Bioresource Technology 136 (2013) 574–581
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Biore source Tec hnology
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Reactor performance and microbial community dynamics during solid-state anaerobic digestion of corn stover at mesophilic and thermophilic conditions
0960-8524/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.biortech.2013.02.073
⇑ Corresponding author. Tel.: +1 330 263 3855.E-mail address: [email protected] (Y. Li).
1 Currently with Sandia National Laboratories /Joint BioEne rgy Institute (JBEI),Emeryville, CA 94608, USA.
Jian Shi a,1, Zhongjiang Wang a, Jill A. Stiverson b, Zhongtang Yu b, Yebo Li a,⇑a Department of Food, Agricultural and Biological Engineering, The Ohio State University, Ohio Agricultural Research and Development Center,1680 Madison Ave., Wooster, OH 44691, USA b Department of Animal Sciences, The Ohio State University, Columbus, OH 43210, USA
h i g h l i g h t s
" Microbial community in solid state anaerobic digestion (SS-AD) of corn stover." Thermophil ic SS-AD led to faster reduction s of cellulose and hemicelluloses." Thermophilic SS-AD also led to higher accumulation of VFAs." Reactor performance correlated well with populations of microbes." Bacterial and archaeal communities underwent considerable successions in SS-AD.
a r t i c l e i n f o
Article history:Received 5 August 2012 Received in revised form 12 February 2013 Accepted 21 February 2013 Available online 5 March 2013
Keywords:Anaerobic digestion Solid state BiogasDenaturing gradient gel electrophoresis 16S rRNA
a b s t r a c t
Reactor performance and microbial community dynamics were investigated during solid state anaerobic digestion (SS-AD) of corn stover at mesophilic and thermophilic conditions. Thermophilic SS-AD led tofaster and greater reductions of cellulose and hemicelluloses during the first 12 days compared to mes- ophilic SS-AD. However, accumulation of volatile fatty acids (VFAs) was 5-fo ld higher at thermoph ilic than mesophilic temperatures, resulting in a large pH drop during days 6–12 in the thermophilic reactors.Culture -based enumeration revealed 10–50 times greater populations of cellulolytic and xylanolytic microbes during thermophilic SS-AD than mesophilic SS-AD. DGGE analysis of PCR amplified 16S rRNA genes showed dynamic shifts, especially during the thermophilic SS-AD, of bacter ial and archaeal com- munities over the 38 days of SS-AD as a result of acclima tion of the initial seed microbial consortia tothe lignocellulos ic feedstock. The findings of this study can guide future studies to improve efficiencyand stability of SS-AD.
� 2013 Elsevier Ltd. All rights reserved.
1. Introduction
Anaerobic digestion (AD) is one of the few sustainab le technol- ogies that both produce energy and treat waste streams. Driven bya complex and diverse community of microbes (Yu and Schanbach- er, 2010 ), AD is affected by a host of factors, many of which also af- fect the biodiversity and activity of the microbial community.Temperatur e is one of the key operational factors that affect the microbial diversity and biogas production in AD systems. While the majority of commerc ial AD systems in the US are operated atmesophilic temperatures (�20–40 �C), interest in thermophili c
(50–60 �C) digestion has increased over the past several years.The advantages of thermophili c AD include a greater degree ofpathogen reduction, decreased retention time, and a higher rate of biogas production compared to mesophilic AD (Ahring, 2003 ).Addition ally, thermophili c conditions are favorable for AD of ligno- cellulosic feedstocks as the high operating temperature can facili- tate degradation of recalcitrant cellulose (Frigon and Guiot, 2010;Li et al., 2011a ). One of the challenges of thermop hilic AD systems is a higher likelihood of system failure due to the accumulati on ofvolatile fatty acids (VFAs), especially propionate, which causes asevere decrease in biogas production (Kim and Speece, 2002 ).
Depending on the total solid (TS) content in digesters , AD sys- tems can be categorized as either liquid AD (L-AD), operated with 0.5–15% TS, or solid-state AD (SS-AD), generally operated with 15–40% TS. SS-AD systems have been developed for treating the organ- ic fraction of municipal solid waste (OFMSW) over the past decades
J. Shi et al. / Bioresource Technology 136 (2013) 574–581 575
(Fantozzi and Buratti, 2011; Mata-Alvare z et al., 2000; Rapport et al., 2008 ) and recently gained attention due to the potential application of SS-AD to treat lignocellulos ic biomass for energy production (Li et al., 2011b ). Recent studies demonstrated that inoculating SS-AD with L-AD effluent improved biogas production from a variety of lignocellulos ic biomass wastes (Li et al., 2011b;Liew et al., 2011 ). However, SS-AD of lignocellulosic biomass faces a few challenges, including slow start-up and acclimation of start- ing microbial communi ties to lignocellulos ic feedstock s, and acid- ification at high organic loading (Liew et al., 2012; Xu et al., 2013 ).Recent studies showed that microbial activities and the chemical composition of the inoculum can greatly affect SS-AD performanc e,especially during start-up of SS-AD of lignocellulosic biomass (Grif-fin et al., 1998; Xu et al., 2013 ). Furthermore, the presence of lignin,crystalline cellulose, and surface availability significantly reduced the biodegradab ility of lignocellul osic biomass, making the hydro- lysis step one of the bottlenecks that limit methane production (Liew et al., 2011; Frigon and Guiot, 2010 ). Slow degradation of lig- nocellulose and biogas production require larger reactor volumes and higher capital investments than otherwise required, a main barrier hindering commerc ial implementati on of SS-AD. Moreover,prompt acclimation of the microbial community in the seed inoc- ulum to recalcitrant lignocellulosic feedstocks to be digested iscrucial for high biogas yield.
