Biotechnology and Bioprocess Engineering 17: 1284-1293 (2012)
DOI 10.1007/s12257-012-0044-z
RESEARCH PAPER
Optimization of Biogas Production from Co-digestion of Whey and
Cow Manure
Andrea Hublin, Tatjana Ignjatió
c Zokió
c, and Bruno Zelió
c
Received: 20 January 2012 / Revised: 10 June 2012 / Accepted: 2 July 2012
© The Korean Society for Biotechnology and Bioengineering and Springer 2012
Abstract Anaerobic co-digestion is effective and envi-
ronmentally attractive technology for energy recovery from
organic waste. Organic, agricultural and industrial wastes
are good substrates for anaerobic co-digestion because they
contain high levels of easily biodegradable materials. In
this paper enhancement of biogas production from co-
digestion of whey and cow manure was investigated in a
series of batch experiments. The influence of whey ratio on
specific biogas production in a mixture with cow manure
was analyzed at 35 and 55oC, for different initial pH values
and for different concentrations of supplemental bicarbonate
in experiments carried out over 12 days. Good biogas
production (6.6 dm3/dm3), methane content (79.4%) in a
biogas mixture and removal efficiencies for total solids (16%)
were achieved at optimum process conditions (temperature
of 55oC, 10% v/v of whey and 5 g/dm3 NaHCO3 in the
initial mixture). In order to validate optimized conditions
for co-digestion of whey and cow manure in the one-stage
batch process, the experiments were performed within 45
days. The high biogas production (21.8 dm3/dm3), a good
methane content (78.7%) in a biogas mixture as well as
maximum removal efficiencies for total solids (32.3%),
and chemical oxygen demand (56.3%), respectively indicate
that whey could be efficiently degraded to biogas in a one-
stage batch process when co-digested with cow manure.
Keywords: anaerobic co-digestion, whey, cow manure,
biogas production, methane
Andrea Hublin
EKONERG - Energy and Environmental Protection Institute, Ltd., Koranska
5, HR-10000 Zagreb, Croatia
Tatjana Ignjatió c*
c, Bruno Zelió
c Zokió
Faculty of Chemical Engineering and Technology, University of Zagreb,
Maruliócev trg 19, HR-10000 Zagreb, Croatia
Tel: +385-1459-7146/+385-1459-7281; Fax: +385-1459-7261
E-mail: bzelic@fkit.hr
1. Introduction
The most important property of biowaste as an alternative
energy source is its environmental compatibility. Renewable
waste materials from different sources, such as agriculture
(plant and animal waste), industries (dairy waste, sugar
refinery waste, slaughter waste…) and residential (kitchen
and garden waste), are convertible to useful energy forms
like biohydrogen, biogas, bioalcohols, etc. [1]. The produc-
tion of biogas through anaerobic digestion offers significant
advantages over other forms of waste treatment [2]. The
major advantages of this process are high-energy efficiency
and process simplicity compared to other waste treatment
methods [3]. Therefore, the anaerobic digestion of whey
offers an approach in terms of both energy saving and
pollution control. Additionally, biogas as a renewable energy
source plays an important role in reducing greenhouse
gases because it is a carbon neutral fuel.
The main issue for the co-digestion process lies in
balancing several parameters in the co-substrate mixture:
macro- and micronutrients, C:N ratio, pH, inhibitors/toxic
compounds, biodegradable organic matter and dry matter
[4]. Co-digestion has been reported to enhance the
anaerobic digestion process by creating a better nutrient
balance from the materials mixed to feed the digester and/
or provide positive synergism for bacterial growth [5,6].
Different organic substrates are combined to generate a
homogenous mixture as input to the anaerobic reactor in
order to increase process performance, realize a more
efficient use of equipment and cost-sharing by processing
multiple waste streams in a single facility [7].
The dairy industry generates residues of which whey is
the most important wastewater produced with an extremely
high organic content [8]. The strong polluting potential of
dairy wastewater is characterised by high biological oxygen
demand (BOD) and chemical oxygen demand (COD) [9].
Optimization of Biogas Production from Co-digestion of Whey and Cow Manure
The principal components of whey are lactose, proteins and
mineral salts [10,11]. Whey is highly biodegradable (~ 99%)
with a very high organic content (up to 70 g COD/dm3)
and a low alkalinity content (50 meq/dm3 or 2,500 mg/dm3
as CaCO3) [12-14]. Therefore, whey has a very strong
polluting potential, approximately one hundred times greater
than that caused by an equivalent volume of domestic
wastewater [15].
