Enzyme and Microbial Technology 38 (2006) 493503

Kinetic studies of biogas generation using municipal

waste as feed stock
J. Biswas, R. Chowdhury , P. Bhattacharya
Chemical Engineering Department, Jadavpur University, Kolkata 700 032, India

Received 21 March 2005; received in revised form 30 June 2005; accepted 8 July 2005

Abstract

A comprehensive study has been made on biogas kinetics from municipal waste. A 10 dm3 anaerobic batch digester equipped with a
mechanical agitator has been used under controlled environment (pH 6.8, temperature = 40 C) for this purpose. Influent slurry concentration
varied from 20 to 56 kg/m3 . At 20 kg/m3 the concentration of carbohydrate, protein and fat varied from 0.83 to 2.0 kg/m3 , 0.25 to 0.70 kg/m3
and 0.26 to 0.56 kg/m3 , respectively. The biogas generated from the digester had the methane concentration as high as 80% (v/v) at an influent
slurry concentration of 20 kg/m3 on the 15th operating day of the digester. The calorific value of biogas obtained from the digester is in
the range of 27.9831.46 MJ/(N m3 ). An attempt has been made to separate both acidogenic and methanogenic bacterial consortia. It has
been noted that classical Monod-type substrate uninhibited model equation can be used to predict the growth kinetics of separated bacterial
consortia of both types. Biogas generation kinetics was studied using initial influent slurry, carbohydrate, protein and fat concentrations as
parameters. By judiciously coupling differential mass balance equations, mathematical model equations were obtained.
2005 Elsevier Inc. All rights reserved.

Keywords: Kinetic studies; Biogas; Municipal waste

1. Introduction Literature survey [6,7] shows that batch mode of operation

Efficient disposal of municipal market waste (both vegeta-
bles and non vegetables) is always a sensitive issue to civic
authorities since the presently available disposal processes
like sanitary landfill, incineration, pyrolysis, etc., are always
associated with pollution hazards posing a serious threat to
public health [1]. This problem demands necessity of a green
technology for disposal of such municipal wastes, which
should be cost effective in one hand and pollution free on the
other. Anaerobic biological treatment of market wastes may
probably be one of the promising routes which can satisfy
the above two criteria [24]. Successful application of such
anaerobic digestion technology is, however, critically depen-
dent on proper understanding of the degradation kinetics,
judicious selection of mode of operation- batch, semi-batch
continuous, etc., and optimization of process parameter [5].
Corresponding author. Tel.: +91 33 2414 6378; fax: +91 33 2414 6378.
E-mail address: ftbe bon@yahoo.com (R. Chowdhury).
has been used for such anaerobic treatment of market waste
mainly due to high digestion time. It is also observed that all
the pioneering works in this line have been directed towards
studying overall performance of the digester [810]. How-
ever, it is now well established that biodigestion of market
waste follows a sequence of several distinctly identified steps
namely acidogenic and methanogenic reactions, each having
a separate kinetics and characteristics. Thus, understanding
of reaction engineering behaviour of individual step is abso-
lutely necessary for evaluation of design parameters of the
bioreactor.
In order to investigate the kinetics of anaerobic digestion
of market solid waste and more specifically to evaluate intrin-
sic kinetic parameters of different sequential steps, namely
acidogenic and methanogenic reactions as well, as to predict
global reaction rate, the present study has been undertaken.
By proper manipulation of experimental data an attempt has
been made to determine the intrinsic kinetic parameters for
the individual steps namely acidogenic and methanogenic
reactions. Finally the kinetics of these two steps have been

0141-0229/$ see front matter 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.enzmictec.2005.07.004
494 J. Biswas et al. / Enzyme and Microbial Technology 38 (2006) 493503

2.2. Methods
Nomenclature
2.2.1. Analytical
c concentration (kg/m3 )
2.2.1.1. Determination of bacterial mass concentration.
d days
The concentration of bacterial mass in the reaction broth was
Ks saturation constant (kg/m3 )
determined by dry weight method [11]. The culture broth was
M molecular weight
centrifuged at 10,000 rpm for 20 min. The cells were washed
Qc cumulative concentration
twice with distilled water and were taken in aluminium cups.
r reaction rate
Aluminium cups containing the cell mass were left for 24 h
Sl slurry concentration
at 80 C for drying. Finally, the weight of cell mass was
S solubility
measured using a sensitive Mettler-AE 240 (Switzerland) bal-
Y yield coefficientsexpressed as gram
ance.
material substrate/product
consumed/produced per gram of
2.2.1.2. Proximate analysis of solid waste and whey. The
biomass formed
total solid (TS), moisture content and the volatile matter (VM)
Greek letters of vegetable waste, oil cake and whey were determined by
max maximum specific growth rate (d1 ) standard methods [12].
Two grams of the sample was taken in a silica crucible and
Subscripts was dried in an air oven to constant weight at 105110 C for
IVII reaction numbers IVII 5 h. The weight of the residue was reported as total solid (TS)
Bio of biogas content of the material. The difference between the weight
carb of carbohydrate of sample and the residue gave the moisture content. Volatile
CH4 of methane matter and the ash content of the waste were determined using
fat of fat a muffle furnace maintained at 925 C and at 775 25 C,
i concerned with any reaction i respectively. Covered and open petridishes were used to hold
LCFA of long chain fatty acid the samples in the furnace for the determination of volatile
pr of protein matter and ash content, respectively. Volatile matter and ash
s of substrate content were determined by taking the differences between
VFA of volatile fatty acid weight of the samples taken and the residues left in respective
x of biomass experiments.

