Bioresource Technology 102 (2011) 94479455

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Anaerobic digestion and co-digestion processes of vegetable and fruit residues: Process and microbial ecology
E.I. Garcia-Pea a,, P. Parameswaran b, D.W. Kang b, M. Canul-Chan a, R. Krajmalnik-Brown b,
a b

Bioprocesses Department, Unidad Profesional Interdisciplinaria de Biotecnologa, IPN P.O. Box 07340, Mexico City, Mexico 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
This study evaluated the feasibility of methane production from fruit and vegetable waste (FVW) obtained from the central food distribution market in Mexico City using an anaerobic digestion (AD) process. Batch systems showed that pH control and nitrogen addition had signicant effects on biogas production, methane yield, and volatile solids (VS) removal from the FVW (0.42 m3 biogas =kg VS, 50%, and 80%, respectively). Co-digestion of the FVW with meat residues (MR) enhanced the process performance and was also evaluated in a 30 L AD system. When the system reached stable operation, its methane yield was 0.25 (m3/kg TS), and the removal of the organic matter measured as the total chemical demand (tCOD) was 65%. The microbial population (general Bacteria and Archaea) in the 30 L system was also determined and characterized and was closely correlated with its potential function in the AD system. 2011 Elsevier Ltd. All rights reserved.

Article history: Received 31 March 2011 Received in revised form 18 July 2011 Accepted 20 July 2011 Available online 27 July 2011 Keywords: Methane production Co-digestion of FVW Microbial ecology

1. Introduction 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 landll, the only landll in the area, which is approaching capacity. Because no other locations exist for solid waste disposal, the application of efcient 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 vegetablefruitgarden or VFG fraction) could be a good candidate for bioenergy production. Fruit and vegetable waste (FVW) is produced in large quantities in markets in many large cities (MataAlvarez et al., 1992; Misi and Forster, 2002; Bouallagui et al.,
Corresponding authors. Tel.: +52 5557 296000x56386; fax: +52 5557 296000x56305 (E.I. Garcia-Pea), tel.: +1 480 727 7574; fax: +1 480 727 0889 (R. Krajmalnik-Brown). E-mail addresses: egarciap@ipn.mx (E.I. Garcia-Pea), Dr.Rosy@asu.edu (R. Krajmalnik-Brown).
0960-8524/$ - see front matter 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2011.07.068

2003, 2005) and constitute a nuisance in municipal landlls because 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 solid 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 organic waste, and more than 50% of that is from the fruit and vegetable 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 production. 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 landlls, 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 (MataAlvarez et al. 1992; Bouallagui et al., 2003, 2005, 2009). The

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FWV material is usually collected from food markets and has a volatile solid (VS) content of between 8% and 18% (Bouallagui et al., 2005). The organic fraction includes approximately 75% sugars and hemicellulose, 9% cellulose and 5% lignin (Verrier et al., 1987; Bouallagui et al., 2005). For most digestion processes, depending on the substrate used, hydrolysis is the rate limiting step (Vavilin et al., 1997; Mata-Alvarez et al., 2000). Hydrolysis constants were obtained from carbohydrates, protein and lipids, with the highest constant observed for carbohydrates, and these rates were determined to be pH-dependent (Mata-Alvarez et al., 2000). The anaerobic processing of cellulose-poor waste such as FVW is limited by methanogenesis rather than by hydrolysis (Bouallagui et al., 2005). The rate and extent of degradation are intrinsic properties of the waste characteristics and the microorganisms involved in the process. According to Mata-Alvarez et al. (1992), the FVW contains cellulose (32%), hemicelluloses (15%) and lignins (15%), and under mesophilic conditions, up to 32%, 86% and 0% of these compounds are removed, respectively. Gunaseelan (2004) reported the methane yields (B0) of several fractions of FVW, sorghum and napiergrass. The methane potential depends on the organic components in the FVW used as feedstock, which are mainly carbohydrates, proteins and lipids. The theoretical methane yields (B0) from acetic acid, carbohydrates, proteins and lipids are 370, 415, 496 and 1014 L CH4/ kg VS, respectively (Moller et al., 2004). B0 could also be estimated considering that 1 kg of COD reduction is equivalent to 0.35 m3 CH4 (STP) (Gunaseelan, 2007). The high biodegradability of the FVW promotes the rapid production of volatile fatty acids (VFAs) resulting in a rapid decrease in pH, which in turn could inhibit the methanogenic activity (Mata-Alvarez et al., 1992; Bouallagui et al., 2003, 2009). An interesting option to avoid the acidication of the system when FVW is used is the addition of co-substrates with high nitrogen contents, which could result in a natural pH regulation and also constitute a source of nitrogen. This strategy, known as co-digestion, results in a more efcient digestion process, improving the methane yields obtained from certain organic materials due to the positive synergistic effects of the mixed materials with complementary characteristics and the supply of missing nutrients by the co-substrate (Agdag and Sponza, 2005). Co-digestion also presents economic advantages, such as minimizing equipment needs by sharing the same equipment for different residues and easier handling of mixed waste (Mata-Alvarez et al., 2000). Habiba et al. (2009) studied co-digestion as a novel solution to adjust unbalanced nutrient constituents and reported that the anaerobic digestion of activated sludge (AS) with substrates containing high levels of C/N, such as FVW, overcame the difculties of digesting AS. The addition of high nitrogen content co-substrates to adjust the nutrient 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. The aim of this study was to evaluate the potential use of FVW as a substrate for methane production and to examine various conditions that allow for anaerobic systems the optimal performance using FVW. A mixture of FVW from the biggest market in Mexico was characterized to assess its potential as a feedstock for an anaerobic digestion process. Additionally, the effects of: (1) pH, (2) nitrogen addition, and (3) inoculation of the FVW were evaluated to enhance methane production in batch systems. Co-digestion of the FVW with meat residues (MR) was also evaluated. The performance of a 30 L reactor was assessed under the most effective conditions obtained in the batch systems to determine the feasibility of converting the FVW and MR into biogas. The microbial ecology of the 30 L system when operating at steady state conditions was evaluated and its links to process performance were assessed using molecular methods.

