Co-digestion of different waste mixtures from agro-industrial activities: Kinetic

evaluation and synergetic effects

Jhosan Pags Daz
a,b,
, Ileana Pereda Reyes
a
, Magnus Lundin
b
, Ilona Srvri Horvth
b
a
Process Engineering Centre, High Technical Institute Jos Antonio Echeverra (CUJAE), 11901, 114 Street, Marianao, Havana, Cuba
b
School of Engineering, University of Bors, 501 90 Bors, Sweden
a r t i c l e i n f o
Article history:
Received 8 July 2011
Received in revised form 6 September 2011
Accepted 8 September 2011
Available online 18 September 2011
Keywords:
Agro-industrial waste streams
Co-digestion
Methane yield
Specic methanogenic activity
Kinetic evaluation
a b s t r a c t
Several wastes from agro-industrial activities were mixed in different ratios to evaluate the co-digestion
process. Methane yield Y
CH
4
, specic methanogenic activity (SMA) and a kinetic parameter (k
0
) were
determined. A second feeding was also performed to examine the recovery of bacterial activity after
exhaustion.
Mixture ratios of 1:1:1:1 and 1:3:4:0.5 (w/w) showed the best performance, with Y
CH
4
of 664; 582 NmL
CH
4
/gVS
substrate
, as well as SMA of 0.12; 0.13 gCOD
NmLCH
4
=gVS
inoculum
=d, respectively, during the digestion
of the rst feed. It was possible to relate synergetic effects with enhancement in Y
CH
4
by up to 43%,
compared with values calculated from Y
CH
4
of the individual substrates. All batches started up the biogas
production after an exhaustion period, when a second feed was added. However, long lag phases (up to
21 days) were observed due to stressed conditions caused by the substrate limitation prior to the second
feed.
2011 Elsevier Ltd. All rights reserved.
1. Introduction
Energy productionfrombiomass, suchas biogas production, pro-
vides a renewable alternative to fossil fuels, considering the huge
amount of organic residues, such as agro-industrial residues, muni-
cipal solid wastes and animal wastes produced around the world,
estimated at around 140 kg per capita per year (Wilson et al., 2011).
Many of these residues are still unexploited and contribute to
environmental pollution in both urban and rural areas. Anaerobic
digestion of this biodegradable waste will provide a solution for
reducing both this environmental problem and the consumption
of fossil fuels. An additional advantage of the anaerobic treatment
is that beside the produced biogas a mineralized efuent is
obtained, which can be utilized as a biofertilizer with high NPK
concentrations.
Biogas technology in Cuba today focuses on treatment of waste
from the animal and sugar industries. The utilization of cow man-
ure would provide a potential for the production of about 1.7 mil-
lion m
3
biogas per day, while an additional amount of about
60,000 m
3
could be produced from pig manure (ONE, 2010).
So far, there are no measures taken for co-digestion of these
waste streams with residues from other agro-industrial activities.
The development of a co-digestion process for the utilization of
these residues will lead to both economical and environmental
benets not only in Cuba, but also in other countries, since the
main part of theses waste streams are still deposited, leading to
several disadvantages, including but not limited to, high transpor-
tation costs and high organic content at the disposal sites. By pro-
cessing multiple waste streams in a single facility co-digestion will
contribute to an efcient utilization of both the waste and the
equipments (Alatriste-Mondragon et al., 2006). Additionally, this
technology provides a better nutritional balance for the microbio-
logical degradation process improving the efciency of biogas
production by utilizing several types of waste streams that in
themselves may have inadequate nutritional composition.
There are several studies performed during the past years which
investigate anaerobic co-digestion processes (lvarez and Lidn,
2008; Deublein and Steinhauser, 2008; Palatsi et al., 2011). A mix-
ture of fruit wastes was reported to produce 0.250.50 m
3
CH
4
/kg VS (Seadi et al., 2008). When mixing fruit and vegetable
wastes with cattle manure with a ratio of 1:1, a methane yield of
0.45 m
3
CH
4
/kg VS was reported (Callaghan et al., 2002). Municipal
solid waste (MSW), which is a mixture of different organic substrate
streams by itself, is reported to produce between 0.445 and
0.486 m
3
CH
4
/kg VS (Edelmann et al., 1999), although higher values
of methane production(i.e. up to 0.5 m
3
CH
4
/kg VS) fromMSWhave
also been obtained (Angelidaki and Ellegaard, 2003).