2.2.1.3. Determination of protein, carbohydrate and fat.

judiciously coupled in a mathematical model to predict the
overall performance of the biodigester.
2. Materials and methods
2.1. Materials
Sucrose (Alfa Aesar), casein (Alfa Aesar), hydrogenated
oil (Dalda), peptone (Loba Chem Pvt. Ltd.), beef extract
(Loba Chem Pvt. Ltd.), distilled water, tap water, amylo-
glucosidase (Celestial Labs Ltd.), anhydrous sodium sulphate
(E. Merck India Ltd.), copper sulphate (E. Merck India Ltd.)
selenium dioxide (E. Merck India Ltd.), concentrated sulfuric
acid (E. Merck India Ltd.), boric acid (E. Merck India Ltd.),
sodium hydroxide (E. Merck India Ltd.), hydrochloric acid
(E. Merck India Ltd.), light petroleum ether (E. Merck India
Ltd.), sodium acetate (Aldrich), sodium propionate (Aldrich),
sodium butyrate (Aldrich) sodium valerate (Aldrich), sodium
oleate (Aldrich), amyl alcohol (Chempure Pvt. Ltd.), veg-
etable waste, mustard oil cake and sweet meat whey were
used during the experiments.
Protein, carbohydrate and fat content of vegetable waste,
whey and oil cake were determined by standard methods
[12,13].
Protein. A portion (containing approximately 0.03
0.04 g N) of the sample was weighed and transferred to a
Kjeldahl digestion flask. Eight grams of the catalyst mixture
(90% anhydrous sodium sulphate, 3.5%copper sulphate and
0.5% of selenium dioxide) and 20 ml of concentrated sulfuric
acid were added. The flask was gently heated in an inclined
position. When the initial frothing had ceased, the top of the
flask was fitted with a loose pear shaped stopper and was
heated more strongly to ensure that the liquid boiled at a
moderate rate. The flask was shaken from time to time and
the heating was continued for another one hour till the liq-
uid became clear. Then the flask was cooled and was washed
with 400 ml ammonia free water in a distilling flask contain-
ing granulated zinc. To the receiving flask 50 ml of boric acid
solution and methyl red indicator were added. The distillation
apparatus was then connected with the delivery tube dipped in
boric acid solution. The diluted digest was made alkaline with
50% sodium hydroxide solution. After 300 ml had distilled
over, the distillate was titrated with 0.05 M sulfuric acid. The
%N2 in the sample (1 ml 0.05 M sulfuric acid 0.0014 g N)
was calculated. The crude protein figure was calculated
 J. Biswas et al. / Enzyme and Microbial Technology 38 (2006) 493503
using the following relation: crude protein = sample N
6.25.
Fat. For the estimation of the fat content of solid waste,
the material was first digested with acid. The material was
heated with hydrochloric acid to destroy the protein and the
fat separated as a layer on the top of the acidic liquid. The
acid concentration during extraction was approximately 6 M.
One to two grams of solid waste was mixed with 89 ml of
water and 10 ml of acid. The protein got dissolved in the acid
and the separated fat was extracted with light petroleum ether
in a continuous extraction apparatus.
The fat content in whey was measured with the help of
a milk butyrometer. The following liquids, namely 10 ml of
H2 SO4 (density 1.8121.818 at 20 C), 10.94 ml of whey and
1 ml of fat free amyl alcohol (density 0.8090.813 at 20 C)
were pipetted in the butyrometer ensuring that they did not
mix with one another. The tube was closed with a stopper.
The contents were mixed thoroughly and were immediately
centrifuged at 1100 rpm for 4 min. The tube was transferred
(stopper downwards) to a thermostatic bath maintained at
65 C and was held for at least 3 min and the percentage fat
was read directly from the scale.
Total, degradable and non-degradable carbohydrates.
Total: The total carbohydrate, including fibre and soluble
ones, was determined by the method of difference (total car-
bohydrate = 100 %(moisture + ash + crude protein + fat)).
Degradable and non-degradable: The degradable carbo-
hydrate in the vegetable waste and in the oil cake was deter-
mined using amyloglucosidase [14]. Duplicate 1g analytical
portions were taken for the analysis. Enzymatic digestion was
carried out for 30 min at pH 4.5 and at a temperature of 60 C
with continuous agitation. The mass consumed during the
digestion was considered the degradable carbohydrate. The
non-degradable carbohydrate was calculated by subtracting
this value from that of the total carbohydrate.
2.2.1.4. Gas chromatographic analysis. Determination of
volatile and long chain fatty acids. Volatile fatty acids were
analysed in a gas chromatograph (CHEMITO Model No.
8510) equipped with a flame ionization detector (CHEMITO
Model No. 8510) with 60/80 carbopack carbowax column
[15]. Long chain fatty acid was analysed by the same gas
chromatograph using flame ionisation detector (CHEMITO
Model No. 8510) and SP 2330 capillary column [15].
Table 1
Composition of different constituents of slurry
Constituents of Moisture (%) Total solid Volatile solid Protein (%)
slurry (TS) (%) (VS) (%)
Vegetable waste 89.14 0.5 10.76 0.5 9.78 0.5 2.42 0.5
Oil cake 8.50 0.5 91.5 0.5 81.20 0.5 30.8 0.5
Whey 93.5 0.5 6.5 0.5 5.85 0.5 0.7995 0.5


