Bioresource Technology 175 (2015) 430–435

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Bioresource Technology

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Comparison of premixing methods for solid-state anaerobic digestionof corn stover

http://dx.doi.org/10.1016/j.biortech.2014.10.0950960-8524/� 2014 Elsevier Ltd. All rights reserved.

⇑ Corresponding author. Tel.: +1 330 263 3855; fax: +1 330 263 3670.E-mail address: li.851@osu.edu (Y. Li).

1 Authors contributed equally to this work.

Jiying Zhu a,b,1, Liangcheng Yang a,1, 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, OH44691-4096, USAb School of Agricultural and Food Engineering, Shandong University of Technology, Zibo, Shandong 255049, China

h i g h l i g h t s

� Three premixing methods were compared for SS-AD of corn stover.� Two-layer partial premixing method showed the highest methane yield.� Partial premixing methods increased regional inoculation ratio.� VFA concentrations were affected by premixing methods.� Adding extra inoculum to failed digesters partially recovered methane production.

a r t i c l e i n f o

Article history:Received 25 August 2014Received in revised form 12 October 2014Accepted 18 October 2014Available online 25 October 2014

Keywords:Solid-state anaerobic digestionComplete premixingPartial premixingDigester recovery

a b s t r a c t

The development of solid-state anaerobic digestion (SS-AD) has prompted studies to resolve practicalchallenges such as mixing of feedstock and inoculum. This study compared the performance of SS-ADusing three premixing methods. Results showed that at feedstock to inoculum (F/I) ratios of 4 and 6,the two-layer partial premixing method obtained the highest methane yield, followed by one-layer par-tial premixing and complete premixing methods. Partial premixing methods also showed wider dailymethane yield peaks than the complete premixing method. The volatile fatty acid (VFA) concentrationwas affected by the premixing method, and was highly correlated to methane yield; while the concentra-tion of remaining holocellulose was correlated to pH and alkalinity. SS-AD digesters failed at an F/I ratioof 8, regardless of the premixing method. Adding extra inoculum to the top of failed digesters resulted inrecovery of methane production.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction One major concern of SS-AD is the low mass transfer rate result-

More than 60% of the recently built anaerobic digesters in Eur-ope have adopted solid-state anaerobic digestion (SS-AD) technol-ogy that can handle materials with higher than 15% total solids (TS)content (Karthikeyan and Visvanathan, 2013). Compared to con-ventional liquid anaerobic digestion (L-AD,<15% TS), SS-AD fea-tures high organic loading rate, small digester volume, lowenergy demand for heating, and high volumetric methane produc-tion (Li et al., 2011; Yang and Li, 2014). Particularly, SS-AD is suit-able for digestion of lignocellulosic biomass as it alleviates thefloating and stratification issues that occur in L-AD (Brown et al.,2012).

ing in low inoculation efficiency (Bollon et al., 2013; Kalyuzhnyiet al., 2000). Therefore, effective mixing of feedstock and inoculumis required. Mechanical mixing using a stirrer during the digestionprocess has been tested (Verma, 2002), but it was difficult to stirfeedstocks with high total solids content and high viscosity. Afew other practices have tried to enhance mixing without usingstirrers. Examples are recirculating leachate from the bottom tothe top of the digester (Zhu et al., 2014) and recirculating producedbiogas to the bottom of the digester (Karagiannidis and Perkoulidis,2009). Besides in-situ mixing during the digestion process, premix-ing feedstock and inoculum prior to digestion may be a usefulalternative that can offer benefits such as space flexibility, highmixing efficiency, and reduced digester maintenance costs. Com-plete premixing of feedstock and inoculum is common for lab scalestudies (Li et al., 2011; Lin et al., 2014) and previous modelingstudies also suggested that uniform inoculum distribution(complete mixing) would outperform non-uniform inoculum

Table 1Properties of feedstock and inoculum.

