Enhancing the anaerobic digestion of lignocellulose of municipal solid

waste using a microbial pretreatment method

Xufeng Yuan
1
, Boting Wen
1
, Xuguang Ma, Wanbin Zhu, Xiaofen Wang, Shaojiang Chen, Zongjun Cui

College of Agronomy and Biotechnology, Center of Biomass Engineering, China Agricultural University, Beijing 100193, China
h i g h l i g h t s
Effect of microbial pretreatment on methane production of LMSW was evaluated.
Soluble substrates in hydrolysate increased obviously after microbial pretreatment.
CH
4
production yields and rates signicantly increased after microbial pretreatment.
a r t i c l e i n f o
Article history:
Received 23 September 2013
Received in revised form 25 November 2013
Accepted 28 November 2013
Available online 12 December 2013
Keywords:
Lignocellulose of municipal solid waste
(LMSW)
Microbial consortium
Anaerobic digestion
Biological pretreatment
Hydrolysate
a b s t r a c t
The use of biological pretreatment in anaerobic digestion systems has some potential; however, to date,
these methods have not been able to effectively increase methane production of lignocellulose of muni-
cipal solid waste (LMSW). In this study a thermophilic microbial consortium (MC1) was used as a pre-
treatment method in order to enhance biogas and methane production yields. The results indicated
that sCOD concentration increased signicantly in the early stages of pretreatment. Ethanol, acetic acid,
propionic acid, and butyric acid were the predominant volatile organic products in the MC1 hydrolysate.
Biogas and methane production yields of LMSW signicantly increased following MC1 pretreatment. In
addition, the methane production rate of the treated LMSW was greater than that observed from the
untreated sample.
2013 Elsevier Ltd. All rights reserved.
1. Introduction
The quantity of municipal solid waste (MSW) generated in
China has increased by 810% per year over the past several
decades (Shi et al., 2008). For example, in 2007 alone, 150 million
tons of MSW were produced in China (Dong et al., 2010) Anaerobic
digestion (AD) is often considered one of the more economically,
and environmentally sound technologies currently used in the
treatment of MSW (Jun et al., 2009). However, use of this technol-
ogy is not without limitation, especially for MSW. Since approxi-
mately 4050% of landll space is occupied by paper and
cardboard waste (Suita et al., 1992), of which lignocellulose of
municipal solid waste (LMSW) is a signicant component (Bguin
and Aubert, 1994). In addition, the solubilisation of cellulose and
hemicellulose (both primary components of LMSW) is the rate-
limiting step during the anaerobic digestion of lignocellulose of
MSW (OSullivan and Burrell, 2007). As a result, a number of
studies have examined the use of different pretreatment methods,
in an effort to maximize LMSW digestion.
Mechanical pretreatment has been successful in reducing parti-
cle size and disrupting the crystalline structure of LMSW (Pommier
et al., 2010). Thermal and chemical pretreatments are also effective
at enhancing anaerobic digestion of LMSW (Clarkson and Xiao,
2000; Fox and Noike, 2004; Fox et al., 2003; Teghammar et al.,
2010; Xiao and Clarkson, 1997). However, these pretreatment
methods often require signicant energy inputs, and therefore
may not be the most economically and environmentally sound
technologies (Binod et al., 2010; Sun and Cheng, 2002). To remedy
this, the use of biological pretreatment is currently being explored.
Biological pretreatment, which is a safe and environmentally-
friendly method by using microorganisms, offers some conceptu-
ally important advantages such as low chemical and energy use
(Binod et al., 2010). However, to date, few biological pretreatment
methods have been demonstrated to improve methane production
of LMSW. Previous studies have shown that many pure cultures,
such as anaerobic bacteria, fungi, and actinomycetes, were effec-
tively able to degrade lignocellulose (Desvaux et al., 2000; Xu
and Goodell, 2001). The solubilization of lignocellulose occurs
0960-8524/$ - see front matter 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.biortech.2013.11.090

Corresponding author. Tel.: +86 10 62733437; fax: +86 10 62731857.

