Bioresource Technology 110 (2012) 18–25

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Bioresource Technology

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Biochemical methane potential (BMP) of food waste and primary sludge:Influence of inoculum pre-incubation and inoculum source

Elsayed Elbeshbishy a, George Nakhla a,⇑, Hisham Hafez b

a Dept. of Civil and Environmental Engineering, University of Western Ontario, London, Ontario, Canada N6A 5B9b GreenField Ethanol Inc., Chatham, Ontario, Canada N7M 5J4

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

Article history:Received 5 December 2011Received in revised form 5 January 2012Accepted 7 January 2012Available online 16 January 2012

Keywords:Anaerobic digestionBiochemical methane potentialWaste-to-inoculum ratioFood wastePre-incubated inoculum

0960-8524/$ - see front matter � 2012 Elsevier Ltd. Adoi:10.1016/j.biortech.2012.01.025

Abbreviations: BMP, biochemical methane potendemand; CV, coefficient of variation; DOPF, DufferinHRT, hydraulic retention time; JWPCP, Joint Water Pomaximum methane production rate; MMPRs, maximuMPR, methane production rate; S/X, waste to inocchemical oxygen demand; SRT, solid retention tiorganics; TCOD, total chemical oxygen demand;suspended solids; TVS, total volatile solids; VFAs, vosolids; VSS, volatile suspended solids.⇑ Corresponding author. Tel.: +1 519 661 2111x854

E-mail addresses: gnakhla@eng.uwo.ca, gnakhla@f

Biochemical methane potential tests were conducted to evaluate the effect of using a blank versus a pre-incubated inoculum in digestion of primary sludge at different waste to inoculum ratios (S/X). In addition,this study explored the influence of using two different anaerobic inoculum sources on the digestion offood waste: digested sludge from a municipal wastewater treatment plant and from a digester treatingthe organic fraction of municipal solid wastes. The results revealed that although there was no significantdifference in methane yield (on average 114 mL CH4/g TCODsub) or biodegradability (on average 28.3%) ofprimary sludge using pre-incubated or non-incubated inocula, the maximum methane production ratesusing non-incubated inoculum were higher than those using pre-incubated inoculum at all S/X ratios.Moreover, interestingly the inoculum from an anaerobic digester treating municipal wastewater sludgewas superior over the inoculum from anaerobic digester treating food waste in digesting food waste.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

The recycling and treatment of the organic fraction of municipalsolid waste is rapidly emerging as an effective waste managementstrategy that diverts wastes from landfills and recovers energy(Fernandez et al., 2001). In recent years, interest in biochemicalmethane potential (BMP) tests has increased as reflected by thewide range of research papers dealing with the BMP assays(Raposo et al., 2011). The BMP assay is best suited when used toelucidate what types of substrates, from an array of potentialsubstrates, have the highest biomethane potential (Labatut et al.,2011). In addition, BMP assays can be used to estimate the opti-mum ratios between co-substrates when co-digestion is intended.Lastly, BMP assay results can be used to determine the extent ofanaerobic biodegradability of substrates, and thus, relative resi-dence times required for complete digestion (Labatut et al., 2011).

ll rights reserved.

tial; COD, chemical oxygenOrganics Processing Facility;llution Control Plant; MMPR,m methane production rates;ulum ratios; SCOD, solubleme; SSO, source separatedTS, total solids; TSS, total

latile fatty acids; VS, volatile

70; fax: +1 519 850 2991.es.engga.uwo.ca (G. Nakhla).

Although there is no standard detailed procedure for the BMPtest, the various BMP studies followed very similar procedures;the only two main differences between BMP tests relate to consid-eration of the methane production from the inoculum and the inoc-ulum source. For methane production from the inoculum, manyresearchers used the blank assay (Neves et al., 2004; NallathambiGunaseelan, 1995) approach described below, while someresearchers prescribe the German guideline for fermentation tests(VDI-Handbuch, 2006), which entails pre-incubating the inoculumfor about 5 days without substrate before using it, thus eliminatingthe need for the continued testing of both the seed blank and thesample waste. For the blank assay method, the background meth-ane production from the inoculum (determined in blank assayswith medium or water and no substrate) is subtracted from themethane production obtained in the substrate assays (Angelidakiet al., 2009). For the pre-incubated inoculum (German guideline),the inoculum should be ‘‘degassed’’ in order to deplete the residualbiodegradable organic material present. Degassing should beprotracted until no significant methane production is observed:typically 2–5 days of incubation (Raposo et al., 2011) are prescribedin the method. In some cases, e.g. when the inoculum is taken froma reactor fed with relatively high fat/oil concentration, longerperiods of pre-incubation may be required, in order to eliminateall the residual (adsorbed/entrapped) substrate (Angelidaki et al.,2009).

A wide range of biomass has been considered as potential seedmaterials for methane production in BMP tests (Neves et al., 2004;

Table 1aSubstrates characteristics.

Parameter Units Food waste Primary sludge

TCOD mg/L 113000 ± 2800a 42800 ± 180SCOD mg/L 60300 ± 350 8000 ± 470TSS mg/L 48400 ± 2700 26300 ± 260VSS mg/L 27900 ± 1300 20000 ± 250TVFA mg COD/L 260 ± 20 1680 ± 220NH4 mg/L 1670 ± 40 80 ± 20pH – 4.6 ± 0.2 5.0 ± 0.1Alkalinity mg CaCO3/L N.A. 2900 ± 180

a Values represents the average ± STD of three samples.

E. Elbeshbishy et al. / Bioresource Technology 110 (2012) 18–25 19

Hashimoto, 1989; Chynoweth et al., 1993; Owen et al., 1979). It hasbeen observed that the results of the biodegradation tests couldvary with the methodology followed. One of the most importantvariables, which influence BMP results, is the origin of the inocu-lum (Nyholm et al., 1984), since it determines the initial activityof the microorganisms used for the test. Furthermore, the inocu-lum source brings about differences in bacterial populations(Thouand et al., 1995), substrate adaptation (Barkay and Pitchard,1988; Thouand and Block, 1993), and residual anaerobically-biode-gradable substrate.

