Bioresource Technology 158 (2014) 149–155

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Bioresource Technology

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Effect of inoculum sources on the anaerobic digestion of rice straw

http://dx.doi.org/10.1016/j.biortech.2014.02.0110960-8524/� 2014 Elsevier Ltd. All rights reserved.

Abbreviations: DM, digested dairy manure; SM, digested swine manure; CM,digested chicken manure; MS, digested municipal sludge; AGS, anaerobic granularsludge; PS, paper mill sludge.⇑ Corresponding author. Tel./fax: +86 21 65982503.

E-mail addresses: zhouxuefei@tongji.edu.cn (X. Zhou), zhangyalei@tongji.edu.cn(Y. Zhang).

Yu Gu, Xiaohua Chen, Zhanguang Liu, Xuefei Zhou ⇑, Yalei Zhang ⇑Key Laboratory of Yangtze Water Environment of Ministry of Education, State Key Laboratory of Pollution Control and Resource Reuse, Tongji University, Shanghai 200092, China

h i g h l i g h t s

� Digested manures were more suitable than sludge for rice straw anaerobic digestion.� The reactors inoculated with DM achieved the highest biogas production of 3348 mL.� The highest lignocellulose degradation was observed in reactors inoculated with DM.� The DM had the highest cellulase and xylanase activities among all inoculums.� The micronutrients content in DM were higher than other inoculums.

a r t i c l e i n f o

Article history:Received 9 December 2013Received in revised form 1 February 2014Accepted 4 February 2014Available online 12 February 2014

Keywords:Anaerobic digestionBiogasEnzyme activityInoculumNutrients

a b s t r a c t

The aim of this study was to evaluate the effect of different inoculum sources on the rice straw anaerobicdigestion. Six different digestates (DM, SM, CM, MS, AGS and PS) were applied as inoculums and theireffects were evaluated in batch reactors. The results indicated that digested manures were more suitablethan sludge. Reactors inoculated with digested manures achieved higher, biogas production and lignocel-lulose degradation. The better adaptability of digested manures had relationship with its higher cellulaseand xylanase activities and sufficient nutrients content. DM had the best effect among all three digestedmanures. Reactors inoculated with DM achieved the highest biogas production (325.3 mL/g VS) andenzymes activities. The synergism between cellulase and xylanase activities played an important rolein lignocellulose degradation.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

With the development of China, its energy consumption has in-creased rapidly. Fossil fuels such as coal and oil, which are consid-ered unsustainable, aggravate global warming; therefore, thegovernment is searching for renewable energies to allow for multi-ple options for energy production (Yang et al., 2013).As one of thebiggest agricultural countries, China produces more than 800 mil-lion metric tons of waste agriculture straw every year (Bi et al.,2009). However, the methods that can be applied to treat suchlarge-scale waste straw are limited, and more than 60% of thestraw was open burned (Zheng et al., 2009). Agriculture straw,composed mainly of lignocellulose, can be transformed into

renewable energies, such as biogas and ethanol, by anaerobicdigestion (Chandra et al., 2012b). In contrast to fossil fuels, the bio-gas and ethanol produced from agriculture waste straw will not in-crease the greenhouse gases in the atmosphere and will protect theearth from global warming (Chandra et al., 2012b). China has beensearching for this anaerobic digestion technology since the 1980s.Currently, the government hopes that anaerobic digestion technol-ogy can help solve the energy problem in rural areas, and the gov-ernment has set a goal of 60 million household-scale digesters by2020 (Pang et al., 2008).

