Bioresource Technology 157 (2014) 188–196

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Bioresource Technology

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Effects of microbial and non-microbial factors of liquid anaerobicdigestion effluent as inoculum on solid-state anaerobic digestionof corn stover

http://dx.doi.org/10.1016/j.biortech.2014.01.0890960-8524/� 2014 Elsevier Ltd. All rights reserved.

⇑ Corresponding author. Tel.: +1 330 263 3855.E-mail address: li.851@osu.edu (Y. Li).

1 Authors contributed equally to this work.

Jian Shi a,1, Fuqing Xu 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, OH44691, USAb Department of Animal Science, The Ohio State University, Columbus, OH 43210, USA

h i g h l i g h t s

� Solid state anaerobic digestion (SS-AD) using liquid AD effluent as inoculum.� Effect of microbial and non-microbial factors of effluent on performance of SS-AD.� Non-microbial factor of effluent was more influential on methane yield of SS-AD.� Both bacterial and archaeal communities underwent considerable successions.

a r t i c l e i n f o

Article history:Received 18 November 2013Received in revised form 16 January 2014Accepted 21 January 2014Available online 31 January 2014

Keywords:Anaerobic digestionBiogasMicrobial communityDry fermentationInoculum

a b s t r a c t

The microbial activity of the inoculum (liquid anaerobic digestion effluent) was altered by autoclavingpart of the effluent to study the effect of feedstock to active effluent ratio (F/Ea, 2.2–6.6) and the feedstockto total effluent ratio (F/Et, 2.2 and 4.4) on reactor performance in solid state anaerobic digestion (SS-AD)of corn stover. When the F/Ea ratio was increased from 2.2 to 6.6, methane yield was not significantlyreduced; however, reactors became acidified when the F/Et ratio was increased from 2.2 to 4.4. It wasconcluded that F/Et had a greater effect on methane yields than F/Ea for the range studied in this paper.As analyzed by denaturing gradient gel electrophoresis using PCR amplified 16S rRNA genes, the micro-bial community underwent dynamic shifts under acidified conditions over 38 days of SS-AD. These shiftsreflected the acclimation, both adaptive selection and diversification, of the initial inoculated microbialconsortia.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

According to the ‘‘3Rs’’ (reduce, reuse and recycle) hierarchy,the aim of waste management is to generate the minimum amountof waste, while extracting the maximum uses and benefits from it(EPA, 2013). Anaerobic digestion (AD) is one of the most attractivewaste treatment technologies for organic wastes as it both stabi-lizes it and produces energy (biogas) (Karthikeyan and Visvana-than, 2013). Although the dominant type of AD facilities arethose fed with liquid waste streams, the need for treating solidmaterials, including municipal and agricultural wastes, hasfostered the development of solid state AD (SS-AD) systems that

operate at more than 20% solids (Li et al., 2011a). Over the pastfew decades, SS-AD systems have been used to treat the organicfraction of municipal solid waste (OFMSW), diverting it fromlandfills (Fantozzi and Buratti, 2011), and has recently gainedattention due to its potential application to process lignocellulosicbiomass for energy production (Li et al., 2011a).

More recent studies demonstrated that inoculating SS-AD witheffluent from liquid AD (L-AD) systems can successfully initiatebiogas production from a variety of lignocellulosic biomass wastes(Li et al., 2011a; Liew et al., 2011; Zhu et al., 2010). L-AD effluentcan provide sufficient microbes, moisture, micronutrients andnitrogen for the SS-AD process (Li et al., 2011b; Wang et al.,2013). However, SS-AD of lignocellulosic biomass faces challengessuch as slow acclimation of inoculum to lignocellulosic feedstocksand acidification of the reactor at high organic loading (Xu et al.,2013). A few key factors have been identified that affect SS-AD

J. Shi et al. / Bioresource Technology 157 (2014) 188–196 189

performance: inoculum size, carbon/nitrogen (C/N) ratio, type offeedstocks, organic loading, operating temperature, and total solids(TS) content (Karthikeyan and Visvanathan, 2013; Motte et al.,2013; Shi et al., 2013). Recent studies showed that microbial activ-ities and the chemical composition of the AD effluent/digestate asinoculum can greatly affect SS-AD performance, especially duringstart-up of SS-AD of lignocellulosic biomass (Griffin et al., 1998;Ma et al., 2013; Motte et al., 2013; Xu et al., 2013). Furthermore,reactors fed with cellulosic biomass sometimes become acidified(‘‘sour’’) due to the accumulation of volatile fatty acids (VFAs) asa result of imbalanced C/N ratios and inhibited microbial activities.However, the operating parameters and microbial communitystructures that relate to the ‘‘sour’’ phenomena during SS-AD arenot well understood.

