Bioresource Technology 154 (2014) 1–9

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

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.  CH4 production yields and rates significantly 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 municipal solid waste (LMSW). In this study a thermophilic microbial consortium (MC1) was used as a pretreatment method in order to enhance biogas and methane production yields. The results indicated that sCOD concentration increased significantly 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 significantly increased following MC1 pretreatment. In addition, the methane production rate of the treated LMSW was greater than that observed from the untreated sample. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction The quantity of municipal solid waste (MSW) generated in China has increased by 8–10% 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 technology is not without limitation, especially for MSW. Since approximately 40–50% of landfill space is occupied by paper and cardboard waste (Suflita et al., 1992), of which lignocellulose of municipal solid waste (LMSW) is a significant component (Béguin and Aubert, 1994). In addition, the solubilisation of cellulose and hemicellulose (both primary components of LMSW) is the ratelimiting step during the anaerobic digestion of lignocellulose of MSW (O’Sullivan and Burrell, 2007). As a result, a number of ⇑ Corresponding author. Tel.: +86 10 62733437; fax: +86 10 62731857. 1

E-mail addresses: [email protected], [email protected] (Z. Cui). These authors contributed equally to this article and are joint first authors.

0960-8524/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2013.11.090

studies have examined the use of different pretreatment methods, in an effort to maximize LMSW digestion. Mechanical pretreatment has been successful in reducing particle 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 significant 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 environmentallyfriendly method by using microorganisms, offers some conceptually 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 effectively able to degrade lignocellulose (Desvaux et al., 2000; Xu and Goodell, 2001). The solubilization of lignocellulose occurs

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X. Yuan et al. / Bioresource Technology 154 (2014) 1–9

naturally via the action of multiple microorganisms (Wongwilaiwalin 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 efficiency 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 office 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 office paper, newspaper, and cardboard was 1:1:1. All paper waste was first 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 degrading various cellulosic materials (e.g. filter paper, cotton and rice straw) under aerobic static conditions was constructed via a succession of enrichment cultures as in Haruta et al. (2002). The high stability of the consortium’s 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 frozen at 80 °C (Haruta et al., 2002). MC1 was cultured in a peptone cellulose solution (PCS) containing 1% (w/v) filter 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, Pseudoxanthomonas 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 composed of 2 g peptone, 1 g yeast extract, 2 g CaCO3, 5 g NaCl, and 1 L H2O (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. Previously prepared and frozen MC1 was inoculated into 125 ml sterile peptone cellulose solution (PCS) with a 1% (w/v) carbon source (filter 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 (final 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 medium 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 sampled 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 final weight (each measurement 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 taken 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 anaerobic digesters were purged with N2 for 5 min to remove O2, 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 biogas and methane yield of the 400 ml PCS medium and anaerobic sludge alone. The biogas and methane yields of LMSW were calculated as follows:

Biogas yield; ðml=g VSÞ ¼

ðBiogas volumeÞtotal  ðBiogas volumeÞCK VS of substrates added

Methane yield; ðml=g VSÞ ¼

ðMethane volumeÞtotal  ðMethane volumeÞCK VS of substrates added

Table 1 Characteristics of the substrates used in the experiments. Parameter

Office paper

Newspaper

Cardboard

Mixture (LMSW)

TS (%) VS (%TS) Ash (%TS) Lignin (%TS) Cellulose (%TS) Hemicellulose (%TS) CODsubstrate (g O2/g TS)

95.3 ± 0.2 98.6 ± 0.2 1.4 ± 0.0 1.4 ± 0.5 84.9 ± 1.3 12.3 ± 0.6 1.07 ± 0.02

93.2 ± 0.4 96.1 ± 0.3 3.9 ± 0.1 23.4 ± 0.5 68.5 ± 1.1 13.1 ± 0.3 1.21 ± 0.03

95.4 ± 0.3 87.2 ± 0.2 12.8 ± 0.2 17.8 ± 0.5 56.9 ± 0.8 10.7 ± 0.3 1.10 ± 0.03

94.6 ± 0.3 94.0 ± 0.2 6.0 ± 0.1 14.2 ± 0.5 70.1 ± 1.1 12.0 ± 0.4 1.13 ± 0.03

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X. Yuan et al. / Bioresource Technology 154 (2014) 1–9 Table 2 Experiments Settings of pretreatment and anaerobic digestion. Substrate concentration (%)