E-mail addresses: acuizj@cau.edu.cn, B08010004@cau.edu.cn (Z. Cui).
1
These authors contributed equally to this article and are joint rst authors.
Bioresource Technology 154 (2014) 19
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j our nal homepage: www. el sevi er . com/ l ocat e/ bi or t ech
naturally via the action of multiple microorganisms (Wongwilaiw-
alin et al., 2010). As such, the use of microbial consortia is often
regarded as the most likely successful approach to increasing the
methane production rate of LMSW. This is likely a result of the lack
of feedback regulation, and metabolite repression that commonly
occurs in single strain anaerobic digesters. (Haruta et al., 2002;
Soundar and Chandra, 1987). In fact, several studies have directly
demonstrated the efciency of constructed microbial consortia in
the hydrolysis of lignocellulose (Guo et al., 2011; Haruta et al.,
2002; Wongwilaiwalin et al., 2010; Yang et al., 2011). However,
to our knowledge such microbial consortia have not been directly
used in the pretreatment of LMSW. Therefore, the objective of this
present study was to develop and demonstrate a novel microbial
pretreatment method for the effective anaerobic digestion of
LMSW. To meet this objective we analyzed the effectiveness of a
thermophilic cellulose-degrading consortium (MC1) in enhancing
LMSW anaerobic digestion.
2. Methods
2.1. Materials
The lignocellulose from municipal solid waste (LMSW) was
obtained by mixing waste ofce paper, newspaper, and cardboard,
all of which were collected from a refuse collection point at the
China Agricultural University (Haidian District, Beijing City, China).
The mass-mixing ratio of ofce paper, newspaper, and cardboard
was 1:1:1. All paper waste was rst cut into 20 20 mm squares,
and oven dried at 80 C for 48 h. The lignin, cellulose, and
hemicellulose content of this waste were 14.2%, 70.1%, and 12.0%,
respectively (Table 1).
2.2. Microbial consortium and culture medium
The microbial consortium (MC1) capable of effectively degrad-
ing various cellulosic materials (e.g. lter paper, cotton and rice
straw) under aerobic static conditions was constructed via a suc-
cession of enrichment cultures as in Haruta et al. (2002). The high
stability of the consortiums degradation ability was demonstrated
by its ability to tolerate several rounds of subculture in medium
with/without cellulosic material, and being heated to 95 C or fro-
zen at 80 C (Haruta et al., 2002). MC1 was cultured in a peptone
cellulose solution (PCS) containing 1% (w/v) lter paper for three
days at 50 C, and stored at 20 C in 20% glycerol. Although
MC1 has not been fully characterized, it is known to contain
Clostridium straminisolvens CSK1, Clostridium sp. FG4b, Pseudoxan-
thomonas sp. train M1-3, Brevibacilus sp. M1-5, and Bordetella sp.
M1-6 (Kato et al., 2005).
Culture medium: The peptone cellulose solution (PCS) was com-
posed of 2 g peptone, 1 g yeast extract, 2 g CaCO
3
, 5 g NaCl, and 1 L
H
2
O (pH 8.0). All medium was autoclaved at 121 C for 20 min and
cooled prior to inoculation.
2.3. Pretreatment with the microbial consortium MC1
The primary purpose of pretreatment with MC1 was to increase
cellulose and hemicellulose availability, and thus digestibility. Pre-
viously prepared and frozen MC1 was inoculated into 125 ml ster-
ile peptone cellulose solution (PCS) with a 1% (w/v) carbon source
(lter paper), and allowed cultured at 50 C for 3 days. Following
this 3 days culture, 2, 4, 10 and 20 g of LMSW were mixed with
400 ml PCS medium (nal LMSW concentrations = 0.5%, 1.0%,
2.5% and 5.0%, respectively) and each inoculated with 20 ml of this
3-day-old MC1 culture. The ratio of inoculum to PCS culture med-
ium was 1:20 (Table 2). All mixtures were subsequently incubated
at 50 C for 14 days.
Samples were obtained at: 0 (immediately after inoculation), 1,
2, 4, 6, 8, 10, and 14 days (Table 2). The pretreatment experiment
consisted of 62 digesters. Experimental digesters (32) were sam-
pled at the eight pretreatment post-inoculation times for the four
substrate concentrations of 0.5%, 1.0%, 2.5% and 5.0%. Samples were
analyzed for soluble chemical oxygen demand (sCOD), pH, volatile
organic products (VOPs), and substrate nal weight (each mea-
surement was repeated three times). The remaining 30 digesters
were used for subsequent anaerobic digestion for only the 2.5%
and 5.0% substrate concentrations.
2.4. Anaerobic digestion
The residual LMSWs with 400 ml hydrolysates pretreated by
MC1 were respectively digested in batch anaerobic digesters at
the pretreatment times of 2, 4, 6, 8, and 10 days for the 2.5%
and 5.0% substrate concentrations (Table 2). Untreated LMSWs
with 400 ml PCS medium were used as the control. The volume
of each anaerobic digester was 1 L, with a working volume of
750 ml. Each digester was seeded with the anaerobic sludge ta-
ken from a mesophilic anaerobic digester from the Deqinyuan
Biogas Plant (Beijing, China). The sludge contained 57.2 g/l total
solids (TS), 31.5 g/l volatile solids (VS), and 39.6 g/l mixed liquor
suspended solids (MLSS). The ratio of substrate to inoculum
(anaerobic sludge) was 1:1 in each anaerobic digester. All anaer-
obic digesters were purged with N
2
for 5 min to remove O
2
, and
then sealed with a rubber stopper. Each digestion was repeated
three times at mesophilic temperature (35 C) Average values
were used in the blank test (CK) in which biogas production only
resulted from the 400 ml PCS medium, and the seeded anaerobic
sludge. The purpose of the blank test (CK) was to obtain the bio-
gas and methane yield of the 400 ml PCS medium and anaerobic
sludge alone. The biogas and methane yields of LMSW were cal-
culated as follows:
Biogas yield; ml=g VS
Biogas volume
total
Biogas volume
CK
VS of substrates added
Methane yield; ml=g VS
Methane volume
total
Methane volume
CK
VS of substrates added
Table 1
Characteristics of the substrates used in the experiments.
Parameter Ofce paper Newspaper Cardboard Mixture (LMSW)
TS (%) 95.3 0.2 93.2 0.4 95.4 0.3 94.6 0.3
VS (%TS) 98.6 0.2 96.1 0.3 87.2 0.2 94.0 0.2
Ash (%TS) 1.4 0.0 3.9 0.1 12.8 0.2 6.0 0.1
Lignin (%TS) 1.4 0.5 23.4 0.5 17.8 0.5 14.2 0.5
Cellulose (%TS) 84.9 1.3 68.5 1.1 56.9 0.8 70.1 1.1
Hemicellulose (%TS) 12.3 0.6 13.1 0.3 10.7 0.3 12.0 0.4
COD
substrate
(g O
2
/g TS) 1.07 0.02 1.21 0.03 1.10 0.03 1.13 0.03
2 X. Yuan et al. / Bioresource Technology 154 (2014) 19
2.5. COD, sCOD, pH, and volatile organic products (VOPs) of
hydrolysates
Chemical oxygen demand of substrate (COD
substrate
) before pre-
treatment, and COD of substrate residue (COD
substrate residue
) during
pretreatment were analyzed using potassium dichromate as an
oxidant using 25 mg samples previously milled into a 1 mm pow-
der (Pommier et al., 2010). The soluble chemical oxygen demand
(sCOD) of hydrolysate, and COD of PCS medium (COD
PCS medium
)
were analyzed using a COD analyzer (Model ET99731, Lovibond,
Germany) following centrifugation at 8000g for 10 min, and sam-
pling of supernatant only. During the pretreatment, the hydroly-
sate pH was recorded at 0, 1, 2, 4, 6, 8, 10, and 14 days using a
pH meter (Model B-212, Horiba, Inc., Japan).
The determination of VOPs was conducted using GC-MS. Briey,
all hydrolysate samples were ltered through an 0.22 lm aperture
and analyzed using a GCMS (Model QP-2010, Shimadzu, Japan)
on-line with a capillary column, CP-Chirasil-Dex CB
(25 0.25 mm). The analytical conditions were as described previ-
ously (Yuan et al., 2011).
2.6. Final substrate weight and lignocellulose component
Final substrate weights were determined using the hydrolysate
(including both the fermentation broth and the residual LMSW)
following centrifugation at 8000g for 10 min. The resulting precip-
itate was washed with acetic acid/nitric acid reagent followed by a
water rinse to remove non-cellulosic materials. The un-inoculated
medium served as the control. Residual LMSW nal weight was
determined as in Yuan et al., 2011). Residual LMSW components
were analyzed using a ber analyzer (Model ANKOM220, ANKOM
Technology, USA) using the methods as described in Guo et al.
(2010).
2.7. Methane analyses
Biogas volume was monitored daily using the water displace-
ment method, and the corresponding cumulative biogas volume
was calculated. Using the ideal gas law, the measured volume
was then converted to a gas volume at standard temperature and
pressure. Methane content was analyzed daily using a biogas ana-
lyzer (Biogas check, Geotech, Britain).
2.8. Microbial community analyses using PCR-DGGE
The microbial consortium (MC1) was constructed using rice
straw as the sole carbon source; it had not been directly used in
the pretreatment of LMSW. In order to verify the stability or the
changes in composition of MC1 for the LMSW pretreatment, PCR-
DGGE was used during the pretreatment period at different
substrate concentrations. Hydrolysate (7 ml) was centrifuged at
15,000g for 20 min, and total genomic DNA was extracted from
samples obtained on days 2, 4, 8, 10 and 14 of 2.5% substrate con-
centration, and also on day 6 of the four substrate concentrations
using a benzyl chloride method as in Zhu et al. (1993).
PCR amplication of the bacterial 16S rRNA gene was
performed using the GeneAmp PCR System (Model 9700, Applied
Biosystems, USA). The primers for bacterial 16S rRNA gene PCR
amplication were 357F-GC, 5
0
CCTACGGGAGGCAG CAG-3
0
(Escherichia coli positions, 341-357), which was attached to a GC-
clamp (5
0
-CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGCACGGGGG
G-3
0
) at the 5
0
-terminus, and 517R, 5
0
-ATTACCGCGGCTGCTGG-3
0
(E. coli positions, 517534) (Guo et al., 2010). Primers were pur-
chased from Sangon Biotech Co., Ltd. (Beijing, China). Initial DNA
denaturation was performed at 95 C for 10 min, followed by 30
cycles of denaturation at 93 C for 1 min, annealing at 48 C for
1 min, and elongating at 72 C for 1 min 30 s, followed by a nal
elongation step at 72 C for 5 min. The products were examined
by electrophoresis on a 2% agarose gel.
DGGE (denaturing gradient gel electrophoresis) analysis of PCR
products was carried out using the DCode Universal Mutation
Detection System (Bio-Rad Laboratories, Hercules, CA, USA) using
polyacrylamide gels with a 3560% denaturing gradient (where
100% is dened as 7 M urea with 40% formamide) (Yuan et al.,
2011). Gels were run at a constant voltage of 200 V and tempera-
ture of 61 C for 5 h in 0.5 TAE electrophoresis buffer. Following
electrophoresis, gels were stained with SYBR Green I (Molecular
Probes, Eugene, OR, USA) and photographed under UV (302 nm)
using the Alpha Imager 2200 Imaging System (Alpha Innotech,
USA). The images and UPGMA cluster were analyzed using Quan-
tity One Software (Bio-Rad, USA). The DNA was recovered, and
re-amplied with the primers 357F (5
0
-CCTACGGGAGGCAGCAG-
3
0
) and 517R (5
0
-ATTACCGCGG CTGCTGG-3
0
) (Haruta et al., 2002)
as described above. Amplied fragments were puried using the
high purity PCR product purication kit (Tiangen Biotech Co.,
LTD, China) and sequenced using the ABI 3730XL DNA Sequencer
(Perkin Elmer) at SunBiotech Developing Center. Sequence similar-
ity searches were performed in the GenBank data library using the
BLAST Program.
