Research Article

Received: 25 July 2018 Revised: 24 October 2018 Accepted article published: 31 October 2018 Published online in Wiley Online Library:

(wileyonlinelibrary.com) DOI 10.1002/jctb.5859

Methane potential and metagenomics of

wastewater sludge and a methane-producing
landfill solid sample as microbial inocula for
anaerobic digestion of food waste
María V Sillas-Moreno, Carolina Senés-Guerrero, Adriana Pacheco*
and Alejandro Montesinos-Castellanos

Abstract
BACKGROUND: Anaerobic digestion of food waste is influenced by the selection of an adequate microbial inoculum. In this study,
wastewater sludge, a methane (CH4 )-producing landfill solid sample and the combination of both [1:1 volatile solid (VS)] were
tested as inocula to treat food waste modelled to the Mexican diet. Hence, biochemical methane potential assays at inoculum to
substrate (I:S) ratios of 1–3 and each inoculum metagenome were determined.

RESULTS: Methane production was optimal at I:S = 1 for sludge and the combined inoculum, whereas for landfill solid sample
increasing the proportion of inoculum was beneficial up to I:S = 2. The landfill solid sample produced more CH4 than commonly
used sludge (311.5 ± 11.1 and 282.5 ± 18.1 mL CH4 g−1 VS, respectively) but presented an adaptation phase (four days).
Combining inocula (I:S = 1) produced a synergistic effect in CH4 yield (374.5 ± 10.5 mL CH4 g−1 VS), biogas quality (58.0 ± 0.3%
CH4 ) and COD removal (90.0 ± 0.6%) without start-up time. Moreover, an enriched microbial community high in Archaea and
Bacteria was observed, probably because of the incorporation of predominant members of each inoculum (Bacteroidetes,
Firmicutes and Euryarchaeota in sludge, and Proteobacteria and Actinobacteria in landfill solid sample). Archaea was represented
mainly by the genus Methanosaeta (order Methanosarcinales), suggesting that methanogenesis occurred by the acetoclastic
pathway.

CONCLUSION: Food waste from Mexican diet was a suitable feedstock for biogas generation. Optimal conditions were observed
when combining both inocula. Further studies on population dynamics during digestion would help understand the synergistic
effect for effective industrial application.
© 2018 Society of Chemical Industry

Supporting information may be found in the online version of this article.

Keywords: biogas; anaerobic digestion; genomics; industrial microbiology; sludge; waste treatment and waste minimisation

INTRODUCTION time, which is associated to the acclimation of the inoculum to

Food waste is an important proportion of municipal solid waste. In
Mexico, it accounts for 52.4% (w/w) and is estimated to increase
annually as a result of higher living standards and population
growth (INEGI (http://www.inegi.org.mx). This biomass represents
a suitable low-cost feedstock for biogas production because of
its high organic content and biodegradability.1 However, com-
position varies according to human diet and impacts biogas
production2,3 ; thus, it is of relevance to determine the potential of
a particular feedstock.
The biochemical methane potential (BMP) assay is an inexpen-
sive and simple procedure to evaluate a particular feedstock or the
effect of operational conditions such as the type of inoculum for
biogas generation.4–6 Microbial composition can greatly influence
biogas production and process stability, because it determines
the equilibrium among different functional groups required to
sequentially degrade the waste to CH4 . It also affects start-up
the substrate.6,7 For food waste digestion, municipal wastewater
sludge6,8 , other industrial wastewaters6,9 , animal manure7,10 and
grass silage11 have all been tested as microbial inocula. These
studies all concluded that the inoculum source had a significant
effect on biogas production. For example, Neves et al.9 reported
better performance using granular sludge from brewery efflu-
ents than suspended sludge from wastewater because of the
high concentration of microorganisms attached to the granules.
Another interesting inoculum, associated with municipal waste
treatment, is landfill that intrinsically possesses methanogenic
∗ Correspondence to: A Pacheco, Tecnologico de Monterrey, Escuela de Inge-
nieria y Ciencias, Ave. Eugenio Garza Sada 2501, Monterrey, N.L. 64849, Mexico.
E-mail: adrianap@itesm.mx
Tecnologico de Monterrey, Escuela de Ingenieria y Ciencias, Monterrey, Mexico

J Chem Technol Biotechnol (2018) www.soci.org © 2018 Society of Chemical Industry

 www.soci.org MV Sillas-Moreno et al.

activity. Landfill leachate has been used with satisfactory results;