Waste agricultural straw, such as rice straw, cannot be digestedby itself, thus extra methane-producing bacteria are needed tostart the digestive process. Inoculums should contain active micro-bial communities, which are needed for anaerobic digestion. Thedigestates produced in anaerobic engineering projects have beensources for these inoculums (Bayane and Guiot, 2011; Elbeshbishyet al., 2012; Li et al., 2011; Suwannoppadol et al., 2011; Zhou et al.,2011). Digested manures and sludge are the two main types of dig-estates available in most cities in China. The chemical and physicalstructure of waste agricultural straw, which is mainly composed of

150 Y. Gu et al. / Bioresource Technology 158 (2014) 149–155

lignin, cellulose and hemicellulose, is difficult for bacterialdegradation (Himmel et al., 2007); therefore, the source of an inoc-ulum will affect the digestion results of these molecules. A suitableinoculum can increase the degradation rate, enhance biogas pro-duction, shorten the starting time, and make the digestion processmore stable (Quintero et al., 2012). For example, the biogas andmethane production from corn stover were enhanced by 15.5%and 10.8% by selecting a suitable inoculum, respectively (Li et al.,2011). Furthermore, digestates also can serve as nitrogen andmicronutrients sources during the waste agriculture straw anaero-bic digestion process (Xu et al., 2013). Some researchers havenoted that rumen fluid has high activity as an inoculum in organicwaste digestion (Lopes et al., 2004), including lignocellulosefermentation (Prochazka et al., 2012). However, the extraction ofrumen fluid is complicated and the fluid produced by goats cannotsatisfy the needs of engineering at the appropriate scale for biofuelproduction.

Interestingly, when comparing the effects of different inocu-lums in biomass digestion, most research has focused on substratedegradation and biogas production but not on the inoculumsthemselves, such as their enzyme activities and nutrient contents.Cellulase and xylanase are the main enzymes that convert lignocel-lulose to reducing sugars, and higher enzyme activities lead tohigher substrate degradation and biogas production (Hu et al.,2011); however, the enzyme activities of the different inoculumsare rarely compared. Another aspect of an inoculum is the nutri-ents it contains. The micronutrients contained in an inoculumcan enhance the enzyme activity and biogas production (Pobeheimet al., 2010; Zhang et al., 2011). However, the concentrations ofmicronutrients in different inoculums were rarely mentioned. Lig-nocellulose has high carbon content and low nitrogen content,which leads to a decrease in biogas (Chandra et al., 2012b). Whenusing digested manures or sludge as the inoculum, extra nitrogencan be provided to meet the needs of the methanogens.

The main purpose of this work was to evaluate the effect of dif-ferent inoculum sources on waste agriculture straw anaerobicdigestion. Waste rice straw was chosen as the substrate and six dif-ferent inoculums including three digested manures and threesludge were chosen as inoculum. The biogas production and ligno-cellulose degradation were treated as the main indicators. Enzymeactivities and nutrients contained in different inoculum also havebeen compared to explain their different effect.

2. Methods

2.1. Inoculum

The inoculums used in this study were obtained from six differ-ent sources. The digested dairy manure (DM), digested swine man-ure (SM) and digested chicken manure (CM) were obtained fromthe waste digestate of a livestock and poultry farm in Chongming.The digested municipal sludge (MS) was acquired from the Songji-ang sewage plant. The anaerobic granular sludge (AGS) was ob-tained from a beer factory in Wuxi. The paper mill sludge (PS)was obtained from a paper mill in Wujiang. All of the digestershave been stably operated for many years. To maintain freshnessand microbial activity, all inoculums were stored at 4 �C and reac-tivated at 37 �C for 3 days prior to use.

2.2. Substrate

The rice straw used for digestion was acquired from a rice fieldin Chongming. The rice straw was then chopped by an electricalgrinder (Yili, QE-200) and the resulting particles were screenedby an 8-mesh sieve, which removed the coarse particles. Then,the straw was air dried at 40 �C to reduce the moisture content

to less than 5%. Finally, the rice straw was stored in vacuum bagsat room temperature to prevent possible hydrolysis.

