Bioresource Technology 102 (2011) 9447–9455

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Bioresource Technology
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Anaerobic digestion and co-digestion processes of vegetable and fruit residues:

Process and microbial ecology
E.I. Garcia-Peña a,⇑, P. Parameswaran b, D.W. Kang b, M. Canul-Chan a, R. Krajmalnik-Brown b,⇑
a
Bioprocesses Department, Unidad Profesional Interdisciplinaria de Biotecnología, IPN P.O. Box 07340, Mexico City, Mexico
b
Swette Center for Environmental Biotechnology at Arizona State University, P.O. Box 875701, Tempe, AZ 85287-5701, USA

a r t i c l e i n f o a b s t r a c t

Article history: This study evaluated the feasibility of methane production from fruit and vegetable waste (FVW)
Received 31 March 2011 obtained from the central food distribution market in Mexico City using an anaerobic digestion (AD)
Received in revised form 18 July 2011 process. Batch systems showed that pH control and nitrogen addition had significant effects on biogas
Accepted 20 July 2011
production, methane yield, and volatile solids (VS) removal from the FVW (0.42 m3biogas =kg VS, 50%, and
Available online 27 July 2011
80%, respectively). Co-digestion of the FVW with meat residues (MR) enhanced the process perfor-
mance and was also evaluated in a 30 L AD system. When the system reached stable operation, its
Keywords:
methane yield was 0.25 (m3/kg TS), and the removal of the organic matter measured as the total
Methane production
Co-digestion of FVW
chemical demand (tCOD) was 65%. The microbial population (general Bacteria and Archaea) in the
Microbial ecology 30 L system was also determined and characterized and was closely correlated with its potential func-
tion in the AD system.
Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction 2003, 2005) and constitute a nuisance in municipal landfills be-

In recent years, concern has increased about waste disposal
from mega cities, such as Mexico City, which has a population of
more than 20 million people and produces a tremendous amount
of solid waste, more than 12,000 tons per day. Large volumes of
organic waste are disposed of in the Bordo Poniente sanitary land-
fill, the only landfill in the area, which is approaching capacity.
Because no other locations exist for solid waste disposal, the appli-
cation of efficient technologies for waste treatment and volume
reduction is becoming increasingly important (Forster-Carneiro
et al., 2008). Interest is also increasing in the production and use
of alternative energy sources due to the limited supply of fossil
fuels and their negative effects on the environment (Rittmann
et al., 2008). The organic fraction of municipal solid wastes that
is mechanically sorted in central plants (OFMSW) or the organics
that are separated at the source, referred to as biowaste (the veg-
etable–fruit–garden or VFG fraction) could be a good candidate
for bioenergy production. Fruit and vegetable waste (FVW) is pro-
duced in large quantities in markets in many large cities (Mata-
Alvarez et al., 1992; Misi and Forster, 2002; Bouallagui et al.,
⇑ Corresponding authors. Tel.: +52 5557 296000x56386; fax: +52 5557
296000x56305 (E.I. Garcia-Peña), tel.: +1 480 727 7574; fax: +1 480 727 0889
(R. Krajmalnik-Brown).
E-mail addresses: egarciap@ipn.mx (E.I. Garcia-Peña), Dr.Rosy@asu.edu
(R. Krajmalnik-Brown).
cause of their high biodegradability (Misi and Forster, 2002). The
central market for food distribution in Mexico City, Central de
Abasto (CEDA), is the second largest market in the world, receiving
24,000 tons of food products and producing 895 tons of organic so-
lid waste each day (Central de Abastos de la Ciudad de Mexico,
2011). Occasionally, food products in perfectly good condition
are discarded because of the high cost of refrigeration storage.
Approximately 84% of the total solid waste produced in CEDA is or-
ganic waste, and more than 50% of that is from the fruit and vege-
table fraction (Silva-Rodriguez, 2007). The most promising
alternative to incinerating or composting this waste material is
to apply an anaerobic digestion process (Bouallagui et al., 2005)
for simultaneous waste treatment and renewable energy produc-
tion. The main advantage of the anaerobic digestion process is
the production of biogas, which can be used to produce electricity.
The stabilized biosolids can be used as a soil conditioner (Bouallagui
et al., 2005). This technology has been successfully applied in
reducing the volume of waste that enters landfills, thereby
decreasing methane emissions produced by decay (Mata-Alvarez
et al., 2000; Forster-Carneiro et al., 2008; Bouallagui et al., 2009).
Some authors have studied the feasibility of using FVW as a
substrate for anaerobic digestion. The easily biodegradable and
highly moist organic matter content of FVW (75%) facilitates
the biological treatment of these wastes and demonstrates the
feasibility of using this material for anaerobic digestion (Mata-
Alvarez et al. 1992; Bouallagui et al., 2003, 2005, 2009). The

0960-8524/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2011.07.068
9448 E.I. Garcia-Peña et al. / Bioresource Technology 102 (2011) 9447–9455