Parameters Feedstock Inoculum

TS, % 89.14 ± 0.43 8.06 ± 0.01VS, % 84.05 ± 0.61 3.94 ± 0.01TC, % 43.40 ± 0.23 2.60 ± 0.07TN, % 0.60 ± 0.00 0.32 ± 0.01C/N ratio 71.86 ± 0.75 8.19 ± 0.59pH 6.41 ± 0.02 8.00 ± 0.01Alkalinity, g-CaCO3/kg 0.19 ± 0.00 10.76 ± 0.21VFAs, g/kg 0.27 ± 0.02 0.93 ± 0.06Cellulosea, % 34.40 ± 0.35 1.75 ± 0.13Hemicellulosea, % 20.61 ± 0.05 1.01 ± 0.00

TS = total solids, VS = volatile solids, TC = total carbon, TN = total nitrogen,C/N = carbon to nitrogen, VFAs = volatile fatty acids.Average ± S.E., n = 2.

a Based on TS. Others are based on total weight.

Table 2Experimental design of SS-AD digesters.

Initial F/I Premixing methods TS, % Recovery F/I after recovery

2 CP 15 No N/A4 CP 21 No N/A4 PP-1 21 No N/A4 PP-2 21 No N/A6 CP 26 No N/A6 PP-1 26 No N/A6 PP-2 26 No N/A8 CP 30 Yes 48 PP-1 30 Yes 48 PP-2 30 Yes 4

CP: complete premixing, PP-1: partial premixing with rest of the inoculum in onelayer, PP-2: partial premixing with rest of the inoculum in two layers. F/I ratio wascalculated based on VS. N/A = not applied.

J. Zhu et al. / Bioresource Technology 175 (2015) 430–435 431

distribution (Vavilin et al., 2002a,b). However, complete premixingmight not be affordable for large scale garage type SS-AD due tohigh energy demand. After complete premixing, the materialsmight become more pumpable, but would be more difficult to loadinto a garage type digester with toe tip buckets. Therefore,researchers are interested in developing partial premixing meth-ods that may resolve this problem and also reduce inoculation sizeand energy costs. One early partial premixing test was carried outby Veeken and Hamelers (2000), who added feedstock and inocu-lum into digesters layer by layer without further mixing and notedthat the slow mass transfer of volatile fatty acids (VFAs) was a lim-iting factor. Later, El-Mashad et al. (2006) tested two partial pre-mixing methods including (1) adding inoculum at the bottom ofthe digester, and (2) dividing the same amount of inoculum inequal doses to be loaded alternatively with feedstocks in two lay-ers without mixing. Their results showed that the average dailymethane production was 0.4 and 0.5 L for methods (1) and (2),respectively. More recently, Zhu et al. (2014) premixed 50% ofthe required inoculum with feedstock and then directly addedthe remaining inoculum onto the top of the feedstock without fur-ther mixing. Similar cumulative methane yields were obtainedwith the 50% premixing method compared to the control methodthat completely premixed inoculum with feedstock. In that study,it was observed that the inoculum, which was added on the top ofthe digester, gradually permeated through the feedstock, leading toa much longer startup period than the complete premixingmethod.

Besides long startup times, another possible risk of premixing isthe accumulation of AD intermediate products due to the low masstransfer rate (Gerardi, 2003; Liew et al., 2012). For partial premix-ing, the overall effectiveness of inoculation in the digester isdependent on inoculum diffusion, which is not likely to be homo-geneous, and thus may cause regional low inoculation ratio envi-ronments (Zhu et al., 2014). Low inoculation ratios can beproblematic or even cause digester failure. Recovery of a malfunc-tioning SS-AD digester, although of practical importance, has rarelybeen studied (Chiew and Cord-Ruwisch, 1991).

The objectives of this study were to (1) compare the perfor-mance of complete premixing with two partial premixing methodsfor methane production via SS-AD of corn stover at various inocu-lation ratios, (2) explore the possible causes of methane yield var-iability, and (3) assess recovery of failed SS-AD digesters by addingextra inoculum to the top of the digester.

2. Methods

2.1. Feedstock and inoculum

Corn stover was used as the feedstock in this study. It was col-lected from a research farm at the Ohio Agricultural Research andDevelopment Center (OARDC) in Wooster, OH, USA, in January2013, and was air dried to less than 15% moisture content and thenground to pass a 6 mm sieve (Mighty Mac, MacKissic Inc., ParkerFord, PA, USA). Effluent taken from a mesophilic liquid anaerobicdigester (fed with municipal sewage sludge and food wastes, oper-ated by quasar energy group in Zanesville, OH, USA) was used as theinoculum and was activated at 37 �C in an incubation room for oneweek prior to use. L-AD effluent provides not only digestionmicrobes, but also macro- and micro-nutrients, and bufferingcapacity (Shi et al., 2014; Wan et al., 2011; Wang et al., 2013);and has been shown to be a better inoculum than activated sludge,manure, or rumen fluid (Forster-Carneiro et al., 2007). CombiningL-AD and SS-AD through the inoculation of solid feedstock withL-AD effluent also provides a solution for the treatment of L-ADeffluent. Characteristics of the feedstock and inoculum are shownin Table 1.