It has been shown that decreasing the feedstock-to-inoculumratio can effectively reduce startup time and increase methaneyield in SS-AD (Forster-Carneiro et al., 2007; Motte et al., 2013).The positive effects of a larger inoculation size on SS-AD are be-lieved to be a combined influence from increased microbial popu-lations (especially methanogens), higher buffering capacity, and insome cases balanced C/N ratios (Griffin et al., 1998; Xu et al., 2013).The effect of microbial factors of L-AD effluent (inoculum) on per-formance and microbial communities of the SS-AD process havebeen reported recently (Motte et al., 2013; Shi et al., 2013); how-ever, the non-microbial factors of the liquid AD effluent the perfor-mance of SS-AD has not been investigated.

Microbial community dynamics can provide valuable informa-tion for understanding the effect of operating conditions on ADsystem performance. Culture independent molecular analysis ofthe microbial communities in environmental samples is the pre-ferred methodology for the investigation of AD systems. Thesemethods are generally based on analysis of certain well character-ized marker genes, the most common of which is the ribosomalRNA (rRNA) gene. Acclimation of archaeal and bacterial communi-ties under various AD conditions and with different feedstocks hasbeen studied in L-AD systems (Lee et al., 2010). The effect of tem-perature variations (ranging from mesophilic to thermophilic) andtemperature shocks on microbial community structures in L-ADsystems were also studied (Gao et al., 2011). However, limitedinformation can be found in the literature for microbial dynamicsin SS-AD systems, especially for those fed with lignocellulosic bio-mass as the major feedstock (Shi et al., 2013). In this study, the ef-fects of inoculation on reactor performance, such as methane yield,pH, VFA production, and reduction of TS, volatile solids (VS, wetbased in this paper), cellulose, and hemicelluloses, were studiedby controlling the feedstock to active effluent ratio (F/Ea) and feed-stock to total effluent ratio (F/Et). The effect of F/Et and F/Ea on thesuccessions of the initial microbial community during SS-AD ofcorn stover was also studied. The shift of archaeal and bacterialcommunities in both ‘‘healthy’’ and ‘‘acidified’’ SS-AD reactorswere investigated using denaturing gradient gel electrophoresis(DGGE) analysis of the archaeal and bacterial communities follow-ing PCR amplification of 16S rRNA.

2. Methods

2.1. Feedstock and effluent

Corn stover was collected from a farm operated by the OhioAgriculture Research and Development Center (OARDC) in Woos-ter, OH, USA in October 2009. Upon receipt, corn stover was airdried to a moisture content of less than 10% and then ground topass a 9 mm sieve (Mighty Mac, MacKissic Inc., Parker Ford, PA,USA). The TS, VS, and C/N ratio of the corn stover were91.8 ± 0.0%, 88.1 ± 0.2%, and 79.7 ± 3.7%, respectively. On a dry

matter basis, the corn stover contained 18.6 ± 0.6% lignin,38.0 ± 0.4% cellulose, and 17.2 ± 0.3% hemi-celluloses. AD effluentwas obtained from a mesophilic liquid anaerobic digester fed withmunicipal sewage sludge and food wastes (operated by quasar en-ergy group, Cleveland, OH, USA). Effluent was dewatered by centri-fugation to increase its TS content from 6.3% to 9.6%, and wasacclimated at 36 ± 1 �C for 1 week prior to inoculation. The charac-teristics of the concentrated effluent were TS of 9.6 ± 0.1%; VS of5.5 ± 0.1%; C/N ratio of 6.6 ± 0.3; pH of 8.3 ± 0.1; alkalinity of14.5 ± 1.2 g CaCO3/kg; total VFA of 3.6 ± 0.6 g/kg; and total ammo-nia nitrogen of 3.8 ± 0.3 g N/kg.