2. Methods 2.1. Set up for the batch experiments The biodegradability of the fruit and vegetable waste (FVW) was determined using batch anaerobic digestion tests. The characteristics of the FVW mixture are depicted in Table 1. FVW (50 g) with an initial total solid (TS) content of 98.9 g TS/kgresidues (10% organic matter) was placed into 125 mL serum bottles that were sealed with butyl rubber septums and aluminum crimps and ushed with N2 to provide anaerobic conditions. Some treatments were inoculated with 5 mL (10% v/v) of cow manure (density of the FWV was of 1.14 g/L). The FVW without inoculation or salt addition was used as a control, and the effects of inoculum (cow manure) addition, salts (to control the pH), and the addition of a nitrogen source were evaluated; the tested conditions are summarized in Table 2. For pH controlled systems, a 100 mM phosphate buffer with an initial pH of 7.0 was used. In the nitrogen supplemented systems, 0.08 g of NH4Cl was used per g of waste, and the experiments were carried out with 50 g of FVW as mentioned above. All experiments were performed in duplicate. The systems were incubated at 30 C for 30 days or until biogas production ceased. Each system was manually mixed once per day. Additionally, two control systems that only contained inoculum were incubated at the same temperature to correct for the amount of biogas produced by the organics in the inoculum. Statistical analysis was carried out with the NCSS statistical system (NCSS, PASS, and GESS, NCSS, Kaysville, UT, http://www.ncss.com). 2.2. Experimental setup (anaerobic digester) The process was also evaluated in an anaerobic digestion system (ADS) consisting of a stainless steel tubular reactor with a total volume of 30 L into which 20 L of a (50:50) mixture of FVW

Table 1 Initial characteristics of the fruit and vegetable waste (FVW). Solid waste Tomato Lettuce Papaya Pineapple Banana Orange Mixture of the solid waste Organic matter (g/kgwaste) 59.1 53.5 85.5 72.7 107.6 115.5 72.7 Total solids (g/kgwaste) 55.7 31.3 116.5 102 181.2 153.2 98.9 Volatile solids (g/kgwaste) 54.9 30.4 114.4 99.2 176.4 149.4 96.4 pH 4.5 5.6 5.5 3.5 5.0 3.8 4.02

Table 2 Conditions established in eight systems (in duplicate) to evaluate the effects of inoculation, pH control and addition of a nitrogen source (0.08 g ammonium chloride/ gwaste, with experiments conducted with 50 g of FVW) on the performance of the anaerobic digestion process. System I IN IpH IpHN wI FVW wIN wIpH wIpHN Conditions FVW inoculated with cow manure (10%) FVW inoculated and supplemented with NH4Cl as a nitrogen source FVW inoculated and salts added (buffer) to control pH FVW inoculated, buffering salts, and NH4Cl added (VSW) without inoculation (WI) (Control) FVW and NH4Cl FVW and buffering salts FVW buffering salts and NH4

I = inoculated systems, wI = systems without inoculum.

