Bioresource Technology 110 (2012) 18–25

Contents lists available at SciVerse ScienceDirect

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

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 5B9
b
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: Biochemical methane potential tests were conducted to evaluate the effect of using a blank versus a pre-
Received 5 December 2011 incubated inoculum in digestion of primary sludge at different waste to inoculum ratios (S/X). In addition,
Received in revised form 5 January 2012 this study explored the influence of using two different anaerobic inoculum sources on the digestion of
Accepted 7 January 2012
food waste: digested sludge from a municipal wastewater treatment plant and from a digester treating
Available online 16 January 2012
the organic fraction of municipal solid wastes. The results revealed that although there was no significant
difference in methane yield (on average 114 mL CH4/g TCODsub) or biodegradability (on average 28.3%) of
Keywords:
primary sludge using pre-incubated or non-incubated inocula, the maximum methane production rates
Anaerobic digestion
Biochemical methane potential
using non-incubated inoculum were higher than those using pre-incubated inoculum at all S/X ratios.
Waste-to-inoculum ratio Moreover, interestingly the inoculum from an anaerobic digester treating municipal wastewater sludge
Food waste was superior over the inoculum from anaerobic digester treating food waste in digesting food waste.
Pre-incubated inoculum Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction Although there is no standard detailed procedure for the BMP

The recycling and treatment of the organic fraction of municipal
solid waste is rapidly emerging as an effective waste management
strategy that diverts wastes from landfills and recovers energy
(Fernandez et al., 2001). In recent years, interest in biochemical
methane potential (BMP) tests has increased as reflected by the
wide range of research papers dealing with the BMP assays
(Raposo et al., 2011). The BMP assay is best suited when used to
elucidate what types of substrates, from an array of potential
substrates, 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 of
anaerobic biodegradability of substrates, and thus, relative resi-
dence times required for complete digestion (Labatut et al., 2011).
Abbreviations: BMP, biochemical methane potential; COD, chemical oxygen
demand; CV, coefficient of variation; DOPF, Dufferin Organics Processing Facility;
HRT, hydraulic retention time; JWPCP, Joint Water Pollution Control Plant; MMPR,
maximum methane production rate; MMPRs, maximum methane production rates;
MPR, methane production rate; S/X, waste to inoculum ratios; SCOD, soluble
chemical oxygen demand; SRT, solid retention time; SSO, source separated
organics; TCOD, total chemical oxygen demand; TS, total solids; TSS, total
suspended solids; TVS, total volatile solids; VFAs, volatile fatty acids; VS, volatile
solids; VSS, volatile suspended solids.
⇑ Corresponding author. Tel.: +1 519 661 2111x85470; fax: +1 519 850 2991.
E-mail addresses: gnakhla@eng.uwo.ca, gnakhla@fes.engga.uwo.ca (G. Nakhla).
test, 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, many
researchers used the blank assay (Neves et al., 2004; Nallathambi
Gunaseelan, 1995) approach described below, while some
researchers prescribe the German guideline for fermentation tests
(VDI-Handbuch, 2006), which entails pre-incubating the inoculum
for about 5 days without substrate before using it, thus eliminating
the need for the continued testing of both the seed blank and the
sample waste. For the blank assay method, the background meth-
ane production from the inoculum (determined in blank assays
with medium or water and no substrate) is subtracted from the
methane production obtained in the substrate assays (Angelidaki
et al., 2009). For the pre-incubated inoculum (German guideline),
the inoculum should be ‘‘degassed’’ in order to deplete the residual
biodegradable organic material present. Degassing should be
protracted until no significant methane production is observed:
typically 2–5 days of incubation (Raposo et al., 2011) are prescribed
in the method. In some cases, e.g. when the inoculum is taken from
a reactor fed with relatively high fat/oil concentration, longer
periods of pre-incubation may be required, in order to eliminate
all the residual (adsorbed/entrapped) substrate (Angelidaki et al.,
2009).
A wide range of biomass has been considered as potential seed
materials for methane production in BMP tests (Neves et al., 2004;

0960-8524/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2012.01.025
 E. Elbeshbishy et al. / Bioresource Technology 110 (2012) 18–25 19