The concentration of microorganisms that are used in the testswill determine the biodegradation rates (Simkins and Alexander,1984), the lag time (Chudoba et al., 1992), and the probability thatthe degradation of the substrate occurs during the test perfor-mance (Thouand et al., 1995).

There are different anaerobic biodegradation test methodolo-gies, which seldom state inoculum characteristics. For example,some methodologies (Shelton and Tiedje, 1984; Birch et al.,1989; Pagga and Beimborn, 1993; ISO 11734, 1995) recommendanaerobic municipal wastewater treatment plant inoculum.

The waste to inoculum ratio (S/X) is an important parameter inbatch high solids anaerobic digestion processes as well as in theassessment of anaerobic biodegradability of solid wastes (Neveset al., 2004). Although theoretically, the S/X ratio has an effect onlyon the kinetics, and not on the ultimate methane yield, which onlydepends on the organic matter content (Nallathambi Gunaseelan,1995; Raposo et al., 2006), it is reported that too high S/X may betoxic while too low S/X may prevent induction of the enzyme neces-sary for biodegradation (Prashanth et al., 2006). This ratio also hasan effect on the lag phase, which is shorter for low ratios (Chenand Hashimoto, 1996).

Each substrate has its optimum S/X ratio, considering the poten-tial amount of volatile fatty acids (VFAs) produced and its capacityto buffer the medium due to the ammonium produced by thehydrolysis of proteins (Lesteur et al., 2010). A small amount ofinoculum is preferred because of the endogenous biogas produc-tion, which can bias the results (Lesteur et al., 2010). Moreover,the increase in the S/X can lead to overloads due to volatile fattyacid accumulation (Neves et al., 2004). From another point of view,the inoculum concentration should always be high compared tothat of the substrate (in term of volatile solids) and the S/X shouldbe recognised as one of the major parameters affecting the resultsof anaerobic assays (Neves et al., 2004). Hashimoto (1989) andLabatut et al. (2011) reported that the minimum S/X ratio of 2 gVS substrate/g VS inoculum was required when digesting wheatstraw at concentrations of 10–40 g VS/L and dairy manure at con-centrations of P3 g VS/L, respectively. However, in the case ofmore recalcitrant wastes (woody feed stocks and municipalwastes), the rate of methane production in BMP assays was opti-mum at S/X of 0.5 g VS substrate/g VS inoculum (Chynowethet al., 1993). The S/X proposed by Owen et al. (1979) as a standardwas approximately 1 g VS substrate/g VS inoculum.

Based on the above mentioned introduction, it is obvious thatthere is a wide range of the optimum or recommended S/X depend-ing on the substrate and inoculum (some reported optimum S/X of0.5 g VS substrate/g VS inoculum while other reported optimum S/Xof 5.7 g VSsubstrate/g VSinoculum). Moreover the BMP test despite itswide use lacks standardization (some researchers recommendedusing blank assays while others recommended pre-incubating theinoculum prior use). Therefore, the primary purpose of the currentwork was to evaluate the two approaches i.e. the widely used blankseed assay versus the pre-incubated inoculum in digestion of pri-mary sludge at different S/X. The secondary goals of this study werethe assessment of the impacts of the seed source and S/X ratio onBMP results. In this study, three seeds from conventional meso-philic digesters, two from municipal wastewater treatment plant

digesters and one from a digester treating the organic fraction ofmunicipal solid wastes, were employed.

2. Methods

2.1. Substrates and inocula

Two substrates were used in this study, food waste i.e. organicfraction of municipal solid wastes and primary sludge from a mu-nicipal WWTP. The food waste was obtained from Dufferin Organ-ics Processing Facility (DOPF) in Toronto, Ontario, Canada. The cityof Toronto’s DOPF receives approximately 25,000 metric tons/yearof source separated organics (SSO) material from Toronto’s residualGreen Bin and the commercial Yellow Bag collection programs. Thepurpose of the DOPF is to separate the film plastic bin finer andcontaminant materials fractions of the SSO from the organic mate-rial and convert the organic fraction into a material that is asuitable feedstock for the anaerobic digester (Van Opstal, 2006).The primary sludge was obtained from Joint Water Pollution Con-trol Plant (JWPCP), Carson, California. The JWPCP provides bothprimary and secondary treatment for approximately 300 milliongallons of wastewater per day. The characteristics of both sub-strates are presented in Table 1a.

Three inocula (anaerobic sludge) were used in this study; thefirst inoculum was collected from the primary mesophilic anaero-bic digester at Guelph’s wastewater treatment plant (Guelph,Ontario), the second inoculum was collected from the mesophilicanaerobic digester treating SSO at DOPF in Toronto, Ontario, andthe third inoculum was obtained from a mesophilic anaerobicdigester treating primary and secondary wastewater at JWPCP,Carson, California. The anaerobic digester at Guelph wastewatertreatment plant is completely mixed reactor with solid retentiontimes (SRTs) in the range of 14–18 days, that achieves VSS destruc-tion efficiency of �45%. The anaerobic digester in DOPF at Torontois a completely mixed reactor with a solids recycling system withhydraulic retention time (HRT) of approximately 17 days, SRT ofapproximately 27 days, VSS destruction efficiency of 62%. Theanaerobic digester at JWPCP is a completely mixed reactor withHRTs (SRTs) in the range of 17–20 days, with VSS destruction effi-ciencies of 49–52%. The characteristics of the three inocula are pre-sented in Table 1b. Inocula from both municipal WWTP were about2.0% solids (w/w) while the DOPF inoculum was about 5.0% solids.

The primary reason for the selection of seed from digesterstreating municipal biosolids and food waste is the difference in sol-uble COD concentrations between the two digesters. The foodwaste digester seed had five times higher soluble COD concentra-tion than the Guelph municipal wastewater treatment plant diges-ter. Thus, it is anticipated that the ratio of fermentative bacteria i.e.hydrolyzers, carbohydrates, proteins, and lipids degraders to acid-formers and methanogens differs between the two seeds. The otherreason for selection of food waste anaerobic digestion inoculumand food waste is the increasing number of these facilities world-wide in light of the push for less land filling and resource recovery.

