 days HRT in parallel and served as
control. (It was observed that biodigesters fail at HRTs
of lower than 25 days at 10 1C [13]. It is mainly due to
lower number of methanogens and their reduced activity
at low temperatures. Hence, control biodigesters were
operated at 25 days HRT throughout this study).
2.3. Evaluation and optimization of amount of coir
For the purpose coir material of different forms were
selected with the following properties: (1) rubberized
coir or packing coir fibres covered with polymeric
material or latex. The material is mainly used to provide
safety during transportation of equipments; (2) Rope
coir is the one that has been made into a rope; (3) raw
coir 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 the
purpose, it was added to 2 L digesters in various
amounts i.e., 25/50/75/100 g (in duplicates), and the
digestion 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. Bottles
were flushed with N2:H2:CO2 (8.5:1.0:0.5) gases and
incubated for 5 days at 10 1C for stabilization of culture.
Subsequently, acetate, propionate and butyrate were
added individually up to 4000 ppm and incubation was
continued. At 15 days intervals, VFA were monitored
and bottles were recharged with individual VFA so that
their concentrations were maintained at the level of
4000 ppm. This cycle of enrichment was repeated five
times. The enriched cultures were subsequently used for
 ARTICLE IN PRESS
R.K. Dhaked et al. / Anaerobe 11 (2005) 217–224
biodegradation studies. Each of these enriched cultures
was used as inoculum in immobilization study and NS
was fed at 15 days HRT in 2 L biodigesters in the
presence of RC after 10 days of stabilization at 10 1C.
2.5. Analysis
Total biogas was measured by water displacement
method. Methane contents and volatile fatty acids
(VFAs )were determined by Shimadzu gas chromato-
graph (GC 9A) equipped with a flame ionization
detector (FID) and free fatty acid phase (FFAP)
column. For methane estimation, nitrogen flow was
50 mL/min and column and injector temperatures were
90 and 110 1C, respectively. VFAs concentrations were
determined after acidification with phosphoric acid and
ether extraction of samples. The conditions used for
VFAs 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 methane
contents of biogas were measured at weekly intervals.
The results reported are the average values of duplicates.
2.6. Microbial counts
Proteolytic, lipolytic, cellulolytic and amylolytic
bacteria were enumerated by inoculating a fixed amount
of the diluted slurry into casein, tributyrin, carboxy-
methyl cellulose (CMC) and starch containing agar [4],
respectively. The media plates were incubated in
anaerobic jars for 3 days at 10 1C under N2, H2, and
CO2 at a ratio of 8.5:1.0:0.5. Methanogenic bacteria
were enumerated by the most probable number (MPN)
method by growing them in the media described by
Balch et al. [8]. Serum vials containing 18 mL of liquid
medium, prepared under anaerobic conditions (N2, H2
and CO2 in the ratio of 8.5:1.0:0.5), were used for
methanogenic counts. Ten fold serial dilutions of the
well-mixed samples were prepared in the dilution
medium containing NaH2PO4
2H2O, 2.964; Na2HPO4,
11.49; Cystine HCl, 0.025; Sodium sulphide, 0.025 and
resazurin, 0.001 g/L. Two millilitre of each dilution was
inoculated in a set of five vials under aseptic conditions.
The bottles along with control were incubated at 37 1C
for 15 days. Gas from the headspace was analysed for
presence of methane on a gas chromatograph. Metha-
nogenic MPN was computed on the basis of bottle
showing positive test using standard table.
2.7. Scanning electron and fluorescent microscopy
Scanning electron microscopy of matrices RC and PC
with immobilized microorganisms was carried out using
15-nm thick gold layer using a JFC-1100 sputter-coating
219
unit (Japan). The specimens were observed at 5 and
10 kV with a JSM-840 JEOL scanning electron micro-
scope (Japan). The matrices (RC and PC) were also
observed before keeping in the digester, for comparison.
The auto-fluorescence of methanogens was ascertained
after eluting the adsorbed bacteria using 0.05 M
phosphate buffer with gentle shaking. Similarly the
bacteria present in the slurry in free form were also
observed under fluorescent microscope (Leica Micro-
scope, Germany).
3. Results
3.1. Screening of matrices
During preliminary screening of various matrices in
2 L biodigesters at 15 days HRT and 10 1C, the highest
biogas production was observed after 6 weeks of
operation in case of coconut coir. The volume of biogas
produced was considerably low in case of other matrices
even lower than control (Fig. 2). Biogas production with
woolen cloth was the least and about half than the
control. After 6 weeks of operation (steady state
conditions) VFAs content in the control digester was
in the range of 6700–6960 ppm and has been much lower
than the test biodigesters (Table 1). The amount of VFA
produced in coir immobilized biodigesters were approxi-
mately 10 000 ppm where as increased VFAs levels were
recorded in slurry of biodigesters with other matrices.
The pH of the slurries of stabilized control and coir
containing biodigesters was in the range of 6.9–7.3. The
pH of other biodigesters was comparatively lower and
was found in between 6.2 and 6.8. The methane content
of biogas was 69% in coir containing biodigesters,
which was almost similar to control (68%). However
biogas contained low amount of methane (45–53%) in
biodigesters with other matrices (data not shown).
control
rubberized coir
PUF
3 polystyrene
WSF
Glass wool
2.5 woolen cloth
2
Biogas (L)
1.5
1
0.5
0
1 2 3 4 5 6 7 8 9
Time (week)
Fig. 2. Biogas production in presence of different matrices.
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220 R.K. Dhaked et al. / Anaerobe 11 (2005) 217–224