Hashimoto, 1989; Chynoweth et al., 1993; Owen et al., 1979). It has digesters and one from a digester treating the organic fraction of
been observed that the results of the biodegradation tests could municipal solid wastes, were employed.
vary with the methodology followed. One of the most important
variables, which influence BMP results, is the origin of the inocu-
2. Methods
lum (Nyholm et al., 1984), since it determines the initial activity
of the microorganisms used for the test. Furthermore, the inocu-
2.1. Substrates and inocula
lum source brings about differences in bacterial populations
(Thouand et al., 1995), substrate adaptation (Barkay and Pitchard,
Two substrates were used in this study, food waste i.e. organic
1988; Thouand and Block, 1993), and residual anaerobically-biode-
fraction of municipal solid wastes and primary sludge from a mu-
gradable substrate.
nicipal WWTP. The food waste was obtained from Dufferin Organ-
The concentration of microorganisms that are used in the tests
ics Processing Facility (DOPF) in Toronto, Ontario, Canada. The city
will determine the biodegradation rates (Simkins and Alexander,
of Toronto’s DOPF receives approximately 25,000 metric tons/year
1984), the lag time (Chudoba et al., 1992), and the probability that
of source separated organics (SSO) material from Toronto’s residual
the degradation of the substrate occurs during the test perfor-
Green Bin and the commercial Yellow Bag collection programs. The
mance (Thouand et al., 1995).
purpose of the DOPF is to separate the film plastic bin finer and
There are different anaerobic biodegradation test methodolo-
contaminant materials fractions of the SSO from the organic mate-
gies, which seldom state inoculum characteristics. For example,
rial and convert the organic fraction into a material that is a
some methodologies (Shelton and Tiedje, 1984; Birch et al.,
suitable feedstock for the anaerobic digester (Van Opstal, 2006).
1989; Pagga and Beimborn, 1993; ISO 11734, 1995) recommend
The primary sludge was obtained from Joint Water Pollution Con-
anaerobic municipal wastewater treatment plant inoculum.
trol Plant (JWPCP), Carson, California. The JWPCP provides both
The waste to inoculum ratio (S/X) is an important parameter in
primary and secondary treatment for approximately 300 million
batch high solids anaerobic digestion processes as well as in the
gallons of wastewater per day. The characteristics of both sub-
assessment of anaerobic biodegradability of solid wastes (Neves
strates are presented in Table 1a.
et al., 2004). Although theoretically, the S/X ratio has an effect only
Three inocula (anaerobic sludge) were used in this study; the
on the kinetics, and not on the ultimate methane yield, which only
first inoculum was collected from the primary mesophilic anaero-
depends on the organic matter content (Nallathambi Gunaseelan,
bic digester at Guelph’s wastewater treatment plant (Guelph,
1995; Raposo et al., 2006), it is reported that too high S/X may be
Ontario), the second inoculum was collected from the mesophilic
toxic while too low S/X may prevent induction of the enzyme neces-
anaerobic digester treating SSO at DOPF in Toronto, Ontario, and
sary for biodegradation (Prashanth et al., 2006). This ratio also has
the third inoculum was obtained from a mesophilic anaerobic
an effect on the lag phase, which is shorter for low ratios (Chen
digester treating primary and secondary wastewater at JWPCP,
and Hashimoto, 1996).
Carson, California. The anaerobic digester at Guelph wastewater
Each substrate has its optimum S/X ratio, considering the poten-
treatment plant is completely mixed reactor with solid retention
tial amount of volatile fatty acids (VFAs) produced and its capacity
times (SRTs) in the range of 14–18 days, that achieves VSS destruc-
to buffer the medium due to the ammonium produced by the
tion efficiency of 45%. The anaerobic digester in DOPF at Toronto
hydrolysis of proteins (Lesteur et al., 2010). A small amount of
is a completely mixed reactor with a solids recycling system with
inoculum is preferred because of the endogenous biogas produc-
hydraulic retention time (HRT) of approximately 17 days, SRT of
tion, which can bias the results (Lesteur et al., 2010). Moreover,
approximately 27 days, VSS destruction efficiency of 62%. The
the increase in the S/X can lead to overloads due to volatile fatty
anaerobic digester at JWPCP is a completely mixed reactor with
acid accumulation (Neves et al., 2004). From another point of view,
HRTs (SRTs) in the range of 17–20 days, with VSS destruction effi-
the inoculum concentration should always be high compared to
ciencies of 49–52%. The characteristics of the three inocula are pre-
that of the substrate (in term of volatile solids) and the S/X should
sented in Table 1b. Inocula from both municipal WWTP were about
be recognised as one of the major parameters affecting the results
2.0% solids (w/w) while the DOPF inoculum was about 5.0% solids.
of anaerobic assays (Neves et al., 2004). Hashimoto (1989) and
The primary reason for the selection of seed from digesters
Labatut et al. (2011) reported that the minimum S/X ratio of 2 g
treating municipal biosolids and food waste is the difference in sol-
VS substrate/g VS inoculum was required when digesting wheat
uble COD concentrations between the two digesters. The food
straw at concentrations of 10–40 g VS/L and dairy manure at con-
waste digester seed had five times higher soluble COD concentra-
centrations of P3 g VS/L, respectively. However, in the case of
tion than the Guelph municipal wastewater treatment plant diges-
more recalcitrant wastes (woody feed stocks and municipal
ter. Thus, it is anticipated that the ratio of fermentative bacteria i.e.
wastes), the rate of methane production in BMP assays was opti-
hydrolyzers, carbohydrates, proteins, and lipids degraders to acid-
mum at S/X of 0.5 g VS substrate/g VS inoculum (Chynoweth
formers and methanogens differs between the two seeds. The other
et al., 1993). The S/X proposed by Owen et al. (1979) as a standard
reason for selection of food waste anaerobic digestion inoculum
was approximately 1 g VS substrate/g VS inoculum.
and food waste is the increasing number of these facilities world-