2.2. Premixing methods

Three premixing methods were examined: (1) complete pre-mixing (CP), in which feedstock and inoculum were premixed out-side of the digesters to achieve designated initial feedstock toinoculum (F/I, based on VS) ratios of 2, 4, 6, and 8; (2) partial pre-mixing of inoculum and feedstock to achieve an F/I ratio of 10 andone layer of inoculum (PP-1) placed on top of the feedstock toattain overall F/I ratios of 4, 6, and 8; and (3) partial premixingof inoculum and feedstock to achieve an F/I ratio of 10 and two lay-ers of inoculum (PP-2), one in the middle of the feedstock and oneon the top, to achieve overall F/I ratios of 4, 6, and 8 (Table 2). Thefirst step of partial premixing was to reduce the chance of digesterfailure. Schematics of digesters prepared with the three premixingmethods are shown in Fig. 1. As indicated, partial premixing digest-ers with one layer of inoculum had one top layer and one bottomlayer, with higher inoculation ratios (lower F/I ratio) in the toplayer; while partial premixing digesters with two layers of inocu-lum had two top layers and two bottom layers, also with higherinoculation ratios in the top layers. Note that the layers had noclear boundaries as the inoculum penetrated through the digestingmaterials; therefore, the layers were only roughly differentiated bymoisture content and color.

2.3. Anaerobic digestion

The SS-AD tests were carried out in 5-L plastic digesters withoutleachate or biogas recirculation in a 37 �C incubation room. The ini-tial TS contents ranged from 15% to 30% as shown in Table 2. Foreach digester, a 10-L Tedlar gas bag (CEL Scientific, Santa FeSprings, CA, USA) was attached to collect biogas every 2–5 daysduring the 60-day experimental period. All tests had two repli-cates. Digestate samples were collected at the beginning and the

(a) (b) (c)

T

B

T1

B1

T2

B2

Fig. 1. SS-AD digesters prepared with different premixing methods. (a): completepremixing (CP), (b): partial premixing with rest of the inoculum in one layer (PP-1),(c): partial premixing with rest of the inoculum in two layers (PP-2). T: top layer, B:bottom layer, T1: top1 layer, B1: bottom1 layer, T2: top2 layer, B2: bottom2 layer.

Cum

ulat

ive

met

hane

yie

ld, L

/kg-

VS

0

60

120

180

240

300F/I=2A

0

20

40

60

80F/I=4B

Operation time, d

0 10 20 30 40 50 600

20

40

60

80F/I=6C

PP-1PP-2

CP

Fig. 2. Effect of premixing methods on cumulative methane yields at F/I ratios of 2,4, and 6. CP: Complete premixing; PP-1: partial premixing with the rest of inoculumin one layer; PP-2: partial premixing with the rest of inoculum in two layers.

432 J. Zhu et al. / Bioresource Technology 175 (2015) 430–435

end of the test. For the failed digesters, digestate samples were alsocollected on day 20 before adding extra inoculum.