FWV material is usually collected from food markets and has a 2. Methods
volatile solid (VS) content of between 8% and 18% (Bouallagui
et al., 2005). The organic fraction includes approximately 75% 2.1. Set up for the batch experiments
sugars and hemicellulose, 9% cellulose and 5% lignin (Verrier
et al., 1987; Bouallagui et al., 2005). For most digestion processes, The biodegradability of the fruit and vegetable waste (FVW)
depending on the substrate used, hydrolysis is the rate limiting was determined using batch anaerobic digestion tests. The charac-
step (Vavilin et al., 1997; Mata-Alvarez et al., 2000). Hydrolysis teristics of the FVW mixture are depicted in Table 1. FVW (50 g)
constants were obtained from carbohydrates, protein and lipids, with an initial total solid (TS) content of 98.9 g TS/kgresidues (10%
with the highest constant observed for carbohydrates, and these organic matter) was placed into 125 mL serum bottles that were
rates were determined to be pH-dependent (Mata-Alvarez et al., sealed with butyl rubber septums and aluminum crimps and
2000). The anaerobic processing of cellulose-poor waste such as flushed with N2 to provide anaerobic conditions. Some treatments
FVW is limited by methanogenesis rather than by hydrolysis were inoculated with 5 mL (10% v/v) of cow manure (density of the
(Bouallagui et al., 2005). The rate and extent of degradation are FWV was of 1.14 g/L). The FVW without inoculation or salt addition
intrinsic properties of the waste characteristics and the microor- was used as a control, and the effects of inoculum (cow manure)
ganisms involved in the process. According to Mata-Alvarez et al. addition, salts (to control the pH), and the addition of a nitrogen
(1992), the FVW contains cellulose (32%), hemicelluloses (15%) source were evaluated; the tested conditions are summarized in
and lignins (15%), and under mesophilic conditions, up to 32%, Table 2. For pH controlled systems, a 100 mM phosphate buffer
86% and 0% of these compounds are removed, respectively. Gun- with an initial pH of 7.0 was used. In the nitrogen supplemented
aseelan (2004) reported the methane yields (B0) of several frac- systems, 0.08 g of NH4Cl was used per g of waste, and the experi-
tions of FVW, sorghum and napiergrass. The methane potential ments were carried out with 50 g of FVW as mentioned above. All
depends on the organic components in the FVW used as experiments were performed in duplicate. The systems were incu-
feedstock, which are mainly carbohydrates, proteins and lipids. bated at 30 °C for 30 days or until biogas production ceased. Each
The theoretical methane yields (B0) from acetic acid, carbohy- system was manually mixed once per day. Additionally, two
drates, proteins and lipids are 370, 415, 496 and 1014 L CH4/ control systems that only contained inoculum were incubated at
kg VS, respectively (Moller et al., 2004). B0 could also be esti- the same temperature to correct for the amount of biogas produced
mated considering that 1 kg of COD reduction is equivalent to by the organics in the inoculum. Statistical analysis was carried out
0.35 m3 CH4 (STP) (Gunaseelan, 2007). with the NCSS statistical system (NCSS, PASS, and GESS, NCSS,
The high biodegradability of the FVW promotes the rapid Kaysville, UT, http://www.ncss.com).
production of volatile fatty acids (VFAs) resulting in a rapid de-
crease in pH, which in turn could inhibit the methanogenic activity 2.2. Experimental setup (anaerobic digester)
(Mata-Alvarez et al., 1992; Bouallagui et al., 2003, 2009). An inter-
esting option to avoid the acidification of the system when FVW is The process was also evaluated in an anaerobic digestion
used is the addition of co-substrates with high nitrogen contents, system (ADS) consisting of a stainless steel tubular reactor with a
which could result in a natural pH regulation and also constitute total volume of 30 L into which 20 L of a (50:50) mixture of FVW
a source of nitrogen. This strategy, known as co-digestion, results
in a more efficient digestion process, improving the methane yields
obtained from certain organic materials due to the positive syner- Table 1
Initial characteristics of the fruit and vegetable waste (FVW).
gistic effects of the mixed materials with complementary charac-
teristics and the supply of missing nutrients by the co-substrate Solid waste Organic matter Total solids Volatile solids pH
(Agdag and Sponza, 2005). Co-digestion also presents economic (g/kgwaste) (g/kgwaste) (g/kgwaste)
advantages, such as minimizing equipment needs by sharing the Tomato 59.1 55.7 54.9 4.5
same equipment for different residues and easier handling of Lettuce 53.5 31.3 30.4 5.6
mixed waste (Mata-Alvarez et al., 2000). Habiba et al. (2009) Papaya 85.5 116.5 114.4 5.5
Pineapple 72.7 102 99.2 3.5
studied co-digestion as a novel solution to adjust unbalanced Banana 107.6 181.2 176.4 5.0
nutrient constituents and reported that the anaerobic digestion Orange 115.5 153.2 149.4 3.8
of activated sludge (AS) with substrates containing high levels of Mixture of the 72.7 98.9 96.4 4.02
C/N, such as FVW, overcame the difficulties of digesting AS. The solid waste
addition of high nitrogen content co-substrates to adjust the nutri-
ent content of FVW was recently evaluated by Bouallagui et al.
(2009), and a methane yield of approximately 0.35 L/g VS was
obtained without the addition of chemical alkali. Table 2
The aim of this study was to evaluate the potential use of FVW Conditions established in eight systems (in duplicate) to evaluate the effects of
as a substrate for methane production and to examine various inoculation, pH control and addition of a nitrogen source (0.08 g ammonium chloride/
conditions that allow for anaerobic systems the optimal perfor- gwaste, with experiments conducted with 50 g of FVW) on the performance of the
anaerobic digestion process.
mance using FVW. A mixture of FVW from the biggest market in
Mexico was characterized to assess its potential as a feedstock System Conditions
for an anaerobic digestion process. Additionally, the effects of: I FVW inoculated with cow manure (10%)
(1) pH, (2) nitrogen addition, and (3) inoculation of the FVW were IN FVW inoculated and supplemented with NH4Cl as a nitrogen
evaluated to enhance methane production in batch systems. source
IpH FVW inoculated and salts added (buffer) to control pH
Co-digestion of the FVW with meat residues (MR) was also evalu- IpHN FVW inoculated, buffering salts, and NH4Cl added
ated. The performance of a 30 L reactor was assessed under the wI (VSW)
most effective conditions obtained in the batch systems to deter- FVW without inoculation (WI) (Control)
mine the feasibility of converting the FVW and MR into biogas. wIN FVW and NH4Cl
wIpH FVW and buffering salts
The microbial ecology of the 30 L system when operating at steady
wIpHN FVW buffering salts and NH4
state conditions was evaluated and its links to process perfor-
mance were assessed using molecular methods. I = inoculated systems, wI = systems without inoculum.
 E.I. Garcia-Peña et al. / Bioresource Technology 102 (2011) 9447–9455
and meat residues (MR) was initially packed. After the initial
period of operation (20 days), the ADS was inoculated with cow
manure (10% v/v) to enrich the methanogenic population. The
reactor was stirred by re-circulating the FVW twice a day. The