( m )2
Definition: (standard deviation) s = n , % deviation = sm 100 where , value of any observation, m , mean value, n, number of replicate
experiments. Percent standard deviation: 0.1% in all the cases. All the experiments were repeated thrice.
495
Determination of Biogas composition. The product bio-
gas composition was analysed by gas chromatograph
(CHEMITO Model No. 8510) using thermal conductivity
detector (CHEMITO Model No. 8510) with a Porapaq-q col-
umn (Netel Chromatographs length 2 m, mesh size range
80/100). Hydrogen was used as a carrier gas at a flow rate
of 20 ml/min. The oven, injector and detector temperatures
were maintained at 60, 80 and 80 C, respectively.
2.2.1.5. Determination of caloric value of biogas. Calorific
value of the gas was determined using Junkers Calorimeter.
2.2.2. Experimental methods
2.2.2.1. Preparation of inuent digester feed. Vegetable
market waste, consisting of potato, spinach, carrot,
cauliflower, cabbage, etc., having seasonal variation, sweet
meat whey and mustard oil cake were collected from local
municipal market. The characteristics of different wastes,
determined following different analytical techniques, have
been given in Table 1. All the components were sterilized
in an autoclave at a temperature and pressure of 121 C and
0.2 MPa to ensure absence of harmful pathogens. Sterile com-
ponents were subsequently taken in different proportion and
were mixed thoroughly using electrically operated mixer.
Slurry concentration was maintained at different levels using
variable quantities of tap water. Concentrations of carbohy-
drate, protein and fat were adjusted at desired values using
their pure sources like sucrose, casein and hydrogenated oil,
respectively.
2.2.2.2. Operation of the digester. A 10 dm3 semi-batch
biodigester having a height to diameter ratio of three, fit-
ted with a mechanical agitator, was employed in the present
investigation. Outside the reactor a jacket was provided
through which water was circulated to ensure isothermal con-
dition at 40 C inside the reactor. The pH of the reaction broth
was always maintained at 6.8. There was a provision for gas
outlet on the top closure from which the generated biogas
was collected continuously.
Initially the digester was fed with influent slurry to fill 80%
of the reactor volume. The volume of the seed culture added
was 10% of the reactant volume. A continuous agitation at
50 rpm was maintained during digestion process. Although
the reaction is an anaerobic one, stirring was provided only to
Fat (%) Carbohydrate Carbohydrate Ash (%)
degradable (%) non-degradable (%)
0.28 0.5 7.18 0.5 0.98 0.5
9.3 0.5 41.0 0.5 10.30 0.5
0.0494 0.5 5.005 0.5 9.2 0.5
496 J. Biswas et al. / Enzyme and Microbial Technology 38 (2006) 493503
maintain uniformity in the reaction broth. The slurry concen-
tration was varied in the range of 2056 kg/m3 . According
to the constituents of vegetable waste the concentration of
soluble carbohydrate, protein and fat varied from 0.83 to
2.0 kg/m3 , 0.5 to 0.7 kg/m3 and 0.46 to 0.56 kg/m3 when the
slurry concentration was varied from 20 to 56 kg/m3 , taking
the contribution of sweet meat whey and mustard oil cake also
into account. At a slurry concentration of 20 kg/m3 , the effects
of concentration of fat, protein and carbohydrate were stud-
ied by varying their concentration from 0.26 to 0.56 kg/m3 ,
0.25 to 0.70 kg/m3 , 0.83 to 3.0 kg/m3 . Biogas coming out of
the digester was collected continuously in a sampling bottle
by the downward displacement of water and was analysed by
a gas chromatograph.
2.2.2.3. Preparation of general bacterial medium. The gen-
eral bacterial medium was used for growth, enrichment and
maintenance of bacterial cultures. The composition of the
medium was as follows:
Component Amount to be taken
Peptone (g) 5.0
Beef extract (g) 5.0
Distilled water (ml) 1000
The three constituents were taken in a conical flask and
the pH was adjusted to 6.8. Sterilization was carried out by
keeping the medium in an autoclave for 0.9 ks under 0.2 MPa
pressure. The medium was then taken out from the autoclave
and was allowed to cool under ambient condition. pH was
adjusted to 6.8.
2.2.2.4. Separation of acidogenic and methanogenic bacte-
rial consortia. Reaction broth was taken out of the digester at
different stages, namely acidogenic and methanogenic ones
and the bacterial consortia of both the stages were enriched
using specific media. The acidogenic and methanogenic
stages of the bioreactor were identified on the basis of time
history of pH change under uncontrolled pH conditions. As it
is evident from the profile shown in Fig. 1 that acid generation
attains the saturation level on second day, reaction broth for
the enrichment of acidogenic bacterial culture were collected
at the end of the second day. Methanogenic bacterial consortia
were enriched from the reaction broth collected on the 14th
day as the methane production rate was maximum on that day.
In this case, however, the digester was run under controlled
pH condition. The enrichment medium used for acidogenic
bacteria contained carbohydrate, namely sucrose, protein,
namely casein and fat, namely hydrogenated oil at a concen-
tration of 0.83, 0.5 and 0.46 kg/m3 , respectively. The medium
for methanogenic culture for the degradation of volatile fatty
acids contained 0.82 kg/m3 volatile fatty acids, namely acetic,
propionic, butyric and valeric acids in equal proportions as
their sodium salts. The medium of methanogenic culture for
the degradation of long chain fatty acids contained sodium
oleate at a concentration of 0.08 kg/m3 . The above composi-
Fig. 1. Uncontrolled variation of pH within the biodigester.
tion of the enrichment media were chosen on the basis of the
values of concentration of carbohydrate, fat, protein, volatile
fatty acids and long chain fatty acids at respective condi-
tions of digester when the influent slurry concentration was
20 kg/m3 . The pH of acidogenic and methanogenic media
were kept at 44.5 and 6.87.0, respectively. Enrichment of
both the consortia was carried out by using repeated culture
method. All the enriched consortia were maintained in gen-
eral bacterial medium.