Although the microbiol ogy of L-AD for wastewater treatment iswell studied, knowledge of the microbial community underpinni ngSS-AD of plant biomass is limited. This knowledge gap hinders de- sign and operation of SS-AD systems. The objective of this study was to investigate reactor performance and microbial community dynamics during SS-AD of corn stover at mesophilic and thermo- philic conditions. Reduction of TS, volatile solids (VS), cellulose,and hemicelluloses were monitored during SS-AD and the relation- ship between biogas production and microbial communities was also explored. The acclimation of archaeal and bacterial communi- ties to lignocellulos ic feedstock at both mesophilic and thermo- philic conditions was investigated using an integrated approach consisting of culture-base d enumeration of cellulolytic microbes,xylanolytic microbes, and acetotrophic methanogens and denatur- ing gradient gel electroph oresis (DGGE) analysis of the archaeal and bacterial communitie s following PCR amplification of 16S rRNA.
2. Methods
2.1. Feedstock and inoculum
Corn stover was collected in October 2009 from a research farm operated by the Ohio Agricultur al Research and Development Cen- ter (OARDC) in Wooster, OH, USA (40�4803300N, 81�5601400W). Upon receipt, corn stover was dried to a moisture content of less than 10% and then ground to pass a 9 mm sieve (Mighty Mac, MacKissic Inc., Parker Ford, PA, USA). Effluent from a mesophilic liquid anaer- obic digester fed with municipal sludge and food wastes (operatedby quasar energy group, Cleveland, OH, USA) was obtained and used as the inoculum for the SS-AD. The effluent was dewatered by centrifugati on to increase its TS content from 6.3% to 9.6%. Char- acteristics of the corn stover and the concentr ated effluent are shown in Table 1. One aliquot of the effluent was incubate d anaer- obically at 36 ± 1 �C for 1 week while another aliquot was incu- bated anaerobicall y at 55 ± 1 �C for 2 weeks before being inoculated into SS-AD reactors at mesophilic and thermophili cconditions, respectively.
2.2. Solid-state anaerobic digestion
SS-AD reactors (1 L working volume) were loaded with a mix- ture of corn stover and the effluent, at a feedstock -to-effluent (F/
E) ratio of 2 (based on VS) to obtain a TS content of 20%. Each reac- tor was sealed with a rubber stopper and placed in a 36 ± 1 �C or a55 ± 1 �C incubator for mesophil ic or thermophilic digestion,respectively , for a duration of 45 days. As a negligible amount ofbiogas was produced from day 38–45, data for the last week are not shown in figures that follow. A 5-L gas bag (CEL Scientific Ted- lar gas bag, Santa Fe Springs, CA, USA) was attached to the outlet ofeach reactor to collect the biogas produced. Composition and vol- ume of the biogas were measure d every 2–3 days during the 38- day period. At predetermined time intervals (day 0, 2, 4, 6, 8, 10,12, and 38), all the content in each reactor was taken out of the reactor and mixed thoroughly by a hand-held homogenize r prior to sampling for compositi on and microbial analysis. An aliquot ofeach digestate sample was stored at �80 �C for PCR-DGGE analysis.All tests were conducte d in duplicate. Reactors that received only the effluent were run in parallel as controls.
2.3. Analytica l methods
Samples of the feedstock, effluents, and reactor contents col- lected during the SS-AD process were characterized. The TS and VS contents were determined accordin g to the Standard Methods for the Examination of Water and Wastewa ter (Eaton et al., 2005 ).Total carbon and nitrogen contents were determined by an elemen- tal analyzer (Vario Max CNS, Elementar Americas, Mt. Laurel, NJ,USA) and were used to calculate the carbon-to- nitrogen (C/N) ratio.Total ammonia nitrogen was determined by the AmVer™ Salicylate Test ‘N Tube™ using a DR 2800™ Portable Spectrophotom eter (Loveland, CO, USA) as described by Hach Method 10031 (Hach,2012). The samples for VFA measurement were prepared by sus- pending 10 g of digestate in 10 mL of distilled water, thoroughly mixing it, and then separating the solids by centrifugation (8000 rpm, 5 min). The supernatant was acidified to pH 2–3 by add- ing hydrochlori c acid and then filtered through a syringe filter with 0.2 lm porosity for GC analysis of individual VFAs, including acetic,propionic , iso-butyric, and butyric acids (Li et al., 2011a ). Total VFAs and alkalinity were measured using an auto-titrator (Mettler Tole- do, DL22 Food & Beverage Analyzer, Columbus, OH, USA) following a titration procedure modified from McGhee (1968).