Raw whey is a quite difficult substrate to treat anaero-
bically because of the lack of alkalinity, the high COD
concentrations and the tendency to acidify very rapidly.
Anaerobic treatment of whey has frequently encountered
difficulties in maintaining stable operation [15,16]. Because
of its low bicarbonate alkalinity supplemental alkalinity
is required to avoid process failure [17-19]. Alkalinity
supplementation can be minimized by using operation
conditions directed at obtaining better treatment efficiency
or dilution of the influent to reduce the instability and low
efficiency problems caused by its high organic content [8].
Most of the reported studies in whey wastewater treatment
were performed at mesophilic (35oC) or thermophilic (55oC)
conditions [19].
Whey, as one of the waste products in dairy industry,
contains lactic acid bacteria Streptococcaceae, Leucono-
stocaceae and Lactobacillaceae (genera Streptococcus,
Lactococcus, Leuconostoc and Lactobacillus) when it is
fermented. These are all Gram positive bacteria. Some of
them are mesophiles which grow fastest at 20 ~ 30oC and
others are moderate thermophiles, growing best at 35 ~
45oC [20]. The dominant methanogens in anaerobic
treatment of dairy waste are assumed to be acetoclastic
Methanosarcinales (Methanosarcinaceae and Methano-
saetaceae) than hydrogenotrophic Methanobacteriales,
Methanomicrobiales and Methanococcales. The acetoclastic
methanogenic activity measured in anaerobic treatment of
dairy wastewater was mostly found to be due to Methano-
saeta species whilst Methanosarcina-like species contributed
insignificantly. However, Methanococcus species seemed
to become the most dominant group towards the end of the
operation [21].
Despite the different possibilities of whey utilization,
approximately half of the world whey production is not
treated and is simply discarded as waste effluent [13,22].
This represents a significant loss of resources and causes
serious pollution problems. Dairy manure is one of the
most polluting agro-industrial wastewater. Intensive dairy
farming produces large amounts of manure that, when not
properly managed due to its high organic content, nitrogen
and phosphorous concentrations can cause numerous envi-
ronmental problems. Dairy manure, which contains too many
suspended solids, presents low anaerobic biodegradability
[23]. The co-digestion of whey, as an easily degradable
1285
substrate with dairy manure, increases the specific methane
yields when compared to manure-only digestion [24,25].
Biogas yields of whey range from 0.35 to 0.80 m3 biogas/kg
of volatile solids, while biogas yields of cattle manure range
from 0.20 to 0.30 m3 biogas/kg of volatile solids [26].
There are many laboratory and pilot-scale studies in
literature on the anaerobic treatment of whey. Various types
of anaerobic reactors have been summarized by Galegenis
et al. [13] and Kavacik and Topaloglu [27]. Likewise,
anaerobic digestion of dairy manure has been demonstrated
to be an attractive treatment that provides benefits such as
pollution control, odour and pathogen level reduction,
nutrient recovery and energy production [23,28-30].
The aim of this study was the optimization of the
anaerobic co-digestion process of whey and cow manure.
The influence of process conditions on whey and cow
manure was investigated in the one-stage batch process in
order to produce maximum biogas yield.
2. Materials and Methods
2.1. Whey and cow manure
The whey and cow manure used in this study were
obtained from the Milk Plant and the Cow Farm located
near the city of Osijek in Eastern Croatia. The whey was
drained directly from the cheese vats, collected in a 10 dm3
tank and transported to the laboratory. The cow manure
was taken directly from the manure tank, collected in a
20 dm3 container and transported to the laboratory. The
fresh whey and dairy manure were stored at 4oC to avoid
a chemical composition modification. The chemical com-
position of whey and cow manure was determined in
accordance to the American Public Health Association
(APHA) standard methods [31] and the standard method of
organic carbon measurement [32]. The composition of
whey and cow manure is shown in Table 1.
2.2. Experimental set-up
Experimental studies were performed in 1 dm3 glass batch
Table 1. The composition of whey and cow manure
Whey Cow manure
pH 3.53 7.11
Total solids, TS (%) 4.69 6.4
Volatile solids, VS (%) 90.85 85.34
Organic carbon (%) 40.95 42.76
Nitrogen (%) 4.68 5.61
C/N (g/g) 8.74 7.62
Chemical oxygen demand, 47,950 43,900
COD (mg/dm3)
1286
anaerobic reactors at set temperatures of 35oC or at 55oC.