Table 1bInocula characteristics.

Parameter Units Guelph’s inoculum Toronto’s inoculum JWPCP’s inoculum

TCOD mg/L 18100 ± 2600a 70500 ± 1800 19400 ± 110SCOD mg/L 6560 ± 280 31200 ± 700 660 ± 70TSS mg/L 18000 ± 3400 47400 ± 2300 18300 ± 450VSS mg/L 10000 ± 720 37100 ± 1800 11600 ± 280TVFA mg COD/L 230 ± 60 210 ± 30 26 ± 6NH4 mg/L 540 ± 80 820 ± 20 470 ± 60pH – 7.6 ± 0.2 6.9 ± 0.2 7.2 ± 0.1Alkalinity mg CaCO3/L 3200 ± 180 5000 ± 220 5400 ± 260

a Values represents the average ± STD of three samples.

20 E. Elbeshbishy et al. / Bioresource Technology 110 (2012) 18–25

2.2. Anaerobic biodegradability test

The batch tests were conducted using two substrates (foodwaste and primary sludge) and three inocula (Toronto, Guelph,and JWPCP inocula) at different S/X of 0.25, 0.5, 1, 2, and 4 witheach test condition run in triplicates. TCOD of the substrate andVSS of the seed were determined prior to the initiation of the batchtest (12 h prior the test). The volumes of inoculum and substraterequired to maintain an S/X ratio of 0.25, 0.5, 1, 2 and 4 on massCOD/mass VSS were then determined. The pH was adjusted to7 ± 0.2 using NaOH and HCl. The volumes of inoculum and thesubstrate were then added to the batch test bottle (total liquid vol-ume of 200 ml and headspace volume of 60 ml). A sample of themixture was then collected for initial analysis. The headspacewas flushed with nitrogen gas at 5–10 psi for a period of 5 minand capped tightly with rubber stoppers. The bottles were thenplaced in a swirling-action shaker (Max Q4000, Incubated andRefrigerated Shaker, Thermo Scientific, CA) operating at 180 rpmand maintained at a temperature of 37 �C. The volume of the gasproduced and the gas composition were analyzed until the testwas completed (cumulative gas curve reached a plateau).

2.3. Blank and pre-incubated inoculum

Three 200 mL bottles were used as blank (inoculum only) foreach inoculum without any substrate. The methane productionfrom the blank (inoculum only) adjusted by the ratio of the inocu-lum volume in the test bottle to the 200 mL in the inoculum blankwas subtracted from the methane production obtained in the sub-strate assays prior to data analysis. For the pre-incubated inoculum,the pre-incubation was done at the same process temperature(37 �C), where the inoculum originated from. The degassing wascontinued until no significant methane production (methane pro-duction per day became less than 1% of the cumulative methane)was observed. Subsequently the pre-incubated seed sludge wasmixed with the substrate at various S/X ratios and the methane pro-duced attributed only to the substrate (feedstocks).

2.4. Analytical methods

The biogas production was measured by releasing the gas pres-sure in the vials using appropriately sized glass syringes (Perfek-tum; Popper & Sons Inc., NY, USA) in the 5–100 mL range toequilibrate with the ambient pressure (Owen et al., 1979). Biogascomposition including hydrogen, methane, and nitrogen wasdetermined by a gas chromatograph (Model 310, SRI Instruments,Torrance, CA) equipped with a thermal conductivity detector (TCD)and a molecular sieve column (Molesieve 5A, mesh 80/100,6 ft � 1/8 in.). The temperatures of the column and the TCD detec-tor were 90 and 105 �C, respectively. Argon was used as the carriergas at a flow rate of 30 mL/min. The VFAs concentrations wereanalyzed after filtering the sample through 0.45 lm using a gas

chromatograph (Varian 8500, Varian Inc., Toronto, Canada) witha flame ionization detector (FID) equipped with a fused silicacolumn (30 m � 0.32 mm). Helium was used as the carrier gas ata flow rate of 5 mL/min. The temperatures of the column anddetector were 110 and 250 �C, respectively. Samples were analyzedfor total solids (TS), volatile solids (VS), total suspended solids(TSS), volatile suspended solids (VSS), and alkalinity using standardmethods (APHA, 1995). Total and soluble chemical oxygen demand(TCOD, SCOD) were measured using HACH methods and test kits(HACH Odyssey DR/2500). Soluble parameters were determinedafter filtering the samples through 0.45 lm filter paper.

2.5. Data analysis

Methane gas production was calculated from headspace mea-surements of gas composition and the total volume of biogasproduced, at each time interval, using the mass balance equation

VCH4 ;i ¼ VCH4 ;i�1 þ CCH4 ;iðVG;i � VG;i�1Þ þ VCH4 ðCCH4 ;i � CCH4 ;i�1Þ ð1Þ

where VCH4 ;i and VCH4 ;i�1 are cumulative methane gas volumes at thecurrent (i) and previous (i � 1) time intervals, VG,i and VG,i�1 are thetotal biogas volumes in the current and previous time intervals,CCH4 ;i and CCH4 ;i�1 are the fractions of methane gas in the headspaceof the bottle measured using gas chromatography in the current andprevious intervals, and VCH4 is the total volume of headspace in thereactor (Lopez et al., 2007).

3. Results and discussion

3.1. Pre-incubation of inocula from different sources

According to the German guidelines of the fermentation tests,the pre-incubation required about 5 days (Raposo et al., 2006). Inthis section, three inocula from different sources (Toronto, Guelph,and JWPCP) were pre-incubated until the methane productioncurves plateaued (methane production per day was less than 1%of the cumulative methane production). Fig. 1 represents the cumu-lative methane production from Toronto, Guelph, and JWPCP inoc-ula with the specific rates depicted in Fig. 2. The highest ultimatemethane production from the 200 mL batches of 816 mL CH4 wasobserved for Toronto’s inoculum while only 96, and 71 mL were ob-tained for the inocula from Guelph and JWPCP, respectively. Thehigher initial TCOD (70500 mg/L for Toronto’s inoculum versus18100 and 19400 mg/L for Guelph and JWPCP inocula, respectively)is the primary reason for the huge difference in methane productionfrom Toronto’s inoculum compared to Guelph and JWPCP inocula.Normalizing the ultimate methane production per unit initial inoc-ulum VSS yielded 22 mL CH4/g VSS, 9.6 mL CH4/g VSS, and 6.1 mLCH4/g VSS from Toronto’s inoculum compared to Guelph andJWPCP inocula, respectively. While the two inocula from municipalwastewater treatment plants are comparable, the inoculum fromthe food waste digester is substantially different, reflecting both

Fig. 1. Cumulative methane production from the three inoculums.