2.3. Experimental set up

The rice straw was inoculated and digested in batch digesters.Glass bottles (500 mL) with a 400 mL working volume and a100 mL head volume were used as reactors. The rice straw wasinoculated separately with six different inoculums in an Inoculumto Substrate (I/S) ratio of 0.5 (based on the VS content). Then, thereactors were closed with a rubber stopper and Vaseline was usedto seal the closure. Every rubber stopper contained a 10 cm longglass tube that was connected to a gasbag with a disposable rubbertube. Reactors with the same amount of inoculum and distilledwater were used as controls. All of the reactors were placed in anincubator shaker at 37 �C and were digested for 40 days.

2.4. Analytical methods

The amount of biogas produced was measured using a glass syr-inge. The biogas production for each reactor was measured dailyand transferred to the standard condition. The biogas samples weretaken from the gas bags by disposable syringes and the methanecontent was analyzed by a gas chromatograph (Agilent, 6890 N)equipped with a thermal conductivity detector (TCD). The temper-atures of the injection port, the column oven and the TCD were120,35 and 250 �C, respectively. Helium gas was used as the carrier gasand the flow rate was 20 mL/min. A standard gas sample consistingof 60% (v/v) CH4 and 40% CO2 was applied to the instrument for cal-ibration of the results. The methane production was calculated bymultiplying the methane content by the biogas production.

Samples were taken from the reactors before and after fermen-tation and stored at 4 �C prior to analysis. The total solids (TS), vol-atile solids (VS) and alkalinity were tested according to thestandard methods (APHA, 2005). The total carbon, nitrogen, andhydrogen content were tested by an elemental analyzer (Elemen-tar, Vario EL III), and the C/N ratio was calculated. The metal con-tent was analyzed using an inductively coupled plasma opticalemission spectrometer (ICP-OES). The cellulase and xylanase activ-ities were analyzed according to the method introduced by Lin andThomson (1991). The cellulose, hemicellulose and lignin contentswere measured according to a previously published method (Linet al., 2009), with modifications.

2.5. Data analysis

Each analytical data point was the mean of three measure-ments. The experimental results were analyzed with SAS 8.0, anda p-value was used to analyze the significant differences.

3. Results and discussion

3.1. Characterization of the rice straw and inoculum

Table 1 summarizes the characterizations of the rice straw andthe inoculums. The rice straw was air-dried, and the TS contentwas 97.3%. The main component of rice straw was lignocellulose,which included 34.3% cellulose, 24.2% hemicellulose and 11.2% lig-nin. The inoculums in this study had a wide range of TS and VS con-tent, which were likely a result of the different operationalconditions of the previous reactors. The rice straw had high carboncontent and low nitrogen content and the C/N ratio was 58.6,which was much higher than the optimal range. The inoculumshad a relatively higher nitrogen content that varied from 1.0% to5.6%. The characteristics of the rice straw used in this study were

Table 1Characteristics of rice straw and inoculum.a

Parameter Rice straw DM SM CM AGS MS PS

TS (%) 97.3 ± 0.3 2.8 ± 0.0 6.1 ± 0.2 22.5 ± 0.2 5.4 ± 0.2 19.2 ± 0.1 17.3 ± 0.1VS (%) 84.0 ± 0.5 1.6 ± 0.0 3.9 ± 0.1 8.2 ± 0.1 3.9 ± 0.2 9.7 ± 0.1 14.0 ± 0.0VS/TS (%) 86.3 59.1 62.7 36.4 71.8 50.4 80.5Ash (%)b 13.6 ± 0.3 41.0 ± 0.0 37.2 ± 0.2 63.6 ± 0.2 28.1 ± 0.2 49.6 ± 0.1 19.5 ± 0.1N (%)b 0.6 ± 0.0 3.1 ± 0.1 2.1 ± 0.1 1.0 ± 0.1 5.6 ± 0.1 2.8 ± 0.1 5.4 ± 0.2C (%)b 35.2 ± 1.1 20.5 ± 1.2 28.1 ± 1.6 16.7 ± 0.8 31.3 ± 1.4 21.8 ± 0.9 37.1 ± 0.0H (%)b 5.3 ± 0.2 3.0 ± 0.1 4.6 ± 0.3 2.6 ± 0.1 5.1 ± 0.2 4.2 ± 0.2 5.7 ± 0.2C/N (%) 58.6 6.7 13.2 17.2 5.6 7.7 6.9

a Data is expressed as means ± SD (n P 3).b Data is based on TS.