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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 temperature and operated in a fed batch mode. To avoid inhibition due to metabolic products and to ensure a sufcient supply of organic matter, 2.5 kg of different compositions of fresh feedstock mixtures were fed periodically (approximately every 1215 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 standard 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 periodically from the gas collection lines prior to the water displacement set up, and the gas composition was analyzed using a gas chromatograph (GowMac Series 550, Bethlehem, PA) equipped with a thermal conductivity detector. A CTR1 packed column (Alltech Co., Beereld, IL) was used for the analysis. The analysis conditions were the same as those reported previously (Garcia-Pea et al., 2009). The measured biogas volume was adjusted at standard temperature (25 C) and pressure (1 atm). VFA samples were analyzed in a gas chromatograph (Buck Scientic, East Norwalk, CT) as previously reported (Garcia-Pea et al., 2009). The acetic and butyric acid concentrations in liquid solution were calculated using the Henrys 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 quantied 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 convenience for quantitative real time PCR (QPCR). The sample was tested for total Archaea and the methanogenic genera Methanomicrobiales, Methanobacteriales, Methanosaetaceae, Methanosarcinaceae, 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 Testing 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 modied

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 community in the anaerobic digester. We aligned and clustered the readouts by using the MOTHUR software (the Silva alignment and Operating Taxonomic Unit (OTU)-based clustering). We classied sequences by the Ribosomal Database Project (RDP) classier software at an 80% condence threshold (Cole et al., 2009). The sequences were 390 bp long in average, which is sufcient to obtain information at the species level. Species level identication was obtained after clustering the aligned sequences using RDP pyro pipeline application and analyzing the sequences with BLAST and SEQMATCH applications provided by National Center for Biotechnology 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 because they have an effect on biogas production and process stability 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 73100 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 candidate 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 prole, 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 methanogenic 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 continuously 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 sufcient 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 inoculation and pH control favored the process, enhancing the VS

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reduction and allowing the methane production to occur over a shorter period of time (15 days), the biogas production was similar for both systems (considering the bar errors, Fig. 1). These results could be explained by the pH values presented in Fig. 1B, showing that the pH in the IpH system was maintained around 6.5. The biogas yields of the wI (FVW, control) and IpH systems were 0.23 and 0.414 m3/kg VS, respectively; only CO2 was determined because no methane was produced in the wI (FVW) control system. The data suggest that in the wI (FVW) system, only the hydrolysis phase of the process occurred, and the methanogenic activity was completely inhibited by the low pH. The biogas yields obtained here are in the range of those reported by other authors for similar solid wastes (Bouallagui et al., 2005). Other reports have demonstrated that pH control is one of the most important parameters to achieve high biogas production (Mata-Alvarez et al., 1992). Data obtained in this study (Fig. 1A and B) corroborated the fact that the pH needs to be regulated throughout the process to allow for good performance of the anaerobic digestion of the FVW. Bouallagui et al. (2005) reported that the limitation of the anaerobic digestion of FVW is due to rapid acidication by the production of large amounts of volatile fatty acids (VFA), which inhibits the biological activity of the methanogens. Some authors buffered the FVW by adding sodium hydroxide (Verrier et al., 1987), and other experiments included

100

No methane was detected

14

12 80
waste

10 60

40

20 2

0 0 5 10 15 20 25 30 35

the use of buffer solutions to regulate the pH (Bouallagui et al., 2005). Another characteristic of the FVW is the absence of a nitrogen source; experiments were also performed to evaluate the effect of adding a nitrogen source, considering that adequate C/N relation is necessary to enhance the anaerobic digestion process. A variety of conditions were tested to promote biogas and methane production using the FVW, including buffered and nitrogen supplemented systems in experimental systems with and without inoculation (Table 2). Cumulative biogas production by the systems, under the tested conditions, is depicted in Fig. 2. Dark lines are used to t the inoculated systems and dashed lines represent the systems without inoculum. The highest biogas production (approximately 10 L/ kgwaste) was reached in the FVW and buffering salts (wIpH) system, and was approximately twice that obtained in the control system (wI, FVW). The systems inoculated with a pH control (IpH) and the system at the same conditions but supplemented with nitrogen (IpHN) attained biogas productions of 7.5 and 6 L/kgwaste, respectively. The lowest biogas production was obtained in the system wIN without inoculation and supplemented with nitrogen, which could be because an excess of nitrogen can lead to toxic ammonium concentrations as demonstrated in previous reports (Fricke et al., 2007; Bouallagui et al., 2009). Table 3 summarizes the results obtained for the TS removal, the biogas productivity (m3/kg VS), the methane percentage (v/v) and the nal pH determined under the various conditions evaluated here. In general, the inoculation, pH control and nitrogen source addition had signicant inuences on the VS reduction, with the I, IpH and IpHN systems showing higher degradation percentages between 70% and 86% of the initial tVS in 28 days. Lower organic VS reductions in the range of 3542% were obtained for the systems without inoculation. The highest biogas yield (0.420 m3/kg VS) was obtained in the inoculated system with pH control and N addition (IpHN), reaching a VS removal percentage of 86% as shown in Table 3. For the IN (inoculated and nitrogen added) and IpH (inoculated + buffer) systems, lower biogas productions of 0.270 and 0.230 m3/kg VS, respectively, were observed, and lower VS reductions were obtained. These results suggest a correlation of pH with biogas productivity, with the higher productivity occurring at pH values close to the optimum pH of 7. Lower biogas productions of 0.150 and 0.080 m3/kg VS were measured in the systems without inoculum (wIpH and wIpHN) with corresponding low VS removals of 42% and 34%, respectively. Methane production (approximately