Digestion of organic matter to methane is a complex microbio-
logical process so that to operate a biogas plant effectively towards
a maximum gas yield, more knowledge is required. Even though
there are many reports on biogas production from different
0960-8524/$ - see front matter 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2011.09.031

Corresponding author at: Process Engineering Centre, High Technical Institute

Jos Antonio Echeverra (CUJAE), 11901, 114 Street, Marianao, Havana, Cuba.
Tel.: +53 537 266 3398; fax: +53 537 267 2964.
E-mail address: jhosane@quimica.cujae.edu.cu (J. Pags Daz).
Bioresource Technology 102 (2011) 1083410840
Contents lists available at SciVerse ScienceDirect
Bioresource Technology
j our nal homepage: www. el sevi er . com/ l ocat e/ bi or t ech
mixtures of substrates, many of the co-digestion plants are today
operating below their full capacity. Poor operational stability lead-
ing to low methane yields is often encountered in anaerobic diges-
tion (Chen et al., 2008).
For the development of cost-efcient technologies, data on
optimal mixture ratios, methane yield and the kinetics of the degra-
dationprocess areneeded. Increasedmethaneyieldandbiodegrada-
tion rate will result in more methane generated per unit fed mass
and will reduce the size of the reactor required, which makes the
process economically more attractive (Kang and Weiland, 1993).
In this work, the utilization of different agro-industrial and slaugh-
terhouse waste mixtures for biogas production was investigated.
The co-digestion process and the synergetic effects in different
waste mixtures were characterized by determining methane yield,
specic methanogenic activity and kinetic development using
anaerobic batch digestion assays.
2. Methods
2.1. Substrates
The different substrates were obtained from sites outside Bors,
Sweden. Manure (M) was obtained froma farmoutside Bors, Swe-
den. Slaughterhouse waste, a well mixed cow ruminal waste with
blood (SB), came from slaughterhouses in the periphery of Bors,
Sweden. Municipal solid waste (MSW) was provided by a large
scale biogas plant, Sobacken in Bors, Sweden. Various crop resi-
dues (VC) were prepared containing straw (37.5%), animal feed
(37.5%) and wastes of fruits and vegetables (25%). Furthermore, dif-
ferent mixtures of these fractions were also prepared in order to
further investigations of the co-digestion process. The different
samples were stored at temperatures below 0 C until usage.
The total solids (TS), volatile solids (VS) and ash content of the
substrates were determined according to Sluiter et al. (2005) after
drying to constant weight at 105 and 575 C, respectively. Addi-
tional analytical tests were performed for the characterization of
the different waste fractions and waste mixtures based on APHA,
AWWA, WPCF Standardized Methods for water and wastewater
analyses (APHA, AWWA, WEF, 1995), and are briey summarized
here: Total organic carbon (TOC) was determined using TOC ana-
lyzer (5000 A, Shimadzu, Japan) equipped with a SSM-5000 solid
module. Total Kjeldhal nitrogen (TKN) was determined by the Kjel-
dahl digestion method and fat content by the Soxhlet extraction
method. The total protein content was then estimated by multiply-
ing TKN with a factor of 6.25 according to Gunaseelan (2009). The
data are presented in Table 1.
2.2. Experimental procedure
The anaerobic batch digestion tests were carried out in tripli-
cates under thermophilic conditions (55 C) according to a method
previously described by Hansen et al. (2004). The thermophilic
inoculum (55 C) was obtained from a large scale co-digestion
plant, Sobacken Bors, Sweden.