Agitation were provided by an agitator (0.3 Hz). The
reactors were fed from their upper part, thereafter were
closed to prevent any air leakage. The biogas collection
from the reactor was made via the displaced liquid from a
graded cylinder filled with a sodium chloride (NaCl)
saturated solution (330 g NaCl/dm3 distilled water) (Fig. 1).
Experiments were performed using a series of batch
reactors whose numbers depended on the duration of the
co-digestion process conducted (each day samples were
taken from one reactor). The substrate samples were taken
at the beginning of the experiment from each reactor in the
series. Each day the process was stopped in one of the
reactors and the corresponding reaction mixture was
analysed. Standard methods were used for pH measurement
and the determination of TS, VS, organic carbon, total
nitrogen and COD [31,32]. The volume of biogas was
measured using a graded cylinder shown in Fig. 1. Gas for
the analysis was transferred from the graded cylinder to a
gas burette by a system of connected vessels. The gas
burette was previously filled with a sodium chloride saturated
solution. Gas from the graded cylinder was transmitted into
the burette by under pressure resulting from the leakage of
fluid. The burette was connected to a sampling valve over
which the gas entered the chromatograph.
2.3. The experimental procedure
Experiments were performed in three phases in order to
investigate the best co-digestion process conditions for
biogas production. The total volume of the reaction mixture
(whey and cow manure) in all experiments was 0.5 dm3.
All experiments were performed in triplicate and in the
95% confidence range the results showed no significant
difference.
In the first phase the influence of different initial whey
content during the batch co-digestion process was analyzed
at mesophilic (35oC) and thermophilic (55oC) conditions
for 12 days. The investigated initial contents of whey were
5, 10 and 15% v/v, respectively. In order to investigate the
Fig. 1. Scheme of the experimental set-up used for anaerobic co-
digestion of whey and cow manure; A-substrate; B-water bath; C-
sodium chloride saturated solution.
Biotechnology and Bioprocess Engineering 17: 1284-1293 (2012)
influence of pH, the initial pH value of whey was adjusted
within the optimum range for methanogenesis (6.5 and 7.5)
during additional experiments of the first phase. The content
of whey was 10% v/v in the initial mixture and NaHCO3
was added to adjust the pH value. The pH and TS were
measured before and after fermentation, for all investigated
conditions. Biogas productivity and biogas composition
were measured daily.
The effect of NaHCO3 addition on the batch co-digestion
process was investigated in the second experimental phase
at thermophilic conditions for 12 days. Experiments were
performed for four different initial concentrations of NaHCO3
(0, 5, 10 and 15 g/dm3). The content of whey was 10% v/v in
the initial mixture. The pH, TS, biogas productivity and
biogas composition were measured daily. Removal effici-
encies of COD were measured daily for the experiment
with initial NaHCO3 concentration of 5 g/dm3.
In the third phase of investigation experiments were
performed at 55oC, with 10% v/v of whey in the initial
mixture and 5 g/dm3 NaHCO3 was added as a supplemental
bicarbonate. Experiments were performed over a period of
45 days. The pH, TS, VS, organic carbon, total nitrogen,
biogas productivity and biogas composition were measured
daily. The removal efficiency of COD was measured at
specific intervals during the 45 day period.
2.4. Analytical methods
The pH measurements were performed by a pH digital
electrode (Methrom). TS were determined by drying at
105oC to constant weight. TS samples were ignited to
constant weight at 550oC for VS determination. Oxidation
in the potassium dichromate digestion solution was used for
determination of organic carbon. The Kjeldahl method was
used for determination of total nitrogen. COD determination
was based on potassium dichromate as an oxidizing agent
(under acidic conditions by addition of sulphuric acid) and
measuring of absorbance at 605 nm on a HACH DR/2000
spectrophotometer for colorimetric measurements [31,32].
Biogas composition (methane, CH4; nitrogen, N2 and carbon
dioxide, CO2) was determined by gas chromatography
(Varian GC 3900) using 10 m of capillary column CP-
PoraPLOT Q and helium as the carrier gas.