2.2. Solid-state anaerobic digestion

SS-AD reactors (1 L) were loaded with a mixture of corn stoverand L-AD effluent (inoculum). The experimental design and relatedinitial parameters are shown in Table 1. Three F/Ea ratios (2.2, 4.4,and 6.6) and two F/Et ratios (2.2 and 4.4) were selected based onthe range of typical F/Et ratios for SS-AD of corn stover in literature(Li et al., 2011b; Xu et al., 2013). In one set of reactors, the F/Et ratiowas kept at 2.2, and the F/Ea ratio was adjusted to 2.2, 4.4, and 6.6by replacing a portion of the microbial active effluent with auto-claved (at 121 �C for 60 min) effluent. As a result, these reactorshad similar non-microbial properties such as initial C/N ratio andalkalinity, but proportionally decreased microbial populations.The other set of reactors, which were run at an F/Et ratio of 4.4,did not receive autoclaved L-AD effluent, thus the F/Ea ratio wasalso 4.4. Reactors with the same F/Ea ratio of 4.4 but differentF/Et ratios (4.4 and 2.2) had the same microbial populations perfeedstock VS, but different non-microbial properties, such as pH,alkalinity, C/N ratio, and micronutrients. Water was added ifnecessary to obtain a TS content of 20% for all reactors.

After loading with well mixed feedstock and L-AD effluent, eachreactor was sealed by a rubber stopper and connected to a 5-L gasbag (CEL Scientific Tedlar gas bag, Santa Fe Springs, CA, USA) forbiogas collection. The reactor was incubated at 36 ± 1 �C for38 days. Every 2–3 days, biogas collected in the gas bag was mea-sured for composition and volume. At predetermined times (day 0,2, 4, 6, 8, 10, 12, and 38), two of the reactors were terminated astwo replicates and all the contents were taken out of the reactor,mixed thoroughly by a hand-held mixer, and sampled for chemicaland microbial analyses. All tests were conducted in duplicates.

2.3. Analytical methods

Samples of feedstock, effluent, and digested material collectedas described in Section 2.2 were analyzed as described below. TSand VS contents were analyzed according to the Standard Methodsfor the Examination of Water and Wastewater (American PublicHealth Association., 2005). Total carbon and nitrogen contentswere determined by an elemental analyzer (Vario Max CNS, Ele-mentar Americas, Mt. Laurel, NJ, USA) and were used to calculatethe C/N ratio. For the pH and VFA determination, 10 g solid samplewas mixed with 15 mL deionized water and homogenized by a vor-tex mixer for 20 s, then centrifuged at 10,000 rpm for 10 min by aSorvall Legend T Plus Centrifuge (Thermo Scientific, Waltham, MA,USA). Alkalinity was measured following a titration procedure(McGhee, 1968) using an auto-titrator (Mettler Toledo, DL22 Food& Beverage Analyzer, Columbus, OH, USA). Samples for titrationwere prepared by diluting five grams of digestate with 50 mL ofdeionized water, mixing for 2 min, then filtrated with four layersof cheese cloth. The pH of the supernatant was measured and thenadjusted to a pH of 3–4 with 2 M HCl for analysis of VFAs, includingacetic, propionic, butyric, iso-butyric, and valeric acid, using a Shi-madzu GC-2010 Plus (Shimadzu, Columbia, MD, USA). The GC wasequipped with a 25 m � 0.32 mm � 0.5 lm Stabiwax-DA column

Table 1Reactor design and initial parameters (based on 1 kg reactor content)a.

Reactor design Initial parameters

Effluent (g) Autoclaved effluent(g) Corn stover (g) Water (g) TS (%) F/Etb ratio F/Eab ratio pH Alkalinity (mgCaCO3/kg)889 0 111 0 20 2.2 2.2 8.4 ± 0.1 1695.8 ± 41.5445 445 111 0 20 2.2 4.4 9.0 ± 0.0 1731.3 ± 60.2296 593 111 0 20 2.2 6.6 9.2 ± 0.0 1772.9 ± 15.6610 0 152 238 20 4.4 4.4 8.5 ± 0.1 1036.4 ± 2.2

a The +/� values represent standard errors.b F/Et ratios and F/Ea ratios are calculated based on VS.

Fig. 1. (a) Accumulative methane yield, (b) methane content and (c) daily methaneyield during SS-AD of corn stover at different F/Et and F/Ea ratios.

