Reactor performance and microbial community dynamics during solid-state anaerobic digestion of corn stover at mesophilic and thermophilic conditions

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  • Bioresource Technology 136 (2013) 574581Contents lists available at SciVerse ScienceDi rect

    Biore source Tec hnology

    journal homepage: www.elsevier .com/locate /bior techReactor 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.

    Corresponding author. Tel.: +1 330 263 3855.E-mail address: (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 612 in the thermophilic reactors.Culture -based enumeration revealed 1050 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 (2040 C), interest in thermophili c(5060 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 0.515% TS, or solid-state AD (SS-AD), generally operated with 1540% 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) 574581 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 (404803300N, 815601400W). 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 3845, 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 23 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 23 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















    , %


    ly b


    s yi



    g VS


    Time, d

    Daily biogas yield, 36 C Daily biogas yield, 55 C Methane content, 36 C Methane content, 55 C

    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) 574581extract, 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 cysteineHCl (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



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