3. Results and Discussion
3.1. The influence of whey content, temperature, and
pH on biogas production
The temperature inside the digester has a major effect on
the biogas production process because the length of the
fermentation period depends on the temperature. An
increased process temperature in general has a positive
Optimization of Biogas Production from Co-digestion of Whey and Cow Manure 1287

Fig. 2. Influence of whey content on specific volume of biogas produced at mesophilic (35oC) and thermophilic (55oC) conditions in
experiments with and without alkalinity addition (A) mesophilic conditions without alkalinity addition; (B) thermophilic conditions
without alkalinity addition; (C) mesophilic conditions with alkalinity addition; (D) thermophilic conditions with alkalinity addition
( ◆ 0% whey; △ 5% whey, initial pH 3.5; ■ 10% whey, initial pH 3.5; ○ 15% whey, initial pH 3.5; ◇ 10% whey, initial pH 6.5; ● 10%
whey, initial pH 7.5).

effect on the metabolic rate of the microorganisms but also
results in a higher concentration of free ammonia. The
anaerobic fermentation of wastes with a high concentration
of ammonia was more easily inhibited and less stable at
thermophilic temperatures than at mesophilic temperatures
[33]. On the other hand, the role of pH in the stability of the
process, as evidenced by the concentration of volatile fatty
acids, is one of the most important parameters in anaerobic
digesters. The pH of the digester should be kept within a
desired range of 6.8 ~ 7.2. The amount of carbon dioxide
and volatile fatty acids produced during the anaerobic
process affects the pH of the digester contents [34].
In all fermentation conditions studied, specific volume
of biogas produced, V' (Fig. 2) showed dependence on
temperature and pH, which are important parameters
affecting the growth of microorganisms during anaerobic
fermentation. In order to study the influence of whey
content on the biogas production the batch co-digestion
process was analyzed at mesophilic (35oC) and thermophilic
(55oC) conditions over a 12 day period. The results of the
experiments performed with a different initial content of
whey and cow manure at 35oC (Fig. 2A) and 55oC (Fig. 2B)
showed that the production of biogas was diminished and
the process practically stopped without alkalinity addition.
The process collapsed faster in thermophilic conditions
because pH value dropped below the optimum range for
methanogenesis earlier. The initial pH of the whey and cow
manure mixture was 6.8 and it showed a slight increase to
7.4 after 12 days in all of the experiments with different
whey content at mesophilic conditions (Fig. 2A). In ther-
mophilic experiments (Fig. 2B) only the lowest whey content
(5%) provided optimal pH conditions for methanogenesis
(pH 7.1) after 12 days. Higher whey content (10 and 15%)
affects the decline of pH below 5.8, which is too low for
biogas production in thermophilic conditions.
Experiments with alkalinity addition to the mixtures
with different initial content of whey and cow manure at 35
and 55oC were performed and the best fitting results for
initial whey content of 10% v/v were achieved. Correspon-
dingly, following experiments with alkalinity addition at
35oC (Fig. 2C) and 55oC (Fig. 2D) were performed with
the initial whey content of 10% v/v. The initial pH value of
whey was adjusted at 6.5 and 7.5 by the addition of
NaHCO3. The comparatively best results were achieved for
1288
Table 2. Specific biogas volume produced during 12 days and the average biogas production rate for different process conditions
Mesophilic (35οC)
Process conditions V'
(dm3/dm3)
0% whey 6.49
5% whey, initial pH 3.5 4.88
10% whey, initial pH 3.5 2.50
15% whey, initial pH 3.5 1.16
10% whey, initial pH 6.5 2.12
10% whey, initial pH 7.5 6.40
thermophilic conditions in the experiment in which the
initial pH value of whey was adjusted to 7.5. The initial pH
value of mixture of those experiments was 7.2. At the end
of experiments the pH value of mixture was slightly higher
than 8.0.
Removal efficiencies of TS are expressed as a reduction
of TS during the fermentation process. In experiments with
alkalinity addition performed at 35oC and 55oC removal
efficiencies of TS were increased by 16% compared to
experiments without alkalinity addition (for experiments
carried out at 10% v/v of whey in the initial mixture).
Specific biogas volume and the average biogas production
rate for different process conditions are shown in Table 2.
Maximal specific biogas volume (V', dm3/dm3) produced
during 12 days and the average biogas production rate
(r, dm3/dm3/day) were achieved in thermophilic experiments
with 10% v/v of whey in the initial mixture where the
initial pH value of whey was adjusted to 7.5.