190 J. Shi et al. / Bioresource Technology 157 (2014) 188–196

(Restek, Bellefonte, PA, USA) and flame ionization detector (FID).Helium was used as carrier gas at a flow rate of 10.8 mL/min.The temperatures of the column and detector were maintained at150 and 250 �C, respectively.

Cellulose and hemicellulose contents of corn stover and reactorcontents were determined using a two-step acid hydrolysis processaccording to the NREL Laboratory Analytical Procedure (Sluiteret al., 2010). Monomeric sugars (glucose, xylose, galactose,arabinose, and mannose) and cellobiose were measured by HPLC(Shimadzu LC-20AB, Columbia, MD, USA) equipped with a BioradAminex HPX-87P column and a refractive index detector (RID)using deionized water as mobile phase at a flow rate of 0.6 mL/min.

The volume of biogas collected in the Tedlar bags was measuredby a liquid displacement method (Park and Li, 2012) and the compo-sition of the biogas (CO2, CH4, N2, and O2) was analyzed by gas chro-matograph (GC) (Agilent Technologies, HP 6890, Wilmington, DE,USA) equipped with a 30 m � 0.53 mm � 10 lm alumina/KCl deac-tivation column and a thermal conductivity detector (TCD) usinghelium at a flow rate of 5.2 mL/min as carrier gas. The temperaturesof the injector and detector were set at 150 and 200 �C, respectively.

2.4. PCR-DGGE based microbial community analysis

Reactor contents taken during the SS-AD process were stored in a�20 �C freezer. Samples were thawed on ice and two 0.5 g portionsof homogenized sludge were taken from each sample for DNAextraction using the repeated bead beating plus column purification(RBB+C) method (Yu and Morrison, 2004b). Denaturing gradient gelelectrophoresis (DGGE) analysis of the archaeal and bacterial com-munities for each sample was performed by PCR amplification of theV3 hyper-variable region using the extracted community DNA (Yuet al., 2008; Yu and Morrison, 2004a). The DGGE gels were processedusing BioNumerics (v.5.1, Applied Maths, Inc., Austin, TX) to extractthe binary banding patterns for principle component analysis (PCA)as previously described (Cressman et al., 2010).

2.5. Statistical analysis

SAS 9.2 software (SAS Institute Inc., Cary, NC, USA) was used foranalysis of variance (ANOVA) and Tukey’s honestly significantdifference (HSD) tests on experimental results with a thresholdp-value of 0.05. Canonical correlation analysis (CCA) and PCA wereconducted on the binary matrices derived from the archaeal andbacterial DGGE profiles and on the other measurements, which in-cluded F/Et, F/Ea, VFA, pH, daily methane yield, and daily reductionin TS, VS, cellulose, and xylan, using XLSTAT version 2012 (Addin-soft, New York, NY). All detectable bands were included in the PCAand CCA analysis.

3. Results and discussion

3.1. Methane yields and related initial parameters

The accumulative methane yields, methane contents, and dailybiogas yields of reactors during 38 days of SS-AD are shown in

Fig. 1. The accumulative methane yields of all reactors with anF/Et ratio of 2.2 ranged between 100–103 L/kgVSfeed, while thereactors at an F/Et ratio of 4.4 had much lower accumulative

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methane yield of 17 L/kgVSfeed, which was approximately 83% lesscompared with reactors at an F/Et ratio of 2.2 (Fig. 1a). Althoughthe accumulative methane yield was low at the end of 38 days,biogas production peaked earlier in the reactors with an F/Et of4.4, probably because of the higher concentration of feedstockand easily digestible materials at this F/Et ratio (Fig. 1c). However,the biogas production at an F/Et of 4.4 started to decline rapidlyafter day 4 and ceased after day 16 (Fig. 1c). Low methane contentsof about 40% were also observed in reactors with an F/Et of 4.4,indicating possible acidification and reactor failure (Fig. 1b). Incontrast, the daily biogas yields of all reactors at an F/Et of 2.2exhibited trends similar to typical ‘‘healthy’’ batch digesters. Themethane yields obtained from this study at F/Et ratios of 2.2 werecomparable with those obtained in previous studies on SS-AD ofcorn stover where methane yields were 100–130 L/kgVSfeed atF/Et ratios of 2 and 3 and TS contents of about 22–20% (Brownet al., 2012; Xu et al., 2013). An F/Et ratio of 4.4 caused reactorfailure in the present study, which was in accordance with aprevious study that reported failed SS-AD of corn stover at an F/Et ratio of 4.58 and a TS content of 22% (Li et al., 2011b). However,at a TS content of 22%, F/Et ratios of 3.44 and 4.0 did not causereactor failure, but did reduce the methane yield of corn stovercompared with lower F/Et ratios (Li et al., 2011b; Xu et al., 2013).This difference may indicate that when digesting corn stover atabout 20% TS, the critical F/Et ratio may be between 4 and 4.4,and F/Et ratios higher than this value may cause reactor failure.