Lignin, cellulose, and hemicellulose contents in the corn stover and the reactor contents were determined using a two-step acid hydrolysi s process according to the NREL Laboratory Analytical Procedur e (Sluiter et al., 2008 ). Monomeric sugars (glucose, xylose,galactose, arabinos e, and mannose) and cellobiose were measured using HPLC (Shimadzu LC-20AB, Columbia, MD, USA) equipped with a Biorad Aminex HPX-87P column and a refractive index detector (RID) using deionized water as the mobile phase at a flowrate of 0.6 mL/min.
The volume of biogas collected in the Tedlar bags was measured by liquid displacemen t (Park and Li, 2012 ) and the composition ofthe biogas (CO2, CH4, N2, and O2) was analyzed by gas chromato- graph (GC) (Agilent Technologie s, HP 6890, Wilmington , DE, USA)equipped with a 30 m � 0.53 mm � 10 lm alumina/KCl deactiva- tion column and a thermal conductivi ty detector (TCD) using he- lium at a flow rate of 5.2 mL/min as a carrier gas. The temperat ures of the injector and detector were kept at 150 and 200 �C, respectively .
2.4. Microbial analysis
Celluloly tic microbes (CM), xylanolytic microbes (XM), and acetotrophic methano gens (AM) in the effluent and the reactor con- tents were quantified by the most-probable- number (MPN) method in triplicate (Balch et al., 1979 ) using the enumeration medium (EM)medium prepared under anaerobic conditions (Champio n et al.,1988). The EM medium contains (in 1 L): 2.0 g trypticase, 1.0 g yeast
Table 1Characteristics of materials.
Parameters Corn stover Concentrated effluent
Total solids (%)a 91.8 ± 0.0 9.6 ± 0.1 Volatile solids (%)a 88.1 ± 0.2 5.5 ± 0.1 Total carbon (%)a 43.6 ± 1.1 4.3 ± 0.3 Total nitrogen (%)a 0.5 ± 0.0 0.6 ± 0.0 Carbon to nitrogen (C/N) ratio 79.7 ± 3.7 6.6 ± 0.3 pH ND 8.3 ± 0.1 Alkalinity (g CaCO 3/kg) ND 14.5 ± 1.2 Total volatile fatty acid (g/kg) ND 3.6 ± 0.6 Total ammonia nitrogen (gN/kg) ND 3.8 ± 0.3 Lignin (%) 18.6 ± 0.6 NDCellulose (%) 38.0 ± 0.4 NDHemi-cellulose (%) 17.2 ± 0.3 ND
a Based on wet weight; the rests are based on dry weight; ND, not determined.
0 10 20 30 400
5
10
15
20
0
20
40
60
80
100
Met
hane
con
tent
, %
Dai
ly b
ioga
s yi
eld,
L/k
g VS
/d
Time, d
Daily biogas yield, 36 οC Daily biogas yield, 55 οC Methane content, 36 οC Methane content, 55 οC
Fig. 1. Daily biogas yield and methane content during SS-AD of corn stover atmesophilic (36 �C) and thermophilic (55 �C) conditions.
576 J. Shi et al. / Bioresource Technology 136 (2013) 574–581
extract, 25 mL mineral solution #1, 25 mL mineral solution #2,25 mL 40� calcium chloride solution, 10 mL 0.01% w/v resazurin solution, 50 mL of clarified rumen fluid, 20 mL volatile fatty salt solution, 4 g sodium carbonate (Cotta and Russell, 1982 ). The med- ium mixture was adjusted to pH 6.7 and autoclaved at 120 �C for 30 min and then mixed with sterile reducing agent solution con- taining sodium sulfide and cysteine–HCl (Cotta and Russell, 1982 ).Filter paper (Whatman No. 1, milled through a 40 mesh screen bya Thomas Model 4 Wiley � Mill, Swedesboro, NJ), xylan (from birch wood, Catalog No. X0064, TCI America, Portland, OR), and acetic acid, all at a concentr ation of 0.4% (w/w), was used as the sole sub- strate to enumerate CM, XM, and AM, respectivel y. All the proce- dures were conducted in a vinyl anaerobic chamber (Coy Labs,Grass Lake, MI, USA). The contents from each reactor were diluted by 1012 times with anaerobic EM medium using 1:10 serial dilution.An aliquot (0.5 mL) of the diluted effluent was inoculated into 4.5 mL of EM medium and incubated for 30 days at 36 and 55 �Cfor mesophil ic and thermophilic conditions, respectively. After incubation, growth of hydrolytic microbes was determined by vi- sual examination of the disappea rance (degradation) of cellulose or xylan. Growth of acetotrophic methano gens was determined bydetection of the methane content in the headspace of the culture tubes using GC as described above. Microbial counts were calcu- lated from a standardi zed MPN table and expresse d as the number of microbes per gram of VS of the starting digester content.