Fig. 2. Methane production rate of the three inoculums.

a

b

Fig. 3. Cumulative methane production of primary sludge at different S/X ratios; (a)pre-incubated inoculum and (b) non-incubated inoculum.

E. Elbeshbishy et al. / Bioresource Technology 110 (2012) 18–25 21

the high biodegradability of the food wastes relative to municipalbiosolids as well as the high residual biodegradable COD remainingin the digested sludge. As shown in Fig. 1, more than 90% of themethane production was achieved after 12 days regardless of theinocula source, while after the 5 days recommended by Germanguideline only 70%, 56%, and 49% of the methane potential wereobserved for Toronto, Guelph, and JWPCP inocula, respectively. Thisis also substantiated by the specific methane production ratesdepicted in Fig. 2, which show that the rates after 12 days are 6%,10%, and 12% of the maximum rates for Toronto, Guelph and JWPCPinocula, respectively. Therefore based on the findings of this study,it is recommended that a minimum of 10 days are required for thepre-incubation irrespective of the source of the inoculum.

Methane yields of 58, 25, and 18 mL CH4/g TCODinitial wereobtained for inocula from Toronto, Guelph, and JWPCP, respec-tively, while the corresponding methane yields per mass of VSSof 146, 47, and 31 mL CH4/g VSSinitial. The corresponding maximummethane production rates were 34, 6.4, and 3.9 mL CH4/g VSS d.

3.2. Methane production from primary sludge using pre-incubated andnon-incubated inocula

3.2.1. Methane potentialIn this experiment, primary sludge from JWPCP was digested

using inoculum obtained from JWPCP. Two sets of experimentswere conducted, first one using a pre-incubated inoculum (as de-scribed in Section 2) and the other one using the inoculum withoutpre-incubation (non-incubated inoculum). Four S/X ratios of 0.5,1.0, 2.0, and 4.0 g CODsubstrate/g VSSinoculum were used; the volumes

of substrates and inocula were calculated based on the TCOD of thesubstrate and the VSS of the inoculum (for the pre-incubated inoc-ulum, the VSS was measured after the incubation). Fig. 3a and bdepict the cumulative methane production from primary sludgeusing the pre-incubated and non-incubated inocula at the differentS/X ratios in the 200 mL batches. Using both inocula at all S/X ra-tios, a rapid initial methane production (no lag phase) was ob-served. Methane production increased with increasing S/X for thetwo inocula, peaking at 541–551 mL at an S/X of 4 g CODsubstrate/gVSSinoculum. Methane production from primary sludge using thenon-incubated inoculum was generally higher than the pre-incu-bated inoculum at all S/X ratios except at S/X of 4 g CODsubstrate/gVSSinoculum which exhibited no significant difference. The differ-ences in methane production between using the non-incubatedand using the pre-incubated inoculum decreased with increasingS/X ratio i.e. 39%, 23%, and 14% (all as percentage of the lower num-ber) for S/X of 0.5, 1.0 and 2.0 g CODsubstrate/g VSSinoculum,respectively.

Based on the aforementioned results, it is obvious that therewere no significant differences either in the trend (no lag phaseswere observed) or in the ultimate methane production when pre-incubated or non-incubated (with blank correction) inocula at ahigh S/X ratios of 2.0 and 4.0 g CODsubstrate/g VSSinoculum were used.However, the differences between the two methods are pronouncedat the low S/X ratios of 0.5 and 1.0 g CODsubstrate/g VSSinoculum, clearlyemphasizing that pre-incubating the seed and elimination of seedblank control can grossly underestimate biogas production undercertain circumstances. The underlying reason for pre-incubation isto eliminate the methane contributed by the seed to avert skewingBMP results. Pre-incubation is recommended to run until the daily

a

b

Fig. 4. Methane production rate of primary sludge at different S/X ratios; (a) pre-incubated inoculum and (b) non-incubated inoculum.

Table 2aMaximum methane production rate and methane yield of primary sludge.

Inoculum S/X Maximum methaneproduction rate

Methane yield

mL CH4/g VSSinoculum d

mL CH4/g VSSsub

mL CH4/g TCODsub

Pre-incubatedinoculum

0.5 8 241 1131 14 221 1032 31 235 1104 56 273 127

22 E. Elbeshbishy et al. / Bioresource Technology 110 (2012) 18–25

methane production rate is less than 1% of the cumulative methane,and this corresponds to almost complete stabilization of the seedmaterial. Thus, the concentrations of the pathogenic and non-path-ogenic active biomass have been reduced significantly due to pre-incubation. Accordingly, upon addition of particulate and solublesubstrates at the beginning of the BMP test, the biodegradation ratesare very different from the non-incubated seed. Furthermore, dur-ing incubation, microorganisms that degrade soluble substrates(primarily acetogens and methanogens) will proliferate whileothers that hydrolyze and uptake particulate substrates undergodecay, thus affecting the microbial consortium balance. In thenon-incubated samples, due to the addition of both particulateand soluble substrate, all microbial groups function simultaneouslylike in a digester, taking advantage of the synergies between thevarious microbial groups to maintain the microbial consortiumbalance. Although the initial ammonia concentrations of the pre-incubated inoculum (ranged from 260 to 410 mg/L) and the non-incubated inoculum (280–430 mg/L) were comparable, the finalammonia concentrations differed significantly. The final ammoniaconcentrations of the non-incubated inoculum ranging from 1460to 1620 mg/L were about 30–40% higher than those of the non-incu-bated inoculum, and since the main products of the biodegradationof proteins in anaerobic conditions are ammonia and differentamino acid compounds, the higher final ammonia concentrationsfor the non-incubated inoculum batches reflect greater hydrolysisand acidogenic microbial activities. The toxicity of ammonia tomethanogenic bacteria is well established (Soubes et al., 1994).Free ammonia (FA) has been suggested to be the main cause of inhi-bition since it is freely membrane-permeable (Kroeker et al., 1979;De Baere et al., 1984). The concentrations of FA from 100 to140 mg N-FA/L inhibit mesophilic treatment (De Baere et al.,1984). It is possible to calculate FA concentration from the totalammonia concentration in the liquid (TA) and the fraction of FA(fN), using the equation (Omil et al., 1995):