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

control
control
rubberized coir
rope coir 25 g
3
raw coir 50 g
polished coir
2.5 3 75 g
100 g
2 2.5
Biogas (L)

2
Biogas (L)
1.5

1.5
1
1
0.5
0.5
0
1 2 3 4 5 6 7 8 0
Time (week) 1 2 3 4 5 6 7 8
Time (week)
Fig. 3. Biogas production by various types of coir.
Fig. 4. Effect of coir amount on biogas production.

Since immobilization on coconut coir resulted in high
amount of biogas production, its different forms, i.e.
rubberized coir, polished coir, rope coir and raw coir
were evaluated for their performance at 10 1C. The
biogas produced in different types of coir is shown in
Fig. 3. In all biodigesters, biogas production reached to
a maximum after 3 weeks of operation and subsequently
got stabilized. Based on biogas production rubberized
coir was found superior for immobilization than other
matrices. It was followed by raw and rope coirs.
Polished coir with smooth surfaced fibrils was least
suitable for immobilization purpose and biogas was
produced was comparable to control operating at 25
days HRT. The total VFAs concentrations were in
between 6295 and 6663 ppm and the pH was in the range
of 6.9–7.3 at the end of the experiment.
3.2. Immobilization on rubberized coir
The effect of quantity of RC on biogas production
has been shown in Fig. 4. Optimum amount of RC for
immobilization was 25 g/L of biodigester volume, which
produced higher volume of biogas. Biodigesters with
12.5 g/L of RC yielded lower amount of biogas followed
by 37.5 and 50 g/L. RC biodigesters were operated at
9–18 days HRTs and results are shown in Table 2. The
pattern of biogas production, methane content, pH and
VFAs with the time indicates that biodigester operating
at 9 and 12 days HRT tend to sour. There was
continuous decrease in amount of biogas and methane
content with the time. Biodigesters operating at 15 and
18 days HRTs (as well as control) got stabilized by 75
days of operation and maximum biogas was produced at
15 days HRT with 25 g/L of RC used as immobilization
matrix. 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 days
HRT (Table 3). The pH was in the range of 6.9–7.3 and
VFAs were 4593–5711 ppm. The biodigesters operating
at 9 and 12 days HRTs contained higher TS, VS, COD
and accumulated VFAs.
Proteolytic, lipolytic, amylolytic and cellulolytic
bacteria per gram of matrix on the rubberized coir were
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R.K. Dhaked et al. / Anaerobe 11 (2005) 217–224 221