Based on the above mentioned introduction, it is obvious that
wide in light of the push for less land filling and resource recovery.
there is a wide range of the optimum or recommended S/X depend-
ing on the substrate and inoculum (some reported optimum S/X of
Table 1a
0.5 g VS substrate/g VS inoculum while other reported optimum S/X Substrates characteristics.
of 5.7 g VSsubstrate/g VSinoculum). Moreover the BMP test despite its
Parameter Units Food waste Primary sludge
wide use lacks standardization (some researchers recommended
using blank assays while others recommended pre-incubating the TCOD mg/L 113000 ± 2800a 42800 ± 180
SCOD mg/L 60300 ± 350 8000 ± 470
inoculum prior use). Therefore, the primary purpose of the current
TSS mg/L 48400 ± 2700 26300 ± 260
work was to evaluate the two approaches i.e. the widely used blank VSS mg/L 27900 ± 1300 20000 ± 250
seed assay versus the pre-incubated inoculum in digestion of pri- TVFA mg COD/L 260 ± 20 1680 ± 220
mary sludge at different S/X. The secondary goals of this study were NH4 mg/L 1670 ± 40 80 ± 20
the assessment of the impacts of the seed source and S/X ratio on pH – 4.6 ± 0.2 5.0 ± 0.1
Alkalinity mg CaCO3/L N.A. 2900 ± 180
BMP results. In this study, three seeds from conventional meso-
a
philic digesters, two from municipal wastewater treatment plant Values represents the average ± STD of three samples.
20 E. Elbeshbishy et al. / Bioresource Technology 110 (2012) 18–25
Table 1b
Inocula characteristics.
Parameter Units
TCOD mg/L
SCOD mg/L
TSS mg/L
VSS mg/L
TVFA mg COD/L
NH4 mg/L
pH – 7.6 ± 0.2
Alkalinity mg CaCO3/L
a
Values represents the average ± STD of three samples.
2.2. Anaerobic biodegradability test
The batch tests were conducted using two substrates (food
waste and primary sludge) and three inocula (Toronto, Guelph,
and JWPCP inocula) at different S/X of 0.25, 0.5, 1, 2, and 4 with
each test condition run in triplicates. TCOD of the substrate and
VSS of the seed were determined prior to the initiation of the batch
test (12 h prior the test). The volumes of inoculum and substrate
required to maintain an S/X ratio of 0.25, 0.5, 1, 2 and 4 on mass
COD/mass VSS were then determined. The pH was adjusted to
7 ± 0.2 using NaOH and HCl. The volumes of inoculum and the
substrate were then added to the batch test bottle (total liquid vol-
ume of 200 ml and headspace volume of 60 ml). A sample of the
mixture was then collected for initial analysis. The headspace
was flushed with nitrogen gas at 5–10 psi for a period of 5 min
and capped tightly with rubber stoppers. The bottles were then
placed in a swirling-action shaker (Max Q4000, Incubated and
Refrigerated Shaker, Thermo Scientific, CA) operating at 180 rpm
and maintained at a temperature of 37 °C. The volume of the gas
produced and the gas composition were analyzed until the test
was completed (cumulative gas curve reached a plateau).
2.3. Blank and pre-incubated inoculum
Three 200 mL bottles were used as blank (inoculum only) for
each inoculum without any substrate. The methane production
from the blank (inoculum only) adjusted by the ratio of the inocu-
lum volume in the test bottle to the 200 mL in the inoculum blank
was 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 was
continued 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 was
mixed 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 to
equilibrate with the ambient pressure (Owen et al., 1979). Biogas
composition including hydrogen, methane, and nitrogen was
determined 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 carrier
gas at a flow rate of 30 mL/min. The VFAs concentrations were
analyzed after filtering the sample through 0.45 lm using a gas
Guelph’s inoculum Toronto’s inoculum JWPCP’s inoculum
a
18100 ± 2600 70500 ± 1800 19400 ± 110
6560 ± 280 31200 ± 700 660 ± 70
18000 ± 3400 47400 ± 2300 18300 ± 450
10000 ± 720 37100 ± 1800 11600 ± 280
230 ± 60 210 ± 30 26 ± 6
540 ± 80 820 ± 20 470 ± 60
6.9 ± 0.2 7.2 ± 0.1
3200 ± 180 5000 ± 220 5400 ± 260
chromatograph (Varian 8500, Varian Inc., Toronto, Canada) with
a flame ionization detector (FID) equipped with a fused silica
column (30 m  0.32 mm). Helium was used as the carrier gas at
a flow rate of 5 mL/min. The temperatures of the column and
detector were 110 and 250 °C, respectively. Samples were analyzed
for total solids (TS), volatile solids (VS), total suspended solids
(TSS), volatile suspended solids (VSS), and alkalinity using standard
methods (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 determined
after 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 biogas
produced, at each time interval, using the mass balance equation
V CH4 ;i ¼ V CH4 ;i1 þ C CH4 ;i ðV G;i  V G;i1 Þ þ V CH4 ðC CH4 ;i  C CH4 ;i1 Þ ð1Þ
where V CH4 ;i and V CH4 ;i1 are cumulative methane gas volumes at the
current (i) and previous (i  1) time intervals, VG,i and VG,i1 are the
total biogas volumes in the current and previous time intervals,
C CH4 ;i and C CH4 ;i1 are the fractions of methane gas in the headspace
of the bottle measured using gas chromatography in the current and
previous intervals, and V CH4 is the total volume of headspace in the
reactor (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). In
this section, three inocula from different sources (Toronto, Guelph,
and JWPCP) were pre-incubated until the methane production
curves 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 ultimate
methane production from the 200 mL batches of 816 mL CH4 was
observed for Toronto’s inoculum while only 96, and 71 mL were ob-
tained for the inocula from Guelph and JWPCP, respectively. The
higher initial TCOD (70500 mg/L for Toronto’s inoculum versus
18100 and 19400 mg/L for Guelph and JWPCP inocula, respectively)
is the primary reason for the huge difference in methane production
from 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 mL
CH4/g VSS from Toronto’s inoculum compared to Guelph and
JWPCP inocula, respectively. While the two inocula from municipal
wastewater treatment plants are comparable, the inoculum from
the food waste digester is substantially different, reflecting both
 E. Elbeshbishy et al. / Bioresource Technology 110 (2012) 18–25 21