Table 1. Composition of the model food waste on a wet weight basis
its alkaline nature can alleviate organic acid accumulation dur-
ing digestion.12–14 However, start-up time and CH4 production Component Fraction (% WW)
rate from landfill leachate were inferior when compared to
wastewater sludge. Nonetheless, landfill solid samples from active Carbohydrates Tortilla 20.35
CH4 -producing sites have not been tested, even though they are Bread 3.31
expected to carry a methanogen community already adapted to Pasta 0.37
municipal waste. Moreover, in an integrated waste management Proteins (meat) Chicken 5.06
model, hard-to-digest waste could be treated in landfills and Meat 2.21
served as inoculum for food waste digesters, while generating Legumes Beans 1.80
biogas as a renewable energy source. Rice 0.95
Another important parameter in anaerobic digestion is the Vitamins and Orange bagasse 18.03
minerals (fruit) Lemon bagasse 15.96
inoculum to substrate (I:S) ratio as it is suggested that each
feedstock and microbial inoculum possesses an optimum for max- Banana peel 6.48
imum CH4 yield.6,15 Usually, I:S = 1 is an appropiate relation for Apple 0.91
easily degradable biomass, whereas higher ratios are necessary for Vitamins and Onion 4.65
minerals Tomato 7.09
recalcitrant substrates.9 By contrast, lower ratios tend to increase (vegetables) Chili pepper 2.21
start-up time and can overload the system.8 For food waste, a
Lettuce 4.79
wide range of I:S ratios have been tested with optima around
Potato peel 0.86
0.7–3.0.8–10,15–17
Carrot peel 1.06
A consortium of interdependent microorganisms participates
Minerals Egg shell 3.79
in the four well-known metabolic stages of anaerobic digestion.
Bacteria contribute to the first steps of hydrolysis, acidogenesis
and acetogenesis, whereas methanogens execute the last. An
Microbial inocula
imbalance between acidogenesis and acetogenesis can result in
volatile fatty acid (VFA) accumulation and a consequent pH drop Sludge was obtained from a municipal anaerobic wastew-
that compromises methanogen activity.17,18 Hence, understand- ater treatment plant located at the Universidad Autonoma
ing the variations in microbial community structure is essential Metropolitana-Iztapalapa (Mexico City, Mexico). A 1-gallon plastic
for biogas production. Metagenomic analysis by Next Gener- container was loaded with a sample from the mesophilic upflow
ation Sequencing has gained attention as a high-throughput anaerobic sludge blanket (UASB) reactor (50 m3 ), operated at
culture-independent tool for studying microbial communities. 38 ∘ C. The landfill solid sample was collected from the municipal
Particularly, in food waste digestion efforts have been made solid waste treatment facility of Garcia (Nuevo Leon, Mexico).
to determine the biogas microbiome19–21 and increase yields First, a CH4 -producing area of the landfill was identified using a
by feedstock modification and evaluating changes in micro- portable gas analyzer (GEM2000, LandTech, Long Beach, CA, USA);
bial composition.1,22–25 Co-digestion with sewage sludge1 or an subsequently, samples were taken with a tractor shovel from a
increase in total solids (TS) content25 are reported to enhanced depth of 5 m in order to study the anaerobic zone of the stored
CH4 production, whereas microwave1 or autoclave19 pretreatment waste. Six sterile 1-L Nalgene bottles were filled with this material.
generated inhibiting or hard-to-digest compounds as well as a less This area had been closed for c.1 year and, at the moment of
productive and diverse microbial community. All of these studies sampling, soil temperature was around 30 ∘ C (50 cm depth) and
identified Methanosarcina as a typical methanogen member in biogas content was 54% CH4 (3 m depth). All samples were trans-
food waste digestion, indicating that the acetoclastic pathway ported and maintained at 4 ∘ C until use. The combination of both
plays an important role in CH4 generation.1,19–25 inocula was implemented at commencement of experimentation,
Studies particular to the Mexican population are scarce, as is as described in the following section.
the use of inoculum from active CH4 -producing landfill sites.
Therefore, the aim of the present work was to determine the BMP Biochemical methane potential (BMP) assay
at different I:S ratios of food waste modelled to the Mexican diet by The standardized protocols of Angelidaki et al.4 and Elbeshbishy
using different inocula, whose compositions were elucidated by et al.6 were followed with some modifications. Three I:S ratios (1,
metagenomics. Microbial inocula hypothesized to contain highly 2, 3) were established on a VS basis (20 g L−1 ) and tested first for
active biogas-producing communities included: (i) sludge from an each inoculum (sludge, landfill solid sample). Then, the combi-
anaerobic wastewater treatment plant, (ii) a solid sample from a nation of both inocula (sludge+landfill solid sample) at a 1:1 VS
CH4 -producing site in a municipal landfill and (iii) the combination basis was evaluated under the optimized I:S ratio of each inocu-
of both on a 1:1 volatile solids (VS) basis. lum. Before experimentation, refrigerated samples were tempered
at room temperature and shaken vigorously. Experimental glass
flasks (125 mL) were filled with 100 mL of previously mixed compo-
MATERIALS AND METHODS nents which consisted of model food waste, inoculum and sterile
Food waste model mixture water in order to achieve 20 g L−1 of VS at the different I:S ratios
Components of the model mixture were determined according (Supporting Information, Table S1). The mixture was buffered with
to national surveys of the most consumed foods in Mexico and the NaHCO3 (6 g L−1 ) and the pH adjusted to 7.5 ± 0.1 using NaOH
municipal waste associated with these products.26,27 All con- 3 N and HCl 1 N. Also, a trace element solution with concentration
stituents of the modelled waste were hand-mixed and ground to a 1.2 μL g−1 was added to ensure optimal microbial activity, as
paste using a food processor (Hamilton Beach, Glen Allen, VA, USA). suggested by Rincón et al.28 . A sample of the mixture was used to
The composition of the model food waste is shown in Table 1. determine initial chemical oxygen demand (COD). Each flask was

