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, Mexicob 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:Received 31 March 2011Received in revised form 18 July 2011Accepted 20 July 2011Available online 27 July 2011

Keywords:Methane productionCo-digestion of FVWMicrobial ecology

0960-8524/$ - see front matter � 2011 Elsevier Ltd. Adoi:10.1016/j.biortech.2011.07.068

⇑ Corresponding authors. Tel.: +52 5557 2960296000x56305 (E.I. Garcia-Peña), tel.: +1 480 727 7(R. Krajmalnik-Brown).

E-mail addresses: egarciap@ipn.mx (E.I. Gar(R. Krajmalnik-Brown).

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 significant effects on biogasproduction, methane yield, and volatile solids (VS) removal from the FVW (0.42 m3

biogas=kg VS, 50%, and80%, 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, itsmethane yield was 0.25 (m3/kg TS), and the removal of the organic matter measured as the totalchemical demand (tCOD) was 65%. The microbial population (general Bacteria and Archaea) in the30 L system was also determined and characterized and was closely correlated with its potential func-tion in the AD system.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

In recent years, concern has increased about waste disposalfrom mega cities, such as Mexico City, which has a population ofmore than 20 million people and produces a tremendous amountof solid waste, more than 12,000 tons per day. Large volumes oforganic 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 volumereduction is becoming increasingly important (Forster-Carneiroet al., 2008). Interest is also increasing in the production and useof alternative energy sources due to the limited supply of fossilfuels and their negative effects on the environment (Rittmannet al., 2008). The organic fraction of municipal solid wastes thatis mechanically sorted in central plants (OFMSW) or the organicsthat are separated at the source, referred to as biowaste (the veg-etable–fruit–garden or VFG fraction) could be a good candidatefor 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.,

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00x56386; fax: +52 5557574; fax: +1 480 727 0889

cia-Peña), Dr.Rosy@asu.edu

2003, 2005) and constitute a nuisance in municipal landfills be-cause of their high biodegradability (Misi and Forster, 2002). Thecentral market for food distribution in Mexico City, Central deAbasto (CEDA), is the second largest market in the world, receiving24,000 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 conditionare 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 promisingalternative to incinerating or composting this waste material isto 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 isthe production of biogas, which can be used to produce electricity.The stabilized biosolids can be used as a soil conditioner (Bouallaguiet al., 2005). This technology has been successfully applied inreducing the volume of waste that enters landfills, therebydecreasing methane emissions produced by decay (Mata-Alvarezet al., 2000; Forster-Carneiro et al., 2008; Bouallagui et al., 2009).

Some authors have studied the feasibility of using FVW as asubstrate for anaerobic digestion. The easily biodegradable andhighly moist organic matter content of FVW (75%) facilitatesthe biological treatment of these wastes and demonstrates thefeasibility of using this material for anaerobic digestion (Mata-Alvarez et al. 1992; Bouallagui et al., 2003, 2005, 2009). The

Table 1Initial characteristics of the fruit and vegetable waste (FVW).

Solid waste Organic matter(g/kgwaste)

Total solids(g/kgwaste)

Volatile solids(g/kgwaste)

pH

Tomato 59.1 55.7 54.9 4.5Lettuce 53.5 31.3 30.4 5.6Papaya 85.5 116.5 114.4 5.5Pineapple 72.7 102 99.2 3.5Banana 107.6 181.2 176.4 5.0Orange 115.5 153.2 149.4 3.8Mixture of the

solid waste72.7 98.9 96.4 4.02

Table 2Conditions established in eight systems (in duplicate) to evaluate the effects ofinoculation, 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 theanaerobic digestion process.

System Conditions

I FVW inoculated with cow manure (10%)IN FVW inoculated and supplemented with NH4Cl as a nitrogen

sourceIpH FVW inoculated and salts added (buffer) to control pHIpHN FVW inoculated, buffering salts, and NH4Cl addedwI (VSW)FVW without inoculation (WI) (Control)wIN FVW and NH4ClwIpH FVW and buffering saltswIpHN FVW buffering salts and NH4

I = inoculated systems, wI = systems without inoculum.