2.5. Microbial community DNA extraction, PCR, and DGGE
Samples were thawed on ice and homogen ized. Two subsam- ples of 0.5 g each was subjected to DNA extraction using the RBB+C method (Yu and Morrison, 2004a,b ) and the resultant DNA was then combined for each sample. The quality of the DNA was visu- ally assessed using agarose gel (0.8%) electrophoresis and quanti- fied using the Quant-it dsDNA Broad Range assay kit (MolecularProbes, Carlsbad, CA) on a MX3000P real-time PCR system (Agilent,La Jolla, CA, USA).
The V3 hyper-varia ble region of bacterial and archaeal 16S rRNA genes was amplified by PCR from the community DNA using bac- terial and archaeal primers, respectively, and analyzed using DGGE as described previously (Yu and Morrison, 2004b; Yu et al., 2008 ).The DGGE gel images were processed using BioNumerics (v.5.1;Applied Maths, Inc., Austin, TX) and the banding patterns were analyzed using principle component analysis as previously de- scribed (Cressman et al., 2010 ).
2.6. Statistical analysis
The SAS 9.2 software (SAS Institute Inc., Cary, NC, USA) was used for analysis of variance (ANOVA), and means were separated using
Tukey’s honestly significant difference (HSD) tests with a threshold P-value of 0.05 declared significant. Principal component analysis (PCA) was conducted on the binary matrices derived from the archaeal and bacterial DGGE profiles using PC-ORD 5.18 (MjM Soft- ware, Gleneden Beach, OR). XLSTAT version 2012 (Addinsoft, New York, NY) was used to conduct canonical correspondenc e analysis (CCA) of the archaeal and bacterial communi ty profiles as deter- mined by DGGE and the other measureme nts, which included tem- perature , VFA, pH, daily biogas yield, and daily reduction in TS, VS,cellulose, and xylan. All the detectable bands were included in the PCA and CCA analysis.
3. Results and discussion
3.1. Performan ce of SS-AD
3.1.1. Methane yield Corn stover is an abundant lignocellulosic feedstock rich in cel-
lulose and hemicellulos es and has a much higher C/N ratio at 79.7 than that of typical feedstocks or L-AD effluent (Table 1). The use ofL-AD effluent as inoculum and nutrient source provided a balanced C/N ratio of 22, which is more suitable for digestion of corn stover than the initial C/N ratio of the corn stover. However , acclimation of the initial microbial community of L-AD effluent was a key tohigh biogas yield from SS-AD of recalcitrant lignocellulosic bio- mass, such as corn stover. A side-by-side comparis on showed dif- ferent trends in the daily biogas yield and methane content during the SS-AD of corn stover at mesophilic and thermop hilic condition s (Fig. 1). At mesophilic conditions, the biogas production peaked at day 8 with a maximal daily production rate of 14.8 L/kg VS, and then gradually decreased to 5.1 L/kg VS on day 25 and about 2.9 L/kg VS on day 38. Biogas production at thermop hilic condition s peaked on day 12 with a maximal daily rate of 16.2 L/kg VS; however, it decreased quickly to 2.7 L/kg VS on day 25and then to only 1.1 L/kg VS on day 38. It is also noted that the highest biogas production rate at thermophili c condition s was accompani ed by a lower methane content (higher CO2 content)in comparison to that at mesophilic conditions (Fig. 1). These re- sults indicate that thermop hilic conditions may lead to greater hydrolysi s and acidogenes is and subsequent excessive production of VFAs than mesophil ic conditions, eventually resulting in high CO2 content in biogas due to an imbalance between acidogenesi sand methanogenesi s. However, acclimation of the mesophilic starting microbial community to the corn stover feedstock and thermop hilic condition s quickly overcame the imbalance, and the
J. Shi et al. / Bioresource Technology 136 (2013) 574–581 577
biogas production recovered during days 8–12 with the methane content increasing to �60%. The final accumulative biogas produc- tion did not differ significantly between thermophilic and meso- philic conditions. It should be noted, however, that the lack offluidity of the solid digestate and lack of mixing after day 12 ofthe SS-AD experiment probably limited the access of microbes tothe remaining substrate s in the digesters, diminishing the poten- tially greater biogas production at the thermophili c conditions.