pH was set at 7 and was maintained by the addition of a NaOH
(0.8 N) solution when the ADS was started. Later in the process,
the pH was naturally regulated by the metabolic intermediates
produced during digestion. The bioreactor was kept at room tem-
perature and operated in a fed batch mode. To avoid inhibition
due to metabolic products and to ensure a sufficient supply of
organic matter, 2.5 kg of different compositions of fresh feedstock
mixtures were fed periodically (approximately every 12–15 days),
and an equal volume of exhausted sludge was removed.
2.3. Analytical methods
The fruit and vegetable waste samples were analyzed for total
solids (TS) and volatile solids (VS) contents according to the stan-
dard methods of the American Public Health Association (APHA,
2005).
Biogas production in the batch cultures and in the anaerobic
digester was periodically measured using a water displacement
set up in which the biogas was passed through a 5% NaOH solution
(Anaerobic Lab Work, 1992). Biogas samples were taken periodi-
cally from the gas collection lines prior to the water displacement
set up, and the gas composition was analyzed using a gas chro-
matograph (GowMac Series 550, Bethlehem, PA) equipped with a
thermal conductivity detector. A CTR1 packed column (Alltech
Co., Beerfield, IL) was used for the analysis. The analysis conditions
were the same as those reported previously (Garcia-Peña et al.,
2009). The measured biogas volume was adjusted at standard
temperature (25 °C) and pressure (1 atm). VFA samples were ana-
lyzed in a gas chromatograph (Buck Scientific, East Norwalk, CT) as
previously reported (Garcia-Peña et al., 2009). The acetic and buty-
ric acid concentrations in liquid solution were calculated using the
Henry’s Law dimensionless constant (H0 = Concentration in gas
phase/Concentration in liquid phase) for each compound at the
corresponding temperature.
2.4. Molecular microbial ecology
2.4.1. DNA extraction
A 0.5 g sample of the anaerobic digested sludge was taken when
the system was operating at steady state conditions and was used
for DNA extraction following the recommendations of the MOBIO
Powersoil DNA isolation kit. Extracted DNA was quantified with a
Nanodrop Spectrophotometer and stored at 20 °C.
2.4.2. Quantitative PCR and 454 pyrosequencing
The extracted DNA was diluted tenfold for analytical conve-
nience for quantitative real time PCR (QPCR). The sample was
tested for total Archaea and the methanogenic genera Methano-
microbiales, Methanobacteriales, Methanosaetaceae, Methanosarcina-
ceae, and Methanococcales according to the conditions described in
Parameswaran et al. (2009). All assays were carried out using an
Eppendorf Realplex 4S unit (Eppendorf, Germany).
Using extracted DNA as the template, bacterial tag-encoded FLX
amplicon pyrosequencing (bTEFAP) was performed based on the
titanium protocol (Roche, Indianapolis, IN) at the Research and Test-
ing Laboratory (Lubbock, TX) and previously published in Zhang
et al. (2011). Using the FLX-Titanium System Genome Sequencer,
the combined V2 and V3 regions of 16S rDNA were sequenced by
the previously described procedure (Wolcott et al., 2009). After
sequencing, all failed sequence reads, low quality sequence ends,
and tags were removed. Sequences were depleted of any non-bacterial
ribosome sequences and chimeric sequences by using modified
9449
ChimeraSlayer in the MOTHUR software (Gontcharova et al., 2010)
at the Research and Testing Laboratory. We also excluded sequences
shorter than 250 bp and longer than 450 bp. After qualifying 954
sample readouts, we obtained 611 readouts for the bacterial com-
munity in the anaerobic digester. We aligned and clustered the read-
outs by using the MOTHUR software (the Silva alignment and
Operating Taxonomic Unit (OTU)-based clustering). We classified
sequences by the Ribosomal Database Project (RDP) classifier soft-
ware at an 80% confidence threshold (Cole et al., 2009). The se-
quences were 390 bp long in average, which is sufficient to obtain
information at the species level. Species level identification was ob-
tained after clustering the aligned sequences using RDP pyro pipe-
line application and analyzing the sequences with BLAST and
SEQMATCH applications provided by National Center for Biotech-
nology Information (NCBI) and RDP, respectively.
3. Results and discussion
3.1. Batch experiments
The physical and chemical characteristics of the organic waste
are important for designing and operating anaerobic digesters be-
cause they have an effect on biogas production and process stabil-
ity during anaerobic digestion. The residues were characterized
individually and as a mixture (equal proportions of each residue
w/w), the composition of which was chosen based on the products
most frequently and consistently sold in the market, i.e., excluding
seasonal products. The characteristics of the FVW mixture were as
follows: a total solid (TS) content of approximately 73–100 g/kg
waste (approximately 10%), a pH of 4, and a moisture content of
90%. Table 1 presents the initial characterization of the FVW. The
FVW mixture used in this study had a higher soluble carbohydrate
content than protein content because it only contained fruits and
vegetables and did not include a source of protein. Additionally,
this FVW had a high moisture content and can be considered a
highly degradable substrate, both of which make it an ideal candi-
date for CH4 production. The main drawback of the anaerobic
digestion process with FVW could be the low pH, which negatively
affects the methanogenesis phase. Two experimental setups were
simultaneously assessed: (1) only the fruit and vegetable waste
(wI, FVW) as a control, and (2) a system with the FVW amended
with salts (phosphate buffer) for pH control and inoculated with
the cow manure (IpH). The results of the two systems during
35 days of operation are presented in Fig. 1A and B. Fig. 1A shows
the VS removal profile, the biogas production and the pH evolution
for the control system (FVW), in which 100 VS/kgwaste were rapidly
consumed by the natural anaerobic microbial community over
5 days and biogas production started at the time of consumption.
Then, the degradation of organic matter (OM) stopped after 5 days
due to the low pH (approximately 4) that inhibits the methanogen-
ic activity. No methane was produced under these conditions. In
this system, 85% VS removal was obtained in 19 days with a biogas
yield of 13.3 L/kgwaste. Fig. 1B shows the evolution of the inoculated
system with pH control (IpH), demonstrating that the OM was con-
tinuously degraded and transformed into biogas, attaining the
maximum gas production (11.8 L/kgwaste) in approximately
15 days. In this time, the IpH system reached 82% conversion of
the initial OM to methane, and the pH was maintained around
6.5, which is sufficient for methanogenesis to proceed. The total
alkalinity of the system could also have limited methanogenesis
(Rittmann and McCarty, 2001). A methane percentage of 30% (v/
v) was obtained with this system. In the control wI (FVW) system,
only 65% of the VS was removed in 15 days, while at the same time
a VS removal of 82% was obtained in the IpH system. The inocula-
tion and pH control favored the process, enhancing the VS
9450 E.I. Garcia-Peña et al. / Bioresource Technology 102 (2011) 9447–9455