Pretreatment

Anaerobic digestion

LMSW (g)

PCS medium (ml)

Inoculum (ml)

Sample point (day)

0.5 1.0 2.5 5.0

2 4 10 20

400 400 400 400

20 20 20 20

0, 0, 0, 0,

1, 1, 1, 1,

2, 2, 2, 2,

4, 4, 4, 4,

6, 6, 6, 6,

8, 8, 8, 8,

10, 10, 10, 10,

2.5. COD, sCOD, pH, and volatile organic products (VOPs) of hydrolysates Chemical oxygen demand of substrate (CODsubstrate) before pretreatment, and COD of substrate residue (CODsubstrate residue) during pretreatment were analyzed using potassium dichromate as an oxidant using 25 mg samples previously milled into a 1 mm powder (Pommier et al., 2010). The soluble chemical oxygen demand (sCOD) of hydrolysate, and COD of PCS medium (CODPCS medium) were analyzed using a COD analyzer (Model ET99731, Lovibond, Germany) following centrifugation at 8000g for 10 min, and sampling of supernatant only. During the pretreatment, the hydrolysate 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. Briefly, all hydrolysate samples were filtered through an 0.22 lm aperture and analyzed using a GC–MS (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 previously (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 precipitate 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 final weight was determined as in Yuan et al., 2011). Residual LMSW components were analyzed using a fiber 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 displacement 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 analyzer (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, PCRDGGE 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-

14 14 14 14

Substrate

The Ratio of substrate to inoculum

– – 2, 4, 6, 8, and 10 days treated LMSW and untreated LMSW 2, 4, 6, 8, and 10 days treated LMSW and untreated LMSW

– – 1:1 1:1

centration, and also on day 6 of the four substrate concentrations using a benzyl chloride method as in Zhu et al. (1993). PCR amplification 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 amplification were 357F-GC, 50 –CCTACGGGAGGCAG CAG-30 (Escherichia coli positions, 341-357), which was attached to a GCclamp (50 -CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGCACGGGGG G-30 ) at the 50 -terminus, and 517R, 50 -ATTACCGCGGCTGCTGG-30 (E. coli positions, 517–534) (Guo et al., 2010). Primers were purchased 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 final 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 35–60% denaturing gradient (where 100% is defined as 7 M urea with 40% formamide) (Yuan et al., 2011). Gels were run at a constant voltage of 200 V and temperature 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 Quantity One Software (Bio-Rad, USA). The DNA was recovered, and re-amplified with the primers 357F (50 -CCTACGGGAGGCAGCAG30 ) and 517R (50 -ATTACCGCGG CTGCTGG-30 ) (Haruta et al., 2002) as described above. Amplified fragments were purified using the high purity PCR product purification kit (Tiangen Biotech Co., LTD, China) and sequenced using the ABI 3730XL DNA Sequencer (Perkin Elmer) at SunBiotech Developing Center. Sequence similarity 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 Biotech Co., Ltd., Beijing, China) and used as the PCR template. For quantitative real-time PCR, the following specific primer sets and 50 -nuclease probes (TaqMan) were used: Msc (Methanosarcinaceae; Msc380F, Msc492F, Msc828R; amplicon size: 408 bp); Mst (Methanosaetaceae; Mst702F, Mst753F, Mst862R; amplicon size: 164 bp); MBT (Methanobacteriales; MBT857F,

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X. Yuan et al. / Bioresource Technology 154 (2014) 1–9