3.1.2. VFA content and pH of the digestate The total VFAs remained below 4 g/kg digestate at mesophilic
conditions, and the pH values remained between 8 and 8.5 during the entire 38-day of SS-AD (Fig. 2). The total VFAs were 5-fold high- er during the thermophilic SS-AD than during the mesophilic SS- AD, lowering the pH of thermophilic digestate to 7.3 between days 4 and 10. As expected, acetic acid was the major VFA at thermo- philic conditions. Acetic acid concentrations peaked on days 8–10at �16 g/kg digestate probably due to promoted hydrolysis and ra- pid acidogenesis at the thermophili c conditions. The pH drop and VFA accumulation correlated well with the rapid biogas production and high CO2 content and the sudden decrease in biogas yield dur- ing the early stage of thermop hilic SS-AD. However , the acetic acid concentratio n declined quickly to less than 5 g/kg digestate after day 12, possibly as a result of acclimation of the mesophilic micro- bial populations, especially acetoclastic methanogens , to the operating conditions of the thermop hilic SS-AD, particular ly the elevated temperature . The decrease in acetate concentration coin- cided with the recovery of biogas production and high methane content at day 12, which supports the above premise. Additionally ,
0
5
10
15
20
25
Mesophilic, 36 οC
VFA,
g/k
g di
gest
ate
Time, d
Acetic Propionic Isobutyric Butyric
0
5
10
15
20
25
Tota
l VFA
, g/k
g di
gest
ate
Time, d
Mesophilic, 36 οC Thermophilic, 55 οC
0 10 20 30 40
0 10 20 30 40
((c)
((a)
Fig. 2. Changes of VFAs and pH during SS-AD of corn stover. Compositions of VFAs
the decrease in VFAs might be attributed partially to the lack of flu-idity and mixing after day 12, which resulted in un-availa bility ofsubstrate s for continued hydrolysi s and acidogenesis. It is also noted that there were accumulations of non-acetate VFAs, i.e. pro- pionate and butyrate, at both mesophilic and thermophili c condi- tions, suggesting a lack of sufficient syntrophi c acetogenes is.Previous studies have shown that the growth and metabolism offermentative bacteria can exceed those of syntrophic bacteria and methanogens, leading to a buildup of VFAs and acidification of digesters when the organic loading rate was high (Nielsen and Ahring, 2007 ). Propionate as low as 2.2 g/L has been shown to beinhibitory to methanogenes is in L-AD (Barredo and Evison, 1991;de Bok et al., 2001 ). Although, such a threshold is not known inthe SS-AD systems, overfeeding carbohydrat e-rich biomass, i.e.,corn stover, to SS-AD may lead to high levels of propionate.
3.1.3. Reduction of TS, VS, cellulose, and xylan At the end of 38-day SS-AD, similar TS and VS reduction were
observed for both the mesophil ic and thermophili c condition s(P > 0.05). Thermop hilic SS-AD led to a 42.6% reduction in cellulose and a 38.0% reduction in xylan on day 38; however, they were not significantly greater (P > 0.05) compared to the mesophil ic SS-AD (Table 2). Reduction of TS, VS, cellulose, and xylan positivel y corre- lated with the accumulative biogas yields for both conditions.Although the microbial communities were very different between the two temperatures (Fig. 3), there was no significant improve- ment in biogas yield for thermop hilic conditions. However, com- pared with the mesophilic SS-AD, faster and greater reduction ofTS was observed during the first 12 days of thermop hilic SS-AD
0
5
10
15
20
25
Thermophilc, 55 οC
VFA,
g/k
g di
gest
ate
Time, d
Acetic Propionic Isobutyric Butyric
0 10 20 30 40
0 10 20 30 406
7
8
9
10
pH
Time, d
Mesophilic, 36 οC Thermophilic, 55 οC
d)
b)
at (a) mesophilic and (b) thermophilic conditions; (c) total VFAs; and (d) pH.
Table 2Reduction of TS, VS, cellulose and hemicellulose over time: 36 �C vs. 55 �C.
Time TS reduction (%) VS reduction (%) Cellulose reduction (%) Xylan reduction (%)
36 �C 55 �C 36 �C 55 �C 36 �C 55 �C 36 �C 55 �C
0 �0.1A 1.0A 0.0A 1.0A 0.0A 0.0A 0.0A 0.4A
2 2.0 A 4.1B 6.0A 5.9A 10.8A 11.5A 9.3A 8.8A
4 4.7 A 6.0B 6.1A 8.1B 13.4A 15.0A 12.0A 15.6B
6 5.9 A 7.2B 8.2A 10.9B 17.5A 21.7B 16.6A 22.3B
8 9.0 A 10.9B 10.7A 12.6B 19.3A 27.2B 25.0A 25.1A
10 10.4 A 12.6B 12.2A 13.2A 26.4A 30.7B 28.2A 29.0A
12 12.8 A 14.5B 16.0A 18.2A 30.8A 34.6B 33.3A 33.9A
38 17.1 A 18.5A 20.8A 20.9A 40.8A 42.6A 36.3A 38.0A
Note: Letters ‘A’ and ‘B’ denote to the levels of significance (P < 0.05).