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Table 2. Physicochemical characteristics of the model food waste and microbial inocula

Model Sludge Landfill solid 1:1 Sludge+landfill

Parameters food waste inoculum (S) sample inoculum (L) solid sample inoculum (SL)

Moisture content (%) 70.1 ± 0.7 87.9 ± 1.1 10.2 ± 0.8 38.5 ± 1.0
TS (%) 29.9 ± 0.7 12.1 ± 1.9 89.8 ± 1.4 61.5 ± 0.9
VS (%) 24.6 ± 0.5 7.0 ± 0.6 4.6 ± 0.3 6.1 ± 0.5
VS/TS (%) 82.2 ± 0.5 57.5 ± 1.0 5.1 ± 0.3 9.9 ± 0.7
pH 5.07 to 5.30 7.25 7.45 7.40
COD (g L−1 ) 87.5 ± 4.1 42.5 ± 1.0 18.6 ± 0.3 29.7 ± 3.3
Carbon (% DW) 46.23 ND ND ND
Nitrogen (% DW) 1.65 ND ND ND
Hydrogen (% DW) 17.44 ND ND ND
Oxygen (% DW) 34.42 ND ND ND
Sulfur (% DW) 0.23 ND ND ND
Ash (% DW) 8.80 ND ND ND
C/N 28.00 ND ND ND

Values represent average and standard deviation (n = 3).

COD, chemical oxygen demand; DW, dry weight; ND, not determined; TS, total solids; VS, volatile solids; VS/TS, volatile solids out of total solids.

flushed with N2 gas for 3 min, sealed with a Mininert valve (VICI,
Baton Rouge, LA, USA) and placed in a shaker at 60 rpm under
mesophilic conditions (37 ∘ C). Biogas composition was measured
every 48 h by gas chromatography (GC) as described in the follow-
ing section. After taking the GC sample (100 μL), gas production
was assessed by the liquid displacement method using a saturated
saline solution (pH 2), as an effective barrier to avoid errors due to
gas dissolution.29 All tests were conducted in triplicate and a blank
of each inoculum (20 g L−1 VS) alone was considered to subtract
endogenous CH4 production. Incubation proceeded for 30 days
until CH4 production reached steady state.
Analytical methods
The physicochemical characterization of the model food waste
and microbial inocula was according to the standard methods
of APHA-AWWA-WEF30 : 2540B moisture content, 2540B TS, 2540E
VS and 5220D chemical oxygen demand (COD). The pH was
measured using a HANNA probe (HI8424 Woonsocket, RI, USA).
Elemental composition of the food waste was determined from
a previously dried (105 ∘ C) and milled sample through ultimate
analysis using an elemental analyzer (Leco TruSpec 630-100-400,
CA, USA). The oxygen content was assessed by balance of the
other elements.
Biogas composition (CH4 , H2 , CO2 ) during BMP assays was deter-
mined every 48 h from the undisturbed gas phase (100 μL) into a
GC (Hewlett Packard Model 5890 series II, Wilmingtory, DE, USA)
with a thermal conductivity detector. Helium was the carrier gas
and the temperature of the detector and oven was 200 and 225 ∘ C,
respectively. A gas standard with the following analytical compo-
sition was used (in cmol mol−1 ): 4.00 He, 5.04 CO, 10.21 N, 10.06
H, 1.00 H2 S, 29.74 CO2 and 39.95 CH4 as a balance gas (Praxair,
Durham, CT, USA).
was centrifuged (3000 rpm, 5 min, 25 ∘ C), before the supernatant
was collected and centrifuged again (12 000 rpm, 10 min, 25 ∘ C) to
generate a cell pellet. For the landfill solid sample and the com-
bined inoculum, 20 g (wet weight) of the sample were first mixed
with 10 g of sterile glass beads (0.5 mm diameter) in 25 mL of phos-
phate buffer and agitated for 30 min at 200 rpm to disaggregate
cells. Then a cell pellet was generated as mentioned above. All sam-
ples were processed using a FastDNA Spin Kit for Soil (MP Biomed-
icals, OH, USA) according to the manufacturer’s protocol. The DNA
quality was evaluated by spectrophotometry using a NanoDrop
1000 (ThermoFisher Scientific, Waltham, MA, USA), agarose gel
(1%) electrophoresis and quantified by fluorometry using a Qubit
2.0 (Life Technologies, Carlsbad, CA, USA).
Sequencing and bioinformatic analysis
Library preparation of each DNA sample was done with the Nex-
tera XT DNA Library Prep Kit (Illumina, San Diego, CA, USA) accord-
ing to the manufacturer’s protocol. Libraries were sequenced
(2 × 151 bp paired-end) with the MiSeq Reagent Kit v.3 (300 cycles)
using the MiSeq system (Illumina). Sequencing data were trimmed
of adapters and reads below a mean quality of Q30 (unpaired)
or <32 bp of length were removed using FASTQ Toolkit v.2.2.0
(BaseSpace Labs, Illumina). Microbial taxonomy was determined
using the metagenomics classifier Kaiju, which employs the NCBI
microbial subset of nonredundant protein database (nr), including
fungi and microbial eukaryotes.31 A principal component analysis
(PCA) of the samples was executed through the web server
ClustVis. Results from each sample were filtered using 0.05% and
1% as minimum abundance for whole community and taxa anal-
ysis, respectively. Relative abundance was calculated taking into
account the proportion of reads of certain taxa in a sample from
the total amount of reads from that sample.