of substrates and inocula were calculated based on the TCOD of the

substrate and the VSS of the inoculum (for the pre-incubated inoc-
ulum, the VSS was measured after the incubation). Fig. 3a and b
depict the cumulative methane production from primary sludge
using the pre-incubated and non-incubated inocula at the different
S/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 the
two inocula, peaking at 541–551 mL at an S/X of 4 g CODsubstrate/g
VSSinoculum. Methane production from primary sludge using the
non-incubated inoculum was generally higher than the pre-incu-
bated inoculum at all S/X ratios except at S/X of 4 g CODsubstrate/g
VSSinoculum which exhibited no significant difference. The differ-
ences in methane production between using the non-incubated
and using the pre-incubated inoculum decreased with increasing
S/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,
Fig. 1. Cumulative methane production from the three inoculums.
respectively.
Based on the aforementioned results, it is obvious that there
were no significant differences either in the trend (no lag phases
were observed) or in the ultimate methane production when pre-
incubated or non-incubated (with blank correction) inocula at a
high S/X ratios of 2.0 and 4.0 g CODsubstrate/g VSSinoculum were used.
However, the differences between the two methods are pronounced
at the low S/X ratios of 0.5 and 1.0 g CODsubstrate/g VSSinoculum, clearly
emphasizing that pre-incubating the seed and elimination of seed
blank control can grossly underestimate biogas production under
certain circumstances. The underlying reason for pre-incubation is
to eliminate the methane contributed by the seed to avert skewing
BMP results. Pre-incubation is recommended to run until the daily

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

the high biodegradability of the food wastes relative to municipal

biosolids as well as the high residual biodegradable COD remaining
in the digested sludge. As shown in Fig. 1, more than 90% of the
methane production was achieved after 12 days regardless of the
inocula source, while after the 5 days recommended by German
guideline only 70%, 56%, and 49% of the methane potential were
observed for Toronto, Guelph, and JWPCP inocula, respectively. This
is also substantiated by the specific methane production rates
depicted in Fig. 2, which show that the rates after 12 days are 6%,
10%, and 12% of the maximum rates for Toronto, Guelph and JWPCP
inocula, respectively. Therefore based on the findings of this study,
it is recommended that a minimum of 10 days are required for the
pre-incubation irrespective of the source of the inoculum. b
Methane yields of 58, 25, and 18 mL CH4/g TCODinitial were
obtained for inocula from Toronto, Guelph, and JWPCP, respec-
tively, while the corresponding methane yields per mass of VSS
of 146, 47, and 31 mL CH4/g VSSinitial. The corresponding maximum
methane production rates were 34, 6.4, and 3.9 mL CH4/g VSS d.