2.9. Quantitative PCR of different groups of methanogens
Sludge samples were collected from the seed sludge of both
treated and untreated digesters, 4-days pretreated and 10-days
pretreated LMSW of 2.5% substrate concentration after 40 days
anaerobic digestion. Samples were centrifuged at 8000 r/min for
10 min, and the supernatant was decanted to obtain the sediment
samples (0.3 g net weight) for subsequent DNA extraction. The VS
concentration of each sediment sample was used to estimate the
amount of biomass used for DNA extraction. Genomic DNA was
extracted using an automated nucleic acid extractor (Bioteke Bio-
tech Co., Ltd., Beijing, China) and used as the PCR template.
For quantitative real-time PCR, the following specic primer
sets and 5
0
-nuclease probes (TaqMan) were used: Msc (Methano-
sarcinaceae; Msc380F, Msc492F, Msc828R; amplicon size:
408 bp); Mst (Methanosaetaceae; Mst702F, Mst753F, Mst862R;
amplicon size: 164 bp); MBT (Methanobacteriales; MBT857F,
Table 2
Experiments Settings of pretreatment and anaerobic digestion.
Substrate
concentration
(%)
Pretreatment Anaerobic digestion
LMSW
(g)
PCS
medium
(ml)
Inoculum
(ml)
Sample point
(day)
Substrate The Ratio of
substrate to
inoculum
0.5 2 400 20 0, 1, 2, 4, 6, 8, 10, 14
1.0 4 400 20 0, 1, 2, 4, 6, 8, 10, 14
2.5 10 400 20 0, 1, 2, 4, 6, 8, 10, 14 2, 4, 6, 8, and 10 days treated LMSW and untreated LMSW 1:1
5.0 20 400 20 0, 1, 2, 4, 6, 8, 10, 14 2, 4, 6, 8, and 10 days treated LMSW and untreated LMSW 1:1
X. Yuan et al. / Bioresource Technology 154 (2014) 19 3
MBT929F, MBT1196R; amplicon size: 343 bp); and MMB (Methan-
omicrobiales; MMB282F, MMB749F, MMB832R; amplicon size:
506 bp) (Zhang et al., 2011). The TaqMan probes were labeled with
the FAM (reporter), and BHQ-1 (quencher). Quantitative PCR
(Q-PCR) reactions were performed using a ABI 7500 system (Model
7500, Applied Biosystems, USA). The Q-PCR mixture (20 lL) was
prepared using the 2 TaqMan Universal PCR Master mix (Applied
Biosystems, USA): 5 lL of PCR-grade water, 1 lL of each primer
(nal concentration, 10 lM), 2 lL of the TaqMan probe (nal
concentration, 1 lM), 10 lL of 2 reaction solution, and 1 lL of
template DNA. The two-step amplication protocol was performed
as follows: denaturation for 10 min at 94 C, followed by 40 cycles
of 10 s at 94 C, and combined annealing and extension for 30 s at
60 C (63 C was used for only primer set MMB).
To generate standards for real-time PCR, genomic DNA was
extracted from ve species of the Archaea genera Methanosarcina-
ceae (Methanosarcina acetivorans NBRC100939), Methanosaetaceae
(Methanosaeta thermophila NCBR101360), Methanobacteriales
(Methanobrevibacter arboriphilus NBRC101200), Methanomicrobi-
ales (Methanospirillum hungatei NBRC100397) provided by the
NITE Biological Research Center (NBRC, Chiba, Japan). The target
rRNA gene sequences were amplied from each strain using con-
ventional PCR with the corresponding primer sets as described
above, and cloned into the pGEM-T Easy Vector following purica-
tion with the TIANgel Midi Purication Kit. For each plasmid, a
10-fold serial dilution ranging from 10
2
to 10
9
copies per lL was
used as a standard for all real-time PCR assays. The 16S rRNA gene
copy concentrations of target groups were estimated against the
corresponding standard curves within the linear range. The
volume-based concentration (copies/lL) was then converted to
the granule biomass-based concentration (copies/g granule VS)
using the VS concentration of each granular sludge sample previ-
ously used for DNA extraction. All DNA samples were analyzed
with each primer/probe set in duplicate.
2.10. Chemical composition
The TS, VS, and MLSS of the LMSW, ofce paper, newspaper,
cardboard, anaerobic sludge, and their mixture were all measured
according to APHA standard methods (APHA, 1998).
3. Results and discussion
3.1. Changes in hydrolysate pH during pretreatment
The pH of MC1 hydrolysates for the four substrate concentra-
tions of 0.5%, 1.0%, 2.5% and 5.0% all declined signicantly before
day 2 (Fig. 1A). The lowest pH values occurred on day 2, day 4,
day 6 and day 8 for the four substrate concentrations 0.5%, 1.0%,
2.5% and 5.0%, respectively. The lowest pH values of the MC1
hydrolysates were as follows: 0.5% (6.5) > 1.0% (6.1) > 2.5%
(5.8) > 5.0% (5.4). The pH for the three lower substrate concentra-
tions of 0.5%, 1.0% and 2.5% all increased rapidly after day 6, and
reached at 8.8, 8.1, and 7.2 respectively following pretreatment.
However, the lowest pH observed in the 5.0% substrate occurred
on day 8, but then stabilized at between 5.4 and 5.5. This observed
change in pH is consistent with observed by Liu et al. (2006), and
suggest that pH of the MC1 was able to recover when the substrate
concentration was not more than 2.5%. The autorecovery of the
hydrolysate pH is due to the presence of acidophilus strains in
MC1 (Kato et al., 2005), which prevents pH from declining too
rapidly to inhibit the degrading activities of the bacteria at the
lower substrate concentrations. When substrate concentration
was above 5.0%, the lowest pH (5.4) was observed on day 8, and
remained at this low level for the remainder of the pretreatment
periods. This may be an indication that microbial activity was
inhibited by imbalance in pH late in the pretreatment process at
higher substrate concentration (Juhasz et al., 2004).
3.2. Changes in hydrolysate sCOD and volatile organic products (VOPs)
during pretreatment
Similar trends in the sCOD concentrations were observed at the
0.5%, 1.0% and 2.5% concentrations (Fig. 1B). The sCOD concentra-
tions all increased rapidly in the early stages of pretreatment at
the lower substrate concentrations of 0.5%, 1.0% and 2.5%. There
was a rapid decrease of sCOD after day 4 for the 0.5%, 1.0% and
2.5% substrate concentrations. Kato et al. (2005) demonstrated that
there are both hydrolytic microbe and fermentative microbe with-
in MC1. Clostridium straminisolvens CSK1 is regarded as the main
hydrolytic microbe within MC1, and Pseudoxanthomonas sp. train
M1-3, Brevibacilus sp. M1-5 are the main fermentative microbes.
This above phenomenon of sCOD might be due to soluble organic
products being generated by hydrolytic microbes within MC1,
but at the same time consumed by fermentative microbes. Prior
to day 4, these fermentative microbes were also consuming soluble
products but at a lower rate than the production by hydrolytic ones
(Yuan et al., 2011, 2012). However, at the substrate concentration
of 5.0%, the sCOD concentration peaked on day 8, and then
remained stable for the remainder of the pretreatment. This likely
indicated a balance was reached between soluble organic produc-
tion, and consumption. It was also found that if the higher the
substrate contents, the higher the peak values of the sCOD concen-
trations, and the later the peak values of the sCOD concentrations
appeared.
Ethanol, acetic acid, propionic acid, and butyric acid were the
predominant VOPs in the MC1 hydrolysates at the four substrate
concentrations of 0.5%, 1.0%, 2.5% and 5.0% during pretreatment
(Fig. 2). At 0.5%, 1.0% and 2.5% concentrations, the concentration
of these four VOPs all increased in the early stages, followed by a
gradual decline. Peak VOPs concentration occurred on day 4
(2.23 g/l), day 4 (3.64 g/l), and day 6 (6.75 g/l) respectively at the
lower substrate concentrations of 0.5%, 1.0% and 2.5%; then the
total VOPs decreased at 0.79, 0.92, and 2.84 g/l respectively on
day 14. The peak VOPs of 5.0% substrate concentrations occurred
on day 10 (8.95 g/l), and then no signicant change of the total
VOPs occurred at the remainder of the pretreatment.
The peak value of ethanol all occurred on day 4 at 0.5%, 1.0% and
2.5% substrate concentrations, and then the concentration of etha-
nol decreased sharply (Fig. 2AC). However, at 5% substrate con-
centration, the ethanol concentration reached 3.87 g/l on day 6
and no signicant change occurred between day 6 and day 14
(Fig. 2D). In addition, the higher the substrate concentration during
pretreatment, the later the peak value of acetic acid appeared. The
acetic acid concentrations peaked at day 4 (0.68 g/l), day 4 (0.96 g/
l), day 6 (1.85 g/l) and day 10 (2.37 g/l) respectively at 0.5%, 1.0%,
2.5% and 5.0% concentrations. It is worth mentioning that the con-
centration of propionic acid was markedly lower than that of the
other three VOPs during pretreatment at all substrate contents. A
previous research has shown that the high concentration of propi-
onic acid was detrimental to subsequent methane fermentation.
This was because propionate-assimilating microbes were among
the slowest growing, due to low free-energy gain from conversion
of propionate to acetate, and the complicated syntrophic relation
to hydrogen-utilizing methanogens (Yuan et al., 2012). During
the anaerobic digestion process, ethanol, acetic acid, and butyric
acid can be easier to use than propionic acid, because acetic acid
can be directly used by methanogens, and the acetogenic rate of
ethanol and butyric acid is higher than that of propionic acid
(Ren et al., 1997). Conversion of lignocellulose to soluble products
was regarded as the rate-limiting step during the anaerobic
4 X. Yuan et al. / Bioresource Technology 154 (2014) 19
digestion of lignocellulose. So from this perspective, the optimal
pretreatment time should equal the time at which the sCOD, or
volatile organic product concentration reaches a maximum. This
requires the consumption of soluble organic material be mini-
mized during pretreatment by microbial consortium.
A comparison of Fig. 2 with Fig. 1A showed that the production
of the three volatile fatty acids (VFAs) of 0.5%, 1.0%, 2.5% and 5.0%
substrate concentrations followed the same trend as the pH as a
function of time. Prior to day 4, the hydrolysates pH of 2.5% and
5.0% substrate concentrations declined more rapidly than that of
0.5% and 1.0% substrate concentrations (Fig. 1A); simultaneously,
the VFAs of 2.5% and 5.0% substrate concentrations increased more
sharply than that of 0.5% and 1.0% substrate concentrations (Fig. 2).
The pH values for the three lower substrate concentrations of 0.5%,
1.0% and 2.5% all increased rapidly after day 6; simultaneously, the
VFAs of 0.5%, 1.0% and 2.5% substrate concentrations all decreased
signicantly. In addition, the pH of 5.0% substrate concentration
stabilized at between 5.4 and 5.5 between day 8 and day 14; simul-
taneously, no signicant change of the total VFAs occurred after
day 8. This indicated that the VFA production was the direct cause
of the pH evolution.
3.3. COD balance analysis during pretreatment
In order to minimize consumption of soluble organic materials
during pretreatment (so they remain available for methane pro-
duction) organic material loss (COD
loss
) and the rate of COD loss
(Loss ratio) were monitored (Table 3). Prior to pretreatment, the
initial COD in system was calculated as the sum of COD
substrate
and COD
PCS medium
, which was also equal to the sum of sCOD in
hydrolysate, COD
substrate residue
and COD
loss
during pretreatment.
Therefore, the COD
loss
was calculated as follows:
COD
loss
COD
substrate
COD
PCS medium
COD
substrate residue
sCOD
hydrolysate