2.2.2.5. Preparation of seed culture for the digester. Seed
culture for the digester was prepared using the reactor broth
of a running cow-dung based biogas plant. One gram of broth
was suspended in 20 ml general sterile bacterial medium and
was allowed to grow for 2 days in an incubator kept at 40 C.
To allow the culture to be adapted to the actual condition
of the digester, selective medium having the composition of
feed slurry of 20 kg/m3 concentration was inoculated with
the suspended bacterial culture grown in the general bacterial
medium. Enrichment was done by repeating the inoculation
in the selective medium for five times. The ultimate culture,
adapted to the actual slurry condition was used as the seed
culture to inoculate the digester feed. Using the pour plate
method, dry weight analysis and by microscopic analysis
the concentration of acidogenic and methanogenic bacterial
consortia was always determined to be 0.1 and 0.061 kg/m3 ,
respectively, in the seed culture. The volume of inoculum was
always kept at 10% (v/v) of the reactant volume. Thus, the
final value of concentrations of acidogenic and methanogenic
cultures in the digester were 0.01 and 0.0061 kg/m3 ,
respectively, at the initial condition of each experimental
run.
2.2.2.6. Determination of growth kinetics of isolated con-
sortia. Acidogenic consortia. Batch experiments were con-
ducted in 50 ml Erlenmeyer flasks containing 20 ml of the
specific media with 2 ml of the inoculum containing the stock
 J. Biswas et al. / Enzyme and Microbial Technology 38 (2006) 493503
culture of acidogenic bacteria maintained in general bacte-
rial medium. To replicate the real digester condition, same
slurry having 20 kg/m3 concentration was used as the main
specific medium. Growth kinetics of acidogenic consortia
with respect to any one of the limiting substrate, namely
carbohydrate, fat and protein was determined by varying
their concentrations in the range of 0.833.0, 0.250.70,
0.260.56 kg/m3 , respectively. The concentrations of carbo-
hydrate, fat and protein were adjusted by using their pure
sources, namely sucrose, hydrogenated oil and fat. Aseptic
condition was maintained throughout the process. All the
experiments were conducted at optimum values of growth
temperature, namely (40 C) and pH (44.5) for 48 h under
unstirred condition. The control medium was separately inoc-
ulated with the stock acidogenic culture. The medium of
20 kg/m3 slurry concentration was used as the control. Bacte-
rial mass was determined by standard dry weight method after
filtering both the control and the actual media to exclude the
insoluble vegetable matter. The bacterial growth at different
time intervals corresponding to different initial concentration
of carbohydrate, fat and protein was determined along with
that in the control. The bacterial growth, only due to the sup-
ply of corresponding limiting substrate, was determined by
the method of difference.
Methanogenic consortia. Batch experiments were con-
ducted in 50 ml Erlenmeyer flasks containing 20 ml of the
specific medium with 2 ml of the inoculum. To ensure the
closeness with the digester condition, the culture of acido-
genic bacteria obtained after 2 days of incubation was used
to prepare the control medium for methanogenic bacteria. The
supernatant obtained after the centrifugation of this 2-day-old
acidogenic culture was used as the control medium for the
methanogenic culture. Growth kinetics of methanogenic con-
sortia was measured with respect to any one of the limiting
nutrients, namely volatile fatty acid and long chain fatty acids,
whose concentration was varied in the range of 0.821.80
and 0.0760.081 kg/m3 , respectively. The initial concentra-
tions of the acids were adjusted using their pure sources as
used during the separation of methanogenic consortia. All the
experiments were conducted at optimum values of growth
temperature (40 C) and pH (6.87.0). Bacterial mass was
determined by following standard dry weight method for the
497
filtrates of growth medium and the control. The growth, due to
the supply of limiting substrate was calculated by the method
of difference.
3. Theoretical analysis
Since the degradation process occurs following a highly
complex route, the reaction scheme for simulation and mod-
eling purpose can be described in the following manner:
Soluble carbohydrate and amino acids are formed through
enzymatic hydrolysis whereas volatile and long chain fatty
acids are formed through acidogenic degradation [16]. In a
separate set of reactions long chain and volatile fatty acids, so
formed via endogenous step, undergo decomposition through
methanogenic bacterial fermentation to produce the desired
biogas. The enzymatic solubilization reactions involve extra-
cellular enzymes of different types, namely cellulase, pro-
tease, etc., produced by the bacterial culture during the lag or
non-growth period. Since practically no information is avail-
able on the exact nature of enzymes produced by the cells in
the lag phase and keeping in mind the fact that the solubi-
lization reaction by extracellular enzymes mentioned above
may be very fast in nature compared to the other reactions
involving cell growth, it is logical to assume that the enzy-
matic solubilization reactions are always in equilibrium and
plays no role on the overall kinetics of the process. While pro-
cessing experimental data, results obtained from exponential
phase of growth have been considered since this is the only sit-
uation where one gets balanced cell growth condition. Thus,
while developing kinetic models the enzymatic reaction steps
for the formation of soluble carbohydrate and amino acid have
been considered as instantaneous. In the present investigation
in order to understand the reaction engineering behaviour of
complex biodegradation process, Monods substrate uninhib-
ited model has been tested to represent the exponential growth
for simulation purpose.
For the prediction of cell growth occurring in the various
reaction steps in the overall reaction scheme, the following
Monod cell growth equation has been used:
max i cxi csi
ri = (1)
Ksi + csi
498 J. Biswas et al. / Enzyme and Microbial Technology 38 (2006) 493503