2.4. Analytical methods

The volume of collected biogas was measured using a drum-type gas meter (Ritter, Bochum, Germany) and its composition(i.e. CH4, CO2, N2, and O2) was analyzed with a gas chromatograph(Agilent, HP 6890, Wilmington, DE, USA) equipped with a30 m � 0.53 mm � 10 lm Rt� -Alumina Bond/KCl deactivation col-umn and a thermal conductivity detector (TCD). Helium gas wasused as a carrier gas at a flow rate of 5.2 mL/min. The temperatureof the detector was maintained at 200 �C, while the initial temper-ature of the oven was set at 40 �C and then increased to 60 �Cwithin one minute. TS, VS, pH, and alkalinity of digesting materialswere measured based on the modified standard methods for exam-ination of water and wastewater (APHA, 2005). Specifically, a 5 gsample was diluted with 50 ml deionized (DI) water, then the pHand alkalinity were measured using an auto-titrating pH meter(Mettler Toledo, DL22 Food & Beverage Analyzer, Columbus, OH,USA). Total carbon and total nitrogen contents were determinedusing an elemental analyzer (Elementar Vario Max CNS, ElementarAmericas, Mt. Laurel, NJ, USA). VFAs, which include propionic, ace-tic, isovaleric, butyric, isobutyric, and valeric acids, were analyzedusing a gas chromatograph (Shimadzu, 2010PLUS, Columbia, MD,USA) equipped with a 30 m � 0.32 mm � 0.5 lm Stabilwax� polarphase column and a flame ionization detector (FID) according to amethod described previously (Shi et al., 2013). Digestate washydrolyzed at 121 �C for 1 h according to the NREL standard(Sluiter et al., 2008), and then the hydrolyzed monomeric sugars(i.e. glucose, xylose, galactorse, arabinose, and mannose) were ana-lyzed using a high-performance liquid chromatograph (Shimadzu,LC-20AB, Columbia, MD, USA) equipped with a Biorad AminexHPX-87P column and a refractive index detector (RID). HPLC gradewater was used as the mobile phase at a flow rate of 0.3 mL/min,and temperatures of the column and detector were maintainedat 60 and 55 �C, respectively.

2.5. Data analysis

Student’s t test and ANOVA were used to compare results. Datawere analyzed using R Studio software with a significance level of0.95.

3. Results and discussion

3.1. Comparison of partial premixing methods

3.1.1. Methane yieldBoth F/I ratio and premixing method affected methane yields

from SS-AD of corn stover. At an F/I ratio of 2, an average cumulative

methane yield of 222 L/kg-VS was achieved for the digesters usingcomplete premixing (Fig. 2A). This result was comparable to aprevious study that showed methane yields of 110–230 L/kg-VSusing three different inocula for mesophilic anaerobic digestion ofcorn stover with an F/I ratio of 2 and using the complete premixingmethod (Xu et al., 2013). However, the methane yields decreasedsignificantly (p < 0.05) when the F/I ratio was increased to 4 or 6,at which conditions, the premixing methods were shown to beinfluential. The highest methane yields, 86 and 58 L/kg-VS at F/Iratios of 4 and 6, respectively, were obtained in partially premixeddigesters with two layers of inoculum (PP-2), followed by thepartially premixed digesters with one layer of inoculum (PP-1),while the methane yields from the completely premixed digesters(CP) were negligible (Fig. 2B and C). These results indicated thatinoculation ratio was critical for the SS-AD of corn stover and partialpremixing was effective in improving methane yield at high F/Iratios. Partial premixing lowered the F/I ratio in regions of thefeedstock, e.g. in the proximate region of the inoculum layers in thepartially premixed digesters, improving performance. Relativelyhigher methane yields were obtained in partially premixeddigesters with two layers of inoculum than that with one layer ofinoculum, indicating that the feedstock was more accessible tothe inoculum in the digesters with two layers of inoculum.

Premixing method also affected the peaks of daily methaneyield. At an F/I ratio of 2, the daily methane yields of the digesterswith complete premixing peaked on day 8 and then quicklydecreased (Fig. 3A); while for the partially premixed digesters withtwo layers of inoculum and an F/I ratio of 4, the daily methaneyield peaks were much wider, starting on day 8 and continuingto day 20 (Fig. 3B). Likewise, the peaks of the partially premixeddigesters with one layer of inoculum lasted for 8 days (Fig. 3B).

Dai

ly m

etha

ne y

ield

, L/k

g-V

S 0

3

6

9

12F/I=2A

0

1

2

3

F/I=4B

Operation time, d

0 10 20 30 40 50 600

1

2

3

F/I=6C

PP-1PP-2

CP

Fig. 3. Effect of premixing methods on daily methane yield at F/I ratios of 2, 4, and6. CP: Complete premixing; PP-1: partial premixing with the rest inoculum in onelayer; PP-2: partial premixing with the rest of inoculum in two layers.