The loss ratio of organic materials was calculated as follows:

Loss Ratio % COD
loss
=COD
substrate
COD
PCS medium
100%
Greater organic material loss occurred at each substrate con-
centration as the pretreatment time was extended. For the three
lower substrate concentrations of 0.5%, 1.0% and 2.5%, the higher
substrate contents, the more COD loss. At the end of pretreatment,
the COD
loss
varied in the following order: 2.5% (15,580 mg/L) > 1.0%
(11,132 mg/L) > 0.5% (7036 mg/L). However, the COD
loss
of 5.0%
substrate concentration was lower than that of 2.5% substrate
concentration after day8. This phenomenon was consistent with
changes of sCOD, which stabilized between 15,680 and 15,750
mg/L after day 8 at 5.0% substrate concentration. This might be be-
cause the pH of the hydrolysate at 5.0% substrate concentration
was between 5.5 and 5.6 after day 8; the degradation ability of mi-
crobes could be inhibited at lower pH. Therefore, there were no
obvious changes. It was also found that the higher substrate con-
centrations, the lower loss ratio. At the end of pretreatment, loss
ratio varied in the following: 0.5% (74.4%) > 1.0% (73.7%) > 2.5%
(48.6%) > 5.0% (19.5%).
3.4. Change in nal weight of total dry matter, hemicellulose, cellulose
and lignin
Final weight losses of dry matter, cellulose, hemicellulose, and
lignin of LMSW were monitored during the 14-day pretreatment
process. For the four substrate contents of 0.5%, 1.0%, 2.5% and
5.0%, the weight losses of LMSW dry matter were 86.8%, 76.9%,
44.9% and 25.0% on Day 4 respectively; at the end of pretreatment,
the nal weight losses were 91.2%, 89.7%, 58.6% and 40.1%, respec-
tively (Fig. 3). This was indicated that the total dry matter of LMSW
was degraded most expeditiously in the rst 4 days. This phenom-
enon was consistent with changes of pH values that declined rap-
idly before Day 4. After Day 8, LMSW was degraded more slowly,
and the pH became neutral at the lower substrate concentrations,
or became stable between 5.4 and 5.5 at the substrate concentra-
tion of 5.0% simultaneously.
Cellulose, hemicellulose, and lignin are the main components of
LMSW; they are also the main carbon sources for anaerobic micro-
organisms. The availability and digestibility of cellulose and hemi-
cellulose, as well as the association of lignin with carbohydrates
signicantly affected methane production. At the end of pretreat-
ment, cellulose, hemicellulose and lignin were all degraded signif-
icantly by MC1 at each substrate concentrations (Table 4). It was
also found that the MC1 degraded cellulose more strongly than it
did hemicellulose and lignin. Previous studies on the biological
pretreatment of lignocellulosic materials mainly focused on the
pure culture of fungi and bacteria (Cheng et al., 2012; Guo et al.,
2010). However, the pure culture of fungi and bacteria was rarely
applied to the pretreatment in a large scale of biogas production;
this might be because that the degradation activity and ability of
pure culture were generally limited. The pure-culture isolates only
degraded the substrates with a relatively simple structure and
composition, such as articial xylan and pure cellulose (Desvaux
5.0
5.5
6.0
6.5
7.0
7.5
8.0
8.5
9.0
9.5
0 2 4 6 8 10 12 14
Pretreatment time (day)
p
H
0.5%
1.0%
2.5%
5.0%
0
4000
8000
12000
16000
20000
24000
0 2 4 6 8 10 12 14
Pretreatment time (day)
s
C
O
D