An inoculum with 2.24% TS and 1.39% VS was used. The initial
volatile solid (VS) ratio of substrate to inoculum was kept at 1:2
throughout all the experimental setups. Each reactor with 2 L
capacity contained 400 mL of inoculum and appropriate amounts
of VS substrate. The compositions in the different batch setups
are summarized in Table 2. After the setup all the reactors were
sealed with rubber septum and metallic cover (Apodan Nordic,
Copenhagen, Denmark) and then ushed during 3 min with an
anaerobic gas mixture containing 80% N
2
and 20% CO
2
. In order
to investigate the response of the bacteria under stress conditions
(exhaustion) a second feeding with the same amount of substrate
as the initial one was performed after 31 days of incubation (Table
2) and the reactors were then sealed and ushed again and kept for
an additional incubation period of 31 days. Volume corrections for
both feedings were taken into account in order to determine the
headspace volume in each batch.
During the whole investigation period the reactors were kept in
an incubator at 55 C (1 C) and were shaken once every day.
Gas samples were regularly collected from the headspace of
each bottle with a 0.25 mL pressure-tight syringe (VICI, Precision
Sampling Inc., Baton Rouge, LA, USA), making it possible to take
gas samples at the actual pressure. The methane and carbon diox-
ide content of the samples were then directly determined by a gas
chromatograph (Auto System Perkin Elmer, USA) equipped with a
packed column (Column 8000 PKD, Perkin Elmer, USA) and a ther-
mal conductivity detector (Perkin Elmer, USA) with the inject tem-
perature of 150 C. Nitrogen with a ow rate of 20 mL/min at 60 C
was used as carrier gas. Assuming ideal gas mixtures and using the
ideal gas law, the methane and carbon dioxide content in the reac-
tor head space can be calculated using the data from the GC mea-
surements without measuring the actual pressure in the bottle
(Teghammar et al., 2010). All the results of gas volumes are pre-
sented at normal conditions. i.e. 0 C, 101.325 kPa according to
ISO 10780 (ISO, 1994).
2.3. Methane yield
Methane yield characterizes metabolic activity in a methano-
genic ecosystem (Michaud et al., 2002) and can be calculated
according to Eq. (1):
Y
CH
4

V
CH
4
gVS
NmL CH
4
=gVS
substrate
1
where Y
CH
4
is methane yield (NmL CH
4
/gVS
substrate
), V
CH
4
is the
accumulated volume of produced methane (NmL), and VS is the vol-
atile solids content in the substrate added to the reactors (g).
Table 1
Chemical characterization of substrates (mean values and standard deviations from triplicate samples).
Residues pH Bulk density
(g/L)
TS (%) VS
a
(%) Organic carbon
a
(%)
Ash
a
(%) TKN
a
(%) C/N Fat
a
(%) Protein
a
(%)
SB 6.8 1075 13 0.1 92 1.7 55 1.9 8 0.1 4 0.3 14.4 43 2.8 24 1.6
M 7.9 552 23 0.2 80 1.6 N.D. 20 0.4 N.D. N.D. N.D. N.D.
VC 6.9 801 47 0.9 91 0.9 41 3.8 9 0.9 4 0.5 10.0 11 0.1 25 2.8
MSW 5.9 1011 11 0.1 85 0.2 39 2.4 15 0.2 2 0.1 19.5 14 0.0 12 0.7
1:1:1:1
b
7.1 860 20 0.5 90 1.2 43 3.0 10 0.2 3 0.03 12.7 16 0.7 21 0.2
1:3:4:0.5
b
7.0 757 29 0.8 90 0.6 42 2.9 10 0.3 4 0.5 11.8 12 0.8 22 2.9
1:1:2:0.5
b
6.6 830 27 0.3 91 0.2 43 3.6 9 0.2 3 0.5 13.7 18 0.5 20 3.2
2:3:3:0
b
6.9 776 29 0.6 90 0.3 43 2.7 10 0.3 4 0.3 10.7 13 0.9 25 2.2
N.D., non determined.
a
Dry basis.
b
Different substrate mixtures of SB:M:VC:MSW, respectively. The mixing ratios are based on the w/w of the different components.
J. Pags Daz et al. / Bioresource Technology 102 (2011) 1083410840 10835
2.4. Specic methanogenic activity
Specic methanogenic activity (SMA) is used to estimate the
capacity of methanogenic microorganisms to convert substrate
into CH
4
in any anaerobic system, i.e. to evaluate the kinetics of
the system (Field et al., 1988; Pereda et al., 2006). SMA can be cal-
culated according to the following expression:
SMA
R
CF V VS
gCOD
NmL CH
4
=gVS
inoculum
=d 2
where R (NmL CH
4
/d) is the methane production rate; CF is a con-
version factor, converting the produced methane to equivalent
COD and is equal to 350 (Field et al., 1988); V is the volume (mL)
and VS is the concentration of volatile solids (g/mL) of the inoculum.