J. Zhu et al. / Bioresource Technology 175 (2015) 430–435 433

The wide peaking time in the partially premixed digesters couldhave been caused by the slow diffusion of the inoculum from con-centrated regions to poorly inoculated regions. However, the diffu-sion of inoculum may have been limited, as the high solid contentin SS-AD may result in a high mass transfer barrier. The previouslyobserved lower cumulative methane yields from the partially pre-mixed digester with one layer of inoculum than from that with twolayers also suggest that the availability of inoculum was limited tosurrounding areas only. Therefore, to further increase methaneyield, multiple layers of inoculum may minimize poorly inoculatedregions.

3.1.2. Concentration of VFAs in digestateThe concentration of total VFAs varied with premixing method

and location in the digester. For the complete premixing digesters,the concentration of total VFAs was 0.4 g/kg at an F/I ratio of 2, but

VFA

con

c., g

/kg

0

5

10

15

20

F/I = 2

A:complete premixing

B

T T

4 6 4 6

B: partial premixin one-layer inocul

Fig. 4. Effect of premixing methods on the concentrations of VFAs in digestate. T: top lalayer. Shown in Fig. 1.

increased dramatically to 16.7 and 20.1 g/kg at F/I ratios of 4 and 6,respectively (Fig. 4A). These high VFA levels are inhibitory to meth-anogenesis (Gerardi, 2003) as indicated by the cumulative meth-ane yields, showing that the critical F/I ratio in this test withcomplete premixing was between 2 and 4. With regard to partiallypremixed digesters with one layer of inoculum, significantly differ-ent (p < 0.05) total VFA concentrations were observed between thetop and bottom layers at F/I ratios of both 4 and 6 (Fig. 4B). Simi-larly, significantly different (p < 0.05) total VFA concentrationsamong the four layers was observed for partially premixed digest-ers with two layers of inoculum at both F/I conditions (Fig. 4C), andthe total VFA concentrations in the two bottom layers (B1 and B2)were higher at an F/I ratio of 6 than that at 4. Among the VFAs, ace-tic acid, propionic acid, and butyric acid were the dominant organicacids, and may have been responsible for the inhibition. Forinstance, the acetic acid in the complete premixing digestersreached 13.4 g/kg at an F/I ratio of 6, which was much higher thanthe threshold value (3 g/L) that will inhibit methanogenesis(Stronach et al., 1986). The inhibition of VFAs very likely causedthe low methane yields in digesters with complete premixing(Gerardi, 2003).

Partial premixing was shown to reduce regional VFA concentra-tions. In the partial premixing digesters, the total VFA concentra-tions in the top layers (T, T1, and T2) were less than 0.3 g/kg,which were not inhibitory to the methanogenesis process(Stronach et al., 1986). The low VFA concentrations in these toplayers indicated a balanced degradation process that would con-tribute to methane production. Besides, partial premixing withtwo layers of inoculum also showed lower total VFA concentra-tions in the two bottom layers (B1 and B2 layers) compared tothe bottom layer (B layer) in partially premixed digesters withone layer of inoculum. This phenomenon supported the observa-tion that relatively higher methane yields were from the partialpremixing digesters with two layers of inoculum than from thatwith one layer.

3.1.3. Degradation of holocelluloseDegradation of holocellulose (cellulose and hemicellulose) lar-

gely determines the methane yield of corn stover, as the otherorganic components, such as protein and extractives, are usuallylow in corn stover (Xu et al., 2013). However, the degradation rateof cellulose and hemicellulose in each layer in the digesters wasdifficult to obtain due to the penetration of inoculum in digestersduring the digestion process. Instead, the concentrations ofremaining holocellulose in the digestate were reported in thisstudy. Results showed that holocellulose contents in most of thedigesters were in the range of 8–15% TS, with a few exceptionshigher than 20% (Fig. 5). About 56–64% of holocellulose was cellu-lose, while 36–44% was hemicellulose.

The content of the remaining holocellulose was correlated withdigestate pH and alkalinity (Fig. 5). With regard to pH, most sam-ples were in the range of 7.5–8.5 with a holocellulose content

B

B2T2

B1T1

aceticpropionicbutyricisobutyricisovalericvaleric

4 6

g with um

C:partial premixing with two-layer inoculum

B2

T2B1

T1

yer, B: bottom layer, T1: top1 layer, B1: bottom1 layer, T2: top2 layer, B2: bottom2

Alkalinity, g-CaCO3/kg4 6 8 10 12

holo

cellu

lose

, %TS

0

6

12

18

24

30

pH values in digestates5 6 7 8 9

alkalinity pH

Fig. 5. Effects of pH and alkalinity on the concentration of remaining holocellulose.