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FWV material is usually collected from food markets and has avolatile solid (VS) content of between 8% and 18% (Bouallaguiet al., 2005). The organic fraction includes approximately 75%sugars and hemicellulose, 9% cellulose and 5% lignin (Verrieret al., 1987; Bouallagui et al., 2005). For most digestion processes,depending on the substrate used, hydrolysis is the rate limitingstep (Vavilin et al., 1997; Mata-Alvarez et al., 2000). Hydrolysisconstants were obtained from carbohydrates, protein and lipids,with the highest constant observed for carbohydrates, and theserates were determined to be pH-dependent (Mata-Alvarez et al.,2000). The anaerobic processing of cellulose-poor waste such asFVW is limited by methanogenesis rather than by hydrolysis(Bouallagui et al., 2005). The rate and extent of degradation areintrinsic properties of the waste characteristics and the microor-ganisms 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. Gun-aseelan (2004) reported the methane yields (B0) of several frac-tions of FVW, sorghum and napiergrass. The methane potentialdepends on the organic components in the FVW used asfeedstock, which are mainly carbohydrates, proteins and lipids.The theoretical methane yields (B0) from acetic acid, carbohy-drates, proteins and lipids are 370, 415, 496 and 1014 L CH4/kg VS, respectively (Moller et al., 2004). B0 could also be esti-mated considering that 1 kg of COD reduction is equivalent to0.35 m3 CH4 (STP) (Gunaseelan, 2007).

The high biodegradability of the FVW promotes the rapidproduction of volatile fatty acids (VFAs) resulting in a rapid de-crease in pH, which in turn could inhibit the methanogenic activity(Mata-Alvarez et al., 1992; Bouallagui et al., 2003, 2009). An inter-esting option to avoid the acidification of the system when FVW isused is the addition of co-substrates with high nitrogen contents,which could result in a natural pH regulation and also constitutea source of nitrogen. This strategy, known as co-digestion, resultsin a more efficient digestion process, improving the methane yieldsobtained from certain organic materials due to the positive syner-gistic effects of the mixed materials with complementary charac-teristics and the supply of missing nutrients by the co-substrate(Agdag and Sponza, 2005). Co-digestion also presents economicadvantages, such as minimizing equipment needs by sharing thesame equipment for different residues and easier handling ofmixed waste (Mata-Alvarez et al., 2000). Habiba et al. (2009)studied co-digestion as a novel solution to adjust unbalancednutrient constituents and reported that the anaerobic digestionof activated sludge (AS) with substrates containing high levels ofC/N, such as FVW, overcame the difficulties of digesting AS. Theaddition 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 wasobtained without the addition of chemical alkali.

The aim of this study was to evaluate the potential use of FVWas a substrate for methane production and to examine variousconditions that allow for anaerobic systems the optimal perfor-mance using FVW. A mixture of FVW from the biggest market inMexico was characterized to assess its potential as a feedstockfor an anaerobic digestion process. Additionally, the effects of:(1) pH, (2) nitrogen addition, and (3) inoculation of the FVW wereevaluated to enhance methane production in batch systems.Co-digestion of the FVW with meat residues (MR) was also evalu-ated. The performance of a 30 L reactor was assessed under themost effective conditions obtained in the batch systems to deter-mine the feasibility of converting the FVW and MR into biogas.The microbial ecology of the 30 L system when operating at steadystate conditions was evaluated and its links to process perfor-mance 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 charac-teristics 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 weresealed with butyl rubber septums and aluminum crimps andflushed with N2 to provide anaerobic conditions. Some treatmentswere inoculated with 5 mL (10% v/v) of cow manure (density of theFWV was of 1.14 g/L). The FVW without inoculation or salt additionwas used as a control, and the effects of inoculum (cow manure)addition, salts (to control the pH), and the addition of a nitrogensource were evaluated; the tested conditions are summarized inTable 2. For pH controlled systems, a 100 mM phosphate bufferwith an initial pH of 7.0 was used. In the nitrogen supplementedsystems, 0.08 g of NH4Cl was used per g of waste, and the experi-ments were carried out with 50 g of FVW as mentioned above. Allexperiments were performed in duplicate. The systems were incu-bated at 30 �C for 30 days or until biogas production ceased. Eachsystem was manually mixed once per day. Additionally, twocontrol systems that only contained inoculum were incubated atthe same temperature to correct for the amount of biogas producedby the organics in the inoculum. Statistical analysis was carried outwith 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 digestionsystem (ADS) consisting of a stainless steel tubular reactor with atotal volume of 30 L into which 20 L of a (50:50) mixture of FVW