fN ¼ FA=TA ¼ 1=½1þ ðkb � 10�pHÞ=kw� ð4Þ

where kb and kw are the dissociation constants for ammonia andwater, respectively (1:855 � 10�5 and 2:355 � 10�14 mol/l at37 �C). The highest final concentrations of FA computed using Equa-tion (4) observed in the batches (both pre-incubated and non-incu-bated) was less than 45 mg/L, substantially below the inhibitionthreshold level.

Non-incubatedinoculum

0.5 11 283 1321 25 231 1082 57 230 1084 87 235 110

Table 2bMaximum methane production rate and methane yield of food waste.

Inoculum S/X Maximum methaneproduction rate

Methane yield

mL CH4/g VSSinoculum d

mL CH4/g VSSsub

mL CH4/g TCODsub

Toronto’s inoculum 1 19 660 1600.5 26 790 1900.25 9 440 110

Guelph’s inoculum 1 69 1000 1500.5 72 940 2300.25 53 1400 340

3.2.2. Methane production rate (MPR) and methane yieldFig. 4a and b depict the MPR of primary sludge using the pre-

incubated inoculum (Fig. 4a) and using the non-incubated inocu-lum (Fig. 4b). As depicted from the Figures, the maximum MPRs(MMPRs) using the non-incubated inoculum were higher thanthose using the pre-incubated inoculum at all S/X ratios. Usingboth inocula, the MMPR increased with increasing the S/X ratio;the maximum MMPR of 60 and 86 mL CH4/d were achieved atS/X of 4 g CODsubstrate/g VSSinoculum using the pre-incubated andthe non-incubated inocula, respectively. The minimum MMPRwere observed at S/X of 0.5 g CODsubstrate/g VSSinoculum for bothinocula (15 mL CH4/d using the pre-incubated inoculum versus22 mL CH4/d using non-incubated inoculum). The MMPR of pri-mary sludge normalized per initial mass of inoculum (as VSS) ispresented in Table 2a. The normalized MMPRs using the non-incu-bated inoculum were higher than those of the pre-incubated inoc-ulum at all S/X ratios. The highest MMPR of 56 mL CH4/gVSSinoculum d was obtained using the pre-incubated inoculum atan S/X ratio of 4 g CODsubstrate/g VSSinoculum compared to 87 mLCH4/g VSSinoculum d using the non-incubated inoculum at S/X of4.0 g CODsubstrate/g VSSinoculum. As shown in Table 2a, the differencebetween the normalized MMPR (as a percentages of the lower

numbers) using the non-incubated and the pre-incubated inoculaincreased with increasing S/X and peaked at 84% at S/X of 2.0 gCODsubstrate/g VSSinoculum and then decreased to 56% at S/X of4.0 g CODsubstrate/g VSSinoculum.

E. Elbeshbishy et al. / Bioresource Technology 110 (2012) 18–25 23

The methane yield can be normalized either per volume of sub-strate (mL CH4/Lsub), substrate mass VSS (mL CH4/g VSSsub), or sub-strate mass COD (mL CH4/g CODsub), with the last method preferredas it permits direct conversion of the results into percent organicmatter converted to methane using the theoretical 0.350 m3 CH4

at STP per kilogram COD converted (McCarty, 1964). The methaneyields of the primary sludge at different S/X using pre-incubatedand non-incubated inocula are presented in Table 2a. For thepre-incubated inoculum, the maximum methane yield of 127 mLCH4/g TCODsub (or 273 mL CH4/g VSSsub) was achieved at S/X of4.0 g CODsubstrate/g VSSinoculum, while the lowest methane yield of103 mL CH4/g TCODsub (or 221 mL CH4/g VSSsub) was observed atS/X of 1.0 g CODsubstrate/g VSSinoculum. On the other hand, for thenon-incubated inoculum, the max methane yield of 132 mL CH4/gTCODsub (or 283 mL CH4/g VSSsub) was obtained at S/X of 0.5 gCODsubstrate/g VSSinoculum, and there was no difference in the meth-ane yields at S/X ratios of 1, 2, and 4 g CODsubstrate/g VSSinoculum

(about 108 mL CH4/g TCODsub (or 232 mL CH4/g VSSsub)). Moreover,at the low S/X of 0.5 g CODsubstrate/g VSSinoculum, the methane yield ofthe non-incubated inoculum was higher than that of the pre-incu-bated inoculum (283 versus 241 mL CH4/g VSSsub), while at thehigher S/X of 4 g CODsubstrate/g VSSinoculum, the opposite was ob-served as the methane yield of pre-incubated inoculum was higherthan that of the non-incubated inoculum (127 versus 110 mL CH4/gTCODsub). In general, there was no significant difference in themethane yield between pre-incubated or non-incubated inoculumas the average methane yield of 113 mL CH4/g TCODsub wasachieved using pre-incubated inoculum compared to 114 mLCH4/g TCODsub using non-incubated inoculum.

3.2.3. KineticsIn general, the rate limiting step of anaerobic digestion of par-

ticulate wastes is the first step of hydrolysis or solubilization,where the cell wall is broken down allowing the organic matterinside the cell to be available for biological degradation (Wanget al., 1997; Noike et al., 1985). Since the most widely used hydro-lysis model is the first order, anaerobic digestion is generallydescribed as a first order reaction with respect to substrate concen-tration (Eq. (2a)), and methane production (Eq. (2b)):

dS=dt ¼ �kS ð2aÞd½ðBo � BÞ=Bo�=dt ¼ �k½ðBo � BÞ=Bo� ð2bÞ

Table 3aKinetic constants and biodegradability (BDCH4 ) of primary sludge.