434 J. Zhu et al. / Bioresource Technology 175 (2015) 430–435

lower than 20%; while only four samples had pH values of 5.5–6.1with holocellulose content higher than 20%. Two of the four sam-ples were collected from complete premixing digesters with F/Iratios of 4 and 6, while the other two were from the bottom layerof partially premixed digesters with one layer of inoculum. As theoptimum pH for methanogenesis is 6.8–7.2 (Vavilin et al., 2008;Veeken et al., 2000), the low pH conditions in these locationswould have inhibited methanogenesis and further negativelyaffected the degradation of organics, thus reducing methane yield.All layers in partially premixed digesters with two layers of inocu-lum had normal pH values of 7.7–8.4, indicating this premixingmethod was more favorable than the other two. Similarly, lowalkalinity (5.1–6.9 g-CaCO3/kg) was observed for digestate withhigh holocellulose content; while the digestate with holocellulosecontent lower than 20%, had alkalinities in the range of 8.7–11.5g-CaCO3 (Fig. 5). Similar to previous observations about thedistribution of VFA concentration, the content of the remainingholocellulose in the digestate was also affected by premixingmethod and was differentiated by layers (data not show).

3.2. Performance of AD recovery process

According to this experimental design, digesters with a highoverall F/I ratio of 8 (low inoculation ratio) were highly likely tofail. As expected, during days 1–20, methane yields were almostnegligible from these digesters, regardless of the premixing meth-ods (Fig. 6A), although their methane contents were about 10–15%(Fig. 6B). These results indicated that these digesters had failed,possibly caused by the very high VFA levels (Table 3) and/or highsolid content (Table 2). To recover these digesters, extra inoculumwas added to the top of the digesters to bring the overall F/I ratio

Operation time, d0 10 20 30 40 50 60

Cum

ulat

ive

met

hane

yie

ld, L

/kg-

VS

0

15

30

45

60 CPPP-1PP-2

Extra inoculum added on day 20

A

Fig. 6. Performance of the recovery step. A: cumulative methane yields, and B: methanefailed digesters before recovery. Only positive standard error is shown to avoid overlap

down to 4, without further mixing (Table 2). This action was con-sidered to be a simple ‘‘recovery’’ process. As a result of the addi-tion of extra inoculum, the inoculation ratio for the completelypremixed digesters varied from high at the top to low at the bot-tom during the recovery process; thus, at the end of the recoverytest, the digested materials were mixed prior to taking samples.The partial premixed digesters with one layer of inoculum (todecrease F/I ratio from 10 to 8, PP-1 digesters) also had one layerof high inoculation ratio and one layer of low inoculation ratio dur-ing the recovery process, however, their top layers should havebeen thicker than that in the completely premixed digesters afterrecovery; The materials in these digesters were not mixed at theend of the recovery test. The partial premixing digesters withtwo layers of inoculum (to decrease F/I ratio from 10 to 8, PP-2digesters) still had four layers after recovery and were not mixedin the end of the recovery test; their T1 layers were much thickerthan their T2 layers.

During the following 40 days, the methane yields from thesedigesters were highly dynamic as indicated by the large standarddeviation (Fig. 6A). These large variations could have been causedby the uneven diffusion and distribution of inoculum in the digest-ers. It was noticed that inoculum diffusion near the edge of thedigester penetrated deeper than in the central area. Due to theslow diffusion of inoculum, the methane content increased slowly,and took about 20 days to be stabilized (Fig. 6B). This phenomenonwas quite similar to the results shown by Zhu et al. (2014). Thecumulative methane yields of the premixed digesters during the40-day recovery ranged from 21 to 56 L/kg-VS (Fig. 6A), whichwas comparable to the partially premixed digesters with one layerof inoculum at an F/I of 4 (36 L/kg-VS, Fig. 2B). Compared to thepartially premixed digesters with two layers of inoculum and F/Iratio of 4 (Fig. 2B), the T2 layer in the failed digesters with two lay-ers of inoculum was much thinner as, initially, only a limitedamount of inoculum was added to decrease the F/I ratio from 10to 8; accordingly, the methane yields from these digesters werelower (27 VS 86 L/kg-VS). The recovered digesters with initial com-plete premixing achieved the highest methane yield of 56 L/kg-VSby day 60, which was equivalent to 25% of the methane yields fromdigesters with an F/I ratio of 2 (Fig. 2A). More methane productionwould be expected if a longer retention time were allowed; how-ever, the digesters were terminated on day 60 due to practicalconsiderations.