The comparable results of biogas productivity were
obtained in relation to the study of Venetsaneas et al. [35],
where experiments were performed in a two stage continuous
process by an alternative pH controlling approach. In the
thermophilic experiments with 10% v/v of whey where the
initial pH value of whey was adjusted to 7.5, the average
biogas production rate was significantly higher compared
to the mesophilic experiments (1.01 dm3/dm3/day in com-
parison to 0.53 dm3/dm3/day) due to the fact that the
addition of NaHCO3 ensures optimum pH conditions for
faster and more efficient thermophilic co-digestion. Due to
better utilization and degradation of substrates at higher
temperatures, a considerably higher biogas yield was achi-
eved at 55oC (12.08 dm3/dm3) than at 35oC (6.40 dm3/dm3)
(Table 2).
Maximum CH4 content in a biogas mixture (72.0%) was
achieved in experiments with 10% v/v of whey where the
initial pH value of whey was adjusted to 7.5. A decreasing
trend of CH4 content in a biogas mixture was achieved in
experiments where the initial pH values of whey were not
adjusted for all fermentation conditions studied. A significant
reduction of CH4 content was obtained at thermophilic
Biotechnology and Bioprocess Engineering 17: 1284-1293 (2012)
Thermophilic (55οC)
r V' r
(dm3/dm3/ day) (dm3/dm3) (dm3/dm3/day)
0.54 3.11 0.26
0.41 1.93 0.16
0.21 1.82 0.15
0.10 1.08 0.09
0.18 2.15 0.18
0.53 12.08 1.01
conditions (28% of CH4 in a biogas mixture) in comparison
with mesophilic conditions (56% of CH4 in a biogas
mixture) after 12 days, which was caused by the influence
of pH on the growth of methanogens. As mentioned
before, the process collapsed faster at 55oC because the pH
value dropped below the optimal range for methanogenesis.
The growth rate of methanogens is greatly reduced below
pH 6.6, whereas an excessively alkaline pH value can lead
to the disintegration of microbial granules and a subsequent
failure of the process. Although the optimum pH value of
methanogenesis is around pH 7.0, the optimum pH value
of hydrolysis and acidogenesis has been reported to be
between 5.5 and 6.5 [36]. This is an important indicator as
to why some designers prefer the separation of the
hydrolysis/acidification and acetogenesis/methanogenesis
processes in two-stage processes [2,9,22].
Based on experimental research performed in a one-
stage batch process, optimal conditions for co-digestion of
whey and cow manure were determined as follows: a
temperature of 55oC and 10% v/v of whey in the initial
mixture. Those process conditions are kept constant for all
following performed investigations.
3.2. Influence of different initial concentrations of
bicarbonate on biogas production
The influence of different bicarbonate initial concentrations
on biogas production and its composition are presented
in Fig. 3. Previous experimental results showed that
supplemental alkalinity is required because methanogenesis
was inhibited due to a pH decrease below 6.6 in experiments
without NaHCO3 addition. There was a slight increase in
the pH, from 6.5 to 7.1, in experiments without NaHCO3
addition. The pH values for the other experiments with a
supplemental bicarbonate (5, 10 and 15 g/dm3 NaHCO3)
ranged between 7.1 and 8.1, which is in line with the
optimal process conditions and in accordance with literature
data [2,22,34].
As can be seen from Fig. 3A and Table 3, the biogas
production increases from 2.12 to 3.26 dm3/dm3 (without
NaHCO3), from 0.20 to 6.62 dm3/dm3 (5 g/dm3 NaHCO3),
Optimization of Biogas Production from Co-digestion of Whey and Cow Manure 1289

Fig. 3. Influence of initial bicarbonate concentration on (A) specific biogas production; (B) CH4 content; (C) CO2 content; (D) N2 content
( ◇ without NaHCO3; ■ 5 g/dm3 NaHCO3; △ 10 g/dm3 NaHCO3; ● 15 g/dm3 NaHCO3).