Differential mass balance equations for different com- Methane concentration in the biogas was determined using
pounds involved in the reaction scheme mentioned ear- gas chromatograph. The pattern of methane concentration
lier have been solved numerically using fourth-order (v/v%) time history in the biogas with a change in slurry con-
RungeKutta method. centration is shown in Fig. 2. It is observed that the methane
Computation of cumulative methane and cumulative bio- concentration in the biogas is always lower corresponding to
gas production rates per unit volume of the reactor have been higher slurry concentration. The cumulative methane gen-
done using the following expressions; eration rate, however, increases with the increase in the
  cCH4 SCH4   T  slurry concentration. On the other hand, the absolute value of
MCH4 273 22.4 methane production rate expressed as the vapour daily space
Qc,CH4 =  (2) velocity always increases with the enhancement of slurry con-
ti
centration. Calorific value of biogas was determined using
  cCH4 SCH4   cCO2 SCO2   T  Junkers calorimeter and its time history is plotted with slurry
MCH4 + MCO 273 22.4 concentration as a parameter on Fig. 3. The figure reveals that
Qc,Bio =  2 the calorific value of the biogas decreases with the increase
ti
(3) in the slurry concentration.

4.1.2.2. Concentration of carbohydrate and protein. While

4. Results and discussion
4.1. Results
4.1.1. Determination of kinetic parameters of growth
Growth kinetics of acidogenic bacteria were determined
using the batch type experimental data obtained in the Erlen-
meyer flasks when carbohydrate was used as a limiting
substrate. Non-linear regression was carried out to deter-
mine the parameters for Monod type growth kinetics. Similar
method was followed for the determination of kinetic param-
eters of growth of acidogenic bacteria using fat and protein
as limiting substrates and for methanogenic bacteria grown
on both long chain and volatile fatty acids separately. The
kinetic parameters, namely maximum specific growth rate,
max and saturation constant, Ks were determined for both
acidogenic and methanogenic bacteria. These are shown in
Table 2.
4.1.2. Studies on the effect of different process
parameters on methane concentration, cumulative
methane production rate and caloric value of biogas
4.1.2.1. Slurry concentration. Methane concentration of
biogas generated in the digester is an important parameter.
studying the effect of carbohydrate and protein on the
methane concentration of biogas, sucrose and casein were
used as the pure carbohydrate and protein source, respec-
tively. It is noted that the methane concentration decreases
(plots not shown) with the increase in concentration of protein
and carbohydrate. The methane production rate, however,
increases with the increase of concentration of protein and
carbohydrate. Similarly, the heating value of biogas decreases
when initial concentrations of both carbohydrate and protein
increase.
4.1.2.3. Concentration of fat. In this case hydrogenated oil
was used as the pure source of fat when its effect on the
methane concentration of biogas was studied. Fig. 4 shows the
dependence of concentration time history curves of methane
on the concentration of fat. In contrary to the cases of slurry,
carbohydrate and protein concentration, the methane concen-
tration of biogas increases with the increase in fat concentra-
tion. The methane production rate is found to increase with
the increased fat concentration.
4.1.3. Studies on the effect of different process
parameters on cumulative biogas production rate
Cumulative biogas production rate represents the total
quantity of biogas generated up to a certain period per unit

Table 2
Kinetic parameters determined using batch type (Erlenmeyer flasks) experimental data
Reaction type Kinetic parameters Y (yield coefficients)

max Ks carb pr fat LCFA VFA CO2 CH4

Carbohydrate acidogenesis 5.17 0.518 13.15 9.559 2.412
Amino acid acidogenesis 6.40 0.20 14.49 12.210 1.733
Fat acidogenesis 0.550 0.10 181.81 184.0 1.26
VFA methanogenesis 2.440 0.049 20.83 11.128 9.522
LCFA methanogenesis 0.559 0.019 9.80 18.205 6.289 1.905
Percent standard deviation: 0.1% in all cases. All the experiments were repeated thrice. max : maximum specific growth rate (d1 ), Ks : saturation constant
(kg/m3 ), Y: absolute value of yield co-oeficient (defined in nomenclature). Culture conditions (described in text).
 J. Biswas et al. / Enzyme and Microbial Technology 38 (2006) 493503 499

Fig. 2. Simulated (lines) and experimental time histories of volume % of methane in biogas and cumulative methane generation rate (Qc,CH4 ) in biogas with
slurry concentration (SL) as a parameter. Lines () volumetric methane percent (CH4 %); arrows ( ) represent CH4 % as ordinate and time in days as abscissa;
() cumulative methane generation rate (Qc,CH4 ) in (dm3 /(dm3 d)); arrows ( ) represent Qc,CH4 in (dm3 /(dm3 d)) as ordinate and time in days as abscissa.
Points: (
) 20 kg/m3 ; () 40 kg/m3 ; () 56 kg/m3 .

volume of bioreactor and is expressed in terms of vapour
daily space velocity. Since the total quantum of energy
supplied by biogas depends both on its calorific value and
its quantity, vapour daily space velocity of biogas has been
considered as an important parameter.
4.1.3.2. Concentration of carbohydrate and protein. As
in case of the variation of slurry concentration, increase
in the initial concentration of carbohydrate and protein
results in the increase of cumulative biogas production
rate.