Metagenomic analysis
DNA extraction
Samples were homogenized for DNA extraction. Twenty-five millil-
itres of sludge were diluted in 15 mL of sterile phosphate buffer
(10 mmol L –1 , pH 7.0) and placed in a rotatory shaker (200 rpm,
20 min) in order to dissolve and remove solids. Then, the solution
RESULTS AND DISCUSSION
Physicochemical characterization of the model food waste
and microbial inocula
As shown in Table 2, the model food waste presented high values
of moisture content (70.1%), organic material (82.2%) measured

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 www.soci.org
by the proportion of VS in TS, and COD (87.5 g L−1 ). The pH was
between 5.07 and 5.30, and an elemental analysis showed a low
content of sulfur (0.23%) with a high C:N ratio of 28. Therefore, the
modelled matrix exhibited appropriate conditions of water con-
tent and nutrients for biological activity.3,32,33 However, attention
should be paid to its acidic nature that could compromise effec-
tive digestion and biogas production by methanogens for which
optimal activity occurs at a pH range of 6.5–8.2.17 Overall, physic-
ochemical properties of the model food waste were in accordance
with reported values of real and simulated matrices,3,11,10,18,22
which suggest that the Mexican diet considered in this study was
a representative and comparable food matrix.
Regarding the microbial inocula, sludge showed a higher pro-
portion of VS/TS (57.5%) compared to the landfill solid sample
(5.1%) (Table 2). However, the latter exhibited a higher TS content
(89.8%) that resulted in comparable %VS in the samples (7.0% and
4.6% for sludge and landfill solid sample, respectively). The COD
was 18.6 and 42.5 g L−1 for the landfill solid sample and sludge,
respectively. Both samples possessed a neutral pH (7.25–7.45). As
expected, the combined inoculum presented physicochemical val-
ues in the range of each individual inoculum. Because the propor-
tion of each inoculum was on a VS basis, the combined inoculum
possessed a high TS content (61.5%) as a greater amount of the
landfill solid sample was added to the mixture. As expected, sludge
was characterized as a slurry sample from a wastewater treatment
plant comparable to sludge used in other studies.1,9,34 By contrast,
a solid landfill sample from a CH4 -producing site has not been
studied as a potential inoculum, even though it can be hypothe-
sized that this type of sample possesses a microbial community
pre-adapted to food waste and produces CH4 under uncon-
trolled conditions. In situ CH4 production (c.54%) from where the
sample was taken (5 m depth) indicated the presence of active
methanogens. Reports from shallower landfill samples (1.5 m)35
or leachate36,37 agreed with this study sample characterization,
notably in COD values around 18.0 g L−1 and a slightly basic pH,
which could help neutralize the acidity of the model food waste.
Biochemical methane potential assay
Figure 1 shows CH4 production for the different microbial inocula
tested during the BMP assays at different I:S ratios. The sludge
inoculum at I:S ratios of 2 and 3 peaked in CH4 yield at Day 1 with
47.3 ± 4.2 and 39.5 ± 4.5 mL CH4 day−1 , respectively (Fig. 1(a)). At
I:S = 1, daily rates were lower but activity remained up to Day 22;
by contrast, activity stopped at Day 15 for I:S ratios of 2 and 3.
As a result, the final cumulative CH4 generated in each condition
followed the order from highest to lowest: I:S ratio 1 > 2 > 3
(Fig. 1(b)), with a maximum of 282.5 ± 18.1 mL CH4 g−1 VS for
I:S = 1. By contrast, the landfill solid sample inoculum exhibited a
lag phase of 4–8 days at all I:S ratios, with the I:S = 1 the slowest
(Fig. 1(c)). Consequently, maximum daily rates were observed from
day 7 to 15 with I:S = 2 showing the highest rate (59.3 ± 3.0 mL
CH4 day−1 ) and an activity that continued until Day 15. Hence, CH4
production followed the order I:S ratio 2 > 3 > 1, with a maximum
of 311.5 ± 11.1 mL CH4 g−1 VS (Fig. 1(d)). The combined inoculum
did not show a lag phase in either I:S ratio tested (Fig. 1(e)).
Maximum daily production occurred at days 5 and 7 for I:S ratios
of 2 and 1, respectively, and activity remained relatively high
until days 13 and 18. The highest cumulative CH4 volume was
observed at I:S = 1 with 374.5 ± 10.5 mL CH4 g−1 VS (Fig. 1(f )).
For all samples, CO2 production (Supporting Information, Fig. S1)
occurred at high rates during the first days of incubation and
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MV Sillas-Moreno et al.
resulted in cumulative volumes similar or lower to methane yields.
However, H2 production was only detected in the landfill solid
sample during the first day of incubation and in low quantities
(Supporting Information, Fig. S2).
Table 3 summarizes all parameters evaluated during the BMP
assay. Methane production was significantly higher at I:S = 1 for
sludge and the combined inoculum, whereas for the landfill solid
sample an increase in inoculum proportion up to I:S = 2 was
beneficial (Tukey’s test, P < 0.05, n = 6–9). Gas composition did
not differ significantly among I:S ratios per inoculum tested, except
for the landfill solid sample at I:S = 1 that exhibited the lowest CH4
content of all samples (32.3 ± 2.6% CH4 ). Overall, the quantity and
quality of biogas generated with the combined inoculum was the
highest from a range of 292 to 385 mL CH4 g−1 VS and a biogas
content of 57.7–62.3% CH4 . COD removal was >60% for sludge
and the landfill solid sample but the combined inoculum exhibited
values >84.5%, up to 90.5%. The final pH in all treatments was
close to the established initial value (pH 7.5), excluding landfill
solid sample I:S = 1 that showed a pH of 6.18 ± 0.14.
As expected, I:S ratio affected the performance of the micro-
bial inocula tested. At I:S = 1, all samples showed low initial
methanogenic activity but at the end produced more CH4 because
activity persisted for a longer period of time, except the for land-
fill solid sample that showed a four-fold decrease in productivity
compared to the optimal I:S = 2 (Fig. 1; Table 3). However, high
initial activity as a result of an increase in inoculum proportion
(I:S > 1) compromised CH4 production in sludge and the combined
inoculum (Fig. 1(a) and (e)). A high proportion of an inoculum that
contributes to easily available soluble substrates (as I:S was estab-
lished on a VS basis) and is rich in acetogens and methanogens
that feed on these substrates, might lead to high initial activ-
ity, rapid substrate consumption and an imbalance in the abun-
dance of hydrolytic and acidogenic bacteria, which are needed
to sustain substrate degradation.6 Concurrently, in easily degrad-
able feedstocks such as food waste, acidification could rapidly take
place by the accumulation of VFAs produced by acidogens inhibit-
ing methanogens when the buffering capacity of the system is
consumed.17 Although the pH was adjusted at the start of the
assay, a slight decrease in pH was observed in the sludge as the I:S
ratio increased (Table 3). However, if VFAs are generated at a rate
at which they could be continuously consumed by methanogens,
this condition is reverted;17 this seemed to occur by the diauxic
behaviour observed in daily CH4 yield at the optimal I:S = 1 (Fig. 1).
In batch systems, this curve typically shows high initial rates fol-
lowed by a decline and then a second raise with a final drop during
substrate exhaustion.17
In the landfill solid sample, the presence of a lag phase and a
positive response to an increase in inoculum proportion suggests
low microbial abundance of key microorganisms as methanogens
or that a higher proportion of solids that characterized this sam-
ple (Table 2) were beneficial to improve the buffering capacity
and nutrient supply of the system.25 However, at I:S = 3, higher
amounts of age-related substances such as humic acids14 might
have negatively impacted microbial activity (Fig. 1(c)). Interest-
ingly, the duration of the lag phase is consistent with the dou-
bling time of 3–6 days reported for methanogens.1,38,39 Finally, the
combined inoculum did not show a lag phase at either I:S ratio
tested and, at optimal I:S = 1, CH4 yield improved by 33% and 20%
compared to the sludge and landfill samples at the same I:S ratio,
respectively (Table 3). Also, slight increases in biogas quality (3.4%
and 23.4%) and COD removal (1.8% and 6.8%) were observed, sug-
gesting a synergetic effect between inocula.
J Chem Technol Biotechnol (2018)
Wastewater sludge and municipal landfill as inocula for food waste digestion www.soci.org