MBT929F, MBT1196R; amplicon size: 343 bp); and MMB (Methanomicrobiales; 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 (final concentration, 10 lM), 2 lL of the TaqMan probe (final concentration, 1 lM), 10 lL of 2  reaction solution, and 1 lL of template DNA. The two-step amplification 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 five species of the Archaea genera Methanosarcinaceae (Methanosarcina acetivorans NBRC100939), Methanosaetaceae (Methanosaeta thermophila NCBR101360), Methanobacteriales (Methanobrevibacter arboriphilus NBRC101200), Methanomicrobiales (Methanospirillum hungatei NBRC100397) provided by the NITE Biological Research Center (NBRC, Chiba, Japan). The target rRNA gene sequences were amplified from each strain using conventional PCR with the corresponding primer sets as described above, and cloned into the pGEM-T Easy Vector following purification with the TIANgel Midi Purification Kit. For each plasmid, a 10-fold serial dilution ranging from 102 to 109 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 previously 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, office 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 concentrations of 0.5%, 1.0%, 2.5% and 5.0% all declined significantly 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 concentrations 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 concentrations 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 within 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 production, and consumption. It was also found that if the higher the substrate contents, the higher the peak values of the sCOD concentrations, 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 significant 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 ethanol decreased sharply (Fig. 2A–C). However, at 5% substrate concentration, the ethanol concentration reached 3.87 g/l on day 6 and no significant 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 concentration 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 propionic 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

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X. Yuan et al. / Bioresource Technology 154 (2014) 1–9

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 minimized 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 significantly. In addition, the pH of 5.0% substrate concentration stabilized at between 5.4 and 5.5 between day 8 and day 14; simultaneously, no significant 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 production) organic material loss (CODloss) 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 CODsubstrate and CODPCS medium, which was also equal to the sum of sCOD in hydrolysate, CODsubstrate residue and CODloss during pretreatment. Therefore, the CODloss was calculated as follows:

CODloss ¼ ðCODsubstrate þ CODPCS medium Þ  ðCODsubstrate residue þ sCODhydrolysate Þ The loss ratio of organic materials was calculated as follows:

Loss Ratio ð%Þ ¼ CODloss =ðCODsubstrate þ CODPCS medium Þ  100% Greater organic material loss occurred at each substrate concentration 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 CODloss varied in the following order: 2.5% (15,580 mg/L) > 1.0% (11,132 mg/L) > 0.5% (7036 mg/L). However, the CODloss of 5.0% substrate concentration was lower than that of 2.5% substrate

3.4. Change in final 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 final weight losses were 91.2%, 89.7%, 58.6% and 40.1%, respectively (Fig. 3). This was indicated that the total dry matter of LMSW was degraded most expeditiously in the first 4 days. This phenomenon was consistent with changes of pH values that declined rapidly 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 concentration of 5.0% simultaneously. Cellulose, hemicellulose, and lignin are the main components of LMSW; they are also the main carbon sources for anaerobic microorganisms. The availability and digestibility of cellulose and hemicellulose, as well as the association of lignin with carbohydrates significantly affected methane production. At the end of pretreatment, cellulose, hemicellulose and lignin were all degraded significantly 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 artificial xylan and pure cellulose (Desvaux

B 24000 0.5% 1.0% 2.5% 5.0%

9.0 8.5 8.0 7.5

0.5% 1.0% 2.5% 5.0%

20000 16000

sCOD (mg/l)

A 9.5

pH

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 because the pH of the hydrolysate at 5.0% substrate concentration was between 5.5 and 5.6 after day 8; the degradation ability of microbes could be inhibited at lower pH. Therefore, there were no obvious changes. It was also found that the higher substrate concentrations, 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%).

12000

7.0 6.5 6.0

8000 4000

5.5

0

5.0 0

2

4 6 8 10 12 14 Pretreatment time (day)

0

2 4 6 8 10 12 Pretreatment time (day)

14

Fig. 1. Changes in hydrolysate pH and sCOD during pretreatment. (A) The pH of hydrolysate; (B) The sCOD of hydrolysate.