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and meat residues (MR) was initially packed. After the initialperiod of operation (20 days), the ADS was inoculated with cowmanure (10% v/v) to enrich the methanogenic population. Thereactor was stirred by re-circulating the FVW twice a day. ThepH 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 intermediatesproduced during digestion. The bioreactor was kept at room tem-perature and operated in a fed batch mode. To avoid inhibitiondue to metabolic products and to ensure a sufficient supply oforganic matter, 2.5 kg of different compositions of fresh feedstockmixtures 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 totalsolids (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 anaerobicdigester was periodically measured using a water displacementset 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 displacementset up, and the gas composition was analyzed using a gas chro-matograph (GowMac Series 550, Bethlehem, PA) equipped with athermal conductivity detector. A CTR1 packed column (AlltechCo., Beerfield, IL) was used for the analysis. The analysis conditionswere the same as those reported previously (Garcia-Peña et al.,2009). The measured biogas volume was adjusted at standardtemperature (25 �C) and pressure (1 atm). VFA samples were ana-lyzed in a gas chromatograph (Buck Scientific, East Norwalk, CT) aspreviously reported (Garcia-Peña et al., 2009). The acetic and buty-ric acid concentrations in liquid solution were calculated using theHenry’s Law dimensionless constant (H0 = Concentration in gasphase/Concentration in liquid phase) for each compound at thecorresponding temperature.

2.4. Molecular microbial ecology

2.4.1. DNA extractionA 0.5 g sample of the anaerobic digested sludge was taken when

the system was operating at steady state conditions and was usedfor DNA extraction following the recommendations of the MOBIOPowersoil DNA isolation kit. Extracted DNA was quantified with aNanodrop Spectrophotometer and stored at �20 �C.

2.4.2. Quantitative PCR and 454 pyrosequencingThe extracted DNA was diluted tenfold for analytical conve-

nience for quantitative real time PCR (QPCR). The sample wastested for total Archaea and the methanogenic genera Methano-microbiales, Methanobacteriales, Methanosaetaceae, Methanosarcina-ceae, and Methanococcales according to the conditions described inParameswaran et al. (2009). All assays were carried out using anEppendorf Realplex 4S unit (Eppendorf, Germany).

Using extracted DNA as the template, bacterial tag-encoded FLXamplicon pyrosequencing (bTEFAP) was performed based on thetitanium protocol (Roche, Indianapolis, IN) at the Research and Test-ing Laboratory (Lubbock, TX) and previously published in Zhanget al. (2011). Using the FLX-Titanium System Genome Sequencer,the combined V2 and V3 regions of 16S rDNA were sequenced bythe previously described procedure (Wolcott et al., 2009). Aftersequencing, all failed sequence reads, low quality sequence ends,and tags were removed. Sequences were depleted of any non-bacterialribosome sequences and chimeric sequences by using modified

ChimeraSlayer in the MOTHUR software (Gontcharova et al., 2010)at the Research and Testing Laboratory. We also excluded sequencesshorter than 250 bp and longer than 450 bp. After qualifying 954sample 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 andOperating Taxonomic Unit (OTU)-based clustering). We classifiedsequences 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 obtaininformation 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 andSEQMATCH 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 wasteare 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 characterizedindividually and as a mixture (equal proportions of each residuew/w), the composition of which was chosen based on the productsmost frequently and consistently sold in the market, i.e., excludingseasonal products. The characteristics of the FVW mixture were asfollows: a total solid (TS) content of approximately 73–100 g/kgwaste (approximately 10%), a pH of 4, and a moisture content of90%. Table 1 presents the initial characterization of the FVW. TheFVW mixture used in this study had a higher soluble carbohydratecontent than protein content because it only contained fruits andvegetables and did not include a source of protein. Additionally,this FVW had a high moisture content and can be considered ahighly degradable substrate, both of which make it an ideal candi-date for CH4 production. The main drawback of the anaerobicdigestion process with FVW could be the low pH, which negativelyaffects the methanogenesis phase. Two experimental setups weresimultaneously assessed: (1) only the fruit and vegetable waste(wI, FVW) as a control, and (2) a system with the FVW amendedwith salts (phosphate buffer) for pH control and inoculated withthe cow manure (IpH). The results of the two systems during35 days of operation are presented in Fig. 1A and B. Fig. 1A showsthe VS removal profile, the biogas production and the pH evolutionfor the control system (FVW), in which 100 VS/kgwaste were rapidlyconsumed by the natural anaerobic microbial community over5 days and biogas production started at the time of consumption.Then, the degradation of organic matter (OM) stopped after 5 daysdue to the low pH (approximately 4) that inhibits the methanogen-ic activity. No methane was produced under these conditions. Inthis system, 85% VS removal was obtained in 19 days with a biogasyield of 13.3 L/kgwaste. Fig. 1B shows the evolution of the inoculatedsystem with pH control (IpH), demonstrating that the OM was con-tinuously degraded and transformed into biogas, attaining themaximum gas production (11.8 L/kgwaste) in approximately15 days. In this time, the IpH system reached 82% conversion ofthe initial OM to methane, and the pH was maintained around6.5, which is sufficient for methanogenesis to proceed. The totalalkalinity 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 timea VS removal of 82% was obtained in the IpH system. The inocula-tion and pH control favored the process, enhancing the VS