Figure 1. Daily (a,c,e) and cumulative (b,d,f ) methane production of model food waste at different I:S ratios for the three inoculum sources: sludge,
landfill solid sample and combined inoculum (1:1 sludge:landfill solid sample). Blanks: endogenous production from inoculum (only shown in cumulative
production). Graphs show the corrected values after blank subtraction.

In general, CH4 yield of the studied microbial inocula at opti-
mal I:S ratios (283–375 mL CH4 g−1 VS; Table 3) was in the middle
range of reported values of 160–580 mL CH4 g−1 VS for mesophilic
digestion of food waste using microbial inocula such as sludge,
grass silage, distillers grains and livestock dung.10,11,17,22,25 As men-
tioned previously, there are no reports of the use of solid land-
fill samples as a microbial inoculum for biogas production. Our
results showed a maximum productivity of 311.5 mL CH4 g−1 VS
at I:S = 2, which was 10.3% higher than the sludge inoculum at
optimal I:S ratio (Table 3). However, BMP showed a tendency to
increase as I:S decreased, so testing I:S < 1 might result in higher
yields. Even though less CH4 was produced, biogas quality was
slightly higher from sludge than from the landfill solid sample
(56% and 47% CH4 , respectively), suggesting that both samples
have potential as microbial inocula and are comparable at optimal
I:S ratios. Landfill leachate has been used to test agroindustrial