Inoculum S/X Kinetics constant (k)a d�1 BDCH4 %

Pre-incubated inoculum 0.5 0.11 281 0.12 262 0.14 284 0.13 32

Non-incubated inoculum 0.5 0.15 331 0.19 272 0.17 274 0.18 28

a R2 for the kinetics constants ranged from 0.87 to 0.98.

Table 3bKinetic constants and biodegradability (BDCH4 ) of food waste.

Inoculum S/X Kinetics constant (k)a d�1 BDCH4 %

Toronto’s inoculum 1 0.18 410.5 0.13 490.25 0.12 27

Guelph’s inoculum 1 0.16 630.5 0.13 590.25 0.17 87

a R2 for the kinetics constants ranged from 0.85 to 0.99.

where k is the first order kinetic constant (d�1), t is the digestiontime (d) and S represents the residual substrate (organics) concen-tration (mg/L) at any time t. As S is a difficult parameter to measure,it is preferable to derive the model by using gas measurement (Eq.(2b)) (Chen and Hashimoto, 1978), in which Bo is the ultimatemethane production at the end of the experiment correspondingto the initial substrate concentration (So), B is the methane produc-tion corresponding to the substrate consumed i.e. S = Bo � B. The va-lue of the first order kinetic constant, k, can be obtained as the slopeof the linear curve of ln[(Bo � B)/Bo] versus t.

Table 3a shows the kinetic constants of the primary sludge atdifferent S/X using pre-incubated and non-incubated inocula.The k values ranged from 0.11 to 0.19 d�1. The k values for thenon-incubated inoculum were higher than the pre-incubated inoc-ulum at all S/X ratios. For the pre-incubated inoculum, the k valuesranged from 0.11 to 0.14 d�1. Based on the k value in Table 3a forthe pre-incubated inoculum at the different S/X, the average k va-lue of 0.124 d�1 with coefficient of variation (CV) of 8% reveals thatthere was no significant influence of the S/X ratio on the k values.For the non-incubated inoculum, the highest k value of 0.19 d�1

was observed at S/X of 1.0 g CODsubstrate/g VSSinoculum, while thelowest k value of 0.15 d�1 was achieved at S/X of 0.5 gCODsubstrate/g VSSinoculum. The average k value (using non-incubatedinoculum) of 0.172 d�1 with C.V. of 9% emphasised that the S/Xratio did not have significant effect on k values. The relativeindependence of the k value of S/X ratios clearly indicates thatthere was no substrate limitation at all four S/X ratios explored.

3.2.4. BiodegradabilityThe experimental methane yield can be used to calculate the

level of anaerobic biodegradability (BDCH4 ) under the defined testconditions in comparison with its theoretical value as follows(Raposo et al., 2011):

BDCH4 ð%Þ ¼ ðBoExp=BoThÞ � 100 ð3Þ

where BoExp is the experimental ultimate methane production (mL)and BoTh represents the theoretical methane potential based on theinitial TCOD of the substrate, neglecting the biomass synthesis,which is typically about 5% of the organic matter consumed (Sy-mons and Buswell, 1933).

The biodegradability of the primary sludge at the different S/Xusing both inocula presented in Table 3a reflects no significantdifferences between pre-incubated or non-incubated inoculum,as an average biodegradability of 28.3% was computed for bothinocula. Furthermore, there was no significant impact of the S/Xratio on the biodegradability when any of the two inocula wasused. It must be asserted however that the biodegradability ofthe primary sludge in the batch BMP tests relative to about 50%destruction efficiency in conventional mesophilic digesters athydraulic retention times of 15–20 days is attributed primarily tothe very low food-to-microorganisms ratio in the digesters.

3.3. Methane production from food waste using two inoculum sources

3.3.1. Methane production, MPR, and methane yieldThe food waste was digested using two inocula (Toronto and

Guelph inocula) at different S/X ratios of 0.25, 0.5, and 1.0 gCODsubstrate/g VSSinoculum. Fig. 5a and b show the cumulative meth-ane production from food waste at different S/X (0.25, 0.5, and 1.0)using Toronto’s inoculum (Fig. 5a) and Guelph’s inoculum (Fig. 5b)after correcting for the blank in the 200 mL batches. In general, forboth inocula at all S/X ratios, a quick initial methane production(no lag phase) was observed due to the biodegradation of solublecompounds. As shown in Fig. 5a and b, for both inocula, methanepotential increased with increasing S/X ratios. The highest methaneproduction of 760 mL was obtained using Toronto’s inoculum at

a

b

Fig. 5. Cumulative methane production of food waste at different S/X ratios; (a)Toronto’s inoculum and (b) Guelph’s inoculum.

a

b

Fig. 6. Methane production rate of food waste at different S/X ratios; (a) Toronto’sinoculum and (b) Guelph’s inoculum.

24 E. Elbeshbishy et al. / Bioresource Technology 110 (2012) 18–25

S/X of 1.0 g CODsubstrate/g VSSinoculum compared to 560 mL whenGuelph’s inoculum was used. Ultimate methane production usingToronto’s inoculum was higher than using Guelph’s inoculum atS/X of 0.5 and 1.0 g CODsubstrate/g VSSinoculum, while at S/X of0.25 g CODsubstrate/g VSSinoculum, the methane production usingGuelph’s inoculum (190 mL) was higher than that of Toronto’sinoculum (150 mL).