reduction and allowing the methane production to occur over a the use of buffer solutions to regulate the pH (Bouallagui et al.,
shorter period of time (15 days), the biogas production was similar 2005). Another characteristic of the FVW is the absence of a nitro-
for both systems (considering the bar errors, Fig. 1). These results gen source; experiments were also performed to evaluate the ef-
could be explained by the pH values presented in Fig. 1B, showing fect of adding a nitrogen source, considering that adequate C/N
that the pH in the IpH system was maintained around 6.5. The relation is necessary to enhance the anaerobic digestion process.
biogas yields of the wI (FVW, control) and IpH systems were 0.23 A variety of conditions were tested to promote biogas and methane
and 0.414 m3/kg VS, respectively; only CO2 was determined production using the FVW, including buffered and nitrogen supple-
because no methane was produced in the wI (FVW) control system. mented systems in experimental systems with and without inocu-
The data suggest that in the wI (FVW) system, only the hydrolysis lation (Table 2).
phase of the process occurred, and the methanogenic activity was Cumulative biogas production by the systems, under the tested
completely inhibited by the low pH. The biogas yields obtained conditions, is depicted in Fig. 2. Dark lines are used to fit the inoc-
here are in the range of those reported by other authors for similar ulated systems and dashed lines represent the systems without
solid wastes (Bouallagui et al., 2005). inoculum. The highest biogas production (approximately 10 L/
Other reports have demonstrated that pH control is one of the kgwaste) was reached in the FVW and buffering salts (wIpH) system,
most important parameters to achieve high biogas production and was approximately twice that obtained in the control system
(Mata-Alvarez et al., 1992). Data obtained in this study (Fig. 1A (wI, FVW). The systems inoculated with a pH control (IpH) and
and B) corroborated the fact that the pH needs to be regulated the system at the same conditions but supplemented with nitrogen
throughout the process to allow for good performance of the (IpHN) attained biogas productions of 7.5 and 6 L/kgwaste, respec-
anaerobic digestion of the FVW. Bouallagui et al. (2005) reported tively. The lowest biogas production was obtained in the system
that the limitation of the anaerobic digestion of FVW is due to wIN without inoculation and supplemented with nitrogen, which
rapid acidification by the production of large amounts of volatile could be because an excess of nitrogen can lead to toxic ammo-
fatty acids (VFA), which inhibits the biological activity of the meth- nium concentrations as demonstrated in previous reports (Fricke
anogens. Some authors buffered the FVW by adding sodium et al., 2007; Bouallagui et al., 2009). Table 3 summarizes the results
hydroxide (Verrier et al., 1987), and other experiments included obtained for the TS removal, the biogas productivity (m3/kg VS),
the methane percentage (v/v) and the final pH determined under
the various conditions evaluated here. In general, the inoculation,
A 100
No methane was detected
14 pH control and nitrogen source addition had significant influences
on the VS reduction, with the I, IpH and IpHN systems showing
higher degradation percentages between 70% and 86% of the initial
Biogas production (L/Kg