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 (mg O2/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).
2.0  107, 1.73  108, 1.54  108 and 5.6  105, respec-
tively. However, the corresponding counts in RC
digester slurry in suspended form were 1.2  106,
1.32  105, 1.19  104 and 1.6  102/ml. Similarly, the
methanogenic population on RC was 2.5  107/g where
as methanogens in the slurry and in control biodigesters
were 1.2  105 and 2.0  105/ml, respectively.
Fig. 6. Scanning electron micrograph of polished coir (  2500
magnification).
The SEM of RC and polished coir fibres prior to
immobilization displayed remarkable variation in their
surface characteristics. The RC fibres had uniform
smooth surface with dispersed pores, on the contrary
polished coir fibres had grooves and scaly structure
(Figs. 5 and 6). After reaching the steady state
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222 R.K. Dhaked et al. / Anaerobe 11 (2005) 217–224

conditions, the surface of RC fibres displayed a uniform
layer of coccus type of bacteria (Fig. 7). However, the
bacterial mat was not uniform in case of PC and was
rather discontinuous. In contrast, the PC showed to
adhere with lesser number of rod and coccus shaped of
bacteria on its surface (Fig. 8). Under fluorescent
microscope both rods and cocci shaped bacteria were
found to produce auto-fluorescence indicating metha-
nogenic population.
3.3. Immobilization of VFA utilizing consortium

In order to increase VFA degradation, the microbial

Fig. 7. Scanning electron micrograph of rubberized coir with
immobilized bacteria (  2500 magnification).
population utilizing particular VFA was enriched at
10 1C. Production of biogas and methane content were
observed for 12 weeks and the average values are shown
in Table 4. Presence of coir stimulated biogas produc-
tion in all the cases indicating immobilization of
bacterial consortium, which was more pronounced with
propionate utilizing inoculum and 2.1 fold increase in
biogas production was recorded. Methane content
was between 62% and 71%, which increased in the
presence of coir by 1–5%. This increase has been
noticeable in acetate and propionate utilizing consor-
tium and remained marginal by immobilization of
butyrate degrading inoculum. Total VFA and pH
recorded at the end of 12 weeks indicated stable
fermentation with VFA levels ranging from 3738 to
6931 ppm and pH from 6.8 to 7.1.

4. Discussion and conclusions

High methanogenic biomass is essential to enhance

Fig. 8. Scanning electron micrograph of polished coir with immobi-
lized bacteria (  2500 magnification).
the performance of anaerobic biodigesters. Currently,
digesters operational at lower HRT are based on the
immobilization of bacterial populations over matrices
such as glass beads, wooden pieces, clay, polyurethane
foam and many other porous and fibrous materials
[9,12]. Immobilization of bacterial biomass in the
digester as biofilm or bacterial aggregates allows
efficient interspecies mass transfer with discrimination
between hydrolytic retention time (HRT) and mean
solids retention time (MSRT) [10]. Beside this it helps in
the adaptation of microbial communities to low-
temperatures as their residence time increases in the
biodigester [5,11].
In the present study, we have used various matrices,
which are widely available, and to the best of our
knowledge this is the first study where coconut coir was

Table 4
Biodegradation of night soil with VFA utilizing cultures at 10 1C (HRT-15days)

Parameters Acetate Acetate enriched Propionate Propionate enriched Butyrate Butyrate enriched Control