R was determined as a mean slope during the maximum production
activity of the cumulative methane curve for both feedings.
2.5. The kinetics of the discontinuous process
A rst-order kinetic model (Roediger Model) described previ-
ously by Jimnez et al. (2004) was used in this study:
G G
m
1 e
k
0
t
3
where G is the volume of methane accumulated (NmL) after a time t
(d), G
m
is the maximum volume accumulated (NmL) at an innite
digestion time and k
0
is the observed specic rate constant of the
overall process (per day). A nonlinear least-square (NLLS) regression
procedure was used in order to determine k
0
.
2.6. Statistical analysis
One factor ANOVA (Analysis of Variance) was used to analyze
data and test for signicant treatment effects (substrate mixture
performance) for the response variables Y
CH
4
, SMA and k
0
(p-values
0.05) and estimate the size of experimental errors (pooled standard
deviations). The treatments (substrate mixtures) were then placed
in homogeneous groups. Each homogeneous group, not necessarily
disjoint, contains substrates whose estimated responses cannot be
said to be signicantly different. The calculation of homogeneous
groups was done using pair vise comparisons (95% LSD, Least Sig-
nicant Difference) (Montgomery, 2005). The software package
used was STATGRAPHICS Centurion XV.II.
3. Results and discussion
3.1. Substrate characterization
The results of substrates characterization are shown in Table 1.
The C/N ratio is an important parameter to take into account
when investigating different substrates and substrate mixtures
for anaerobic digestion (Kayhanian, 1999). Carbon and nitrogen
are essential nutrients contributing to formation of new cells. Sev-
eral authors agree in an optimal C/N ratio of between 20 and 35
(Habiba et al., 2009; Kayhanian, 1999) for anaerobic digestion.
However, Schattauer and Weiland (2004) reported a wider optimal
range of between 10 and 30. Thus we can conclude that all the dif-
ferent waste mixtures investigated in this study had a C/N ratio
within a range required for a correctly operating anaerobic diges-
tion process (Table 1).
Moreover, according to Yadvika et al. (2004) the temperature
can also inuence the optimal C/N ratio required. In an anaerobic
digestion process ammonia is produced during the biological deg-
radation of nitrogen containing organic matter. Depending on the
temperature and pH, ammonia can exist in two different forms,
i.e. free ammonia (NH
3
) and its ionized form NH

4
. Several studies
reported that it is the free ammonia nitrogen rather than total
ammonia nitrogen (TAN) which inhibits methanogenesis (Gallert
et al., 1998; Kayhanian, 1999).
pH is thus an important parameter inuencing the process sta-
bility. The pH of individual waste fractions and waste mixtures
investigated in this study are between 5.9 and 7.9 (Table 1). The
nal pH values obtained after the digestions of the rst and the
second feed in the different batches are presented in Table 3. These
values are within the optimal range of 6.58.5 reported previously
by Weiland (2010) for an anaerobic digestion process. Neverthe-
less, the SB fraction has the highest pH compared with that of other
substrates indicating the presence of free ammonia.
How much a digestion process can tolerate changes in pH
depends on the carbonate alkalinity. However, the simultaneous
presence of ammonia and bicarbonate in the digester results in
the formation of another buffer system (Lin et al., 2011). Therefore,
to enhance the buffer capacity in an anaerobic system, an adequate
ammonia concentration would be benecial due to the formation
of NH
4
HCO
3
(Murto et al., 2004).
Previous adaptation of the inoculum was not needed, since the
biogas plant is a large-scale co-digestion plant treating residues as
organic fraction of MSW (50% w/w), food residues from restaurants
of Bors City (2025% w/w) and slaughterhouse residues (2530%
Table 2
Experimental setup: amounts of substrates and inoculum in each batch.