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Table 3. Methane (CH4 ) production, biogas CH4 content, COD removal and final pH of the model food waste with the three inoculum sources during
biochemical methane potential (BMP) assays at different I:S ratios (37 ∘ C, 25 days)
Cumulative CH4 production
Inoculum I:S ratio (mL CH4 g−1 VS)
Sludge (S) 1 282.5 ± 18.1A
2 236.8 ± 10.5B
3 202.5 ± 8.0C
Landfill solid sample (L) 1 78.8 ± 8.8C
2 311.5 ± 11.1A
3 199.0 ± 17.4B
Sludge+landfill solid sample (SL) 1 374.5 ± 10.5A
2 307.7 ± 15.7B
Values represent average and standard deviation (n = 3). Means not connected by the same letter within each inoculum are significantly different
(Tukey’s test, P < 0.05, n = 9). COD, chemical oxygen demand.
waste biodegradability with productivities of 50–314 mL CH4 g−1
VS.13,14 Although comparable at the highest value to our results,
landfill leachate might possess concentrated amounts of heavy
metals that could compromise the use of the digestate as a
by-product. The combined inoculum exhibited satisfactory results
for all performance criteria evaluated; in particular, gas composi-
tion up to 62.3% CH4 and COD removal efficiency up to 90.5%. This
is in agreement with the highest reported values in the literature
of 50–70% CH4 16,25,40 and COD removal efficiencies of up to 90%.8
Microbial community characterization
Whole microbial community
A shotgun metanogenomic approach was used to character-
ize the microbial community of each inoculum tested. After
data trimming, libraries contained 943 252, 744 525 and 1 632 227
high-quality reads for sludge, landfill solid sample and com-
bined inoculum, respectively, which were identified taxonomi-
cally at 43.7%, 61.2% and 61.3% (data not shown). Considering
unclassified reads, the Bacteria domain represented the majority of
reads in all samples (32.2–60.2%) followed by Archaea (0.4–19.6%)
that was represented mainly by the phylum Euryarchaeota (0.3%
to 19.4%). As shown in Fig. 2, the highest proportion of clas-
sified Bacteria was found in the landfill solid sample inoculum
(54.4%), whereas sludge exhibited the highest abundance of
Archaea (10.2%). The combined inoculum showed high abun-
dance of both domains (34.3% and 19.4% for Bacteria and Archaea,
respectively).
Bacterial and methanogen community at higher taxonomic levels
Bacteria members with a relative abundance ≥1% were rep-
resented by 7–11 phyla where Proteobacteria was the most
abundant in all samples (28%, 42% and 37% in sludge, landfill
solid sample and combined inoculum, respectively) followed
by Bacteroidetes (14%, 4% and 7%), Firmicutes (11%, 2% and
8%), Actinobacteria (10%, 36% and 18%), Chloroflexi (6.1%, 0.8%
and 2.3%) and Spirochaetes (2.5%, 0.1% and 3.0%) (Fig. 3(a)).
In particular, the sludge inoculum was characterized by a rich
uniform bacterial community, whereas the landfill solid sample
was dominated by Proteobacteria and Actinobacteria. A PCA plot
showed that each inoculum possessed a distinct bacterial com-
munity (Fig. 3(b)). Archaea abundance, represented mainly by
Euryarchaeota, showed that the order Methanosarcinales dom-
inated sludge and the combined inoculum with 82% and 94%
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MV Sillas-Moreno et al.
Biogas CH4 content COD removal Final
(%) (%) pH
56.1 ± 2.4A 84.3 ± 9.8A 7.25 ± 0.03A
49.3 ± 2.7A 69.1 ± 8.6A 7.19 ± 0.03A
55.8 ± 3.9A 79.8 ± 2.0A 7.17 ± 0.06A
32.3 ± 2.6B 63.1 ± 4.8B 6.18 ± 0.14B
47.0 ± 1.8A 88.4 ± 4.2A 7.32 ± 0.04A
45.4 ± 4.8A 86.8 ± 9.0A 7.30 ± 0.08A
58.0 ± 0.3A 89.9 ± 0.6A 7.34 ± 0.04A
60.1 ± 2.2A 86.4 ± 1.9B 7.31 ± 0.03A
relative abundance, respectively; whereas the landfill sample was
composed by 44% Methanosarcinales and 47% Methanomicro-
biales (Fig. 3(c)). The order Methanobacteriales represented a small
percentage in all samples (3%, 5% and 1% in sludge, landfill solid
sample and combined inoculum, respectively). The PCA analysis
of the methanogen community at the order level showed that
sludge and the combined inoculum had similar compositions
(Fig. 3(d)).
Overall, the microbial community of the sludge inoculum was
characterized by a diverse and uniform bacterial component
represented by 26% of the community (Figs 2(a) and 3(a)) and
a high proportion of Archaea [10.2%; Fig. 2(a)] that seemed to
assist CH4 production from the beginning of the experimental
period (Fig. 1(a)). The most abundant bacterial phyla in sludge
(Proteobacteria, Bacteroidetes, Firmicutes and Actinobacteria) have
been associated with anaerobic digestion of food waste.1,7,25
During the first stage of hydrolysis, Firmicutes can contribute to
digesting lipases and proteases, whereas members of Actinobac-
teria are involved in lignocellulose degradation. Subsequently,
Bacteroidetes transform amino acids into VFAs.40 Meanwhile,
members of Proteobacteria are involved in all stages of anaerobic
digestion and are essential in consumption of VFAs, avoiding irre-
versible acidification and producing substrates for acetotrophic
and hydrogenotrophic methanogens.25 By contrast, the landfill
inoculum presented a lag phase of 4–8 days but produced more
methane than sludge at optimum I:S ratio (Fig. 1(c) and (d));
although it possessed low archaeal abundance [0.3%; Fig. 2(b)], its
bacterial component was almost double that in the sludge [54%;
Fig. 2(b)]. In the landfill solid sample, the bacterial component was
mainly Proteobacteria and Actinobacteria (Fig. 3(a)), which are key
phyla in food waste degradation and VFA consumption. Taking
into account that a high percentage of the model food waste
consists of peels and bagasse of fruit and vegetables (Table 1), an
abundance of hydrolytic Actinobacteria could be crucial for diges-
tion of this type of waste. In fact, the landfill solid sample could
be responsible for improved methane production in the com-
bined inoculum by contributing these bacterial phyla (Fig. 3(a)).
In this sense, sludge could contribute Firmicutes and Chloroflexi
(Fig. 3(a)) in particular, in addition to the methanogens that were
especially high in this inoculum [10.2%; Fig. 2(a)]. Metagenomics
studies of municipal and industrial anaerobic digesters have
shown that Archaea usually represent 4–7% of total reads41 or
around 3–9% considering only classified microorganisms.20,42,43
Nevertheless, the increase of the Archaea component in the
J Chem Technol Biotechnol (2018)
Wastewater sludge and municipal landfill as inocula for food waste digestion www.soci.org