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X. Yuan et al. / Bioresource Technology 154 (2014) 1–9

A

B

4.5

4.0 Concentration of VOPs (g/l)

Concentration of VOPs (g/l)

4.0 3.5 3.0 2.5 2.0 1.5 1.0

0.0

3.0 2.5 2.0 1.5 1.0

0.0 2

4

6 8 10 12 Pretreatment time (day)

14

D

4.5 Ethanol 4.0

Propionic acid

3.0

Butyric acid

2

4

6 8 10 12 Pretreatment time (day)

14

2

4

6 8 10 12 Pretreatment time (day)

14

4.5 4.0

Acetic acid

3.5

Concentration of VOPs (g/l)

Concentration of VOPs (g/l)

3.5

0.5

0.5

C

4.5

2.5 2.0 1.5 1.0 0.5

3.5 3.0 2.5 2.0 1.5 1.0 0.5

0.0

0.0 2

4

6 8 10 12 Pretreatment time (day)

14

Fig. 2. Quantitative analysis of major volatile organic products (VOPs) by GC–MS during pretreatment. (A) 0.5% substrate concentration; (B) 1.0% substrate concentration; (C) 2.5% substrate concentration; (D) 5.0% substrate concentration.

et al., 2000). However, they were unable to use natural lignocelluloses efficiently. 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 community composition was consistent with previous results (Haruta et al., 2002). Band 1 was associated with LMSW during the pretreatment process, which was 100% similar to Clostridium thermosuccinogenes. This bacterium could utilize cellobiose, xylose, glucose and sucrose, and produce acetate, lactate, and H2 (Haruta et al., 2002). The strains of genetic relationship represented by other five 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: Pseudoxanthomounas taiwanenis (100%). This data indicated a strong structural stability of MC1 during the pretreatment period of different lignocellulose materials, but the number of each bacterium may 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 indicated 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 digestion. 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 significantly 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 samples produced more methane than did the untreated LMSW samples. This also suggested that pretreatment by MC1 was capable of enhancing not only biogas yield, but also energy gain from the samples. Maximum biogas and methane yields at 2.5% and 5.0% substrate concentrations occurred after 4 and 8 days pretreatment

7

X. Yuan et al. / Bioresource Technology 154 (2014) 1–9 Table 3 COD balance during pretreatment. Substrate concentration (%)

Pretreatment time (days)

0.5

0 1 2 4 6 8 10 14 0 1 2 4 6 8 10 14 0 1 2 4 6 8 10 14 0 1 2 4 6 8 10 14

1.0

2.5

5.0

Before pretreatment

During pretreatment

CODsubstrate (mg/L)

CODPCS (mg/L)

5650 5650 5650 5650 5650 5650 5650 5650 11,300 11,300 11,300 11,300 11,300 11,300 11,300 11,300 28,250 28,250 28,250 28,250 28,250 28,250 28,250 28,250 56,500 56,500 56,500 56,500 56,500 56,500 56,500 56,500

3810 3810 3810 3810 3810 3810 3810 3810 3810 3810 3810 3810 3810 3810 3810 3810 3810 3810 3810 3810 3810 3810 3810 3810 3810 3810 3810 3810 3810 3810 3810 3810

medium

sCODhydrolysate (mg/L)

CODsubstrate (mg/L)

3810 5433 6320 5990 4767 3510 2610 2123 3810 5940 7750 9010 7310 6730 4927 2853 3810 7207 9533 12,253 11,270 8283 6320 4740 3810 7110 10,937 13,140 15,110 16,137 15,683 15,750

5650 3250 1660 644 479 345 372 301 11,300 8220 5520 2375 1710 1240 1070 1125 28,250 23,640 20,315 15,720 13,190 12,650 12,050 11,740 56,500 51,740 46,520 42,230 36,980 34,070 33,500 32,780

residue

CODloss (mg/L)

Loss ratio (%)

0 777 1480 2826 4214 5605 6478 7036 0 950 1840 3725 6090 7140 9113 11,132 0 1213 2212 4087 7600 11,127 13,690 15,580 0 1460 2853 4940 8220 10,103 11,127 11,780

0.0 8.2 15.6 29.9 44.5 59.2 68.5 74.4 0.0 6.3 12.2 24.7 40.3 47.3 60.3 73.7 0.0 3.8 6.9 12.7 23.7 34.7 42.7 48.6 0.0 2.4 4.7 8.2 13.6 16.8 18.4 19.5

respectively. This was also the same time when sCOD of the 100.0

A

90.0

B

Weight loss (%)

80.0 70.0 60.0

1 2

50.0 40.0

3 5

0.5% 1.0% 2.5% 5.0%

30.0 20.0 10.0

4

0.0 0

2 4 6 8 10 12 14 Pretreatment time (days)

6

Fig. 3. Dynamics of the dry matter weight loss of LMSW during pretreatment.