12
80 tVS in 28 days. Lower organic VS reductions in the range of 35–42%
were obtained for the systems without inoculation. The highest
10
)
waste

biogas yield (0.420 m3/kg VS) was obtained in the inoculated

Volatile Solid (g/Kg

60 system with pH control and N addition (IpHN), reaching a VS

8
removal percentage of 86% as shown in Table 3. For the IN (inocu-
lated and nitrogen added) and IpH (inoculated + buffer) systems,
40
6 lower biogas productions of 0.270 and 0.230 m3/kg VS, respec-
waste

tively, were observed, and lower VS reductions were obtained.

)

4 These results suggest a correlation of pH with biogas productivity,

pH

20 with the higher productivity occurring at pH values close to the

2 optimum pH of 7. Lower biogas productions of 0.150 and
0.080 m3/kg VS were measured in the systems without inoculum
0 0 (wIpH and wIpHN) with corresponding low VS removals of 42%
0 5 10 15 20 25 30 35 and 34%, respectively. Methane production (approximately
Time (days)

B 100 14 12

Methane 30 (%)
Biogas production (L/Kg

12
10
80
Volatile Solids (g/Kgwaste)

10
waste

8
Biogas production (L/kg

60
8

6
6
40
waste
)

4 4
pH

20
2
2

0 0
0 5 10 15 20 25 30 35
0
Time (days) 0 5 10 15 20 25 30 35 40
Time (days)
Fig. 1. VS reduction (d), biogas production () and pH evolution (4) during 35 days
in a batch system. (A) wI (FVW) control; (B) IpH (FVW with inoculum and buffering Fig. 2. Cumulative biogas production under different batch conditions: I (), IN (N),
salts added). IpH (d), IpHN (.), wI (FVW) control (e), wIN (4), wIpH (s), and wIpHN (5).
 E.I. Garcia-Peña et al. / Bioresource Technology 102 (2011) 9447–9455
Table 3
VS removal, biogas production and final pH for the various conditions evaluated in batch experiments.
System Initial VS content (g/ Final VS content (g/
kgwaste) kgwaste)
I 136.7 64.4
IN 123.9 17.0
IpH 104.9 31.3
IpHN 107.5 14.7
wI (FVW) 127.5 78.5
wIN 123.9 80.5
wIpH 104.9 60.5
wIpHN 107.5 70.6
Co-digestion 134.8 14
(FVW:MR)
a
Different superscripts correspond to significantly different values (a = 0.05). The Tukey–Kramer test (NCSS statistical system) showed that the inoculated systems were
significantly different from the systems without inoculums for the biogas productivity and VS removal with an a = 0.05. The positive effect of the pH control and nitrogen
addition in the inoculated systems was also demonstrated.
45–53 ± 0.5% v/v in the biogas mixture) was only obtained in the
inoculated systems, with the highest methane percentage (53% v/
v) observed for the IpHN system.
The results obtained in this work are in agreement with previ-
ous data on biogas and CH4 production using FVW as a feedstock:
0.16 m3/kgVS (Rajeshwari et al., 1998), 0.19 m3/kgVS (Alvarez,
2004), and 0.26 m3/kgVS (Boullagui et al., 2005). Higher biogas
production has been obtained using a continuously stirred tank
reactor (CSTR) and a continuous tubular reactor, where production
yields of 0.47 and 0.45 m3/kgVS, respectively, were determined,
with VS removal percentages of 88% and 76% (Mata-Alvarez
et al., 1992; Bouallagui et al., 2003). In the inoculated systems, both
nitrogen addition and pH control had positive influences on the
biogas production (with a significant term a = 0.05, as shown in
Table 3), allowing biogas productivities in the range of those
reported in the literature.
The highest biogas yield was obtained in the system inoculated
and supplemented with nitrogen and buffering salts; therefore,
these conditions should be used to produce methane from FVW.
However, the addition of salts and/or nitrogen to a waste treat-
ment process could result in high operating costs. To address this
issue, a co-digestion experiment was also performed with a
mixture of FVW and meat residues (from meat packaging opera-
tions at the same market). Meat residues (MR) provide a high
nitrogen concentration, and the protein hydrolysis could result in
natural pH control due to NH4 production. For the co-digestion sys-
tem of FVW and MR, the highest biogas yield of 0.9 m3/kgVS
(methane yield of 0.45 m3/KgVS) was observed, reaching an
organic matter degradation of 93% (Table 3).
3.2. Anaerobic digester experiments
3.2.1. Start up
Based on the results obtained in batch experiments showing
that the co-digestion of FVW and MR enhances biogas production,
the feasibility of an anaerobic digestion process using FVW and MR
was evaluated in a 30 L AD system. To determine the effect of the
MR addition on biogas and methane production, experiments were
carried out using different MR proportions. The anaerobic digestion
process was performed in a semi-continuous regime. The biogas
productivity, methane percentages and VS removal obtained under
different conditions after 130 days of operation are presented in
Fig. 3. Biogas production started 24 h after the reactor was packed
with the FVW and MR, and anaerobic conditions and pH regulation
were established. Initially, biogas production (a maximum of
1.03 m3/kg VS) resulted from the hydrolysis of the easily degrad-
able components of the feedstock and the activity of the natural
microbial populations of the FVW. The highest TS removal (89%)
9451
Removal percentage Biogas productivity (m3/ Final Final methane percentage
(28 days) kg VS) pH (%, v/v)
53a 0.07a 4.5 0
86b 0.27b 6.5 45
70c 0.23c 6.9 45
86b 0.42d 6.9 53
38d 0.07a 3.7 0
35e 0.05a 3.7 0
42f 0.15e 6.9 0
34e 0.08a 6.9 0
90g 0.9f 7.1 55
and biogas production were obtained during the startup of the
ADS, during which time methane was not produced, suggesting
that the biogas production (mainly CO2) resulted from the initial
hydrolysis of the highly biodegradable fraction of the feedstock
(Fig. 3).
After 20 days of operation and when the biogas productivity
started to decrease, the anaerobic digestion system (ADS) was
inoculated with 13% (v/v) of cow manure and fed with a mixture
of FVW (3 L), and a new strong biological activity was observed
(Fig. 3) reaching an average biogas production of approximately
0.64 ± 0.1 m3/kg VS. In this period, the CH4 content in the biogas
was 16% (v/v) (methane productivity of 0.1 m3/kg VS) due to the
initial activity of the methanogenic population introduced into
the ADS with the inoculums. The strong microbial activity allowed
for a VS removal of approximately 80%. The biogas production was
lower than that obtained during the start-up of the system, how-
ever, the methanogenic activity started during this period (Fig. 3).
When a mixture of FVW and MR (75:25) was fed to the ADS, the
biogas productivity decreased to a value of approximately 0.5 m3/
kg VS, and the methane percentage increased to 28% (v/v). The VS
removal in this period was of 70%. The co-digestion of 50:50
FVW:MR yielded the highest methane percentage of 30%, which
occurred during the initial stages of the reactor operation, and
which corresponds to a methane production of 0.12 m3/kg VS
and a VS removal of 73%. To evaluate the response of the system