578 J. Shi et al. / Bioresource Technology 136 (2013) 574–581
(P < 0.05). Similarly, VS reductions during days 4–8 were signifi-cantly higher at thermop hilic conditions than at mesophilic condi- tions (P < 0.05). The larger TS and VS reduction s during the early stage of thermophili c SS-AD might be attributed to the greater cel- lulose and xylan reduction, which is in agreement with many pre- vious studies showing promoted hydrolysis at thermop hilic conditions. It is also noted that significantly greater xylan reduc- tion was achieved during days 4–6 at thermophili c conditions,which was followed by significantly higher cellulose reduction ondays 6–12, indicating that microbes utilized xylan before attacking cellulose. We attribute this to the 10–50 times greater populations of cellulolytic and xylanolytic microbes at thermop hilic than atmesophilic conditions as shown in Fig. 3. The greater xylan degra- dation corrobor ates the finding of previous studies which showed that the less crystalline hemicellulos e structures are more readily degradable than the more recalcitrant cellulose structures in ligno- cellulosic biomass (Maki et al., 2009; Shi et al., 2012 ). As men- tioned above, the lack of fluidity and mixing after day 12probably hindered further degradat ion of cellulose and hemicellu- lose due to limited translocatio n of cellulolyt ic or hemicellulol ytic bacteria to remaining substrates. In future studies, frequent mixing or leachate percolation is needed to maximize access of microbes to the solid substrates to maximiz e conversion of lignocellulosic biomass to biogas at either mesophil ic or thermop hilic condition s.
3.2. Quantification of cellulolytic and xylanolyti c microbes and acetoclastic methanogen s
While the AD process can be conceptu ally divided into four dis- tinct phases (hydrolysis, acidogenes is, syntrophic acetogenesi s,and methanogenesi s), all the phases are carried out concurrently by a diverse community of bacteria and archaea in digesters.Hydrolytic microbes, especially cellulolytic and xylanoly tic mi- crobes, are of particular importance in SS-AD of corn stover (andother types of lignocellulosic feedstocks), as cellulose and hemicel- luloses (mainly xylan) are the main carbon sources available tomicrobial growth and methane production. As shown in Fig. 3aand b, the populations of cellulolytic and xylanolytic microbes were dynamic at both thermophili c and mesophil ic conditions,increasing in abundance and then decreasing towards the end ofthe SS-AD process. This dynamic trend might be attributed to accli- mation of the initial microbial communitie s to the lignocellulos iccorn stover feedstock and the environmental conditions character- istic of SS-AD. The decrease in the cellulolytic and xylanolytic pop- ulations during the late stages of SS-AD is probably due to the depletion of digestible cellulose or hemicelluloses in localized spaces in the digestate, which was not fluidic or mixed after day 12. It is also evident that the populations of cellulolytic and xylan- olytic microbes were 10–50 times greater at thermop hilic than atmesophilic conditions, corroborating the greater reduction in TS,VS, cellulose, and xylan during the first 12 days of thermop hilic
SS-AD (Table 2). The cellulolytic and the xylanoly tic microbes exhibited a similar dynamic trend, suggesting possible possession of both cellulolytic and xylanolytic activities by some fibrolytic mi- crobes. This premise is consistent with the ability of many anaero- bic bacteria and fungi that possess both cellulases and xylanases (Kumar et al., 2008; Shi et al., 2012 ).
Acetoclas tic methanogenes is represents the main pathway for methane production in AD systems. The MPN analysis showed agradual increase in acetoclastic methano gen populations at both thermop hilic and mesophilic condition s compared with that ofday 0 (Fig. 3c). Under both conditions, acetotrophic methanogen populations initially increased and then persisted after day 10. Itwas also found that SS-AD of corn stover led to a larger population of acetoclasti c methanogens at thermophilic condition s than atmesophil ic condition s, probably because of the high acetate con- centration observed at the thermophilic conditions. It remains tobe determined why the greater acetotrophic methanogen popula- tion observed during the thermophili c SS-AD did not produce more methane than during the mesophilic digestion. However, given the similar reduction of TS, VS, cellulose, and xylan, similar amounts ofacetate were probably produced at both thermophilic and meso- philic conditions, providing similar amounts of acetate for methano genesis.
3.3. PCR-based DGGE analysis of the archaeal and bacterial communi ties
3.3.1. Achaea The archaeal communi ty underwent obvious temporal shifts, as
evidenced by the disappearan ce and appearance of DGGE bands and changes in band intensities during the mesophil ic and thermo- philic SS-AD processes, with the shifts observed primarily during the first few days (Supplement al material, Fig. S1). The DGGE anal- ysis also suggests selection for and against certain methanogens inthe mesophil ic SS-AD reactors. Under the thermop hilic condition s,major shifts were evident during the first two days. Thereafter,small shifts in archaeal populations continued until day 6. Two in- tense DGGE bands (bands 5 and 6) were seen in all the thermo- philic samples, indicating the survival and potential functioning of two predominant archaeal populations from the mesophilic inoculum during the thermop hilic SS-AD process. As suggested by increasing DGGE band intensity (bands 8 and 10), some metha- nogens were enriched over the course of the thermop hilic SS-AD digestion of corn stover. Under the mesophil ic condition s, fiveDGGE bands (bands 3–7) persisted from days 0 to 38, suggesting that more methanogen populations survived and probably func- tioned during the mesophil ic than the thermophilic SS-AD of corn stover. Under mesophilic condition s, the archaeal community shifted from days 0 to 6, and little succession was observed there- after, reflecting stabilization of the archaeal community.