Reactor Substrate (mL) Substrate (g) Inoculum (mL) V
effective
(mL) V
headspace
(mL)
1st feeding
SB 18 19 400 418 1582
MSW 30 30 400 430 1570
VC 8 6 400 408 1592
1:1:1:1
a
18 15 400 418 1582
1:3:4:0.5
a
14 11 400 414 1586
1:1:2:0.5
a
14 11 400 414 1586
2:3:3:0
a
14 11 400 414 1586
Substrate (mL) V
headspace
(mL)
2nd feeding
SB 18 1564
MSW 30 1540
VC 8 1584
1:1:1:1
a
18 1564
1:3:4:0.5
a
14 1572
1:1:2:0.5
a
14 1572
2:3:3:0
a
14 1572
a
Different substrate mixtures of SB:M:VC:MSW, respectively. The mixing ratios are based on the w/w of the
different components.
10836 J. Pags Daz et al. / Bioresource Technology 102 (2011) 1083410840
w/w), similar to the analyzed substrates and mixtures used in this
study.
3.2. Methane yield
Different substrates, slaughterhouse waste together with blood
(SB), manure (M), various crops residues (VC) and municipal solid
waste (MSW) as well as their mixtures in different ratios (Table 2)
were subjected to anaerobic digestion in batch digestion series.
After 31 days of incubation a second feeding of each reactor was
also performed and the batch reactors were incubated for an addi-
tional period of 31 days, in order to investigate the response of the
bacteria under stress conditions (exhaustion) as explained above
(Table 2). The results obtained during the experiments are summa-
rized in Table 3. Fig. 1a and b show the accumulated methane pro-
duction after the rst and the second feeding, respectively. During
a period of 31 days after the rst feeding all batches behaved as
expected.
An ANOVA analysis of the obtained results shows signicant
differences between substrate mixtures (p = 0.007) and a pooled
Table 3
Summary of results, methane yield YCH4
, specic methanogenic activity (SMA) and specic rate constant (k
0
) for methane production rate, during 1st and 2nd feeding (mean
values and standard deviations from triplicate samples).
Substrates pH Ymax NmL
CH
4
=gVS
substrate

SMA (gCOD NmL

CH
4
=gVS
inoculum
=d)
k
0
(d
1
) R
2
1st feeding
SB 7.94 582 44 0.06 0.000 0.09 0.01 0.99
MSW 7.82 537 2 0.13 0.005 0.33 0.02 0.98
VC 7.80 504 25 0.11 0.004 0.29 0.03 0.98
1:1:1:1 7.84 664 80 0.12 0.002 0.20 0.03 0.99
1:3:4:0.5 7.80 582 59 0.13 0.003 0.30 0.03 0.99
1:1:2:0.5 7.88 491 33 0.12 0.004 0.34 0.06 0.99
2:3:3:0 7.84 499 23 0.11 0.001 0.32 0.03 0.99
2nd feeding
SB 8.12 681 6 0.07 0.003 N.D.
MSW 8.01 596 1 0.04 0.01 N.D.
VC 8.02 603 7 0.05 0.01 N.D.
1:1:1:1 8.01 562 18 0.05 0.009 N.D.
1:3:4:0.5 8.02 532 2 0.03 0.001 N.D.
1:1:2:0.5 8.06 462 13 0.03 0.001 N.D.
2:3:3:0 8.01 595 27 0.05 0.009 N.D.
N.D., not determined.
Fig. 1. Accumulative methane production (NmL CH
4
/gVS
substrate
) determined during the digestion of the rst (a) and second (b) feed.
Table 4
Test of Multiple Ranges for methane yield YCH4
, specic methanogenic activity
(SMA) and specic rate constant (k
0
) for methane production rate obtained during the
digestion of the rst feed. The method used was 95.0% LSD.
Y
CH4
SMA k
0
Mixture H.G. Mixture H.G. Mixture H.G.
1:1:2:0.5 X SB X SB X
2:3:3:0 X VC X 1:1:1:1 X
VC X X 2:3:3:0 X VC X
MSW X X 1:1:2:0.5 X 1:3:4:0.5 X
SB X X X 1:1:1:1 X 2:3:3:0 X
1:3:4:0.5 X X MSW X MSW X
1:1:1:1 X 1:3:4:0.5 X 1:1:2:0.5 X
H.G.; Homogeneous groups, each column marks elements of one H.G.