(
m
g
/
l
)
0.5%
1.0%
2.5%
5.0%
A B
Fig. 1. Changes in hydrolysate pH and sCOD during pretreatment. (A) The pH of hydrolysate; (B) The sCOD of hydrolysate.
X. Yuan et al. / Bioresource Technology 154 (2014) 19 5
et al., 2000). However, they were unable to use natural lignocellu-
loses efciently.
3.5. Analysis of microbial consortium during pretreatment
During pretreatment, the changes in composition of MC1 were
showed by the change in band pattern (Fig. 4A). The microbial com-
munity composition was consistent with previous results (Haruta
et al., 2002). Band 1 was associated with LMSWduring the pretreat-
ment process, which was 100% similar to Clostridium thermosuccin-
ogenes. This bacterium could utilize cellobiose, xylose, glucose and
sucrose, and produce acetate, lactate, and H
2
(Haruta et al., 2002).
The strains of genetic relationship represented by other ve DGGE
bands were 2: Uncultured beta proteobacterium-WkB04 (96.7%),
3: Brevibacillus.sp.Riau (94.2%), 4: Uncultured Brevibacillus.sp.-
KL-13-4-10 (100%), 5: Brevibacillus.sp.Riau (99.4%) and 6: Pseudox-
anthomounas taiwanenis (100%). This data indicated a strong
structural stability of MC1 during the pretreatment period of differ-
ent lignocellulose materials, but the number of each bacteriummay
differ during pretreatment. The four lanes of Fig. 4B showed the
microbial community composition of MC1 at the four substrate
concentrations of 0.5%, 1.0%, 2.5% and 5.0% on day 6. This also indi-
cated that the consortium MC1 was stable at different substrate
concentrations during the pretreatment.
3.6. Anaerobic digestion
In order to determine the optimal pretreatment time, LMSW of
2.5% and 5.0% substrate concentrations after the 2, 4, 6, 8, and
10 days pretreatment were used for subsequent anaerobic diges-
tion. The treated LMSW samples yielded more biogas than the
untreated samples (Fig. 5A). The biogas yields for treated LMSW
of 2.5% substrate concentration were 342, 404, 379, 315, and
209 ml/g VS, at the pretreatment times of 2, 4, 6, 8, and 10 d
respectively. These values were 73.6%, 105.1%, 92.4%, 59.9%, and
6.1% higher than those of the untreated sample yields. Biogas
yields from the 5.0% substrate concentration were 278, 347, 386,
419, and 362 ml/g VS at the pretreatment times of 2, 4, 6, 8, and
10 d respectively. These values were 55.3%, 93.9%, 115.6%,
134.1%, and 102.2% higher than those of the untreated sample
yields. This result shows that MC1 pretreatment is capable of
signicantly enhancing the biogas yields of certain LMSWs.
Energy contained in the biogas was determined by both biogas
volume and methane content. Methane content was measured
during anaerobic digestion (Fig. 5C). It was found that there were
no obvious changes of methane contents between the untreated
and treated LMSW by using MC1 at 2.5% substrate concentration.
Methane yields from the 2.5% substrate concentration were 184,
221, 209, 164, and 106 ml/g VS, respectively, at the pretreatment
times of 2, 4, 6, 8, and 10 d. These yield values were 87.8%,
125.5%, 113.3%, 67.3%, and 8.2% higher than those of the untreated
sample yields (Fig. 5B). This indicated that all treated LMSW sam-
ples produced more methane than did the untreated LMSW sam-
ples. This also suggested that pretreatment by MC1 was capable
of enhancing not only biogas yield, but also energy gain from the
samples.
Maximumbiogas and methane yields at 2.5% and 5.0% substrate
concentrations occurred after 4 and 8 days pretreatment
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
2 4 6 8 10 12 14
Pretreatment time (day)
C
o
n
c
e
n
t
r
a
t
i
o
n