3.2. Methane production from primary sludge using pre-incubated and

non-incubated inocula

3.2.1. Methane potential

In this experiment, primary sludge from JWPCP was digested
using inoculum obtained from JWPCP. Two sets of experiments
were conducted, first one using a pre-incubated inoculum (as de-
scribed in Section 2) and the other one using the inoculum without
pre-incubation (non-incubated inoculum). Four S/X ratios of 0.5, Fig. 3. Cumulative methane production of primary sludge at different S/X ratios; (a)
1.0, 2.0, and 4.0 g CODsubstrate/g VSSinoculum were used; the volumes pre-incubated inoculum and (b) non-incubated inoculum.
22 E. Elbeshbishy et al. / Bioresource Technology 110 (2012) 18–25

methane production rate is less than 1% of the cumulative methane,

a
and this corresponds to almost complete stabilization of the seed
material. 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 soluble
substrates at the beginning of the BMP test, the biodegradation rates
are very different from the non-incubated seed. Furthermore, dur-
ing incubation, microorganisms that degrade soluble substrates
(primarily acetogens and methanogens) will proliferate while
others that hydrolyze and uptake particulate substrates undergo
decay, thus affecting the microbial consortium balance. In the
non-incubated samples, due to the addition of both particulate
and soluble substrate, all microbial groups function simultaneously
like in a digester, taking advantage of the synergies between the
various microbial groups to maintain the microbial consortium
balance. Although the initial ammonia concentrations of the pre- b
incubated inoculum (ranged from 260 to 410 mg/L) and the non-
incubated inoculum (280–430 mg/L) were comparable, the final
ammonia concentrations differed significantly. The final ammonia
concentrations of the non-incubated inoculum ranging from 1460
to 1620 mg/L were about 30–40% higher than those of the non-incu-
bated inoculum, and since the main products of the biodegradation
of proteins in anaerobic conditions are ammonia and different
amino acid compounds, the higher final ammonia concentrations
for the non-incubated inoculum batches reflect greater hydrolysis
and acidogenic microbial activities. The toxicity of ammonia to
methanogenic 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 to Fig. 4. Methane production rate of primary sludge at different S/X ratios; (a) pre-
140 mg N-FA/L inhibit mesophilic treatment (De Baere et al., incubated inoculum and (b) non-incubated inoculum.
1984). It is possible to calculate FA concentration from the total
ammonia concentration in the liquid (TA) and the fraction of FA
Table 2a
(fN), using the equation (Omil et al., 1995): Maximum methane production rate and methane yield of primary sludge.

fN ¼ FA=TA ¼ 1=½1 þ ðkb  10pH Þ=kw  ð4Þ Inoculum S/X Maximum methane Methane yield
production rate
where kb and kw are the dissociation constants for ammonia and mL CH4/ mL CH4/ mL CH4/
water, respectively (1:855  105 and 2:355  1014 mol/l at g VSSinoculum d g VSSsub g TCODsub
37 °C). The highest final concentrations of FA computed using Equa- Pre-incubated 0.5 8 241 113
tion (4) observed in the batches (both pre-incubated and non-incu- inoculum 1 14 221 103
bated) was less than 45 mg/L, substantially below the inhibition 2 31 235 110
4 56 273 127
threshold level.
Non-incubated 0.5 11 283 132
inoculum 1 25 231 108
3.2.2. Methane production rate (MPR) and methane yield 2 57 230 108
Fig. 4a and b depict the MPR of primary sludge using the pre- 4 87 235 110
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 than Table 2b
those using the pre-incubated inoculum at all S/X ratios. Using Maximum methane production rate and methane yield of food waste.

both inocula, the MMPR increased with increasing the S/X ratio; Inoculum S/X Maximum methane Methane yield
the maximum MMPR of 60 and 86 mL CH4/d were achieved at production rate
S/X of 4 g CODsubstrate/g VSSinoculum using the pre-incubated and mL CH4/ mL CH4/ mL CH4/
the non-incubated inocula, respectively. The minimum MMPR g VSSinoculum d g VSSsub g TCODsub
were observed at S/X of 0.5 g CODsubstrate/g VSSinoculum for both Toronto’s inoculum 1 19 660 160
inocula (15 mL CH4/d using the pre-incubated inoculum versus 0.5 26 790 190
22 mL CH4/d using non-incubated inoculum). The MMPR of pri- 0.25 9 440 110