J. Pags Daz et al. / Bioresource Technology 102 (2011) 1083410840 10837
standard deviation of 46.7. The sorted results were then placed in
homogeneous groups (Table 4). Two substrate combinations of
SB:M:VC:MSW with the mixing ratios of 1:1:1:1 and 1:3:4:0.5
were shown to have best performance with methane yields of
664 and 582 NmL CH
4
/gVS
substrate
, respectively. Moreover, two of
the mixtures with the mixing ratios of 2:3:3:0, and 1:1:2:0.5,
showed the lowest values of Y
CH
4
, corresponding to 499 and
491 NmL CH
4
/gVS
substrate
, respectively. The SB fraction alone, with
a methane production of 582 NmL CH
4
/gVS
substrate
is placed in both
high and intermediate groups (Table 4).
Among the systems, the SB fraction has a lower degradation
rate during the digestion of the rst feed compared with those
for the other samples (Fig. 1a). The statistical analyses showed that
the SB fraction had signicantly lower values of the kinetic param-
eters, SMA and k
0
compared to these for the other substrates,
though the observed total yield is not signicantly lower (Table
4). Due to its high protein and fat content, SB has a high potential
to produce methane. However, the hydrolysis step has shown to be
the rate limiting step in the anaerobic digestion process of SB (Pal-
atsi et al., 2011). A two-stage system, in which the hydrolysis step
is separated from the methane production, would therefore be
preferable for the digestion of these kinds of waste streams.
The stressed systems, with stress being caused by long periods
without feeding, reacted as expected (Table 3 and Fig. 1b). There
was a lag phase observed for all batches after the second feeding
and among them the SB fraction presented the longest one
(21 days).
Moreover, the methane yield was increased in four batches after
the second feeding. The SB fraction produced 681 NmL CH
4
/gVS
sub-
strate
, and VC, MSW as well as the mixture with a mixing ratio of
2:3:3:0 (SB:M:VC:MSW) resulted in 603, 596 and 595 NmL CH
4
/
gVS
substrate
methane production, respectively (Table 3). These
increased levels of methane productions were observed in those
batches which showed lower productions of methane (Tables 3
and 4) during the rst feed. The observation can be explained by
the adaptation of bacteria to these substrates.
The methane yield can be used to compare the potential of uti-
lizing different waste streams and waste mixtures for methane
production under given conditions. It depends on substrate com-
position, system congurations and operational parameters.
The theoretical or stoichiometric production of methane in
anaerobic digestion can be calculated according to Deublein and
Steinhauser (2008):
C
a
H
b
O
d
N
c
S
e
yH
2
O ! xCH
4
cNH
3
eH
2
S a xCO
2
in which x 4a b 2d 3c 2e=8 and y 4a b 2d
3c 2e=4.
According to this equation, the theoretical methane production
from starch, cellulose, fat and proteins can be calculated as 415,
415, 636 and 397 L/kg, respectively.
These values can be compared to the obtained methane yields,
even though a precise theoretical estimate cannot be given for the
different substrates due to the lack of data on the composition
regarding carbohydrates, fat and proteins. For mixtures like the
ones studied in this work, no further values of Y
CH
4
appear to be
reported so far.
Comparing mesophilic and thermophilic operations, there are
several advantages when using a thermophilic system. Due to
the higher operation temperature the degradation rate is higher,
and moreover the pathogenic organisms can be eliminated. These
are important factors to take into the consideration especially
when slaughterhouse residues are utilized in the co-digestion
process.