o
f

V
O
P
s

(
g
/
l
)
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
2 4 6 8 10 12 14
Pretreatment time (day)
C
o
n
c
e
n
t
r
a
t
i
o
n

o
f

V
O
P
s

(
g
/
l
)
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
2 4 6 8 10 12 14
Pretreatment time (day)
C
o
n
c
e
n
t
r
a
t
i
o
n

o
f

V
O
P
s

(
g
/
l
)
Ethanol
Acetic acid
Propionic acid
Butyric acid
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
2 4 6 8 10 12 14
Pretreatment time (day)
C
o
n
c
e
n
t
r
a
t
i
o
n

o
f

V
O
P
s

(
g
/
l
)
A B
C D
Fig. 2. Quantitative analysis of major volatile organic products (VOPs) by GCMS during pretreatment. (A) 0.5% substrate concentration; (B) 1.0% substrate concentration;
(C) 2.5% substrate concentration; (D) 5.0% substrate concentration.
6 X. Yuan et al. / Bioresource Technology 154 (2014) 19
respectively. This was also the same time when sCOD of the
hydrolysates reached its maximum; it was not the time when
the TVOPs reached a maximum (Figs. 1B, 2, 5A and B). The higher
sCOD
concentration indicates that there are more soluble substrates in
the hydrolysates, and these are more available for subsequent
anaerobic digestion than lignocelluloses. Otherwise, the high
Table 3
COD balance during pretreatment.
Substrate
concentration
(%)
Pretreatment
time (days)
Before pretreatment During pretreatment
COD
substrate
(mg/L)
COD
PCS medium
(mg/L)
sCOD
hydrolysate
(mg/L)
COD
substrate residue
(mg/L)
COD
loss
(mg/L)
Loss ratio
(%)
0.5 0 5650 3810 3810 5650 0 0.0
1 5650 3810 5433 3250 777 8.2
2 5650 3810 6320 1660 1480 15.6
4 5650 3810 5990 644 2826 29.9
6 5650 3810 4767 479 4214 44.5
8 5650 3810 3510 345 5605 59.2
10 5650 3810 2610 372 6478 68.5
14 5650 3810 2123 301 7036 74.4
1.0 0 11,300 3810 3810 11,300 0 0.0
1 11,300 3810 5940 8220 950 6.3
2 11,300 3810 7750 5520 1840 12.2
4 11,300 3810 9010 2375 3725 24.7
6 11,300 3810 7310 1710 6090 40.3
8 11,300 3810 6730 1240 7140 47.3
10 11,300 3810 4927 1070 9113 60.3
14 11,300 3810 2853 1125 11,132 73.7
2.5 0 28,250 3810 3810 28,250 0 0.0
1 28,250 3810 7207 23,640 1213 3.8
2 28,250 3810 9533 20,315 2212 6.9
4 28,250 3810 12,253 15,720 4087 12.7
6 28,250 3810 11,270 13,190 7600 23.7
8 28,250 3810 8283 12,650 11,127 34.7
10 28,250 3810 6320 12,050 13,690 42.7
14 28,250 3810 4740 11,740 15,580 48.6
5.0 0 56,500 3810 3810 56,500 0 0.0
1 56,500 3810 7110 51,740 1460 2.4
2 56,500 3810 10,937 46,520 2853 4.7
4 56,500 3810 13,140 42,230 4940 8.2
6 56,500 3810 15,110 36,980 8220 13.6
8 56,500 3810 16,137 34,070 10,103 16.8
10 56,500 3810 15,683 33,500 11,127 18.4
14 56,500 3810 15,750 32,780 11,780 19.5
0.0
10.0
20.0
30.0
40.0
50.0
60.0
70.0
80.0
90.0
100.0
0 2 4 6 8 10 12 14
Pretreatment time (days)
W
e
i
g
h
t