105
106
107
108C
ellu
loly
tic m
icro
bes,
MPN
/g T
S Mesophilic, 36 οC Thermophilic, 55 οC
105
106
107
108
109
1010
Xyla
noly
tic m
icro
bes,
MPN
/g T
S Mesophilic, 36 οC Thermophilic, 55 οC
Day 0 2 4 6 8 10 12 38
107
108
109
1010
1011
Acet
ecla
stic
met
hano
gens
, MPN
/g T
S
Time, d
Mesophilic, 36 οC Thermophilic, 55 οC
(a)
(b)
(c)
Fig. 3. Concentration of (a) cellulolytic microbes; (b) xylanolytic microbes; and (c)acetoclastic methanogens enumerated by MPN.
J. Shi et al. / Bioresource Technology 136 (2013) 574–581 579
Several DGGE bands (bands 4–8) were observed at both the mesophilic and the thermophilic conditions (Supplemental mate- rial, Fig. S1). These DGGE band might represent the predominant methanogen populations that functioned under both sets of condi- tions. However , several DGGE bands (bands 5, 6, and 8) were more intense under the thermophilic conditions than under the meso- philic conditions. These intense DGGE bands suggest strong selec- tion for a few thermotolerant and/or thermophili c methanogen populations that were initially present in the mesophilic inoculum.The emerging of band 10 and changes in band intensity (e.g., bands 7 and 8) during thermophili c SS-AD probably also reflect both the dynamic evolution of initial methanogen populations in response to temperat ure and the substrate . The very high level of acetic acid accumulation during day 6–8 at thermophilic condition s was indeed caused by the imbalanc ed hydrolysis/a cidogenesis and methanogenes is, as evidenced from the significantly greater popu- lations of cellulolytic and xylanolytic microbes (Fig. 3). MPN
analysis also revealed a larger population of acetoclastic methano- gens at thermophilic conditions than at mesophilic condition s,probably because of the high acetate concentratio n, and thus great- er availability of acetate, observed at the thermophilic conditions.The PCA also separated the samples largely based on the SS-AD temperat ures, primarily along the PC1, which explained 43% ofthe total variation (Supplement al material, Fig. S1). Temporal shifts seemed to be separated along PC2, but the separation was limited,corrobor ating the degree of the shifts in the archaeal community.Because the primary substrates for methanogens in digesters are acetate, H2, and CO2 irrespective of operational conditions, the shifts observed in archaea could be attributed primarily to physico- chemical processes, such as reduced water activity and mass trans- fer. Of course, the shifts noted in the thermophili c SS-AD process may also be attributed to the high temperature . Further metage- nomic studies should help determine the specific archaea that con- tribute to methanogenesi s over the course of SS-AD of corn stover.
3.3.2. Bacteria The bacterial community also underwent considerable succes-
sions under both SS-AD conditions (Supplement al material,Fig. S2). Under the thermophilic conditions, dramatic shifts were noted during the first two days, but further small shifts were also observed from days 2 to 8. Although a number of DGGE bands were detected from day 2 to the end of the SS-AD process, only a few DGGE bands were detected in the day 0 samples, which make itdifficult to assess the survival of the predominant bacterial popula- tions present in the initial inocula. However, these common bands detected among the samples collected from day 2 and thereafte rsuggest the persistence of many bacteria during thermop hilic SS- AD. It is also interesting to note that several DGGE bands (e.g.,bands 3 and 5) in the upper part of the gel intensified during the thermop hilic SS-AD process. This might reflect the enrichme nt ofthermotol erant or thermophilic bacteria that have a relatively low G + C content in their genomes. Under the mesophilic condi- tions, the bacterial community was also subjected to successions,especiall y during the first two days. Unlike under the thermop hilic condition s, however, the major DGGE bands were detected in the lower portion of the DGGE gel, suggestin g the presence of high G + C bacteria and lack of low G + C bacteria in the mesophil icsamples.
The bacterial community shifts that occurred in the early stage of the SS-AD processes can be attributed to several factors, includ- ing change and availability of substrates (from aqueous to solid,from municipal sludge to corn stover), reduced water activity, tem- perature (mesophilic versus thermop hilic SS-AD), and reduced mass transfer. Because there was no lag phase in methane produc- tion, hydrolysis of the corn stover and the other phases of the ADprocess might have started without much delay. This premise was consistent with the data on solid reduction and fiber degrada- tion (Table 2) and the MPN results of fibrolytic bacteria and aceto- trophic methanogens. Distinctive DGGE patterns of bacterial communi ties were noted between the mesophilic and the thermo- philic samples (Supplemental material, Fig. S2), indicating that the predomin ant bacteria in each of the two SS-AD processes might behighly dependent on the operational temperature s. The bacterial communi ties in thermop hilic L-AD reactors are typically domi- nated by thermophilic genera, such as Thermoana erobacter , Anaer-olinea, and members of the phylum Thermotogae (Balk et al., 2002;Hernon et al., 2006; Yamada et al., 2006 ). Future sequencing-ba sed metagen omic studies will help reveal the predominant genera se- lected during the SS-AD.