Biogas production (L/Kg

Volatile Solid (g/Kg

waste

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Time (days)

100 Methane 30 (%)

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12 80

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10 60

) Biogas production (L/kg

40

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20 2

10

15

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35

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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 salts added).

10

15

20

25

30

35

40

Time (days)
Fig. 2. Cumulative biogas production under different batch conditions: I (), IN (N), IpH (d), IpHN (.), wI (FVW) control (e), wIN (4), wIpH (s), and wIpHN (5).

E.I. Garcia-Pea et al. / Bioresource Technology 102 (2011) 94479455 Table 3 VS removal, biogas production and nal pH for the various conditions evaluated in batch experiments. System I IN IpH IpHN wI (FVW) wIN wIpH wIpHN Co-digestion (FVW:MR) Initial VS content (g/ kgwaste) 136.7 123.9 104.9 107.5 127.5 123.9 104.9 107.5 134.8 Final VS content (g/ kgwaste) 64.4 17.0 31.3 14.7 78.5 80.5 60.5 70.6 14 Removal percentage (28 days) 53a 86b 70c 86b 38d 35e 42f 34e 90g Biogas productivity (m3/ kg VS) 0.07a 0.27b 0.23c 0.42d 0.07a 0.05a 0.15e 0.08a 0.9f Final pH 4.5 6.5 6.9 6.9 3.7 3.7 6.9 6.9 7.1

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Final methane percentage (%, v/v) 0 45 45 53 0 0 0 0 55

a Different superscripts correspond to signicantly different values (a = 0.05). The TukeyKramer test (NCSS statistical system) showed that the inoculated systems were signicantly 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.

4553 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 previous 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 inuences on the biogas production (with a signicant 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 treatment 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 operations 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 system 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 degradable components of the feedstock and the activity of the natural microbial populations of the FVW. The highest TS removal (89%)

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, however, 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 1520 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

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Methane (%)

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VFW:MR 100:0

FVW:MR 100:0 FVW:MR 50:50

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FVW:MR 75:25

0 0 20 40 60 80 100 120

Time (days)
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 conditions. 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 conditions 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 hydrolysis 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 methanogenesis 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 6:74H2 O ! 4:10CH4 3:12CO2 0:36C 5 H7 O2 N 1:97NH 4 1:97HCO 3 1

conditions (70% of removal efciency and an initial substrate concentration 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 acidication of the ADS and the high buffering capacity allows and stable operation 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 proteins 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 methane 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 increased 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 eld 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 efciency is lower and higher retention times are required (Bohn et al., 2007). 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 efuent was determined, with this amount of CO2 corresponding to the initial hydrolysis of the FVW, and a low CH4 percentage was measured

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

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80 MY =0.258 m /KgCOD 70
3

9453

Bacteroidetes 2.3%

Others 1.3%

60

Biogas composition (%, v/v)

50

40

30

Firmicutes 89.5%

20

10

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Time (days)
Fig. 4. Biogas composition of the current ADS operation (v/v): H2% . MY = methane yield. CH4% CO2%

a) Phylum level distribution

Anaerofilum Akkermansia 1% 1% Prevotella 1% Lachnospiraceae 2% Clostridium 2.5% Bacteroides 1%