l
o
s
s

(
%
)
0.5%
1.0%
2.5%
5.0%
Fig. 3. Dynamics of the dry matter weight loss of LMSW during pretreatment.
Table 4
Weight loss of hemicellulose, cellulose and lignin of LMSW at the end of
pretreatment.
Substrate concentration (%) Hemicellulose (%) Cellulose (%) Lignin (%)
0.5 91.8 2.6 97.5 0.7 66.4 3.1
1.0 92.3 4.2 96.7 1.4 55.2 1.2
2.5 45.7 2.4 73.5 3.7 23.9 1.5
5.0 37.7 2.1 50.4 2.7 12.8 0.3
1
Day
2
Day
4
Day
8
Day
10
Day
14
4
3
2
5
6
A
0.5% 1.0% 2.5% 5.0%
B
Fig. 4. DGGE prole of the 16S rDNA fragments of MC1 during pretreatment.
(A) DGGE prole during pretreatment at 2.5% substrate concentration; (B) DGGE
prole of the four substrate concentrations on day 6 during MC1 pretreatment.
X. Yuan et al. / Bioresource Technology 154 (2014) 19 7
concentration of TVOPs was based on a longer pretreatment, which
resulted in a part of soluble substrates being transformed into
VOPs and CO
2
by MC1, leaving less carbon for subsequent anaero-
bic digestion. The 2-d pretreated LMSW of 2.5% substrate concen-
tration achieved lower biogas and methane yields than the 4-d
pretreated sample. This might be because the short pretreatment
time could not effectively degrade the lignocellulose into soluble
substrates.
In addition, the methane production rate of the treated LMSW
was obviously faster than that of the untreated sample. The meth-
ane yields of treated LMSW of 2.5% substrate concentration during
the former 8 days of anaerobic digestion were 93, 155, 136, 108,
and 77 ml/g VS respectively, at the pretreatment times of 2, 4, 6,
8, and 10 d. These yield values were 2.51, 4.19, 3.68, 2.92, and
2.08 times greater than the values obtained by the untreated sam-
ple yields on corresponding days (Fig. 5B). The signicant increase
in the methane production rate further indicated that the LMSW
had become more readily biodegradable following pretreatment.
Moreover, a rapid methane production rate means a short diges-
tion time. This could be of signicant economic benet through
the increase of methane production efciency or via the treatment
capacity of one existing digester that uses a shortened digestion
time (Zheng et al., 2009).
The real-time PCR results showed clear changes in the quantita-
tive composition of the methanogenic community in the anaerobic
digester with the untreated, 4-d pretreated and 10-d pretreated
LMSW of 2.5% substrate concentration (Fig. 5D). Within the seed
sludge, the Methanosaetaceae (53.0%) and Methanomicrobiales
(31.3%) were the dominant methanogenic families. This is consis-
tent with earlier study of high abundance of Methanosaetaceae-re-
lated and Methanomicrobiales-related species in stable anaerobic
digesters (Zhang et al., 2011). After the 40 days digestion, the 16S
rRNA levels of methangens and dominant methangens were all
changed by different degrees comparing with the seed sludge.
Within the digesters treating untreated and 10-d pretreated
LMSW, 16S rRNA levels of methangens decreased 28.0% and
35.3% than that in the seed sludge; Methanomicrobiales became
dominant methanogenic population, and respectively accounted
for 50.2% and 40.3% of the measured methanogenic population.
This might be because that hydrolysis rate of untreated LMSW
was lower under anaerobic condition, and the main volatile fatty
acids were consumed by consortium MC1 with 10-d pretreatment.
Therefore the methangens cannot get enough of volatile fatty acids
for growth when treating untreated and 10-d pretreated LMSW.
Within the digester treating 4-d pretreated LMSW, the dominant
methangens (Methanosaetaceae) sharply increased 212.0% than
that in the seed sludge. This corresponds well to the fact that
aceticlastic methanogen favors high organic acids levels environ-
ment (Zhang et al., 2011). The real-time PCR results were consis-
tent with the results of biogas yields and methane yields.
Previous research concerning the anaerobic digestion of LMSW
has mainly focused on different pretreatment methods and
co-digestion. Pommier et al. (2010) showed that shredding did
not improve the methane potential or the methane production
rates of waste paper and cardboard. Xiao and Clarkson (1997)
showed that the addition of nitric acid during acetic acid pretreat-
ment had a tremendous effect on the solubilization of lignin of
newspaper. Further, the pretreatment signicantly increased
methane production. Teghammar et al. (2010) found that explosive
pretreatment with sodium hydroxide improved the methane yield
of paper tubes by 70107%, from 238 to 403493 N ml/g VS.
Further, the methane production rate was increased by 68132%.
Recently, anaerobic co-digestion of organic wastes for methane
production has attracted more interest. There are some poorly
0
50
100
150
200
250
300
350
400
450
500
u
n
t
r
e
a
t
e
d
2
d
4