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reduction and allowing the methane production to occur over ashorter period of time (15 days), the biogas production was similarfor both systems (considering the bar errors, Fig. 1). These resultscould be explained by the pH values presented in Fig. 1B, showingthat the pH in the IpH system was maintained around 6.5. Thebiogas yields of the wI (FVW, control) and IpH systems were 0.23and 0.414 m3/kg VS, respectively; only CO2 was determinedbecause no methane was produced in the wI (FVW) control system.The data suggest that in the wI (FVW) system, only the hydrolysisphase of the process occurred, and the methanogenic activity wascompletely inhibited by the low pH. The biogas yields obtainedhere are in the range of those reported by other authors for similarsolid wastes (Bouallagui et al., 2005).

Other reports have demonstrated that pH control is one of themost important parameters to achieve high biogas production(Mata-Alvarez et al., 1992). Data obtained in this study (Fig. 1Aand B) corroborated the fact that the pH needs to be regulatedthroughout the process to allow for good performance of theanaerobic digestion of the FVW. Bouallagui et al. (2005) reportedthat the limitation of the anaerobic digestion of FVW is due torapid acidification by the production of large amounts of volatilefatty acids (VFA), which inhibits the biological activity of the meth-anogens. Some authors buffered the FVW by adding sodiumhydroxide (Verrier et al., 1987), and other experiments included

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Fig. 1. VS reduction (d), biogas production (�) and pH evolution (4) during 35 daysin a batch system. (A) wI (FVW) control; (B) IpH (FVW with inoculum and bufferingsalts added).

the use of buffer solutions to regulate the pH (Bouallagui et al.,2005). Another characteristic of the FVW is the absence of a nitro-gen source; experiments were also performed to evaluate the ef-fect of adding a nitrogen source, considering that adequate C/Nrelation is necessary to enhance the anaerobic digestion process.A variety of conditions were tested to promote biogas and methaneproduction using the FVW, including buffered and nitrogen supple-mented systems in experimental systems with and without inocu-lation (Table 2).

Cumulative biogas production by the systems, under the testedconditions, is depicted in Fig. 2. Dark lines are used to fit the inoc-ulated systems and dashed lines represent the systems withoutinoculum. 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) andthe system at the same conditions but supplemented with nitrogen(IpHN) attained biogas productions of 7.5 and 6 L/kgwaste, respec-tively. The lowest biogas production was obtained in the systemwIN without inoculation and supplemented with nitrogen, whichcould be because an excess of nitrogen can lead to toxic ammo-nium concentrations as demonstrated in previous reports (Frickeet al., 2007; Bouallagui et al., 2009). Table 3 summarizes the resultsobtained for the TS removal, the biogas productivity (m3/kg VS),the methane percentage (v/v) and the final pH determined underthe various conditions evaluated here. In general, the inoculation,pH control and nitrogen source addition had significant influenceson the VS reduction, with the I, IpH and IpHN systems showinghigher degradation percentages between 70% and 86% of the initialtVS in 28 days. Lower organic VS reductions in the range of 35–42%were obtained for the systems without inoculation. The highestbiogas yield (0.420 m3/kg VS) was obtained in the inoculatedsystem with pH control and N addition (IpHN), reaching a VSremoval percentage of 86% as shown in Table 3. For the IN (inocu-lated and nitrogen added) and IpH (inoculated + buffer) systems,lower biogas productions of 0.270 and 0.230 m3/kg VS, respec-tively, 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 theoptimum pH of 7. Lower biogas productions of 0.150 and0.080 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

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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).

Table 3VS removal, biogas production and final pH for the various conditions evaluated in batch experiments.