Day Day Day Day Day 4 8 10 14 2

Table 4 Weight loss of hemicellulose, cellulose and lignin of LMSW at the end of pretreatment. Substrate concentration (%)

Hemicellulose (%)

Cellulose (%)

Lignin (%)

0.5 1.0 2.5 5.0

91.8 ± 2.6 92.3 ± 4.2 45.7 ± 2.4 37.7 ± 2.1

97.5 ± 0.7 96.7 ± 1.4 73.5 ± 3.7 50.4 ± 2.7

66.4 ± 3.1 55.2 ± 1.2 23.9 ± 1.5 12.8 ± 0.3

0.5% 1.0% 2.5% 5.0%

Fig. 4. DGGE profile of the 16S rDNA fragments of MC1 during pretreatment. (A) DGGE profile during pretreatment at 2.5% substrate concentration; (B) DGGE profile of the four substrate concentrations on day 6 during MC1 pretreatment.

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

8

X. Yuan et al. / Bioresource Technology 154 (2014) 1–9

Methane yield (ml CH 4/g VS)

B

500 450 400 350 300 250 200 150 100 50 0

2.50% 5.00%

30 d 200

8d

150 100 50

d 10

d 8

d 6

d 4

d 2

un tre ate d

d 10

d 8

d 6

d 4

d 2

un tre

C

250

0 ate d

Biogas yield (ml/g VS)

A

D

60

Methane content (%)

50 40 30 20 10

d

d 8

d 6

d 4

d 2

10

un tre

ate d

0

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.

concentration of TVOPs was based on a longer pretreatment, which resulted in a part of soluble substrates being transformed into VOPs and CO2 by MC1, leaving less carbon for subsequent anaerobic digestion. The 2-d pretreated LMSW of 2.5% substrate concentration 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 methane 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 sample yields on corresponding days (Fig. 5B). The significant 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 digestion time. This could be of significant economic benefit through the increase of methane production efficiency 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 quantitative 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 consistent with earlier study of high abundance of Methanosaetaceae-related 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 environment (Zhang et al., 2011). The real-time PCR results were consistent 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 pretreatment had a tremendous effect on the solubilization of lignin of newspaper. Further, the pretreatment significantly increased methane production. Teghammar et al. (2010) found that explosive pretreatment with sodium hydroxide improved the methane yield of paper tubes by 70–107%, from 238 to 403–493 N ml/g VS. Further, the methane production rate was increased by 68–132%. Recently, anaerobic co-digestion of organic wastes for methane production has attracted more interest. There are some poorly

X. Yuan et al. / Bioresource Technology 154 (2014) 1–9

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 efficient biological pretreatment used to pretreat LMSW for anaerobic digestion. In the present study, a microbial consortium was used for pretreatment of lignocellulose of MSW. The results of anaerobic digestion indicated that LMSW methane production yields and rates significantly increased after microbial consortium pretreatment. Previous studies on the microbial pretreatment of lignocelluloses mainly focused on the pure culture of microorganisms (Desvaux et al., 2000; Xu and Goodell, 2001). However, the pure culture of microorganisms is difficult to culture in an open system. Our previous research indicated that MC1 can be culture continuously 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 efficient 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 fermentation stage. This also decreased cellulose concentration and hemicellulose 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-scientific 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. Béguin, P., Aubert, J.P., 1994. The biological degradation of cellulose. FEMS Microbiol. Rev. 13, 25–58. 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, 4767–4774. Clarkson, W.W., Xiao, W., 2000. Bench-scale anaerobic bioconversion of newsprint and office paper. Water Sci. Technol., 93–100. 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-flow anaerobic sludge bed. Bioresour. Technol. 114, 327–333. Desvaux, M., Guedon, E., Petitdemange, H., 2000. Cellulose catabolism by Clostridium cellulolyticum growing in batch culture on defined medium. Appl. Environ. Microbiol. 66, 2461–2470. 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, 2722–2728.

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