Table 3. Specific biogas volume produced during 12 days and the of the other constituents of biogas, CO2 and N2, also
average biogas production rate for different initial bicarbonate demonstrated a dependence on the bicarbonate concentra-
concentration
tion, which correlated with CH4 content in a biogas
V' r mixture (Figs. 3C and 3D). Increase of CH4 content during
Addition of NaHCO3
(dm3/dm3) (dm3/dm3/day)
the co-digestion process with bicarbonate concentration of
Without NaHCO3 3.26 0.27
5 g/dm3 NaHCO3 were followed by decrease of CO2 and
5 g/dm3 NaHCO3 6.62 0.55
N2 content in a biogas mixture. Lower CH4 content was
10 g/dm3 NaHCO3 7.38 0.62
achieved throughout the co-digestion process with higher
15 g/dm3 NaHCO3 4.22 0.35
bicarbonate concentration (15 g/dm3 NaHCO3) which causes
a higher content of CO2 and N2 in a biogas mixture.
from 2.00 to 7.38 dm3/dm3 (10 g/dm3 NaHCO3) and from Specific biogas volume produced during 12 days and
2.02 to 4.22 dm3/dm3 (15 g/dm3 NaHCO3) during the the average biogas production rate for different initial
period of 12 days. It becomes obvious that the best results bicarbonate concentrations are shown in Table 3.
were achieved with supplemental bicarbonate concentrations The maximum specific biogas volume (V', dm3/dm3)
of 5 g/dm3 NaHCO3 and 10 g/dm3 NaHCO3. Maximum produced during the 12 days and the average biogas
CH4 content in a biogas mixture was achieved in the production rate (r, dm3/dm3/day) were achieved in experi-
experiment with 5 g/dm3 NaHCO3 during the whole ferm- ments with the addition of 5 and 10 g/dm3 NaHCO3 due to
entation process (from 0.1% to nearly 80%, Fig. 3B). Good the fact that optimum pH conditions for efficient co-
CH4 content in a biogas mixture was realized from the 8th digestion were ensured. In the case of 15 g/dm3 NaHCO3,
to the 11th day (from 57.2 to 79.4%). These results were the higher bicarbonate concentration probably inhibited co-
comparable with results for other process conditions digestion.
presented in Fig. 3B (without NaHCO3, with 10 g/dm3 Research results confirmed that CH4 content in a biogas
NaHCO3 and 15 g/dm3 NaHCO3), which showed lower mixture is dependent on bicarbonate concentration. Because
CH4 content during the whole fermentation process. Content CH4 is considered as the most important constituent of biogas
1290
regarding energy capacity, the influence of bicarbonate
concentration on CH4 content, compared to the other
constituents of biogas, is the most important. Therefore,
addition of 5 g/dm3 NaHCO3 is determined as preferable
for efficient co-digestion of whey and cow manure.
A particularly strong reason for co-digestion of feedstock
is the adjustment of the carbon to nitrogen ratio (C:N).
Microorganisms generally utilise carbon and nitrogen in
the ratio 25 ~ 30:1, but C:N ratio can often be considerably
lower than this ideal [37]. Feedstock can vary widely in
their C:N ratios. Waste material that is low in C can be
combined with materials high in N to attain the desired
C:N ratio [34]. Indeed, the two stage reactor with biomass
retention has been reported to be considered as the only
type capable of reliable activity with C:N ratios less than
20 [2]. Our research showed that in all fermentation
conditions studied in the one-stage batch process the C:N
ratio fluctuated from the range of 4.2 ~ 4.6 at the beginning
of the fermentation to the range of 3.2 ~ 4.1 at the end of
fermentation (Fig. 4). Contrary to literature data, such a
low C:N ratio did not limit the microorganisms growth,
which would affect the methanogenesis. Good CH4 content
in a biogas mixture (Fig. 3B) shows that methanogenesis
was performed optimally. Also, a decrease of COD (Fig. 5),
which amounted to 53.5% after 12 days, shows that the
anaerobic treatment of whey and cow manure is quite
effective.
Comparable results for removal efficiencies of COD
were obtained in relation to the study of Kavacik and
Topaloglu [27], were experiments were performed in a
specially designed anaerobic reactor, at different hydraulic
retention times, HRTs (5, 10 and 20 days), total solid
matters (8 and 10%) and temperatures (25 and 34oC). In
this study, maximum removal efficiencies for COD (54%)
Fig. 4. Influence of bicarbonate concentration on C:N ratio
( ◇ without NaHCO3; ■ 5 g/dm3 NaHCO3; △ 10 g/dm3 NaHCO3;
● 15 g/dm3 NaHCO3).
Biotechnology and Bioprocess Engineering 17: 1284-1293 (2012)
were achieved at HRT of 10 days in the mixture containing
8% total solids matters at 34oC.
3.3. Biogas production from co-digestion of whey and
cow manure during 45 days
The aim of the experiments performed in 45 days was to
validate optimized conditions for co-digestion of whey and
cow manure in the one-stage batch process. According
to experimental research performed during preliminary
investigations optimal conditions for co-digestion of whey
and cow manure were determined to be a temperature of
55oC, 10% v/v of whey in the initial mixture and 5 g/dm3
NaHCO3 as a supplemental bicarbonate.