Figure 2. Taxonomic distribution of the microbial community of the three inoculum sources: sludge (a), landfill solid sample (b) and combined inoculum
(1:1 sludge:landfill solid sample) (c). Graphs show relative abundance ≥0.05%.

J Chem Technol Biotechnol (2018) © 2018 Society of Chemical Industry wileyonlinelibrary.com/jctb

 www.soci.org MV Sillas-Moreno et al.

Figure 3. Relative abundance and principal component analysis of bacterial phyla (a, b) and methanogen orders (c, d) of the three inoculum sources (S,
sludge; L, landfill solid sample; SL, sludge+landfill solid sample). Graphs show relative abundance ≥1%.

combined inoculum up to 19.4% (Fig. 2(c)) also could be
associated with a better recovery of DNA when both matrices
were combined.
Bacterial and methanogen community at the genus level
As the most promising inoculum, the bacterial composition in the
combined inoculum possessed a high diversity of genera (Sup-
porting Information, Table S2). Abundances ranged from 0.19%
to 1.74% considering the whole microbial community and unclas-
sified reads. As expected, the majority of genera belonged to the
phylum Proteobacteria, where Smithella (1.08%) and Lysobacter
(0.64%) constituted most of the reads. However, the most repre-
sented genus was Streptomyces of the phylum Actinobacteria with
an abundance of 1.74%. All other genera showed abundances that
were three- to nine-fold lower, with the phyla Bacteroidetes and
Firmicutes the least represented. Interestingly, the most frequent
members within Proteobacteria were particular to an inoculum.
Smithella (1.08%), Desulfomicrobium (0.36%), Syntrophus (0.28%),
Syntrophorabdus (0.24%), Desulfovibrio (0.23%) and Syntrophobac-
ter (0.19%) were present only in the sludge inoculum, whereas
Lysobacter (0.64%), Pseudomonas (0.32%), Rhizobium (0.23%),
Methylocaldum (0.23%) and Luteimonas (0.20%) were detected
only in the landfill solid sample. For the phyla Bacteroidetes, Firmi-
cutes and Actinobacteria, the genera were present in both inocula
with the exception of Clostridium (0.25%) and Actinomadura
(0.30%), found in sludge and the landfill solid sample, respectively.
The most frequent microbial genera found in the combined
inoculum are reported to be involved in all four stages of anaerobic
digestion. Lysobacter and Pseudomonas, also found in the landfill
solid sample inoculum, play major roles as hydrolysers,36,44
whereas Clostridium, Bacillus and Paenibacillus contributed by
both inocula are cellulolytic microorganisms.37,42,45,46 Clostridium
together with Streptomyces and Bacteroides also participate in
acidogenesis.41 In addition, Clostridium, Bacteroides and Smithella
play important roles as acetate producers in syntrophic relation-
ships with methanogens.1,24,40,47–49 Other syntrophic bacteria, only
found in the sludge inoculum, were Syntrophus, Syntrophorhabdus
and Syntrophobacter that are frequently detected in anaerobic
sludge samples.23,45,48 An adequate equilibrium between syn-
trophic bacteria and methanogens promotes CH4 production
because this relationship is essential to maintain nontoxic con-
centrations of VFAs and H2 that affect both microbial groups.50
Desulfomicrobium and Desulfovibrio are sulfate-reducing bac-
teria that play an important role in organic matter recycling
and produce H2 S.36 Rhizobium, Luteimonas and Rhodococcus
are usually found in landfills and contribute to the removal of
toxic waste products such as aromatic compounds, promoting
CH4 production.45,51 In addition, a methanotroph of the genus
Methylocaldum and pathogens such as Mycobacterium and