during the rst days of the process. H2 production was also observed, with the highest H2 production (11%, v/v) observed 48 h after feeding the ADS, and then the microbial activity was shifted to CH4 production after 11 days (63% v/v). The removal rate 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 of substrate was carried out on day 11. Before the new addition, biogas was removed from the system to reduce the pressure in the reactor. During the second feedstock addition, a methane percentage 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 acclimation 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 methane yields of 0.41 m3/kg COD and 0.258 m3/kg COD were determined in the ADS, respectively. These data are similar to the methane yield obtained by Dinsdale et al. (2000) for the co-digestion of FVW and WAS in two stage tubular digesters and are lower compared with the values reported (0.350.4 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 waste activated sludge (WAS). Work is underway to determine the performance of the system under mesophilic conditions to achieve the highest biogas production and methane yields obtained in batch experiments. 3.2.3. Stoichiometric analysis of the anaerobic digestion system (ADS) The stoichiometric balance was formulated based on the empiric formula for waste water C8H17O3N reported by Rittmann and McCarty (2001), which is similar to the formula reported for the vegetable and fruit organic waste. The biomass formula used for the balance was the same formula reported for methanogenic systems using organic matter as electron donor: C5.1H8.5O2.5N (Rittmann and McCarty, 2001). Thus, the cell synthesis half reaction is:

Bifidobacterium 6% Lactobacillus 72%

b) Genus level distribution

Fig. 5. (a) Phylum and (b) genus level distribution of the bacterial population in the full-scale anaerobic digester operated at steady state. Firmicutes (89.5%) constituted the major bacterial phylum. Lactobacillus (72% of total reads) was the predominant bacterial genus in the anaerobic digester.

the reaction for the electron donor is:

0:175CO2 0:025NH 4 0:025HCO3 H e

! 0:048C5:1 H8:5 O2:5 N 0:415H2 O

and the reaction for the electron acceptor is:

0:125CO2 H e ! 0:125CH4 0:125H2 O

fs0

A typical value for 1 (the fraction of electrons from the donor substrate that are used toward the synthesis of biomass) and for fs0 0:9. The overall reaction is given by:

R fe Ra fs Rc Rd
Then, the balanced reaction is:

0:025C8 H17 O8 N 0:196H2 O ! 0:0048C51 H8:5 O2:5 N 0:1125CH4 0:0427CO2

0:02NH 4 0:02HCO3

0:198CO2

0:048HCO 3

0:048NH 4

H e

! 0:048C5:1 H8:5 O2:5 N 0:415H2 O

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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 Strain match only Lactobacillus 89.5 8.2 0.7 0.5 62 18 15 88 26 20 20 62
a

SEQMATCH analysis results All included

b

Type + strain match onlya L. plantarum L. manihativorans L. vaccinostercus L. pentosus B. minimum B. subtile B. longum F. prausnitzii C. orbiscindens strain 17 A. cellulolyticus E. eligens ATCC 27750 B. intestinalis

All includedb L. plantarum L. manihativorans L. vaccinostercus L. plantarum B. minimum B. subtile B. longum F. sp. N567 C. orbiscindens Acetovibrio sp. 613 Eubacterium sp. AD 17 Firmicutes oral clone CK030

Bidobacterium

Faecalibacterium Clostridium

Lachnospiraceae
a b

L. plantarum L. manihativorans L. vaccinostercus L. plantarum str. J108 B. minimum B. sp. LUCL-W4 B. longum F. sp. DJF_VR20 C. orbiscindens strain 17 Acetivibrio sp. 613 Eubacterium sp. AD 17 Butyrivibrio brisolvens

L. plantarum L. manihativorans L. vaccinostercus L. plantarum B. minimum B. sp. LUCL-W4 B. longum F. prausnitzii ATCC 27768 C. orbiscindens Acetovibrio sp. 613 E. eligens Butyrivibrio hungatei strain JK614

Represents both database search results for type strains only. Includes both type strains and environmental samples.

Using 1 mol of organic matter to calculate the theoretical methane and CO2 production expected during the process and the corresponding molar coefcients,

1 mol OM ! 4:5 mol of CH4 ; 1 mol OM ! 1:708 mol of CO2

According to the ideal gas equation, PV 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 efuent 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 Firmicutes 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 Clostridia (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 implications 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 Bidobacterium species (6% of total reads), which are major components of the

intestinal ora and have also been isolated from anaerobic digesters 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 4. In summary, the structure of the bacterial community sequenced is typical of a bacterial community degrading plant- and animalderived wastes. QPCR analysis targeting Archaea and specic groups of methanogens 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 Methanobacteriales accounted for greater than 93% of the archaeal presence 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 Methanobacterium congolense. Hydrogentrophic methanogens dominated the methanogenic community despite the fact that the digester was inoculated with cow manure, which usually contains acetoclastic 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 contributors 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 evaluated; 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 community, 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 supported through funding provided by the CONACYT Grant 60976

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and Instituto Politcnico Nacional, Grant SIP 20101854, and by start-up funds provided by the Arizona State University Fulton Schools of Engineering. Reference
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