The relationship between the 38-day accumulative methaneyields and initial parameters, including F/Et ratio, F/Ea ratio, pH,and alkalinity, were analyzed by linear regression. The resultsshowed that within the studied range of F/E ratios, the F/Et ratiowas negatively related to accumulative methane yield (R2 = 0.9993).Alkalinity was positively related to methane yield, with an R2

of 0.9871. The initial pH had a weak positive relationship withmethane yield (R2 = 0.2513). However, the F/Ea ratio appeared tohave no relationships with methane yield (R2 = 0.0005), indicatingthat it may not have a significant influence on the final methaneyield compared to the F/Et ratio.

This study found that the influence of F/Et ratio on methaneyield agreed with most of the previous research on batch SS-ADsystems: increasing the inoculation size (decreasing F/Et ratio) re-sulted in higher methane yields from the same feedstock (Forster-Carneiro et al., 2008; Li et al., 2011b). However, in those previousstudies, especially that used AD effluent as inoculum, the influenceof F/Et ratio was actually a combined effect of the microbial prop-erties (indicated by F/Ea ratio) and non-microbial properties, suchas alkalinity, C/N ratio, and micronutrients. With more L-AD efflu-ent additions (decreased F/Et ratio), the inoculated microbial pop-ulations (indicated by the F/Ea ratio) and alkalinity of the reactorboth proportionally increased. Because the acidification (pH drop)during startup is one of the major challenges in the AD process(Griffin et al., 1998), the high methanogenic population and highalkalinity can both contribute to stabilizing pH, reducing inhibi-tions and improving methane production (Prochazka et al., 2012;Xu et al., 2013). The initial pH values in this experiment wereslightly different. Usually the F/E ratio will not alter the initialpH of the reactor when using neutral feedstocks, such as lignocel-lulosic biomass. However, due to the release of CO2 during auto-claving, the autoclaved L-AD effluent had a slightly higher pH(from 8.4 to 9.2) but similar alkalinity (Table 1). The most suitablepH for methanogenesis is usually considered to be 7–7.5 (Chenet al., 2008). However, higher pH values of 8–8.7 are commonlyfound in many L-AD and SS-AD reactors (Moller and Muller,2012). In this experiment, although the initial pH of some reactorswas around 9, the pH values decreased on the second day to below8.5 due to the VFA production (Fig. 2a). Thus, the high initial pHwas not likely to affect methane production at high F/Et ratios.

This study found that the tested F/Ea ratio had a minor effect onmethane yield, which is different from many previous studies, inmost of which the F/Ea ratio was the same as the F/Et ratio. How-ever, after the F/Ea ratio was separated from the F/Et ratio by ster-ilizing part of the inoculated L-AD effluent, the initial microbialpopulation might not have been a decisive factor for the methaneyield at F/Et ratios of 2–6. In this research, an F/Et ratio lower than2.2 was not tested since it resulted in high water content and thusfell out of the SS-AD category, while F/Et and F/Ea ratios of greaterthan 6.6 were not explored because F/Ea ratios (and F/Et ratios) ofhigher than 6 usually results in reactor failure if no alkali or bufferis added (Liew et al., 2011). Some research suggested that F/Etratios as high as 40–50 can successfully start up reactors, butmay need a much longer startup time ranging from one week tomonths, and buffer addition is also required (Motte et al., 2013).However, these high F/Et ratios are usually not for practical use,but for the convenience of research so that changes in themicrobial communities during the process can be observed(Forster-Carneiro et al., 2008). One study found that SS-AD ofmunicipal solid waste (MSW) achieved a quick and successfulstartup even without any external inoculum when sufficientalkalinity was provided (Lai, 2001).