System Initial VS content (g/kgwaste)

Final VS content (g/kgwaste)

Removal percentage(28 days)

Biogas productivity (m3/kg VS)

FinalpH

Final methane percentage(%, v/v)

I 136.7 64.4 53a 0.07a 4.5 0IN 123.9 17.0 86b 0.27b 6.5 45IpH 104.9 31.3 70c 0.23c 6.9 45IpHN 107.5 14.7 86b 0.42d 6.9 53wI (FVW) 127.5 78.5 38d 0.07a 3.7 0wIN 123.9 80.5 35e 0.05a 3.7 0wIpH 104.9 60.5 42f 0.15e 6.9 0wIpHN 107.5 70.6 34e 0.08a 6.9 0Co-digestion

(FVW:MR)134.8 14 90g 0.9f 7.1 55

a Different superscripts correspond to significantly different values (a = 0.05). The Tukey–Kramer test (NCSS statistical system) showed that the inoculated systems weresignificantly 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 nitrogenaddition in the inoculated systems was also demonstrated.

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45–53 ± 0.5% v/v in the biogas mixture) was only obtained in theinoculated 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 biogasproduction has been obtained using a continuously stirred tankreactor (CSTR) and a continuous tubular reactor, where productionyields of 0.47 and 0.45 m3/kgVS, respectively, were determined,with VS removal percentages of 88% and 76% (Mata-Alvarezet al., 1992; Bouallagui et al., 2003). In the inoculated systems, bothnitrogen addition and pH control had positive influences on thebiogas production (with a significant term a = 0.05, as shown inTable 3), allowing biogas productivities in the range of thosereported in the literature.

The highest biogas yield was obtained in the system inoculatedand 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 thisissue, a co-digestion experiment was also performed with amixture of FVW and meat residues (from meat packaging opera-tions at the same market). Meat residues (MR) provide a highnitrogen concentration, and the protein hydrolysis could result innatural 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 anorganic matter degradation of 93% (Table 3).

3.2. Anaerobic digester experiments

3.2.1. Start upBased 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 MRwas evaluated in a 30 L AD system. To determine the effect of theMR addition on biogas and methane production, experiments werecarried out using different MR proportions. The anaerobic digestionprocess was performed in a semi-continuous regime. The biogasproductivity, methane percentages and VS removal obtained underdifferent conditions after 130 days of operation are presented inFig. 3. Biogas production started 24 h after the reactor was packedwith the FVW and MR, and anaerobic conditions and pH regulationwere established. Initially, biogas production (a maximum of1.03 m3/kg VS) resulted from the hydrolysis of the easily degrad-able components of the feedstock and the activity of the naturalmicrobial populations of the FVW. The highest TS removal (89%)

and biogas production were obtained during the startup of theADS, during which time methane was not produced, suggestingthat the biogas production (mainly CO2) resulted from the initialhydrolysis of the highly biodegradable fraction of the feedstock(Fig. 3).

After 20 days of operation and when the biogas productivitystarted to decrease, the anaerobic digestion system (ADS) wasinoculated with 13% (v/v) of cow manure and fed with a mixtureof FVW (3 L), and a new strong biological activity was observed(Fig. 3) reaching an average biogas production of approximately0.64 ± 0.1 m3/kg VS. In this period, the CH4 content in the biogaswas 16% (v/v) (methane productivity of 0.1 m3/kg VS) due to theinitial activity of the methanogenic population introduced intothe ADS with the inoculums. The strong microbial activity allowedfor a VS removal of approximately 80%. The biogas production waslower 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, thebiogas productivity decreased to a value of approximately 0.5 m3/kg VS, and the methane percentage increased to 28% (v/v). The VSremoval in this period was of 70%. The co-digestion of 50:50FVW:MR yielded the highest methane percentage of 30%, whichoccurred during the initial stages of the reactor operation, andwhich corresponds to a methane production of 0.12 m3/kg VSand a VS removal of 73%. To evaluate the response of the systemto the composition of the feedstock, the proportion of FVW andMR was again reduced and only FVW was added to the ADS. Asexpected, the CH4 content in the resulting biogas was low, at only14% (v/v). For the next stage, on the 70th day of operation a 50:50mixture of FVW:MR was added, and the CH4 percentage recoveredto 30%. Once stable operation was achieved after 83 days, thebiogas production showed a constant value of approximately0.25 m3/kg VS and a methane percentage of 53%, correspondingto a methane production of 0.135 m3/kg VS and a VS removal of78%. The reactor was regularly fed with a mixture of FVW:MR(75:25), and under these conditions the CH4 percentage was stableat 53 ± 2%, and the pH was stable at 6.9 ± 0.5% (naturally regulatedduring this last stage of the process). An appropriate bufferingcapacity and a highly stable experimental system were observewith Organic Loading Rates (OLRs) in the range of 2.4 and2.7 g COD/L day (Hydraulic retention times HRT in the range of15–20 days). The pH was only regulated during the start-up ofthe reactor, when a total volatile fatty acids (VFAs) concentrationof 4000 mg/L (acetate and butyrate 2:1) in the liquid phase wasobserved. After the second stage, the VFA concentration was lower,approximately 2000 mg/L. The average concentration of the totalVFAs was 1300 mg/L (mainly acetic and butyric acids) at thesteady-state of the ADS (data not shown). In a well balanced