Similar to previous observations, VFAs can be related to meth-ane yields. For the complete premixing digesters, VFA levelsdecreased significantly (p < 0.05) from day 20 to day 60 (Table 3);whereas, for the partially premixed digesters, VFAs in the toplayers (T, T1, and T2) decreased, while in the bottom layers (B,B1, and B2), VFAs remained high on day 60 (Table 3). The highVFA concentrations on day 20 showed that hydrolysis was carried

Operation time, d0 10 20 30 40 50 60

Met

hane

con

tent

, %

0

15

30

45

60 B

F/I=4

F/I= 8

contents. CP, PP-1, and PP-2 are originally employed premixing methods for these.

Table 3Concentrations of total VFAs in failed digesters on days 20 and 60, g/kg. Aver-age ± SEM, n = 4.

Initial premixing method Location Day 20 Day 60

Complete premixing Overall 13.17 ± 0.54 1.55 ± 0.03

Partial premixing withone layer inoculum

Top layer 12.33 ± 0.02 0.59 ± 0.07Bottom layer 13.65 ± 0.36

Partial premixing withtwo layers of inoculum

T1 layer 12.87 ± 0.04 0.02 ± 0.00B1 layer 3.43 ± 0.44T2 layer 0.24 ± 0.01B2 layer 16.00 ± 0.54

J. Zhu et al. / Bioresource Technology 175 (2015) 430–435 435

out; while the low methane yields from these digesters suggestedthat methanogens were inhibited by the high VFA levels, especiallyin bottom layers.

3.3. Application of partial premixing and recovery in large scaledigesters

Although energy consumption was not analyzed in this study, alower energy demand was expected for the partial premixingmethods compared to the complete premixing method. Partial pre-mixing was carried out in two steps in this study, with the first stepof completely mixing of feedstock and inoculum to a very low inoc-ulation rate (F/I = 10) conducted mainly to avoid failure. However,for large scale SS-AD digesters, this first step may be not adopted.In that case, partial premixing could be carried out by adding feed-stock and inoculum separately, and layer by layer, as tested byVeeken and Hamelers (2000). Increasing the number of inoculumlayers should improve methane yield as evidenced by the resultsobtained in this study (Fig. 2), but may increase energy demand.For SS-AD systems that use trucks to load feedstock, such as garagestyle digesters, in which it is not feasible to add feedstock and inoc-ulum layer by layer (McKiernan, 2012), injection of inoculum couldbe an option. Adding extra inoculum to the top of failed SS-ADdigesters is a simple recovery method and requires less energycompared to the previous recovery method that completely mixedadditional inoculum with failed feedstock to stimulate biogas pro-duction (Chiew and Cord-Ruwisch, 1991). However, the perfor-mance of this method depends on the amount of addedinoculum and the available headspace in the digester. The recov-ered digester would also need a relatively long time to becomeproductive.

4. Conclusion

Premixing method and F/I ratio affected methane yield fromSS-AD of corn stover. At F/I ratios of 4 and 6, partial premixingmethods showed higher cumulative methane yields and widerdaily methane yield peaks than the complete premixing method,and the two-layer partial premixing method outperformed theone-layer partial premixing method. VFAs accumulated at layerswhere inoculation rates were low, and affected the performanceof digesters. Holocellulose contents in digestate were correlatedto pH and alkalinity. Digesters failed at an F/I ratio of 8, whileadding extra inoculum to the top of failed reactors partially andslowly recovered methane production.

Acknowledgements

This project was funded by USDA NIFA Biomass Research andDevelopment Initiative Program (Award No. 2012-10008-20302).

The authors wish to thank Mrs. Mary Wicks (Department of Food,Agricultural and Biological Engineering, OSU) for critical review.

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