3.2. Changes of pH and VFA profile

The pH value and VFA concentration during the 38 days ofSS-AD were further investigated to gain insights into how differentF/Et and F/Ea ratios affect methane yields and reaction kinetics.Acidification of the reactor at F/Et of 4.4 was clearly demonstratedby the pH drop to below 6 after day 8 (Fig. 2a). While at an F/Et of2.2, the pH values of all reactors were slightly decreased during thefirst few days and then remained around 8–8.5 during the process.It was observed that during the startup period (when VFA produc-tion exceeded consumption), reactors with high F/Et ratios accu-mulated greater amounts of VFAs (Fig. 3b). At an F/Et of 2.2, thepeak values of acetate, butyrate, and isobutyrate were significantlyincreased (p < 0.05) as the F/Ea ratio increased from 2.2 to 6.6(Fig. 2c, e.and f) The propionate peaks did not differ significantlyamong different F/Ea ratios (p > 0.05) (Fig. 2d).

Acetate is the major contributor to the total VFAs (Fig. 2b and c).The acetate concentration indicated the balance between aceto-genesis and methanogenesis during the process. Among all reac-tors, those with an F/Et of 2.2 and F/Ea of 2.2 had the lowestacetate accumulation. Acetate concentration peaked at 2.2 g/kgdigestate at day 2 and, afterward, declined quickly to less than1 g/kg digestate after day 8, indicating balanced acetate productionthrough acidogenesis and consumption through active methano-genesis during the whole process. However, for reactors at F/Ea ra-tios of 4.4 and 6.6, more acetate accumulated during the startupstage (first 4 days), indicating the lack of the above balance, likelyas a result of the smaller inoculum size used. After starting up, theacetate concentrations of reactors at an F/Et ratio of 4.4 and F/Earatio of 4.4 were much higher than those under other conditions.The same trends were also observed for other VFAs (Fig. 2d–f).

By correlating the VFA profiles with daily biogas yield andmethane content (Figs. 1 and 2), the reduced buffering capacitywas found to be a major reason for the acidification in reactorswith an F/Et ratio of 4.4. The lower methanogenic activities couldhave also contributed to the VFA accumulation. The decline of dailybiogas yield in reactors with an F/Et ratio of 4.4 started at day 4,which corresponded with the decline of pH to below 7 (Fig. 2a).Syntrophic acetogenesis and methanogenesis at an F/Et of 4.4might have been inhibited by the low pH after day 4, and almostceased after day 16, corresponding to the daily biogas yield andmethane content (Fig. 1 b and c). Although at day 4 the concentra-tions of total VFAs at an F/Et of 4.4 were not higher than reactors

Fig. 2. Changes of pH and VFAs during SS-AD of corn stover: (a) pH, (b) total VFAs, (c) acetate, (d) propionate, (e) butyrate, and (f) isobutyrate.

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Fig. 3. Reduction of TS, VS, and holocelluloses.

192 J. Shi et al. / Bioresource Technology 157 (2014) 188–196

with an F/Et of 2.2 and F/Ea of 6.6, the pH of reactors with an F/Et of2.2 and F/Ea of 6.6 declined only slightly to 8.4 on day 4, likely dueto sufficient alkalinity. Because of this higher pH, the methaneyield remained above 6 L/kgVSfeed until day 15. In contrast, withoutsufficient alkalinity, greater pH drops were observed after day 4 atan F/Et of 4.4, followed by a declined methane yield, while for allreactors with an F/Et of 2.2, the VFA concentrations started to de-cline after day 4, indicating the success of reactor startup.

Results of VFA production and accumulation supported thewidely accepted theory that the population and activities of meth-anogens can have a pivotal effect on AD reactor startup and meth-ane yield (Angelidaki and Ahring, 1992; Raposo et al., 2012). Thus,large inoculation size is often favored because it can provide moreactive microbes, especially methanogens. Moreover, the ratio ofmethanogens to other microbial communities, such as hydrolyticand syntrophic acetogens, could also influence methane produc-tion (Ma et al., 2013; Xu et al., 2013). However, results obtained

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in this study strongly suggest that the non-microbial conditions, asindicated by the F/Et ratio, might have a greater effect on the suc-cess of reactor startup and methane yield. Thus, non-microbial fac-tors of the L-AD effluent need to be considered and further studiedin addition to the microbial properties.