to the composition of the feedstock, the proportion of FVW and
MR was again reduced and only FVW was added to the ADS. As
expected, the CH4 content in the resulting biogas was low, at only
14% (v/v). For the next stage, on the 70th day of operation a 50:50
mixture of FVW:MR was added, and the CH4 percentage recovered
to 30%. Once stable operation was achieved after 83 days, the
biogas production showed a constant value of approximately
0.25 m3/kg VS and a methane percentage of 53%, corresponding
to a methane production of 0.135 m3/kg VS and a VS removal of
78%. The reactor was regularly fed with a mixture of FVW:MR
(75:25), and under these conditions the CH4 percentage was stable
at 53 ± 2%, and the pH was stable at 6.9 ± 0.5% (naturally regulated
during this last stage of the process). An appropriate buffering
capacity and a highly stable experimental system were observe
with Organic Loading Rates (OLRs) in the range of 2.4 and
2.7 g COD/L day (Hydraulic retention times HRT in the range of
15–20 days). The pH was only regulated during the start-up of
the reactor, when a total volatile fatty acids (VFAs) concentration
of 4000 mg/L (acetate and butyrate 2:1) in the liquid phase was
observed. After the second stage, the VFA concentration was lower,
approximately 2000 mg/L. The average concentration of the total
VFAs was 1300 mg/L (mainly acetic and butyric acids) at the
steady-state of the ADS (data not shown). In a well balanced
9452 E.I. Garcia-Peña et al. / Bioresource Technology 102 (2011) 9447–9455
1 0
Biogas Production (m /kgVS)
0.8
3
0.6
VSR= 89%
0.4
0.2 Start-up
0
0 20
Fig. 3. Biogas production, methane percentage and VS removal determined during the start-up of the ADS at different VFW and MR proportions.
anaerobic digested process, total VFA levels range from 55 to
1800 mg/L (Dinsdale et al., 2000; Misi and Forster, 2002; Bouallagui
et al., 2009).
One of the biggest problems during the anaerobic process is to
maintain the pH above 6.6, because the desired pH for anaerobic
treatment is between 6.6 and 7.6 (Rittmann and McCarty, 2001).
On the other hand, at normal percentages of CO2 in the digester
gas, between 25% and 45%, a total alkalinity of at least 500–
900 mg/L is required to keep the pH above 6.5. Higher CO2 partial
pressure makes alkalinity requirements larger (Rittmann and
McCarty, 2001). At the start up of the ADS only FVW were fed
and high amounts of VFAs were produced, which causes a rapid
pH drop and the inhibition of methane production. Additionally,
under these conditions the CO2 partial pressure was high because
only this compound was produced, both conditions makes the
alkalinity requirements high. For this reason an alkaline material
was added (0.8 M NaOH solution) to provide the adequate buffer
and to prevent the excessive pH drop under this unbalanced condi-
tions. During the second stage, when only FWV 16% of methane
was produced. The requirement of alkalinity was of 4134 mg/L,
and in spite of the fact that alkaline solution was added the condi-
tions were still unfavorable for an adequate methane production.
When, the meat residues were introduced at a ratio of (75:25,
FVW:MR) into the ADS, NH3 started to be released from the hydro-
lysis of the proteins. The alkalinity started to increase, considering
that the moles of bicarbonate alkalinity is equal to the moles of
NH4 according to the stoichiometric equation of the methanogen-
esis for an organic mixture (carbohydrates and proteins) shown
below. This equation was obtained by modifying the equation
presented by Rittmann and McCarty, 2001 for a mixture of volatile
fatty acids and proteins (fs of 0.18).
C11 H17 O5 N2:33 þ 6:74H2 O ! 4:10CH4 þ 3:12CO2
þ 0:36C 5 H7 O2 N þ 1:97NHþ4
þ 1:97HCO3
For a 75:25 ratio of FVW and MR the required alkalinity was of
3500 mg/L, the alkalinity and the pH drop has to be still regulated
and controlled by the NaOH solution addition. At a ratio of 50:50,
the methane percentage increased, the CO2 percentage decrease
and the required alkalinity was of 3445 mg/L. An alkalinity as
CaCO3 of 4804.6 mg/L was calculated under the experimental
Methane (%)
16 28 30 14 30 53
VSR= 81% VSR= 70% 73% 80% 75% VSR= 78%
FVW:MR
FVW:MR
FVW:MR
FVW:MR
VFW:MR FVW:MR
100:0
75:25
50:50
50:50
100:0
75:25
40 60 80 100 120
Time (days)
conditions (70% of removal efficiency and an initial substrate con-
centration of 50 g COD) by using the stoichiometric equation for an
organic mixture of carbohydrates and protein (50:50). This total
alkalinity value was high enough to avoid a possible acidification
of the ADS and the high buffering capacity allows and stable oper-
ation without the external control of the conditions.
The increase in the methane production after the 80th day of
operation could result from an increase in the methanogenic
population and its adaptation to the operating conditions in the
ADS. The high VS removal, the increase in the methane yield, and
the natural pH control during the stable period of the ADS was
due to an adequate ratio of nutrients and the availability of pro-
teins for new cell synthesis.
Methane production yields during the initial period of operation
of the ADS were lower than those reported in other studies, which
ranged from 0.16 to 0.762 m3/kg VS (Mata-Alvarez et al., 1992;
Bouallagui et al., 2009). The low methane yields obtained during
the start up of the ADS in the present work could be partially
explained as a result of the temperature, as the 30 L reactor was
operated at room temperature. The batch experiment data showed
that at a controlled temperature of 30 °C, better biogas and meth-
ane yields (0.9 and 0.45 m3/kg VS, respectively) could be obtained
with the co-digestion of FVW and MR (50:50). Temperature is well
known to have a strong effect on methane production, and recently
Bohn et al. (2007) showed that the methane yield could be in-
creased almost twice to 0.4 m3/kg VS at 30 °C compared with the
yield obtained at 20 °C (0.25 m3/kg VS). The operation of anaerobic
digestion system in the field in developing countries requires
systems with a simple design and process control, robustness to
harsh conditions and lower investment costs. Low cost systems
operated at temperatures below mesophilic conditions have been
shown to be successful in on-farm manure treatment, although
the degradation efficiency is lower and higher retention times
are required (Bohn et al., 2007).
ð1Þ
3.2.2. Long term operation of the anaerobic digestion system (ADS)
Fig. 4 shows the current performance of the ADS after 3 years of
operation. The biogas composition was determined daily during
15 days of operation. After the addition of the feedstock (24 hours),
an initial CO2 production of 71% (v/v) in the biogas effluent was
determined, with this amount of CO2 corresponding to the initial
hydrolysis of the FVW, and a low CH4 percentage was measured
 E.I. Garcia-Peña et al. / Bioresource Technology 102 (2011) 9447–9455 9453