4.1.3.1. Slurry concentration. Fig. 5 shows that the cumu- 4.1.3.3. Concentration of fat. Fig. 6 reveals that the biogas
lative biogas production rate increases with the increase of production rate is insensitive towards the variation of fat con-
slurry concentration. centration.

Fig. 3. Simulated (lines) and experimental time histories of calorific value of biogas with slurry concentration (SL) as a parameter. Points: (
) 20 kg/m3 ; ()
40 kg/m3 ; () 56 kg/m3 .
500 J. Biswas et al. / Enzyme and Microbial Technology 38 (2006) 493503

Fig. 4. Simulated (lines) and experimental time histories of volume % of methane in biogas and cumulative methane generation rate (Qc,CH4 ) in biogas with
fat concentration in the slurry as a parameter. Lines: () volumetric methane percent (CH4 %); arrows ( ) represent CH4 % as ordinate and time in days as
abscissa; () cumulative methane generation rate (Qc,CH4 ) in (dm3 /(dm3 d)); arrows ( ) represent Qc,CH4 in (dm3 /(dm3 d)) as ordinate and time in days as
abscissa. Points signify concentration of fat: (
) 0.26 kg/m3 ; () 0.46 kg/m3 ; () 0.56 kg/m3 .

4.2. Discussion volumetric concentration of methane in biogas. The simu-

4.2.1. Methane concentration, methane production rate
and caloric value of biogas
4.2.1.1. Effect of slurry concentration. Methane concentra-
tion of biogas was determined experimentally using slurry
concentration as a parameter (Fig. 2). The mathematical
model equations, namely the differential mass balance equa-
tions for various components, have been used to simulate the
lated values of time history of methane concentration have
been represented by lines in the plots shown in Fig. 2. The
pattern of the time history curve of methane in biogas needs
to be explained. The first segment has a linear slope and it
continues up to 3 days of operation. The second segment
which continues up to 10th day after the end of the time
period of the first segment has slope lower than the first
segment. The third segment which starts from the 10th day