3.3. Reduction of TS, VS, and holocellulose

The reduction of TS, VS, and holocellulose contents were com-pared among reactors with different F/Et and F/Ea ratios (Fig. 3).Similar levels of TS, VS, and holocellulose (cellulose and xlyan)reductions were observed for reactors operated at an F/Et of 2.2with different F/Ea ratios (p > 0.05), which was in agreement withthe similarity in methane yields. Not surprisingly, significantly lessreduction in TS, VS, and holocellulose was observed for reactorsoperated at an F/Et of 4.4 and F/Ea of 4.4 (p < 0.05), which was in

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Fig. 4. Principal component analysis (PCA) of DGGE profiles of (a) archaeal and (b) bacteand an acidified reactor with F/Et = 4.4, F/Ea = 4.4 ( , open circle) conditions.

accordance with the low biogas yield (Fig. 1). However, comparedwith those reactors operated at an F/Et of 4.4, the differences in TS,VS, and hollocellulose reduction in reactors with an F/Et of 2.2were not as large as the differences in methane yields. The meth-ane yield at an F/Et of 4.4 and F/Ea of 4.4 was only about 17% of thatof reactors with an F/Et of 2.2, while the TS, VS and holocellulosereduction were around 70% of that at an F/Et of 2.2. Thus, for theacidified reactors, the high holocellulose reduction but low meth-ane yield might be attributed to the degradation of holocelluloseto VFAs (Xu and Li, 2012).

3.4. PCR-based DGGE analysis of the archaeal and bacterialcommunity

To gain more insights into how microbial communities were af-fected by the inoculation, PCR-based DGGE analysis was used to

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rial communities from a healthy reactor with F/Et = 4.4, F/Ea = 4.4 (h, open square)

194 J. Shi et al. / Bioresource Technology 157 (2014) 188–196

examine communities of the archaea and bacteria in both normal(healthy) and acidified digesters. The archaeal community under-went obvious temporal shifts, as evidenced by the disappearanceand appearance of DGGE bands and changes in band intensitiesduring the normal and acidified SS-AD processes, with the ob-served shifts being in accordance with the VFA accumulation(Fig. S1a, Table S1). Several DGGE bands (bands A1, A10, A17,A18) were observed at both the acidified (F/Et = 4.4, F/Ea = 4.4)and the normal (F/Et = 2.2, F/Ea = 4.4) conditions (Fig. S1a,Table S1). These DGGE bands might represent the persistent meth-anogen populations that functioned under both sets of conditions.However, several DGGE bands (bands A5, A7, and A11) were moreintense under normal conditions than under acidified conditions.These intense DGGE bands might represent a few methanogenicpopulations that survived under the acidic conditions but theirgrowth and function were compromised. The emergence of bandA14 and changes in band intensity during acidified SS-AD probablyreflected the enrichment of a methanogenic population by the low

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B26

B27

F/Et

pH dYmethdTS

dVS

dCe

-2

-1

0

1

2

-3 -2 -1 0 1

F2 (2

3.22

%)

F1 (34.14 %

CCA Map / Asymmetric (axes F1 and F2: 57.

A1

A2

A8

A9

A11

A12

A13

A14

A15

A16

A19

F/Et

pH

VFA

dTS

dVS

dCe

-2

-1

0

1

2

-4 -3 -2 -1 0

F2

(28.

99 %

)

F1 (33.46

CCA Map / Asymmetri(axes F1 and F2: 6

Fig. 5. Canonical correspondence analysis (CCA) ordination diagrams showing the correlaenvironmental variables and the performance variables for healthy and acidified reacto

pH. The high level of acetic acid accumulation after day 8 at acid-ified conditions was likely caused by the imbalanced hydrolysis,acidogenesis, and methanogenesis, as evidenced from the reduc-tion of holocellulose by hydrolytic microbes (Fig. 3).