mary sludge normalized per initial mass of inoculum (as VSS) is Guelph’s inoculum 1 69 1000 150
presented in Table 2a. The normalized MMPRs using the non-incu- 0.5 72 940 230
0.25 53 1400 340
bated inoculum were higher than those of the pre-incubated inoc-
ulum at all S/X ratios. The highest MMPR of 56 mL CH4/g
VSSinoculum d was obtained using the pre-incubated inoculum at
an S/X ratio of 4 g CODsubstrate/g VSSinoculum compared to 87 mL numbers) using the non-incubated and the pre-incubated inocula
CH4/g VSSinoculum d using the non-incubated inoculum at S/X of increased with increasing S/X and peaked at 84% at S/X of 2.0 g
4.0 g CODsubstrate/g VSSinoculum. As shown in Table 2a, the difference CODsubstrate/g VSSinoculum and then decreased to 56% at S/X of
between the normalized MMPR (as a percentages of the lower 4.0 g CODsubstrate/g VSSinoculum.
 E. Elbeshbishy et al. / Bioresource Technology 110 (2012) 18–25
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 preferred
as it permits direct conversion of the results into percent organic
matter converted to methane using the theoretical 0.350 m3 CH4
at STP per kilogram COD converted (McCarty, 1964). The methane
yields of the primary sludge at different S/X using pre-incubated
and non-incubated inocula are presented in Table 2a. For the
pre-incubated inoculum, the maximum methane yield of 127 mL
CH4/g TCODsub (or 273 mL CH4/g VSSsub) was achieved at S/X of
4.0 g CODsubstrate/g VSSinoculum, while the lowest methane yield of
103 mL CH4/g TCODsub (or 221 mL CH4/g VSSsub) was observed at
S/X of 1.0 g CODsubstrate/g VSSinoculum. On the other hand, for the
non-incubated inoculum, the max methane yield of 132 mL CH4/g
TCODsub (or 283 mL CH4/g VSSsub) was obtained at S/X of 0.5 g
CODsubstrate/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 of
the non-incubated inoculum was higher than that of the pre-incu-
bated inoculum (283 versus 241 mL CH4/g VSSsub), while at the
higher S/X of 4 g CODsubstrate/g VSSinoculum, the opposite was ob-
served as the methane yield of pre-incubated inoculum was higher
than that of the non-incubated inoculum (127 versus 110 mL CH4/g
TCODsub). In general, there was no significant difference in the
methane yield between pre-incubated or non-incubated inoculum
as the average methane yield of 113 mL CH4/g TCODsub was
achieved using pre-incubated inoculum compared to 114 mL
CH4/g TCODsub using non-incubated inoculum.
3.2.3. Kinetics
In 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 matter
inside the cell to be available for biological degradation (Wang
et al., 1997; Noike et al., 1985). Since the most widely used hydro-
lysis model is the first order, anaerobic digestion is generally
described as a first order reaction with respect to substrate concen-
tration (Eq. (2a)), and methane production (Eq. (2b)):
dS=dt ¼ kS
d½ðBo  BÞ=Bo =dt ¼ k½ðBo  BÞ=Bo 
Table 3a
Kinetic constants and biodegradability (BDCH4 ) of primary sludge.
Inoculum S/X Kinetics constant (k)a d1
Pre-incubated inoculum 0.5 0.11
1 0.12
2 0.14
4 0.13
Non-incubated inoculum 0.5 0.15
1 0.19
2 0.17
4 0.18
a
R2 for the kinetics constants ranged from 0.87 to 0.98.
Table 3b
Kinetic constants and biodegradability (BDCH4 ) of food waste.
Inoculum S/X Kinetics constant (k)a d1
Toronto’s inoculum 1 0.18
0.5 0.13
0.25 0.12
Guelph’s inoculum 1 0.16
0.5 0.13
0.25 0.17
a
R2 for the kinetics constants ranged from 0.85 to 0.99.
23
where k is the first order kinetic constant (d1), t is the digestion
time (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 ultimate
methane production at the end of the experiment corresponding
to 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 slope
of the linear curve of ln[(Bo  B)/Bo] versus t.
Table 3a shows the kinetic constants of the primary sludge at
different S/X using pre-incubated and non-incubated inocula.
The k values ranged from 0.11 to 0.19 d1. The k values for the
non-incubated inoculum were higher than the pre-incubated inoc-
ulum at all S/X ratios. For the pre-incubated inoculum, the k values
ranged from 0.11 to 0.14 d1. Based on the k value in Table 3a for
the pre-incubated inoculum at the different S/X, the average k va-
lue of 0.124 d1 with coefficient of variation (CV) of 8% reveals that
there 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 d1
was observed at S/X of 1.0 g CODsubstrate/g VSSinoculum, while the
lowest k value of 0.15 d1 was achieved at S/X of 0.5 g
CODsubstrate/g VSSinoculum. The average k value (using non-incubated
inoculum) of 0.172 d1 with C.V. of 9% emphasised that the S/X
ratio did not have significant effect on k values. The relative
independence of the k value of S/X ratios clearly indicates that
there was no substrate limitation at all four S/X ratios explored.
3.2.4. Biodegradability
The experimental methane yield can be used to calculate the
level of anaerobic biodegradability (BDCH4 ) under the defined test
conditions in comparison with its theoretical value as follows
(Raposo et al., 2011):
BDCH4 ð%Þ ¼ ðBoExp =BoTh Þ  100 ð3Þ
where BoExp is the experimental ultimate methane production (mL)
and BoTh represents the theoretical methane potential based on the