Biogas (ml/week) 164 295 131 277 197 298 116

Methane content (%) 65.1 70.0 62.6 66.1 70.0 71.1 63.4
Total VFA (ppm) 4863 4060 4685 3738 4759 3963 5037
pH 6.8 6.9 6.9 7.0 6.9 7.1 6.9
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R.K. Dhaked et al. / Anaerobe 11 (2005) 217–224
evaluated for the immobilization of anaerobic bacteria.
Rubberized coir is commonly used as packing material
and cheaply available than other matrices like polyester,
coal, dextran, alginate etc. It was observed and reported
that biodigesters fail at HRTs of lower than 25 days at
10 1C [13] as low temperature reduced population and
activity of methanogens. Hence, control biodigesters
were operated at 25 days HRT.
During preliminary screening we found that biogas
production was maximum in RC containing biodigesters
(Fig. 2). Immobilization of bacteria on RC produced
approximately two-fold higher biogas as compared to
other matrices. RC immobilized biodigesters yielded
higher amount of biogas (37.5 mL/g) as compared to
control (31.2 mL/g). The yield has been poor with other
matrices. The analysis of VFAs in different biodigesters
indicated that with increased organic loading, higher
levels of VFAs accumulated than the control (Table 1).
Higher VFAs might have adversely affected the
methanogenic population leading to lower biogas yield.
However, the RC immobilized biodigester contained
large numbers of methanogens leading to higher biogas
yield (Fig. 7).
In our attempts to evaluate different types of coir
none of the other types displayed activity equivalent to
RC. The performance of rope/raw coir was relatively
poor than RC but better than PC (Fig. 3). SEM revealed
porous structure of the RC and smooth surface of PC
(Figs. 5 and 6). The immobilized bacteria were seen in
large numbers on the surface of RC whereas discontin-
uous bacterial mat with much lower number of bacteria
were observed on the PC (Figs. 7 and 8). Higher
biodegradation in presence of RC is because of better
immobilization of desired bacteria that is attributed to
its physiochemical nature and porous structure. Porous
materials have been the choice of immobilization
matrices as it provides much large surface area per unit
volume of matrix [12].
It was observed that amount of RC is crucial for
biodegradation of night soil. Twenty-five grams of coir/
litre volume of biodigester was found to be the most
appropriate (Fig. 4). The lower biogas with less amount
of RC is justified due to lower numbers of immobilized
bacteria at this juncture it is difficult to explain lower
biogas production with higher amount of coir, However,
it may be because of lower free space available for the
biodegradation reaction.
Different group of hydrolytic bacteria were found to
adhere on RC and the population varied from 5.6  105
to 1.73  108/g. Methanogenic population which is
considered to be the most crucial for biomethanation
at psychrophilic temperatures also immobilized in good
numbers and count 2.5  107/g.
Using the immobilization process in the present study,
HRT was considerably reduced from 25 to 15 days at
10 1C, hence biodigesters volume can also be expected to
223
reduce. Lower volume biodigesters will be useful
because of the low cost and ease in transportation in
hilly terrain. The better performance of biodigesters
with immobilization matrix is mainly attributed to
higher methanogenic and other hydrolytic bacterial
population, which is less susceptible to wash out as well
as to physico-chemical fluctuations. Total bacterial
population in immobilized biodigester was 100 times
more in comparison to the control. Based on the
present data be proposed that the surface character-
istics (hydrophilic/hydrophobic) of the matrices play
vital role in addition to the porous nature of the
matrix. The study also emphasizes the significance
of the SEM and fluorescent microscopic observa-
tions to follow the course of bacterial attachment to
matrix surface.
The immobilization efficiency of RC was also
observed with enriched VFA utilizing inoculum. In-
creased biogas and methane content with immobilized
acetate, propionate and butyrate utilizing cultures also
indicates increased methanogenic and other desired
bacterial population. Acetate, propionate and butyrate
are either directly utilized by methanogens or in
association with their syntrophic partners. Propionate
has been reported as most toxic fatty acid among
different VFAs and our experience shows its accumula-
tion at low temperature biomethanation [13,14]. The
immobilized propionate degrading bacteria can be used
to reduce the probability of biodigester souring because
of accumulated propionate at psychrophilic tempera-
tures.
Immobilization of anaerobic bacterial biomass has
become an important aspect for process intensification
and to protect the bacteria from environmental and
chemical perturbations. It enhances the residence time
and simultaneously adaptation of the bacterial commu-
nities. The study indicates the utility of RC for NS
digestion at 10 1C in biodigesters operating with higher
organic load. The material is widely available at lower
cost and has good stability (more than 1 year) at 10 1C.
The matrix is relatively non-settling in the biodigester
slurry and can provide a large surface area for micro-
bial attachment. The surface characteristics of the
abiotic matrix such as hydrophilic and hydrophobic
properties probably play a vital role in the immobiliza-
tion process. Further experiments are required to
demonstrate the effect on the stability and performance
of biodigester at fluctuating pH and temperatures.
The matrix can also be useful in organic over-load-
ing conditions, as short periods of hydraulic overloading
are difficult to avoid in digesters operating at transit
facilities of the field and other locations where number
of users are not fixed. Furthermore, attempts
will be made to utilize the matrix for methanogenic
degradation of various organic wastes at psychrophilic
temperatures.
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224 R.K. Dhaked et al. / Anaerobe 11 (2005) 217–224
Acknowledgments
The authors are thankful to Director, DRDE,
Gwalior, for their support and encouragement. We are
grateful to Dr R.S. Chauhan, Electron Microscopy
Division, who kindly helped in electron microscopy
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