Taken together, larger shifts in the microbial communitie s,especiall y the bacterial communi ties, occurred at mesophilic than at thermophilic conditions, suggesting that diverse bacteria, possi- bly with different substrate spectra and growth temperat ure
A1
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CCA Map / Asymmetric / Objects(axes F1 and F2: 57.88 %)
Objects Variables
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6.79
%)
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CCA Map / Asymmetric / Objects(axes F1 and F2: 77.55 %)
Objects Variables
(B) Bacteria
(A) Archaea
Fig. 4. Canonical correspondence analysis (CCA) ordination diagrams showing the correlation between the DGGE bands of archaea (A) and bacteria (B) and the environmental variables and the performance variables. The environmental variables included in the CCA are represented as red (dash) vectors in the plot; while the performance variables (daily biogas yield: dYbiogas; daily TS reduction: dTS; daily VS reduction: dVS; daily cellulose reduction: dCellulose; daily xylan reduction: dXylan) are shown as blue (solid)vectors. The A1, A2 . . .A32 and B1, B2 . . .B28 represent all the individual DGGE bands identified from DGGE images to the binary matrices.
580 J. Shi et al. / Bioresource Technology 136 (2013) 574–581
requiremen ts, were selected and involved in the SS-AD process.This result is expected given the multitude of inter-relatin g meta- bolic pathways of bacteria compared to those of archaea (Gao et al.,2011). Due to the limited information that DGGE analysis can pro- vide, future studies will examine the microbial community compo- sition, structure , and population shift. It is also noted that SS-AD oflignocellulos ic feedstock can be a very sophisticated system. In this study, we used a single inoculum source to inoculate the same lig- nocellulosic feedstock, corn stover, and carefully controlle dconditions such as solid content, inoculum-to -feedstock ratio, C/N ratio, etc. The differences in microbial community structures be- tween thermop hilic and mesophilic conditions are mainly due tothe operating temperature during SS-AD. However, the microbial
communi ty shifts during the SS-AD processes can be attributed to several other factors, including changes of substrate s and water activity, variation in temperat ure and pH gradients, due to limited heat and mass transfer, which deserve further study.
3.3.3. Correlation to environm ental factors Canonica l correspondenc e analysis (CCA) was performed to
examine potential correlations between the bands of archaea and bacteria with the environmental parameters measured (i.e., pH,temperat ure, and VFA concentratio ns) as well as the performance paramete rs determined, the latter of which included daily biogas yield (dYbiogas), daily TS reduction (dTS), daily VS reduction (dVS),daily cellulose reduction (dCellulose), and daily xylan reduction
J. Shi et al. / Bioresource Technology 136 (2013) 574–581 581
(dXylan). The CCA results provided further evidence of a link be- tween the compositions of the archaeal and bacterial communi ties,as determined by DGGE, and environmental factors such as pH,temperature , and VFA concentr ation (Fig. 4). Significance analysis on the environmental variables revealed that temperat ure ac- counted for the greatest difference in both archaeal and bacterial community composition observed between the thermophilic and the mesophilic conditions and had a statistically significant corre- lation with species composition (P < 0.05). VFA was positively asso- ciated with temperature, while pH was negatively associate d with temperature and VFAs. All the performanc e variables (dYbiogas, dTS,dVS, dCellulose, and dXylan) were positively associated with each other, well supported by the data that cellulose and xylan reduc- tion were positively associate d with TS and VS reduction (Table 2)and greater TS/VS reduction led to greater biogas yield. It is also noted that some DGGE bands of archaea and bacteria were corre- lated with the environm ental factors and performance measure- ments, suggestin g that some methanogens and bacteria are more dynamic and involved in AD processes than others. However, de- tailed speciation of these archaea and bacteria can only beachieved by sequencing analysis.
4. Conclusions
Thermophili c SS-AD led to faster and greater reduction s of TS,VS, cellulose, and hemicelluloses but higher accumulation of VFAs than did mesophil ic SS-AD during the first 12 days, correlating well with the greater populations of cellulolytic and xylanolytic mi- crobes. As revealed by DGGE analysis, both bacterial and archaeal communitie s underwent considerable successions, reflecting the selection for some bacteria and archaea while against other mem- bers of the initial inoculum , especially when mesophilic sludge was used to inoculate thermophili c SS-AD. At least some methanogens and bacteria were dynamic to environmental conditions and corre- lated with performance of SS-AD of lignocellulos ic feedstocks.
Acknowled gements
The authors would like to thank Ohio Third Frontier Program and Quasar Energy Group for the financial support. The authors would like to thank Mrs. Mary Wicks (Department of Food, Agricul- tural and Biological Engineering, OSU) for reading through the manuscript and providing useful suggestions .
Appendix A. Supplemen tary data
Supplement ary data associated with this article can be found, inthe online version, at http://dx .doi.org/10.1016/j .biortech.2013 .02.073.
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