The MPR of food waste using Toronto’s inoculum and Guelph’sinoculum is illustrated in Fig. 6a and b. The MMPR was achieved atan S/X ratio of 0.5 g CODsubstrate/g VSSinoculum for both inocula(175 mL CH4/d for Toronto’s inoculum versus 137 mL CH4/d forGuelph’s inoculum). Using Toronto’s inoculum, MMPRs of 112 mLCH4/d and 110 mL CH4/d were achieved at S/X ratios of 1.0 and0.25 g CODsubstrate/g VSSinoculum, respectively, compared to 124 mLCH4/d and 103 mL CH4/d when Guelph’s inoculum was used. Meth-ane production rates normalized per initial mass of inoculum (asVSS) are presented in Table 2b. After normalization, the MMPRusing Guelph’s inoculum were significantly higher than thoseusing Toronto’s inoculum at all S/X ratios. Moreover, the highestMMPR were observed at S/X of 0.5 g CODsubstrate/g VSSinoculum forboth inocula (26 mL CH4/g VSS d using Toronto’s inoculum versus72 mL CH4/g VSS d using Guelph’s inoculum).

Using Toronto’s inoculum, the highest methane yield of 190 mLCH4/g TCODsub (or 790 mL CH4/g VSSsub) was observed at an S/X of0.5 g CODsubstrate/g VSSinoculum followed by160 mL CH4/g TCODsub

(or 660 mL CH4/g VSSsub) at S/X of 1.0 g CODsubstrate/g VSSinoculum,with the lowest methane yield of 110 mL CH4/g TCODsub (or440 mL CH4/g VSSsub) observed at S/X of 0.25 g CODsubstrate/gVSSinoculum. For Guelph’s inoculum, the methane yield increasedwith decreasing the S/X ratio and reached a maximum value of

340 mL CH4/g TCODsub (or 1400 mL CH4/g VSSsub) at an S/X of0.25 g CODsubstrate/g VSSinoculum.

Based on the above mentioned results, it is clear that the MMPRand highest methane yield using Guelph’s inoculum were higherthan those obtained using Toronto’s inoculum, possibly due to theinitial inoculum concentration in the assays (on average 6.5 g VSS/bottle for Toronto’s inoculum versus 1.9 g VSS/bottle for Guelph’sinoculum). On the other hand, the high initial SCOD in Toronto’sinoculum lead to higher MPR and higher methane production in Tor-onto’s blank inoculum compared to Guelph’s inoculum which mighthave negatively affected the Toronto’s MMPR and methane yield.

3.3.2. Kinetics and biodegradabilityThe kinetic constant (k) values for the digestion of food waste at

different S/X ratios using Toronto and Guelph’ inocula are presentedin Table 3b. As shown in Table 3b, there were no significant differ-ences in the k values using either Toronto or Guelph inocula at S/Xratios of 0.5 and 1 g CODsubstrate/g VSSinoculum, while a 42% difference(0.17 d�1 for Guelph versus 0.12 d�1 for Toronto) was observed atS/X ratio of 0.25 g CODsubstrate/g VSSinoculum. The kinetic constantsobtained in this study which ranged from 0.12 to 0.18 d�1 weremuch higher than the values of 0.016–0.125 d�1 reported byGunaseelan (2004), using more than 50 fruits and vegetable wastesas substrates. On contrast, the k values obtained here were lowerthan the values of 0.21–0.34 d�1 reported by Raposo et al. (2011)using four substrates (starch, cellulose, gelatine, and mung bean).

Table 3b represents the biodegradability of the food waste atdifferent S/X ratios using the two inocula (Toronto and Guelph).As depicted in the Table 3b, the biodegradability of the food waste

E. Elbeshbishy et al. / Bioresource Technology 110 (2012) 18–25 25

using Guelph’s inoculum was higher than that of using Toronto’sinoculum at all S/X ratios. The max biodegradability of 87% wasachieved using Guelph’s inoculum at S/X of 0.25 g CODsubstrate/gVSSinoculum, while the max biodegradability with Toronto’s inocu-lum was 49% at S/X of 0.5 g CODsubstrate/g VSSinoculum. The lowestvalue of biodegradability of 27% was observed at S/X of 0.25 gCODsubstrate/g VSSinoculum with Toronto’s inoculum.

4. Conclusions

The findings of this study clearly demonstrate that pre-incuba-tion of the seed sludge does not offer any advantages over runninga seed control in BMP tests. However, the inoculum source and testS/X conditions play a significant role in test results and can lead toflawed data interpretation and conclusions, with sometimes theobvious seed source for a waste treatability (such as Toronto in thisstudy) grossly underestimating ultimate biodegradability. Thus,comprehensive BMP testing should include a minimum of threeS/X conditions and preferably two seed sludges.

Acknowledgements

The Egyptian Ministry of Higher Education supported the doc-toral candidate, Mr. Elbeshbishy. The financial contribution of Tro-jan UV and NSERC is gratefully acknowledged. The cooperation ofToronto, Guelph, and JWPCP facilities in providing the inoculaand the substrates is greatly appreciated.

References

Angelidaki, I., Alves, M., Bolzonella, D., Borzacconi, L., Campos, J.L., Guwy, A.J., 2009.Defining the biomethane potential (BMP) of solid organic wastes and energycrops: a proposed protocol for batch assays. Water Sci. Technol. 59 (5), 927–934.

APHA, AWWA, WEF, 1995. Standard Methods for Examination of Water andWastewater, 19th ed.

Barkay, T., Pitchard, T., 1988. Adaptation of aquatic microbial communities topollutant stress. Microbiol. Sci. 5 (6), 165–169.

Birch, R., Biver, C., Campagna, R., Gledhill, W., Pagga, U., Steber, J., Reust, H., 1989.Bontinck W. Screening of chemicals for anaerobic biodegradability.Chemosphere 19 (10–11), 1527–1550.

Chen, T.H., Hashimoto, A.G., 1996. Effects of pH and substrate:inoculum ratio onbatch methane fermentation. Bioresour. Technol. 56 (2–3), 179–186.

Chen, Y.R., Hashimoto, A.G., 1978. Kinetics of methane fermentation. Biotechnol.Bioeng. Symp. 8, 269–283.

Chudoba, P., Capdeville, B., Chudoba, J., 1992. Explanation of biological meaning ofthe So/Xo ratio in batch culture. Wat. Sci. Tech. 26 (3-4), 743–751.