initial TCOD of the substrate, neglecting the biomass synthesis,
which is typically about 5% of the organic matter consumed (Sy-
mons and Buswell, 1933).
ð2aÞ
The biodegradability of the primary sludge at the different S/X
ð2bÞ using both inocula presented in Table 3a reflects no significant
differences between pre-incubated or non-incubated inoculum,
as an average biodegradability of 28.3% was computed for both
inocula. Furthermore, there was no significant impact of the S/X
BDCH4 % ratio on the biodegradability when any of the two inocula was
used. It must be asserted however that the biodegradability of
28
26 the primary sludge in the batch BMP tests relative to about 50%
28 destruction efficiency in conventional mesophilic digesters at
32 hydraulic retention times of 15–20 days is attributed primarily to
33 the very low food-to-microorganisms ratio in the digesters.
27
27
3.3. Methane production from food waste using two inoculum sources
28
3.3.1. Methane production, MPR, and methane yield
The food waste was digested using two inocula (Toronto and
Guelph inocula) at different S/X ratios of 0.25, 0.5, and 1.0 g
CODsubstrate/g VSSinoculum. Fig. 5a and b show the cumulative meth-
BDCH4 % ane production from food waste at different S/X (0.25, 0.5, and 1.0)
41 using Toronto’s inoculum (Fig. 5a) and Guelph’s inoculum (Fig. 5b)
49 after correcting for the blank in the 200 mL batches. In general, for
27 both inocula at all S/X ratios, a quick initial methane production
63 (no lag phase) was observed due to the biodegradation of soluble
59 compounds. As shown in Fig. 5a and b, for both inocula, methane
87
potential increased with increasing S/X ratios. The highest methane
production of 760 mL was obtained using Toronto’s inoculum at
24 E. Elbeshbishy et al. / Bioresource Technology 110 (2012) 18–25
a a
b
Fig. 5. Cumulative methane production of food waste at different S/X ratios; (a)
Toronto’s inoculum and (b) Guelph’s inoculum.
S/X of 1.0 g CODsubstrate/g VSSinoculum compared to 560 mL when
Guelph’s inoculum was used. Ultimate methane production using
Toronto’s inoculum was higher than using Guelph’s inoculum at
S/X of 0.5 and 1.0 g CODsubstrate/g VSSinoculum, while at S/X of
0.25 g CODsubstrate/g VSSinoculum, the methane production using
Guelph’s inoculum (190 mL) was higher than that of Toronto’s
inoculum (150 mL).
The MPR of food waste using Toronto’s inoculum and Guelph’s
inoculum is illustrated in Fig. 6a and b. The MMPR was achieved at
an 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 for
Guelph’s inoculum). Using Toronto’s inoculum, MMPRs of 112 mL
CH4/d and 110 mL CH4/d were achieved at S/X ratios of 1.0 and
0.25 g CODsubstrate/g VSSinoculum, respectively, compared to 124 mL
CH4/d and 103 mL CH4/d when Guelph’s inoculum was used. Meth-
ane production rates normalized per initial mass of inoculum (as
VSS) are presented in Table 2b. After normalization, the MMPR
using Guelph’s inoculum were significantly higher than those
using Toronto’s inoculum at all S/X ratios. Moreover, the highest
MMPR were observed at S/X of 0.5 g CODsubstrate/g VSSinoculum for
both inocula (26 mL CH4/g VSS d using Toronto’s inoculum versus
72 mL CH4/g VSS d using Guelph’s inoculum).
Using Toronto’s inoculum, the highest methane yield of 190 mL
CH4/g TCODsub (or 790 mL CH4/g VSSsub) was observed at an S/X of
0.5 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 (or
440 mL CH4/g VSSsub) observed at S/X of 0.25 g CODsubstrate/g
VSSinoculum. For Guelph’s inoculum, the methane yield increased
with decreasing the S/X ratio and reached a maximum value of
b
Fig. 6. Methane production rate of food waste at different S/X ratios; (a) Toronto’s
inoculum and (b) Guelph’s inoculum.
340 mL CH4/g TCODsub (or 1400 mL CH4/g VSSsub) at an S/X of
0.25 g CODsubstrate/g VSSinoculum.
Based on the above mentioned results, it is clear that the MMPR
and highest methane yield using Guelph’s inoculum were higher
than those obtained using Toronto’s inoculum, possibly due to the
initial inoculum concentration in the assays (on average 6.5 g VSS/
bottle for Toronto’s inoculum versus 1.9 g VSS/bottle for Guelph’s
inoculum). On the other hand, the high initial SCOD in Toronto’s
inoculum lead to higher MPR and higher methane production in Tor-
onto’s blank inoculum compared to Guelph’s inoculum which might
have negatively affected the Toronto’s MMPR and methane yield.
3.3.2. Kinetics and biodegradability
The kinetic constant (k) values for the digestion of food waste at
different S/X ratios using Toronto and Guelph’ inocula are presented
in 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/X
ratios of 0.5 and 1 g CODsubstrate/g VSSinoculum, while a 42% difference
(0.17 d1 for Guelph versus 0.12 d1 for Toronto) was observed at
S/X ratio of 0.25 g CODsubstrate/g VSSinoculum. The kinetic constants
obtained in this study which ranged from 0.12 to 0.18 d1 were
much higher than the values of 0.016–0.125 d1 reported by
Gunaseelan (2004), using more than 50 fruits and vegetable wastes
as substrates. On contrast, the k values obtained here were lower
than the values of 0.21–0.34 d1 reported by Raposo et al. (2011)