80 Others
Bacteroidetes
2.3% 1.3%
3
MY =0.258 m /KgCOD
70

60
Biogas composition (%, v/v)

50

40

Firmicutes
30 89.5%

20

10

0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Time (days)
a) Phylum level distribution
Fig. 4. Biogas composition of the current ADS operation (v/v): H2% CH4% CO2%
. MY = methane yield. Anaerofilum Akkermansia Bacteroides
1% 1% 1%
Prevotella
1%
during the first days of the process. H2 production was also Lachnospiraceae
observed, with the highest H2 production (11%, v/v) observed 2%
48 h after feeding the ADS, and then the microbial activity was Clostridium
shifted to CH4 production after 11 days (63% v/v). The removal rate 2.5%
during this operation period (10 days) was approximately 65% of
the total substrate measured as total Chemical Oxygen Demand
(tCOD), which was approximately 50 g COD/L. A second addition Bifidobacterium
of substrate was carried out on day 11. Before the new addition, 6%
biogas was removed from the system to reduce the pressure in
Lactobacillus
the reactor. During the second feedstock addition, a methane per- 72%
centage of 59% (v/v) was determined on day 15, and in the same
fashion, H2 production (3%) was determined on the second day of
the process. After a long period of operation, the gradual acclima-
tion of the biomass and the enrichment of the methanogenic
population could explain the increase in methane production in
the ADS compared with the production observed during the
start-up of the ADS. Under the current conditions, biogas and b) Genus level distribution
methane yields of 0.41 m3/kg COD and 0.258 m3/kg COD were
Fig. 5. (a) Phylum and (b) genus level distribution of the bacterial population in the
determined in the ADS, respectively. These data are similar to the
full-scale anaerobic digester operated at steady state. Firmicutes (89.5%) constituted
methane yield obtained by Dinsdale et al. (2000) for the co-digestion the major bacterial phylum. Lactobacillus (72% of total reads) was the predominant
of FVW and WAS in two stage tubular digesters and are lower bacterial genus in the anaerobic digester.
compared with the values reported (0.35–0.4 m3/kg VS) by Callaghan
et al. (2002) and Bouallagui et al. (2009) for the co-digestion of
FVW and cattle slurry and chicken manure as well as FVW and the reaction for the electron donor is:
waste activated sludge (WAS). Work is underway to determine
the performance of the system under mesophilic conditions to 0:175CO2 þ 0:025NHþ4 þ 0:025HCO3 þ Hþ þ e
achieve the highest biogas production and methane yields ! 0:048C5:1 H8:5 O2:5 N þ 0:415H2 O ð3Þ
obtained in batch experiments.
and the reaction for the electron acceptor is:
3.2.3. Stoichiometric analysis of the anaerobic digestion system (ADS)
0:125CO2 þ Hþ þ e ! 0:125CH4 þ 0:125H2 O ð4Þ
The stoichiometric balance was formulated based on the
empiric formula for waste water C8H17O3N reported by Rittmann A typical value for fs0
¼ 1 (the fraction of electrons from the
and McCarty (2001), which is similar to the formula reported for donor substrate that are used toward the synthesis of biomass)
the vegetable and fruit organic waste. The biomass formula used and for fs0 ¼ 0:9. The overall reaction is given by:
for the balance was the same formula reported for methanogenic
R ¼ fe Ra þ fs Rc  Rd ð5Þ
systems using organic matter as electron donor: C5.1H8.5O2.5N
(Rittmann and McCarty, 2001). Then, the balanced reaction is:
Thus, the cell synthesis half reaction is:
0:025C8 H17 O8 N þ 0:196H2 O ! 0:0048C51 H8:5 O2:5 N
0:198CO2 þ 0:048HCO3 þ 0:048NHþ4 þH þeþ 
þ 0:1125CH4 þ 0:0427CO2
! 0:048C5:1 H8:5 O2:5 N þ 0:415H2 O ð2Þ þ 0:02NHþ4 þ 0:02HCO3 ð6Þ
9454 E.I. Garcia-Peña et al. / Bioresource Technology 102 (2011) 9447–9455

Table 4
Species level distribution of key bacterial genera in the FVW + cow manure co-digestion reactor operated at steady state.

Genus % species within genus BLAST analysis results SEQMATCH analysis results
a b
Strain match only All included Type + strain match onlya All includedb
Lactobacillus 89.5 L. plantarum L. plantarum L. plantarum L. plantarum
8.2 L. manihativorans L. manihativorans L. manihativorans L. manihativorans
0.7 L. vaccinostercus L. vaccinostercus L. vaccinostercus L. vaccinostercus
0.5 L. plantarum str. J108 L. plantarum L. pentosus L. plantarum
Bifidobacterium 62 B. minimum B. minimum B. minimum B. minimum
18 B. sp. LUCL-W4 B. sp. LUCL-W4 B. subtile B. subtile
15 B. longum B. longum B. longum B. longum
Faecalibacterium 88 F. sp. DJF_VR20 F. prausnitzii ATCC 27768 F. prausnitzii F. sp. N567
Clostridium 26 C. orbiscindens strain 17 C. orbiscindens C. orbiscindens strain 17 C. orbiscindens
20 Acetivibrio sp. 6–13 Acetovibrio sp. 6–13 A. cellulolyticus Acetovibrio sp. 6–13
20 Eubacterium sp. AD 17 E. eligens E. eligens ATCC 27750 Eubacterium sp. AD 17
Lachnospiraceae 62 Butyrivibrio fibrisolvens Butyrivibrio hungatei strain JK614 B. intestinalis Firmicutes oral clone CK030
a
Represents both database search results for type strains only.
b
Includes both type strains and environmental samples.