3.3. Synergetic effects
The methane yield can be higher as it is calculated from the
methane potential of each individual waste stream in a co-diges-
tion process due to synergetic effects within the system (lvarez
and Lidn, 2008; Macas-Corral et al., 2008). While performing
the co-digestion of different mixtures of agro-industrial wastes in
this study it was possible to calculate the expected methane yields
for every SB:M:VC:MSW mixture ratio using the values of methane
yields obtained when only the individual fractions were digested
(Table 3). The Y
CH
4
value for the manure (M) fraction was not
determined during this study. However, this substrate was previ-
ously investigated in an anaerobic batch digestion assay performed
at identical conditions, as the conditions used in this study (Aslan-
dazeh et al., submitted for publication). Hence, a value of
250 NmL CH
4
/gVS
substrate
obtained by Aslandazeh et al. (submitted
for publication) was used as Y
CH
4
of M in the calculations. As it is
shown in Table 5 all the mixtures showed higher values of
obtained Y
CH
4
than the calculated levels. The mixture ratio of
1:1:1:1 of SB:M:VC:MSW showed the highest increase of 43%, on
the other hand, the mixture ratio 1:1:2:0.5 had only a slight
increase, compared to the expected values, respectively. However,
further investigations are needed to evaluate the synergetic effects
in detail and for the optimization of mixture ratios leading to sta-
ble and robust operation systems with the highest possible meth-
ane yields.
3.4. Specic methanogenic activity
The determination of SMA provides an important tool for mon-
itoring and characterizing anaerobic digestion (Srensen and Ahr-
ing, 1993). The system with higher values in SMA has the
possibility for higher rate of methane production per volatile solids
active inoculum. As it is shown in Table 3, there was a difference
observed in SMA calculated for the two different feedings. During
the rst feeding, the SMA values were higher compared with that
of the second feeding. Lag phases observed after the second feeding
played an important role in this difference. The bacteria working in
the reactors were already stressed as a result of a period of nutrient
limitation and consequently the substrate degradation after the
second feed was affected.
Fig. 2 shows SMA, calculated from the specic rate of methane
production vs. time along the digestion of the rst feed, i.e. without
stress conditions.
The mean values of SMA obtained over the rst 5 days of the
experimental period are presented in Table 3. An ANOVA analysis
of these initial SMA results reveals signicant differences between
the substrate mixtures (p = 0.000) and a pooled standard deviation
of 0.003. The sorted results were then placed in homogeneous
groups. This shows three distinct groups, with only the SB fraction
in the third group having a very low value of 0.06 gCOD
NmL CH
4
=
gVS
inoculum
=d (Table 4). Mixtures of SB:M:VC:MSW corresponding
to w/w ratios of 1:3:4:0.5; 1:1:1:1, respectively, and the MSW frac-
tion alone achieving SMA values around 0.13 gCOD
NmL CH
4
=
gVS
inoculum
=d (First group). The second group, i.e. mixtures
corresponding to w/w ratios of 1:1:2:0.5, 2:3:3:0 and the VC frac-
tion alone resulted in SMA values between 0.11 and
0.12 gCOD
NmL CH
4
=gVS
inoculum
=d (Tables 3 and 4).
Those mixtures which showed great synergetic effects in the
co-digestion systems also have high SMA values (Tables 3 and 5).
These results correspond to those which show that during the rst
10 days, most of the methane i.e. more than 80% of the maximum
yield was already obtained (Fig. 1a). Furthermore, after 15 days the
best mixtures have reached 9297% of their maximum methane
production (Fig. 1a). These results are in accordance with changes
in SMA, shown in Fig. 2, along the experimental period, where
10838 J. Pags Daz et al. / Bioresource Technology 102 (2011) 1083410840
there are low remaining activities after 15 days of digestion. How-
ever, for determining long termeffects during the co-digestion pro-
cess further investigations using continuous operation are needed.
The results on accumulated methane production and SMA
obtained in this study, support using hydraulic retention time
(HRT) of 15 days within the continuous thermophilic digestion
system.
On the other hand, the investigated substrates and substrate
mixtures showed low SMA values after the second feeding. How-
ever, the same behavior could be observed in general despite of
the stressful conditions within the microbial consortium (Table
3). Although a long lag phase (Fig. 1b); the highest values of Y
CH
4
and SMA could be achieved within the digestion of SB (Table 3).
Due to the high content of proteins and fats, SB can be benecially
utilized in co-digestion processes.
Moreover, the three other reactors (VC, MSW, and the one with
a mixture ratio of 2:3:3:0) which also showed lower values of
methane production during the rst feed, had the capability to
increase the methane yield after the second feeding (Table 3). This
shows that specic groups of bacteria could be developed due to
adaptation to these substrates during the digestion of the rst feed.