The bacterial community also underwent successions to someextent under both SS-AD conditions (Fig. S1b, Table S2). A fewcommon bands (B6, B7, B10 and B11) were detected among thesamples collected during both normal and acidified SS-AD, sug-gesting the persistence of many bacteria under both conditions.However, some distinct bands were only found under normal(B2, B4, B14, B15) or acidified (B1, B8, B17, B23) SS-AD conditions.The aforementioned shifts in the bacterial communities suggestedthat diverse bacteria, possibly with different substrate spectra andgrowth pH requirements, were selected and involved in the SS-ADprocess. This result is expected given the multitude of inter-relat-ing metabolic pathways of bacteria compared to those of archaea(Gao et al., 2011; Weiss et al., 2008). Due to the limited informationthat DGGE analysis can provide, future study is needed to reveal

8720

21

ane

VFA

llulose

dXylan

2 3 4

)

/ Objects35 %)

a) Archaeal

b) Bacterial

A3

A4

A5

A6

A7A10

A17A18

dYmethane

llulose

dXylan

1 2 3 4

%)

c / Objects2.45 %)

tion between the DGGE bands of (a) archaeal and (b) bacterial communities and thers.

J. Shi et al. / Bioresource Technology 157 (2014) 188–196 195

the microbial community composition, structure, and populationshift using other technologies, such as metagenomics. It is alsonoted that SS-AD of lignocellulosic feedstocks can be a very sophis-ticated process. In this study, we used effluent of L-AD as thesource to inoculate the digesters. The microbial community shiftsduring the SS-AD processes can be attributed to several other fac-tors, including changes of substrates and water activity and varia-tions in temperature and pH gradients due to limited heat andmass transfer, which warrant further study.

The principal component analysis (PCA) separated the sampleslargely based on either pH shifts (caused by inoculation size) ortemporal shifts; but the separation was limited, indicating thatneither was the primary factor (F1, F2) that accounted for mostof the total variation (Fig. 5a and b). Canonical correspondenceanalysis (CCA) was also performed to examine potential correla-tions between the bands of archaea and bacteria with the environ-mental variables measured (i.e., F/Et ratio, pH, and VFAconcentrations) as well as the performance parameters deter-mined, the latter of which included daily methane yield (dYmethane),daily TS reduction (dTS), daily VS reduction (dVS), daily cellulosereduction (dCellulose), and daily xylan reduction (dXylan). TheCCA results provided further evidence of a link between the com-positions of the archaeal and bacterial community, as determinedby DGGE, and environmental factors such as pH, inoculation, andVFA concentration (Fig. 5). Significance analysis of the environ-mental variables revealed that inoculation (F/Et ratio) accountedfor much of the difference in both the archaeal and bacterial com-munity compositions observed between the normal and the acidi-fied conditions and had a statistically significant correlation withspecies composition (p < 0.05). VFA was positively associated withthe F/Et ratio, while pH was negatively associated with the F/Et ra-tio and VFA concentration. As expected, all the performance vari-ables (dYmethane, dTS, dVS, dCellulose, and dXylan) werepositively associated with each other: that is cellulose and xylanreductions were positively associated with TS and VS reduction(Fig. 4) and TS/VS reduction was positively correlated with biogasyield. It was also noted that some DGGE bands of archaea and bac-teria were correlated with environmental factors and performancemeasurements, suggesting that some methanogens and bacteriaare more dynamic and involved in AD processes than others. How-ever, detailed speciation of these archaea and bacteria can only beachieved by sequencing analysis.

4. Conclusion

Inoculation is critical to the startup and performance of SS-ADof corn stover and the succession of the microbial communitiesthat underpin the SS-AD process. High F/Et ratios led to low pH(acidification) because of the limited buffering capacity (low alka-linity). The inoculum size, as reflected by the F/Ea ratio, did not sig-nificantly change the reactor performance. DGGE analysis revealedconsiderable successions of both bacterial and archaeal communi-ties, reflecting the selection for some bacteria and archaea by envi-ronmental conditions. The results of the present study may helpunderstand the roles of L-AD effluent in SS-AD of corn stover.

Acknowledgement

This project was funded by the USDA NIFA Biomass Researchand Development Initiative Program (Award No. 2012-10008-20302) and USDA NIFA Hatch Program. The authors would like tothank Mrs. Mary Wicks (Department of Food, Agricultural and Bio-logical Engineering, OSU) for reading through the manuscript andproviding useful suggestions.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.biortech.2014.01.089.

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