Using 1 mol of organic matter to calculate the theoretical meth-
ane and CO2 production expected during the process and the
corresponding molar coefficients,
1 mol OM ! 4:5 mol of CH4 ; 1 mol OM ! 1:708 mol of CO2
According to the ideal gas equation, PV ¼ nRT for 4.5 mol of CH4,
the volume of methane will be 142.3 L CH4, and for 1.708 mol of
CO2, the volume will be 54.02 L CO2. Then, the expected volume
percentages for each compound (CH4 and CO2) in the biogas efflu-
ent according to the global stoichiometric reaction are 72.5% CH4
and 27.5% CO2. These values correspond to the theoretical methane
production when all the initial organic matter has been completely
assimilated; the highest experimental methane volume percentage
observed in the ADS was 63% (v/v), corresponding to 87% of the
theoretical value.
4. Microbial ecology
The bacterial sequences generated from the anaerobic digester
(Fig. 5a) were distributed among three major phyla: Firmicutes
(89.5%), Actinobacteria (6.9%), Bacteroidetes (2.3%), along with other
phyla at minor predominance. Firmicutes are well-known to be
fermenters and syntrophic bacteria that can degrade volatile fatty
acids, such as butyrate and its analogs. The predominance of Firmi-
cutes is a clear indication that these products are readily available
due to the prior fermentation of these simple volatile fatty acids, or
else that the waste has undergone biodegradation before anaerobic
digestion. Within the phylum Firmicutes, Bacilli (76.1%) and Clos-
tridia (13.3%) form the major classes. Lactobacillus species (in the
class Bacilli; 72% of the total reads, as shown in Fig. 5b) constitute
common food fermenters and can grow on various carbohydrates
(Jo et al., 2007). Some Clostridium species (in the class Clostridia),
such as C. aminobutyricum and C. sticklandii, were reported as
microorganisms capable of utilizing amino acids and producing
acetate, butyrate, and ammonia (Shin et al., 2010). The syntrophic
role of Firmicutes involves H2 removal and has immediate implica-
tions for the composition of the methanogenic community.
The phylum Bacteroidetes are proteolytic bacteria (Kindaichi et
al., 2004) and were probably involved in the degradation of meat
residues (MR) used for the co-digestion studies. Prevotella species
(1% of total reads) are associated with proteolytic degradation of
plant residues (Debroas and Blanchart, 1993), while Bacteroides
species (1% of total reads) attack the 1,4a-glycosidic bonds of plant
polysaccharides (Charleston, 2008). The majority of proteolytic
microorganisms are also able to metabolize carbohydrates to
produce VFAs.
The phylum Actinobacteria is mainly represented by Bifidobacte-
rium species (6% of total reads), which are major components of the
intestinal flora and have also been isolated from anaerobic digest-
ers treating bean-curd wastes (Ling et al., 1996). These species are
mainly saccharolytic and cannot perform proteolytic activity.
Species level information for the major genera is shown in Table
ð7Þ
4. In summary, the structure of the bacterial community sequenced
is typical of a bacterial community degrading plant- and animal-
derived wastes.
QPCR analysis targeting Archaea and specific groups of methano-
gens established that genera archaea were present at
approximately 1.3  106 ± 9.64  103 gene copies/mL of the
digested sludge. The hydrogenotrophic methanogenic genus Met-
hanobacteriales accounted for greater than 93% of the archaeal pres-
ence in the digester (1.09  106 ± 8.92  103 gene copies/mL).
Seventy-seven percent of the sequences generated with a clone
library targeting the Archaea 16S rRNA gene were most similar to
Methanobacterium curvum, and 11.5% were most similar to Methan-
obacterium congolense. Hydrogentrophic methanogens dominated
the methanogenic community despite the fact that the digester
was inoculated with cow manure, which usually contains aceto-
clastic methanogens. This is in accordance with previous work
characterizing FVW anaerobic digestion, which established that
acetate-utilizing methanogens, Methanosaeta, were dominant in
seed sludges of both types but decreased drastically during
processing in the digestion tank. Consequently, Methanosarcina
and Methanobrevibacter/Methanobacterium were the main contrib-
utors to methane production in this system (Ike et al., 2010).
5. Conclusions
This study demonstrated the feasibility of methane production
from FWV using an anaerobic digestion system (ADS). The highest
biogas production (0.42 m3/kg VS) and VS removal (80%) were
obtained in batch systems supplemented with buffering salts and
nitrogen (IpHN). Co-digestion of the FVW with MR was also evalu-
ated; biogas production and methane yield were enhanced almost
twofold. Co-digestion in a 30 L ADS allowed for natural pH control
and stable performance. The highest methane percentage achieved
in the ADS was 63% (v/v). The major phylum in bacterial commu-
nity, Firmicutes (89.5%), was responsible for acidogenesis or
syntrophic acid degradation. Methanobacteriales constituted the
main methanogenic population (93%).
Acknowledgements
The authors are grateful for the experimental work conducted
by Sagrario Veyna and Fernando Cisneros. This work was sup-
ported through funding provided by the CONACYT Grant 60976
 E.I. Garcia-Peña et al. / Bioresource Technology 102 (2011) 9447–9455
and Instituto Politécnico Nacional, Grant SIP 20101854, and by
start-up funds provided by the Arizona State University Fulton
Schools of Engineering.
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