Fig. 5. Simulated (lines) and experimental points time histories of cumulative biogas production rate Qc,Bio in dm3 /(dm3 d)) with slurry concentration (SL) as
a parameter. Points: (
) 20 kg/m3 ; () 40 kg/m3 ; () 56 kg/m3 .
 J. Biswas et al. / Enzyme and Microbial Technology 38 (2006) 493503
Fig. 6. Simulated (lines) and experimental points time histories of cumulative biogas production rate (Qc,Bio in dm3 /(dm3 d)) with fat concentration in the
slurry as a parameter. Points signify ratio of carbohydrate:protein:fat: (
) 3.2:1.92:1; () 3.2:1.92:1.76;() 3.2:1.92:2.15.
and continues up to 11 days has again a rising slope and
after that the fourth segment is characterized by constant
methane concentration with time. The first segment is prob-
ably due to methanogenic breakdown of volatile fatty acids
produced from carbohydrate and protein. The second and
the third segments are due to the methanogenic breakdown
of long chain fatty acids formed from available fat follow-
ing a slow reaction path as evidenced from the low value
of max . The fourth segment is evidently representing the
saturation condition where no further methanogenic reaction
takes place. On the basis of the simplistic model assump-
tions, methane is generated both from the volatile fatty acids
and long chain fatty acids generated from carbohydrate, pro-
tein and fatty components, respectively, at different rates. The
kinetic parameters (Table 2) determined from the actual batch
type experiments using simple model carbohydrate (sucrose),
protein (casein) and fat (hydrogenated oil) reveal that the for-
mation of volatile fatty acids is much faster in comparison to
long chain fatty acids. Keeping in mind that the simulated
data have been computed by solving a number of differential
equations instead of a single one, the effects of interactive
as well as non-interactive parameters play an important role
on the overall methane production at a fixed time param-
eter. As a result simulated curves follow a distinct pattern
which cannot be visualized by simple speculation. However,
the success of present simulation work lies in the fact that
experimental results fit well with the simulated values during
a large course of time. This is evident from Fig. 2. The sim-
ulated patterns are obvious from the mathematical equations
used in the model. Accordingly the mathematical model may
be utilized to predict the methane concentration in biogas for
other systems using waste materials consisting of carbohy-
drate, protein and fatty components.
The plots in Fig. 2 also reveal that the methane concen-
tration in biogas gets higher when the slurry concentration
decreases. This trend was also noticed by other investigators
[17] working in the same area. In fact, CO2 is produced in aci-
501
dogenic degradation of carbohydrate and protein (reactions
III and IV) and also during the methanogenic degradation of
volatile fatty acids (reaction VI). On the other hand methane
is generated only during the methanogenic reactions which
are much slower than the acidogenic reactions. As a conse-
quence, as the slurry concentration increases, the production
rates of both CO2 and CH4 increase. However, the produc-
tion rate of CO2 exceeds that of CH4 causing the fall of CH4
concentration with the increase of slurry concentration. The
increase in the absolute value of methane production rate,
represented by vapour daily space velocity, with the increase
in slurry concentration may also be justified by the same fact.
The calorific value of the biogas has been calculated using
the concentration and the calorific value of methane of the
biogas. Therefore, the pattern of time history curves plotted
in Fig. 3 as functions of the slurry concentration is similar to
that of time history curve of methane (Fig. 2).
4.2.1.2. Effect of carbohydrate and protein concentration.
The same set of mathematical model equations as in case of
slurry concentration, have been used to simulate the methane
concentration in biogas as a function of carbohydrate and
protein concentration. It is observed that both in case of car-
bohydrate and protein concentration the nature of the family
of curves (not shown) is similar to that observed during the
variation of initial slurry concentration. This is because of
the fact that the same reaction mechanism similar to that
of initial slurry concentration study is considered in case of