Chynoweth, D.P., Turick, C.E., Owens, J.M., Jerger, D.E., Peck, M.W., 1993.Biochemical methane potential of biomass and waste feedstocks. BiomassBioenergy 5 (1), 95–111.

De Baere, L.A., Devocht, M., Van Assche, P., Verstraete, W., 1984. Influence of highNaCl and NH4Cl salt levels on methanogenic associations. Water Res. 18 (5),543–548.

Fernandez, B., Porrier, P., Chamy, R., 2001. Effect of inoculum-substrate ratio on thestart up of solid waste anaerobic digesters. Water Sci. Tech. 44 (4), 103–108.

Gunaseelan, V.N., 2004. Biochemical methane potential of fruits and vegetable solidwaste feedstocks. Biomass Bioenergy 26 (4), 389–399.

Hashimoto, A.G., 1989. Effect of inoculum/substrate ratio on methane yield andproduction rate from straw. Biol. Waste 28 (4), 247–255.

ISO 11734, 1995. Water quality – Evaluation of the ‘‘ultimate’’ anaerobicbiodegradability of organic compounds in digester sludge – Method bymeasurement of the biogas production. International Organization ofStandardization, Switzerland.

Kroeker, E.J., Schulte, D.D., Sparling, A.B., Lapp, H.M., 1979. Anaerobic treatmentprocess stability. J. Water Pollut. Control Fed. 51 (4), 718–727.

Labatut, R.A., Angenent, L.T., Scott, N.R., 2011. Biochemical methane potential andbiodegradability of complex organic substrates. Bioresour. Technol. 102 (3),2255–2264.

Lesteur, M., Bellon-Maurel, V., Gonzalez, C., Latrille, E., Roger, J.M., Junqua, G., Steyer,J.P., 2010. Alternative methods for determining anaerobic biodegradability: areview. Process Biochem. 45 (4), 431–440.

Lopez, S., Dhanoa, M.S., Dijkstra, J., Bannink, A., Kebreab, E., France, J., 2007. Somemethodological and analytical considerations regarding application of the gasproduction technique. Anim. Feed. Sci. Technol. 135 (1), 139–156.

McCarty, P.L., 1964. Anaerobic Waste Treatment Fundamentals. Part 1. PublicWorksm NY, p. 107.

Nallathambi Gunaseelan, V., 1995. Effect of inoculum/substrate ratio andpretreatments on methane yield from Parthenium. Biomass Bioenergy 8 (1),39–44.

Neves, L., Oliveira, R., Alves, M.M., 2004. Influence of inoculum activity on the bio-methanization of a kitchen waste under different waste/inoculum ratios.Process Biochem. 39 (12), 2019–2024.

Noike, T., Endo, G., Chang, J.E., Yaguchi, J.I., Matsumoto, J.I., 1985. Characteristics ofcarbohydrate degradation and the rate-limiting step in anaerobic digestion.Biotechnol. Bioeng. 27 (10), 1482–1489.

Nyholm, N., Lindgaard-Jorgensen, P., Hansen, N., 1984. Biodegradation of 4-nitrophenol in standardized aquatic degradation test. Ecotoxicol. Environ. Saf.8 (5), 451–470.

Omil, F., Mendez, R., Lema, J.M., 1995. Anaerobic treatment of saline wastewatersunder high sulphide and ammonia content. Bioresour. Technol. 54, 269–278.

Owen, W.F., Stuckey, D.C., Healy, J.B., Young, L.Y., McCarty, P.L., 1979. Biossay formonitoring biochemical methane potential and anaerobic toxicity. Water Res.13 (6), 485–492.

Pagga, U., Beimborn, D.B., 1993. Anaerobic biodegradation test for organiccompounds. Chemosphere 27 (8), 1499–1509.

Prashanth, S., Kumar, P., Mehrotra, I., 2006. Anaerobic degradability: effect ofparticulate COD. J. Environ. Eng. Sci. 132 (4), 488–496.

Raposo, F., Banks, C.J., Siegert, I., Heaven, S., Borja, R., 2006. Influence of inoculum tosubstrate ratio on the biochemical methane potential of maize in batch tests.Process Biochem. 41, 1444–1450.

Raposo, F., Fernandez-Cegri, V., De la Rubia, M.A., Borja, R., Beline, F., et al., 2011.Biochemical methane potential (BMP) of solid organic substrates: evaluation ofanaerobic biodegradability using data from an international interlaboratorystudy. J. Chem. Technol. Biotechnol. 86 (8), 1088–1098.

Shelton, D., Tiedje, J., 1984. General method for determining anaerobicbiodegradation potential. Appl. Environ. Microbiol. 47 (4), 850–857.

Simkins, S., Alexander, M., 1984. Models for mineralization kinetics with thevariables of substrate concentration and population density. Appl. Environ.Microbiol. 47 (6), 1299–1306.

Soubes, M., Muxõ, L., Fernandez, A., Tarlera, S., Quirolo, M., 1994. Inhibition ofmethanogenesis from acetate by Cr.3 and ammonia. Biotechnol. Lett. 16 (2),195–200.

Symons, G.E., Buswell, A.M., 1933. The methane fermentation of carbohydrates. J.Am. Chem. Soc. 55 (5), 2028–2036.

Thouand, G., Block, J.C., 1993. The use of precultured inocula for biodegradabilitytest. Environ. Tech. 14 (7), 601–614.

Thouand, G., Friant, P., Bois, F., Cartier, A., Maul, A., Block, J.K., 1995. Bacterialinoculum density and probability of para-nitrophenol biodegradability testresponse. Ecotoxicol. Environ. Saf. 30 (3), 274–282.

Van Opstal, B., 2006. Evaluating AD system performance for MSW organics. BioCycle47 (11), 35–39.

VDI-Handbuch Landwirtschaft/Landtechnik, 2006. Fermentation of Organicmaterials, German guideline, ISC 13.030.30, 27.190.

Wang, Q., Noguchi, C., Hara, Y., Sharon, C., Kakimoto, K., Kato, Y., 1997. Studies onanaerobic digestion mechanism: influence of pretreatment temperature onbiodegradation of waste activated sludge. Environ. Technol. 18 (10), 999–1008.