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ion

(m3 /k

gVS)

Methane (%)

0 16 28 30 14 30

Start-up VFW:MR 100:0

FV

W:M

R

75:2

5

FV

W:M

R

50:5

0

FV

W:M

R

100:

0F

VW

:MR

50

:50

FVW:MR 75:25

53

VSR= 89% VSR= 81% VSR= 70% 73% 80% 75% VSR= 78%

Fig. 3. Biogas production, methane percentage and VS removal determined during the start-up of the ADS at different VFW and MR proportions.

9452 E.I. Garcia-Peña et al. / Bioresource Technology 102 (2011) 9447–9455

anaerobic digested process, total VFA levels range from 55 to1800 mg/L (Dinsdale et al., 2000; Misi and Forster, 2002; Bouallaguiet al., 2009).

One of the biggest problems during the anaerobic process is tomaintain the pH above 6.6, because the desired pH for anaerobictreatment is between 6.6 and 7.6 (Rittmann and McCarty, 2001).On the other hand, at normal percentages of CO2 in the digestergas, 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 partialpressure makes alkalinity requirements larger (Rittmann andMcCarty, 2001). At the start up of the ADS only FVW were fedand high amounts of VFAs were produced, which causes a rapidpH drop and the inhibition of methane production. Additionally,under these conditions the CO2 partial pressure was high becauseonly this compound was produced, both conditions makes thealkalinity requirements high. For this reason an alkaline materialwas added (0.8 M NaOH solution) to provide the adequate bufferand to prevent the excessive pH drop under this unbalanced condi-tions. During the second stage, when only FWV 16% of methanewas 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, consideringthat the moles of bicarbonate alkalinity is equal to the moles ofNH4 according to the stoichiometric equation of the methanogen-esis for an organic mixture (carbohydrates and proteins) shownbelow. This equation was obtained by modifying the equationpresented by Rittmann and McCarty, 2001 for a mixture of volatilefatty acids and proteins (fs of 0.18).

C11H17O5N2:33 þ 6:74H2O! 4:10CH4 þ 3:12CO2

þ 0:36C5H7O2Nþ 1:97NHþ4þ 1:97HCO�3 ð1Þ

For a 75:25 ratio of FVW and MR the required alkalinity was of3500 mg/L, the alkalinity and the pH drop has to be still regulatedand controlled by the NaOH solution addition. At a ratio of 50:50,the methane percentage increased, the CO2 percentage decreaseand the required alkalinity was of 3445 mg/L. An alkalinity asCaCO3 of 4804.6 mg/L was calculated under the experimental

conditions (70% of removal efficiency and an initial substrate con-centration of 50 g COD) by using the stoichiometric equation for anorganic mixture of carbohydrates and protein (50:50). This totalalkalinity value was high enough to avoid a possible acidificationof 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 ofoperation could result from an increase in the methanogenicpopulation and its adaptation to the operating conditions in theADS. The high VS removal, the increase in the methane yield, andthe natural pH control during the stable period of the ADS wasdue to an adequate ratio of nutrients and the availability of pro-teins for new cell synthesis.