carbohydrate and protein concentrations. Also in all cases
experimental trend can be explained well by the simulated
one.
4.2.1.3. Effect of concentration of fat. Methane concentra-
tion as a function of initial concentration of fat in the slurry
has been simulated using the same set of model equations.
The theoretical time history patterns of methane concentra-
tion have been represented by lines in Fig. 4. Experimental
502 J. Biswas et al. / Enzyme and Microbial Technology 38 (2006) 493503
values represented by points are in good agreement with
the theoretical ones. Accordingly the mathematical model
may be used to predict the effect of fat concentration on the
concentration of methane and the absolute methane genera-
tion rate. Fig. 4 shows that in contrary to the case of initial
slurry, carbohydrate, and protein concentrations, methane
concentration increases with increase in fat concentration.
Moreover the cumulative methane production also increases
with increase in fat concentration. Evidently during acido-
genic step of fat decomposition and methanogenic step of
decomposition of long chain fatty acids CO2 is acting as one
of the reactants and its consumption increases methane con-
centration in biogas.
4.2.2. Biogas generation rate
4.2.2.1. Effects of slurry, carbohydrate and protein concen-
tration. Since cumulative biogas production is an important
parameter of the present investigation, equation (3) has been
used to compute cumulative biogas production rate in terms
of gas daily space velocity for different initial slurry concen-
tration. When these computed values are plotted against time
as shown in Fig. 5, it is observed that as expected gas daily
space velocity increases with generation time. Moreover the
higher the initial slurry concentration the higher is the gas
production rate. Once again this may be attributed to the fact
that increase in slurry concentration ensures more availability
of limiting substrates in each acidogenic and methanogenic
step which in turn results in high production of both CO2 and
CH4 . The experimental data on gas daily space velocity as a
function of reaction time has been plotted on the same figure
to test the validity of the simulated equation (3). It is quite
evident that reasonably good fit between the experimental
and simulated data has been obtained. Similar trends have
been observed (not shown) when carbohydrate and protein
concentrations have been used as the independent parame-
ters.
4.2.2.2. Effect of fat concentration. While studying biogas
production rate as a function of initial fat concentration it is
observed from the Fig. 6 that although the cumulative pro-
duction rate increases with time it remains unaffected by the
variation of fat concentration. This may be due to the fact
that increase in fat concentration increases methane produc-
tion with simultaneous consumption of CO2 as reactant. Due
to this antagonistic effect the overall biogas production rate
remains insensitive towards the change in fat concentration.
Here also experimental and simulated results are in good
agreement with each other.
5. Conclusion
From the simulated and experimental results it is clear that
the variations in the initial concentrations of slurry, carbohy-
drate, protein and fat play a significant role regarding the
performance of anaerobic digester based on feed consisting
of municipal wastes. Present study reveals that methane con-
centration and the calorific value of biogas decrease with the
increase in the concentration of slurry, carbohydrate and pro-
tein, while the variation in fat concentration has the opposite
effect. A mathematical model based on the kinetic parameters
obtained from the growth kinetics of separated acidogenic
and methanogenic bacterial consortia has been developed.
Sufficient agreement between the theoretical and experimen-
tal values indicates that the model should be suitable for
similar bio-reaction systems. However, refinement of the
model is possible by the incorporation of exact enzymatic
reaction kinetics by undergoing further studies. More exper-
imental work in this area is needed for the expansion of the
applicability of the model.
Acknowledgements
Authors highly acknowledge the financial support ren-
dered by University Grants Commission of India to carry out
the Research and Development Project on Biogas Generation
from Food/Vegetable Wastes.
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