The pH values of substrates ranged between 6.9 and 8.2,
which is the optimum working range for methanogenesis.
After the period of 45 days, the amount of TS and VS
decreased, by 29 and 9%, respectively. The specific biogas
production increased from 0.10 to 20.7 dm3/dm3 (Fig. 6A).
Maximum biogas production of 21.8 dm3/dm3 occurred on
the 44th day. The specific biogas volume (V', dm3/dm3) and
the average biogas production rate (r, dm3/dm3/day) for
different time periods are presented in Table 4. The
maximum CH4 content (78.7%) in a biogas mixture was
achieved on the 19th day. Good CH4 content in a biogas
mixture was realized from the 10th to the 45th day (from
67.3 to 71.8%) (Fig. 6B).
The C:N ratio decreased from 8.0 to 4.9 after 45 days
(Fig. 7). As mentioned before, such a low C:N ratio did not
limit the growth of microorganisms [2]. Good CH4 content
in a biogas mixture (Fig. 6B) shows that methanogenesis
was performed optimally. Also, the decrease of COD (Fig. 7),
which amounted to 54.9% after 45 days, shows that
the anaerobic treatment is quite effective. Although the
maximum biogas production rate of 1.01 dm3/dm3/day
occurred on the 15th day, the maximum reduction of COD
Fig. 5. Dynamic change of COD in experiment with initial
bicarbonate concentration of 5 g/dm3 NaHCO3.
Optimization of Biogas Production from Co-digestion of Whey and Cow Manure 1291

Fig. 6. Biogas production and composition during 45 days of co-digestion ( ■ specific volume of biogas produced; ● CH4, %; △ CO2,
%; ◆ N2, %).

Table 4. Specific biogas volume produced during 45 days and the

average biogas production rate for different time periods
Time V' r
(days) (dm3/dm3) (dm3/dm3/day)
5 3.12 0.62
10 8.46 0.85
15 15.16 1.01
20 16.96 0.85
25 13.98 0.56
30 17.83 0.59
35 17.92 0.51
40 19.95 0.50
45 20.65 0.46 Fig. 7. Decreasing of C:N ratio and COD during 45 days of co-
digestion ( △ C/N ratio; ■ COD).

occurred on the 42nd day (56.3%).

The main issue for the co-digestion process lies in
balancing several parameters in the co-substrate mixture:
macro- and micronutrients, C:N ratio, pH, inhibitors/toxic
compounds, biodegradable organic matter and dry matter
[4]. Optimum values of C:N and COD:N ratios of 20
and 70, respectively, have been suggested for the stable
performance of anaerobic digestion [33]. Alkalinity is
necessary to avoid decreasing pH due to the accumulation
of volatile fatty acids when applying a high organic load.
Mixing intensity was also shown to affect digester perfor-
mances and biogas production [38]. Although, the hydrolysis
of particulate organic material has been considered the
rate-limiting step in anaerobic digestion, acetogenesis or
methanogenesis might be the rate-limiting stages in complex
waste [39]. Waste such as whey produced in dairy industries
which carry a high organic load in terms of COD, instead
of requiring a lot of energy to be treated in an aerobic plant,
can produce energy itself. This can be achieved using the
anaerobic treatment technology, which not only removes a
great amount of COD but also produces biogas, a mixture
of methane and carbon dioxide, which can then be used to
produce electricity, steam or both [40].
4. Conclusion
This study shows that whey could be efficiently degraded
to a biogas in a one-stage batch process when co-digested
with cow manure. Optimal conditions for maximum biogas
production and methane content in a biogas mixture are
55oC, 10% v/v of whey in the initial substrate mixture, with
the addition of 5 g/dm3 NaHCO3 for alkalinity control.
From a wastewater treatment point of view, the anaerobic
co-digestion of whey and cow manure offers an excellent
approach.
Although the separation of the hydrolysis/acidogenesis
and acetogenesis/methanogenesis processes in a two-stage
process is preferable, this study presented process conditions
for effective control and performance of the one-stage batch
process.
1292
Acknowledgment
This study was supported by EKONERG - Energy and
Environmental Protection Institute, Ltd. (Project No: I-14-
0090/10). The authors gratefully acknowledge the financial
support from this institution.
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