Methane production yields during the initial period of operationof the ADS were lower than those reported in other studies, whichranged from 0.16 to 0.762 m3/kg VS (Mata-Alvarez et al., 1992;Bouallagui et al., 2009). The low methane yields obtained duringthe start up of the ADS in the present work could be partiallyexplained as a result of the temperature, as the 30 L reactor wasoperated at room temperature. The batch experiment data showedthat at a controlled temperature of 30 �C, better biogas and meth-ane yields (0.9 and 0.45 m3/kg VS, respectively) could be obtainedwith the co-digestion of FVW and MR (50:50). Temperature is wellknown to have a strong effect on methane production, and recentlyBohn 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 theyield obtained at 20 �C (0.25 m3/kg VS). The operation of anaerobicdigestion system in the field in developing countries requiressystems with a simple design and process control, robustness toharsh conditions and lower investment costs. Low cost systemsoperated at temperatures below mesophilic conditions have beenshown to be successful in on-farm manure treatment, althoughthe degradation efficiency is lower and higher retention timesare 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 during15 days of operation. After the addition of the feedstock (24 hours),an initial CO2 production of 71% (v/v) in the biogas effluent wasdetermined, with this amount of CO2 corresponding to the initialhydrolysis of the FVW, and a low CH4 percentage was measured

0

10

20

30

40

50

60

70

80

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

Bio

gas

com

posi

tion

(%, v

/v)

Time (days)

MY =0.258 m3/KgCOD

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

Firmicutes89.5%

Bacteroidetes2.3%

Others1.3%

a) Phylum level distribution

Lactobacillus72%

Bifidobacterium6%

Clostridium2.5%

Lachnospiraceae2%

Prevotella1%

Anaerofilum1%

Akkermansia1%

Bacteroides1%

b) Genus level distribution

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

E.I. Garcia-Peña et al. / Bioresource Technology 102 (2011) 9447–9455 9453

during the first days of the process. H2 production was alsoobserved, with the highest H2 production (11%, v/v) observed48 h after feeding the ADS, and then the microbial activity wasshifted to CH4 production after 11 days (63% v/v). The removal rateduring this operation period (10 days) was approximately 65% ofthe total substrate measured as total Chemical Oxygen Demand(tCOD), which was approximately 50 g COD/L. A second additionof substrate was carried out on day 11. Before the new addition,biogas was removed from the system to reduce the pressure inthe reactor. During the second feedstock addition, a methane per-centage of 59% (v/v) was determined on day 15, and in the samefashion, H2 production (3%) was determined on the second day ofthe process. After a long period of operation, the gradual acclima-tion of the biomass and the enrichment of the methanogenicpopulation could explain the increase in methane production inthe ADS compared with the production observed during thestart-up of the ADS. Under the current conditions, biogas andmethane yields of 0.41 m3/kg COD and 0.258 m3/kg COD weredetermined in the ADS, respectively. These data are similar to themethane yield obtained by Dinsdale et al. (2000) for the co-digestionof FVW and WAS in two stage tubular digesters and are lowercompared with the values reported (0.35–0.4 m3/kg VS) by Callaghanet al. (2002) and Bouallagui et al. (2009) for the co-digestion ofFVW and cattle slurry and chicken manure as well as FVW andwaste activated sludge (WAS). Work is underway to determinethe performance of the system under mesophilic conditions toachieve the highest biogas production and methane yieldsobtained 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 Rittmannand McCarty (2001), which is similar to the formula reported forthe vegetable and fruit organic waste. The biomass formula usedfor the balance was the same formula reported for methanogenicsystems using organic matter as electron donor: C5.1H8.5O2.5N(Rittmann and McCarty, 2001).

Thus, the cell synthesis half reaction is:

0:198CO2 þ 0:048HCO�3 þ 0:048NHþ4 þHþ þ e�

! 0:048C5:1H8:5O2:5Nþ 0:415H2O ð2Þ

the reaction for the electron donor is:

0:175CO2 þ 0:025NHþ4 þ 0:025HCO�3 þHþ þ e�

! 0:048C5:1H8:5O2:5Nþ 0:415H2O ð3Þ

and the reaction for the electron acceptor is:

0:125CO2 þHþ þ e� ! 0:125CH4 þ 0:125H2O ð4Þ

A typical value for f 0s ¼ 1 (the fraction of electrons from the

donor substrate that are used toward the synthesis of biomass)and for f 0

s ¼ 0:9. The overall reaction is given by:

R ¼ feRa þ fsRc � Rd ð5Þ

Then, the balanced reaction is:

0:025C8H17O8Nþ 0:196H2O! 0:0048C51H8:5O2:5N

þ 0:1125CH4 þ 0:0427CO2

þ 0:02NHþ4 þ 0:02HCO�3 ð6Þ

Table 4Species 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

Strain match onlya All includedb Type + strain match onlya All includedb

Lactobacillus 89.5 L. plantarum L. plantarum L. plantarum L. plantarum8.2 L. manihativorans L. manihativorans L. manihativorans L. manihativorans0.7 L. vaccinostercus L. vaccinostercus L. vaccinostercus L. vaccinostercus0.5 L. plantarum str. J108 L. plantarum L. pentosus L. plantarum