However, we can assume that because of the longer starvation per-
iod between the two feedings, lower amounts of bacteria were
available to degrade the substrate. Nevertheless, in the calculation
of SMA, the VS value of active inoculum added with the experi-
mental set up was used, since determinations of available active
VS for the second feed in the different batches were impossible
to perform. This might explain the calculated low SMA values in
the second feed.
No SMA calculations for the kind of substrates investigated in
this work were found in the literature for comparison.
3.5. Kinetics
To study the kinetics of the systems, k
0
values were evaluated.
Most of the kinetic models proposed earlier are focused on the
Table 5
Synergetic effects obtained during the co-digestion of different SB:M:VC:MSW mixtures.
Mixtures VS
a
(g)
Expected
b
Y
CH4
NmL
CH
4
=gVS
substrate

Obtained Y
CH4
NmL
CH
4
=gVS
substrate

1:1:1:1
SB 0.46 463 664 80
M 0.69
VC 1.64
MSW 0.35
1:3:4:0.5
SB 0.15 452 582 59
M 0.68
VC 2.15
MSW 0.06
1:1:2:0.5
SB 0.31 475 491 33
M 0.46
VC 2.19
MSW 0.12
2:3:3:0
SB 0.31 454 499 23
M 0.72
VC 2.19
a
Volatile solids added.
b
Calculated from methane potentials of the individual fractions.
Fig. 2. Specic methanogenic activity (SMA) calculated from the specic rate of methane production vs. time along the digestion of the rst feed.
J. Pags Daz et al. / Bioresource Technology 102 (2011) 1083410840 10839
substrate consumption rate, as an essential issue to describe an
anaerobic system kinetically (Pavlostathis and Giraldo-Gomez,
1991). However, methane is the main metabolite produced in this
system, thus it can be used as a parameter to evaluate the kinetics
of the discontinuous process due to its direct relationship with
substrate consumption and its relative easy determination. The
model originally proposed by Roediger and used by Jimnez et al.
(2004) (Eq. (3)) to determine k
0
was adopted in the current study.
The rst order kinetic model, even though it is not a precise model
of the process, does give useful description of initial reaction rate
and total methane yield that can be tted from on data of only
accumulated methane production as a function of time.
A non-linear least-square (NLLS) regression with a condence
interval of 95% was applied to adjust Roediger model to the
observed values (Fig. 1a and Table 3). The k
0
parameter can be used
for process design to determine parameters as reactor size or
organic loading, and to predict process performance in terms of
methane production. As k
0
is highly dependent on operational fac-
tors such as temperature, stirring and initial substrate concentra-
tion, it was important to keep these process variables constant
during the study.
Comparing the k
0
values of different batches studied, we can
conclude that the anaerobic process displays a better performance
during co-digestion in nearly all of the batches.
An ANOVA analysis of the obtained results shows signicant
differences between substrate mixtures (p = 0.000) and a pooled
standard deviation of 0.036. The sorted results were then placed
in homogeneous groups (Table 4). Three main blocks could be dis-
tinguished, mixtures of SB:M:VC:MSW corresponding to w/w
ratios of 1:1:2:0.5, 2:3:3:0, 1:3:4:0.5, MSW and VC alone showed
k
0
values of around 0.3 d
1
, followed by the mixture with w/w
ratio of 1:1:1:1 with a k
0
value of 0.2 d
1
, while the lowest k
0
value
of 0.09 d
1
was observed in the case of SB.
Due to the very long lag phases (i.e. up to 21 days) in all batches,
as a result of the stressful conditions in the case of second feeding
(Fig. 1b) it was not possible to evaluate the kinetics of digestion
process after the second feed.
4. Conclusions
The present results show that co-digestion of waste mixtures
from different agro-industrial activities is a good option for biogas
production. It was shown that certain mixture ratios perform sig-
nicantly better than others due to synergetic effects giving
improved methane yields compared to the methane potentials of
the individual substrates. It was possible to relate the enhance-
ment in methane yield and in specic methanogenic activity. Fur-
thermore, the digestion process could be restarted with a second
feeding after the rst feed. However, a long lag phase period was
observed due to a long exhaustion period in between the feedings.
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