using four substrates (starch, cellulose, gelatine, and mung bean).
Table 3b represents the biodegradability of the food waste at
different 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
using Guelph’s inoculum was higher than that of using Toronto’s
inoculum at all S/X ratios. The max biodegradability of 87% was
achieved using Guelph’s inoculum at S/X of 0.25 g CODsubstrate/g
VSSinoculum, while the max biodegradability with Toronto’s inocu-
lum was 49% at S/X of 0.5 g CODsubstrate/g VSSinoculum. The lowest
value of biodegradability of 27% was observed at S/X of 0.25 g
CODsubstrate/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 running
a seed control in BMP tests. However, the inoculum source and test
S/X conditions play a significant role in test results and can lead to
flawed data interpretation and conclusions, with sometimes the
obvious seed source for a waste treatability (such as Toronto in this
study) grossly underestimating ultimate biodegradability. Thus,
comprehensive BMP testing should include a minimum of three
S/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 of
Toronto, Guelph, and JWPCP facilities in providing the inocula
and 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 energy
crops: a proposed protocol for batch assays. Water Sci. Technol. 59 (5), 927–
934.
APHA, AWWA, WEF, 1995. Standard Methods for Examination of Water and
Wastewater, 19th ed.
Barkay, T., Pitchard, T., 1988. Adaptation of aquatic microbial communities to
pollutant 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 on
batch 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 of
the 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. Biomass
Bioenergy 5 (1), 95–111.
De Baere, L.A., Devocht, M., Van Assche, P., Verstraete, W., 1984. Influence of high
NaCl 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 the
start up of solid waste anaerobic digesters. Water Sci. Tech. 44 (4), 103–108.
Gunaseelan, V.N., 2004. Biochemical methane potential of fruits and vegetable solid
waste feedstocks. Biomass Bioenergy 26 (4), 389–399.
Hashimoto, A.G., 1989. Effect of inoculum/substrate ratio on methane yield and
production rate from straw. Biol. Waste 28 (4), 247–255.
25
ISO 11734, 1995. Water quality – Evaluation of the ‘‘ultimate’’ anaerobic
biodegradability of organic compounds in digester sludge – Method by
measurement of the biogas production. International Organization of
Standardization, Switzerland.
Kroeker, E.J., Schulte, D.D., Sparling, A.B., Lapp, H.M., 1979. Anaerobic treatment
process stability. J. Water Pollut. Control Fed. 51 (4), 718–727.
Labatut, R.A., Angenent, L.T., Scott, N.R., 2011. Biochemical methane potential and
biodegradability 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: a
review. Process Biochem. 45 (4), 431–440.
Lopez, S., Dhanoa, M.S., Dijkstra, J., Bannink, A., Kebreab, E., France, J., 2007. Some
methodological and analytical considerations regarding application of the gas
production technique. Anim. Feed. Sci. Technol. 135 (1), 139–156.
McCarty, P.L., 1964. Anaerobic Waste Treatment Fundamentals. Part 1. Public
Worksm NY, p. 107.
Nallathambi Gunaseelan, V., 1995. Effect of inoculum/substrate ratio and
pretreatments 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 of
carbohydrate 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 wastewaters
under 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 for
monitoring biochemical methane potential and anaerobic toxicity. Water Res.
13 (6), 485–492.
Pagga, U., Beimborn, D.B., 1993. Anaerobic biodegradation test for organic
compounds. Chemosphere 27 (8), 1499–1509.
Prashanth, S., Kumar, P., Mehrotra, I., 2006. Anaerobic degradability: effect of
particulate COD. J. Environ. Eng. Sci. 132 (4), 488–496.
Raposo, F., Banks, C.J., Siegert, I., Heaven, S., Borja, R., 2006. Influence of inoculum to
substrate 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 of
anaerobic biodegradability using data from an international interlaboratory
study. J. Chem. Technol. Biotechnol. 86 (8), 1088–1098.
Shelton, D., Tiedje, J., 1984. General method for determining anaerobic
biodegradation potential. Appl. Environ. Microbiol. 47 (4), 850–857.
Simkins, S., Alexander, M., 1984. Models for mineralization kinetics with the
variables 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 of
methanogenesis 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 biodegradability
test. Environ. Tech. 14 (7), 601–614.
Thouand, G., Friant, P., Bois, F., Cartier, A., Maul, A., Block, J.K., 1995. Bacterial
inoculum density and probability of para-nitrophenol biodegradability test
response. Ecotoxicol. Environ. Saf. 30 (3), 274–282.
Van Opstal, B., 2006. Evaluating AD system performance for MSW organics. BioCycle
47 (11), 35–39.
VDI-Handbuch Landwirtschaft/Landtechnik, 2006. Fermentation of Organic
materials, German guideline, ISC 13.030.30, 27.190.
Wang, Q., Noguchi, C., Hara, Y., Sharon, C., Kakimoto, K., Kato, Y., 1997. Studies on
anaerobic digestion mechanism: influence of pretreatment temperature on
biodegradation of waste activated sludge. Environ. Technol. 18 (10), 999–1008.