Bifidobacterium 62 B. minimum B. minimum B. minimum B. minimum18 B. sp. LUCL-W4 B. sp. LUCL-W4 B. subtile B. subtile15 B. longum B. longum B. longum B. longum

Faecalibacterium 88 F. sp. DJF_VR20 F. prausnitzii ATCC 27768 F. prausnitzii F. sp. N567Clostridium 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. 6–1320 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.

9454 E.I. Garcia-Peña et al. / Bioresource Technology 102 (2011) 9447–9455

Using 1 mol of organic matter to calculate the theoretical meth-ane and CO2 production expected during the process and thecorresponding molar coefficients,

1 mol OM! 4:5 mol of CH4; 1 mol OM! 1:708 mol of CO2 ð7Þ

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 ofCO2, the volume will be 54.02 L CO2. Then, the expected volumepercentages 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 methaneproduction when all the initial organic matter has been completelyassimilated; the highest experimental methane volume percentageobserved in the ADS was 63% (v/v), corresponding to 87% of thetheoretical 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 otherphyla at minor predominance. Firmicutes are well-known to befermenters and syntrophic bacteria that can degrade volatile fattyacids, such as butyrate and its analogs. The predominance of Firmi-cutes is a clear indication that these products are readily availabledue to the prior fermentation of these simple volatile fatty acids, orelse that the waste has undergone biodegradation before anaerobicdigestion. Within the phylum Firmicutes, Bacilli (76.1%) and Clos-tridia (13.3%) form the major classes. Lactobacillus species (in theclass Bacilli; 72% of the total reads, as shown in Fig. 5b) constitutecommon 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 asmicroorganisms capable of utilizing amino acids and producingacetate, butyrate, and ammonia (Shin et al., 2010). The syntrophicrole of Firmicutes involves H2 removal and has immediate implica-tions for the composition of the methanogenic community.

The phylum Bacteroidetes are proteolytic bacteria (Kindaichi etal., 2004) and were probably involved in the degradation of meatresidues (MR) used for the co-digestion studies. Prevotella species(1% of total reads) are associated with proteolytic degradation ofplant residues (Debroas and Blanchart, 1993), while Bacteroidesspecies (1% of total reads) attack the 1,4a-glycosidic bonds of plantpolysaccharides (Charleston, 2008). The majority of proteolyticmicroorganisms are also able to metabolize carbohydrates toproduce 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 aremainly saccharolytic and cannot perform proteolytic activity.Species level information for the major genera is shown in Table4. In summary, the structure of the bacterial community sequencedis 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 atapproximately 1.3 � 106 ± 9.64 � 103 gene copies/mL of thedigested 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 clonelibrary targeting the Archaea 16S rRNA gene were most similar toMethanobacterium curvum, and 11.5% were most similar to Methan-obacterium congolense. Hydrogentrophic methanogens dominatedthe methanogenic community despite the fact that the digesterwas inoculated with cow manure, which usually contains aceto-clastic methanogens. This is in accordance with previous workcharacterizing FVW anaerobic digestion, which established thatacetate-utilizing methanogens, Methanosaeta, were dominant inseed sludges of both types but decreased drastically duringprocessing in the digestion tank. Consequently, Methanosarcinaand 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 productionfrom FWV using an anaerobic digestion system (ADS). The highestbiogas production (0.42 m3/kg VS) and VS removal (80%) wereobtained in batch systems supplemented with buffering salts andnitrogen (IpHN). Co-digestion of the FVW with MR was also evalu-ated; biogas production and methane yield were enhanced almosttwofold. Co-digestion in a 30 L ADS allowed for natural pH controland stable performance. The highest methane percentage achievedin the ADS was 63% (v/v). The major phylum in bacterial commu-nity, Firmicutes (89.5%), was responsible for acidogenesis orsyntrophic acid degradation. Methanobacteriales constituted themain methanogenic population (93%).

Acknowledgements

The authors are grateful for the experimental work conductedby 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 9455

and Instituto Politécnico Nacional, Grant SIP 20101854, and bystart-up funds provided by the Arizona State University FultonSchools of Engineering.

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