d
6

d
8

d
1
0

d
B
i
o
g
a
s

y
i
e
l
d

(
m
l
/
g

V
S
)
2.50%
5.00%
0
50
100
150
200
250
u
n
t
r
e
a
t
e
d
2

d
4

d
6

d
8

d
1
0

d
M
e
t
h
a
n
e

y
i
e
l
d

(
m
l

C
H
4
/
g

V
S
)
30 d
8 d
0
10
20
30
40
50
60
u
n
t
r
e
a
t
e
d
2
d
4

d
6

d
8

d
1
0

d
M
e
t
h
a
n
e

c
o
n
t
e
n
t

(
%
)
A B
C
D
Fig. 5. Biogas yield, methane yield, methane content and quantitative changes in the 16S rRNA gene concentrations of methanogenic groups during anaerobic digestion.
(A) Biogas yield of treated and untreated LMSW at 2.5% and 5.0% substrate concentrations; (B) Methane yield of treated and untreated LMSW at 2.5% substrate concentration;
(C) Methane content of treated and untreated LMSW at 2.5% substrate concentration; (D) Quantitative changes in the 16S rRNA gene concentrations of methanogenic groups
during anaerobic digestion.
8 X. Yuan et al. / Bioresource Technology 154 (2014) 19
biodegradable organic wastes that cannot be digested alone due to
low solubility or unbalanced carbon to nitrogen ratios. Waste
paper is an example of one of these poorly biodegradable organic
wastes. Yen and Brune (2007) found that adding 50% of waste
paper in algal sludge feedstocks increased the methane production
rate from 573 28 ml/l day (algal sludge digestion alone) to
1170 75 ml/l day. Other research has also proven that anaerobic
co-digestion could effectively improve the methane production of
waste paper (Yusuf and Ify, 2011). Biological pretreatment is
regarded as an eco-friendly method for lignocellulose of anaerobic
digestion. But to date, there is no efcient biological pretreatment
used to pretreat LMSW for anaerobic digestion. In the present
study, a microbial consortium was used for pretreatment of ligno-
cellulose of MSW. The results of anaerobic digestion indicated that
LMSW methane production yields and rates signicantly increased
after microbial consortium pretreatment.
Previous studies on the microbial pretreatment of lignocellu-
loses mainly focused on the pure culture of microorganisms
(Desvaux et al., 2000; Xu and Goodell, 2001). However, the pure
culture of microorganisms is difcult to culture in an open system.
Our previous research indicated that MC1 can be culture continu-
ously and stably for 2 months or more in an open system (Liu et al.,
2006). This characteristic is a good basis for further culture in large
scale. In addition, such large scale experiments (three tons) are
now under way in our laboratory.
4. Conclusion
Pretreatment with the microbial consortium MC1 proved to be
efcient in improving biodegradability and enhancing methane
production from LMSW. Simultaneously, the methane production
rate was obviously faster in the treated LMSW than in the
untreated LMSW. MC1 increased the sCOD concentration of the
hydrolysates of LMSW. It also produced volatile organic products
that could be directly used in the subsequent anaerobic fermenta-
tion stage. This also decreased cellulose concentration and hemi-
cellulose concentration within the LMSW. Maximum biogas and
methane yields occurred at the time when hydrolysate sCOD
reached its maximum.
Acknowledgements
This work was supported by the National Key Technology R&D
Program of China (No. 2012BAD14B06), the National 863 Program
of China (2012AA101803) and the Special Fund for Agro-scientic
Research in the Public Interest (No. 201303080).
References
APHA, 1998. Standard methods for the examination of water and wastewater.
American Public Health Association, Washington DC, USA.
Bguin, P., Aubert, J.P., 1994. The biological degradation of cellulose. FEMS
Microbiol. Rev. 13, 2558.
Binod, P., Sindhu, R., Singhania, R.R., Vikram, S., Devi, L., Nagalakshmi, S., Kurien, N.,
Sukumaran, R.K., Pandey, A., 2010. Bioethanol production from rice straw: an
overview. Bioresour. Technol. 101, 47674774.
Clarkson, W.W., Xiao, W., 2000. Bench-scale anaerobic bioconversion of newsprint
and ofce paper. Water Sci. Technol., 93100.
Cheng, X., Li, Q., Liu, C., 2012. Coproduction of hydrogen and methane via anaerobic
fermentation of cornstalk waste in continuous stirred tank reactor integrated
with up-ow anaerobic sludge bed. Bioresour. Technol. 114, 327333.
Desvaux, M., Guedon, E., Petitdemange, H., 2000. Cellulose catabolism by
Clostridium cellulolyticum growing in batch culture on dened medium.
Appl. Environ. Microbiol. 66, 24612470.
Dong, L., Zhenhong, Y., Yongming, S., 2010. Semi-dry mesophilic anaerobic digestion
of water sorted organic fraction of municipal solid waste (WS-OFMSW).
Bioresour. Technol. 101, 27222728.
Fox, M., Noike, T., 2004. Wet oxidation pretreatment for the increase in anaerobic
biodegradability of newspaper waste. Bioresour. Technol. 91, 273281.
Fox, M., Noike, T., Ohki, T., 2003. Alkaline subcritical-water treatment and alkaline
heat treatment for the increase in biodegradability of newsprint waste. Water
Sci. Technol. 48, 7784.
Guo, P., Mochidzuki, K., Zhang, D., Wang, H., Zheng, D., Wang, X., Cui, Z., 2011.
Effects of different pretreatment strategies on corn stalk acidogenic
fermentation using a microbial consortium. Bioresour. Technol. 102, 7526
7531.
Guo, P., Zhu, W., Wang, H., Lv, Y., Wang, X., Zheng, D., Cui, Z., 2010. Functional
characteristics and diversity of a novel lignocelluloses degrading composite
microbial system with high xylanase activity. J. Microbial. Biotechnol. 20, 254
264.
Haruta, S., Cui, Z., Huang, Z., Li, M., Ishii, M., Igarashi, Y., 2002. Construction of a
stable microbial community with high cellulose-degradation ability. Appl.
Microbiol. Biotechnol. 59, 529534.
Juhasz, T., Szengyel, Z., Szijarto, N., Reczey, K., 2004. Effect of pH on cellulase
production of Trichoderma ressei RUT C30. Appl. Biochem. Biotechnol. 113, 201
211.
Jun, D., Yong-sheng, Z., Mei, H., Wei-Hong, Z., 2009. Inuence of alkalinity on the
stabilization of municipal solid waste in anaerobic simulated bioreactor. J.
Hazard. Mater. 163, 717722.
Kato, S., Haruta, S., Cui, Z.J., Ishii, M., Igarashi, Y., 2005. Stable coexistence of ve
bacterial strains as a cellulose-degrading community. Appl. Environ. Microbiol.
71, 70997106.
Liu, J., Wang, W., Yang, H., Wang, X., Gao, L., Cui, Z., 2006. Process of rice straw
degradation and dynamic trend of pH by the microbial community MC1. J.
Environ. Sci. 18, 11421146.
OSullivan, C.A., Burrell, P.C., 2007. The effect of media changes on the rate of
cellulose solubilisation by rumen and digester derived microbial communities.
Waste Manage. (Oxford) 27, 18081814.
Pommier, S., Llamas, A.M., Lefebvre, X., 2010. Analysis of the outcome of shredding
pretreatment on the anaerobic biodegradability of paper and cardboard
materials. Bioresour. Technol. 101, 463468.
Ren, N., Wang, B., Huang, J.C., 1997. Ethanol-type fermentation from carbohydrate
in high rate acidogenic reactor. Biotechnol. Bioeng. 54, 428433.
Shi, D.Z., Wu, W.X., Lu, S.Y., Chen, T., Huang, H.L., Chen, Y.X., Yan, J.H., 2008. Effect of
MSWsource-classied collection on the emission of PCDDs/Fs and heavy metals
from incineration in China. J. Hazard. Mater. 153, 685694.
Soundar, S., Chandra, T.S., 1987. Cellulose degradation by a mixed bacterial culture.
J. Ind. Microbiol. Biot. 2, 257265.
Suita, J.M., Gerba, C.P., Ham, R.K., Palmisano, A.C., Rathje, W.L., Robinson, J.A., 1992.
Worlds largest landll; a multidisciplinary investigation. Environ. Sci. Technol.
26, 14861495.
Sun, Y., Cheng, J., 2002. Hydrolysis of lignocellulosic materials for ethanol
production: a review. Bioresour. Technol. 83, 111.
Teghammar, A., Yngvesson, J., Lundin, M., Taherzadeh, M.J., Horvth, I.S., 2010.
Pretreatment of paper tube residuals for improved biogas production.
Bioresour. Technol. 101, 12061212.
Wongwilaiwalin, S., Rattanachomsri, U., Laothanachareon, T., Eurwilaichitr, L.,
Igarashi, Y., Champreda, V., 2010. Analysis of a thermophilic lignocellulose
degrading microbial consortium and multi-species lignocellulolytic enzyme
system. Enzyme Microb. Technol. 47, 283290.
Xiao, W., Clarkson, W.W., 1997. Acid solubilization of lignin and bioconversion of
treated newsprint to methane. Biodegradation 8, 6166.
Xu, G., Goodell, B., 2001. Mechanisms of wood degradation by brown-rot fungi:
Chelator-mediated cellulose degradation and binding of iron by cellulose. J.
Biotechnol. 87, 4357.
Yang, H.Y., Wu, H., Wang, X.F., Cui, Z.J., Li, Y.H., 2011. Selection and characteristics of
a switchgrass-colonizing microbial community to produce extracellular
cellulases and xylanases. Bioresour. Technol. 102, 35463550.
Yen, H.W., Brune, D.E., 2007. Anaerobic co-digestion of algal sludge and waste paper
to produce methane. Bioresour. Technol. 98, 130134.
Yuan, X., Wang, H., Cheng, X., 2011. Enhancing the anaerobic digestion of corn stalks
using composite microbial pretreatment. J. Microbiol. Biotechnol. 21, 746752.
Yuan, X., Cao, Y., Li, J., Wen, B., Zhu, W., Wang, X., Cui, Z., 2012. Effect of
pretreatment by a microbial consortium on methane production of waste paper
and cardboard. Bioresour. Technol. 118, 281288.
Yusuf, M.O.L., Ify, N.L., 2011. The effect of waste paper on the kinetics of biogas yield
from the co-digestion of cow dung and water hyacinth. Biomass Bioenergy 35,
13451351.
Zhang, D., Guo, P., Li, J., Suo, Y., Wang, X., Cui, Z., 2011. Dynamic transition of
microbial communities in response to acidication in xed-bed anaerobic
bafed reactors (FABR) of two different ow directions. Bioresour. Technol. 102,
47034711.
Zheng, M., Li, X., Li, L., Yang, X., He, Y., 2009. Enhancing anaerobic biogasication of
corn stover through wet state NaOH pretreatment. Bioresour. Technol. 100,
51405145.
Zhu, H., Qu, F., Zhu, L.H., 1993. Isolation of genomic DNAs from plant, fungi and
bacteria using benzyl chloride. Nucleic Acids Res. 21, 52785280.
X. Yuan et al. / Bioresource Technology 154 (2014) 19 9