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Wastewater sludge and municipal landfill as inocula for food waste digestion
Figure 4. Distribution of methanogen genera by potential methanogen-
esis process of the three inoculum sources (S, sludge; L, landfill solid
sample; SL, sludge+landfill solid sample). Data represent number of reads
per genera. ND, not detected.
Nocardia were found that have not been reported to be involved
in anaerobic digestion.41,51
Methanogen genera and its related potential methanogene-
sis process are shown in Fig. 4. Most reads were assigned to
the acetotroph Methanosaeta followed by Methanosarcina, which
produces CH4 by all known pathways, both genera of the order
Methanosarcinales. These two genera were observed in all micro-
bial inocula tested. However, Methanosaeta dominated in sludge
and the combined inoculum with 61% and 90% of reads, respec-
tively. The second most abundant genus in these inocula was the
methylotroph Methanomethylovorans with an abundance of 16%
and 2%, respectively. Interestingly, these inocula contained repre-
sentatives of all methanogenesis pathways. On the contrary, the
most abundant genera in the landfill solid sample inoculum were
the hydrogenotroph Methanoculleus (20%) followed by the facul-
tative Methanosarcina (17%), with no exclusive methylotroph rep-
resentatives.
As a result, methanogenesis in the sludge and combined inocu-
lum is suggested to occur by the acetotrophic pathway with the
dominance of the genus Methanosaeta (Fig. 4). This also is sup-
ported by no detection of H2 production during the early stages
of the BMP assay (Supporting Information, Fig. S2). This pathway is
commonly reported in the digestion of food waste and municipal
wastewater sludge.1,22,25,40,42,52 The landfill inoculum was almost
equally represented by the hydrogenotroph Methanoculleus
and facultative Methanosarcina that possess a broad metabolic
capacity (Fig. 4). These genera have been described previously
in landfill leachate under the dominance of Methanoculleus.37
The abundance of Methanosaeta and Methanosarcina is reg-
ulated by the concentration of acetate.1,24,25 Methanosaeta
possess high affinity for acetate, whereas Methanosarcina exhibit
low affinity. Therefore, it seems that the sludge and landfill
solid samples presented contrasting conditions for acetoclas-
tic methanogens; sludge favoured low acetate concentrations
whereas landfill solid sample retained high concentrations. In
the combined inoculum, either genus could dominate due to
the contribution of both inocula. A metagenomic study during
CH4 generation would help elucidate which methanogenesis
J Chem Technol Biotechnol (2018) © 2018 Society of Chemical Industry
www.soci.org
pathway dominates in anaerobic digestion of the model food
waste. In addition, the more diverse microbial community in the
sludge inoculum also presented methylotroph methanogens that
could contribute to the metabolic versatility in the combined
inoculum.
Metagenomic analysis showed clear differences among the
microbial inocula tested, which is a key factor for CH4 production.
The microbial composition of the inoculum determines the time
for substrate adaptation and the delicate equilibrium that must
exist among different microbial populations for adequate biogas
production. These differences correlate to different optimum I:S
ratios for each inoculum tested. For example, in the landfill solid
sample an increase in the proportion of inoculum was beneficial
for biogas generation as this community showed low methanogen
abundance.
CONCLUSIONS
In the present study, we proposed and tested food waste modelled
to the Mexican diet as a suitable feedstock for biogas production.
Each microbial inoculum tested possessed different optimum I:S
ratios and distinct microbial compositions. The landfill solid sam-
ple, from active 1-year-old stored waste, which has not been tested
previously as inoculum, produced more CH4 than the commonly
used wastewater sludge at optimum I:S ratios but showed a lag
phase of 4 days. The combination of both inocula produced a syn-
ergistic effect in CH4 yield, biogas quality and COD removal with-
out start-up time. In addition, an enriched microbial community
(high in Archaea and Bacteria) characterized the combined inocu-
lum, probably due to the incorporation of predominant mem-
bers of each inoculum (Bacteroidetes, Firmicutes and Euryarchaeota
from sludge, and Proteobacteria and Actinobacteria from the land-
fill solid sample). Further studies on microbial population dynam-
ics during digestion could help elucidate the synergistic effect
between inocula for industrial application.
ACKNOWLEDGEMENTS
The authors would like to thank the Universidad Autonoma
Metropolitana-Iztapalapa and Promotora Ambiental S.A.B. de C.V.
for providing the inoculum samples used in this study. In addition,
they greatly appreciate the support provided in GC-TCD by Dr
Porfirio Caballero and J. Rodríguez. This work was funded by Tec-
nologico de Monterrey Research Funding Program of Energy
and Climate Change and Emerging Technologies (GIEE EICIM01)
and CONACYT Mexican National Council for Research and Tech-
nology doctoral scholarship (MVS No. 351483).
Supporting Information
Supporting information may be found in the online version of this
article.
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