Y. Gu et al. / Bioresource Technology 158 (2014) 149–155 151

similar to those in other reports (Chandra et al., 2012a; Chenget al., 2010).

3.2. Anaerobic digestion

3.2.1. Biogas productionTwo main widely applied groups of digestate were selected as

inoculums (digested manures, including DM, SM and CM; sludge,including AGS, PS and MS). The biogas and methane yields fromthe anaerobic digestion of rice straw inoculated with these inocu-lums have been presented in Fig. 1 Peak values, starting time andpeak appearance times were the main differences. When usingDM and SM as inoculums, a rapid biogas production of 55 and

Fig. 1. (a) Daily biogas production, (b) cumulative biogas production and (c) specific bio(DM: digested dairy manure, SM: digested swine manure, CM: digested chicken manusludge) (digestion time: 40 days, TS: 5%, I/S ratio: 0.5).

89 mL was observed in the first day, while in other reactor (CM,AGS, MS and PS) it was only 12, 35, 2 and 5 mL, respectively. Fromday 6 to day 16, the daily biogas production in DM inoculated reac-tors was the highest and its peak value (255 mL) was 59.1%, 47.9%and 89.6% higher than with CM, AGS and SM, respectively (Fig. 1a).Reactors inoculated with sludge were not as efficient as those inoc-ulated with digested manures. Although the AGS had the highestbiogas and methane production of the three types of sludge, thestarting time of the AGS inoculated reactors was much longer thanthe DM and SM inoculated reactors. The biogas production in theAGS inoculated reactors did not reach its first peak until day 10,which was later than the DM and SM inoculated reactors. Addition-ally, the daily methane production in AGS inoculated reactors

gas and methane production for rice straw inoculated with six different inoculumsre, MS: digested municipal sludge, AGS: anaerobic granular sludge, PS: paper mill

Fig. 3. Alkalinity for reactors inoculated with different inoculums.

152 Y. Gu et al. / Bioresource Technology 158 (2014) 149–155

experienced significant fluctuations and the biogas productionmaintained a low level after 22 days of incubation (Fig. 1a andb). The methane production measurements were relative to thebiogas production and methane content. DM had the highest totalbiogas production and methane content ratio (54.8%), which led tothe highest specific methane production (178.3 mL/g VS) (Fig. 1c).

Based on the aforementioned information, it is obvious that dif-ferences existed between inoculums. It can be concluded that reac-tors inoculated with digested manures had higher and steadierbiogas production than reactors inoculated with sludge. The biogasproduction of the MS and PS inoculated reactors was significantlydifferent from the other reactors either in the trend or in the finalbiogas production. Without pretreatment, pH adjustment or addi-tional alkalinity, sludge (such as MS and PS) contained a higher le-vel of easily degradable carbon than the digested manures, andmight cause acidification and unsuccessful commencement ofanaerobic digestion (O’keefe and Chynoweth, 2000; Suwannopp-adol et al., 2011). The biogas and methane production from DM,SM and AGS in this study were at the same levels as with other re-ports (Chandra et al., 2012a; Jing et al., 2011). With the exceptionof size reduction, there was no other pretreatment applied in thisstudy; thus, the biogas and methane production could still bepromoted.

3.2.2. pHThe pH in the reactors before and after digestion is shown in

Fig. 2 The optimal pH value for methane producing microorgan-isms is 6.7–7.5, and the pH has toxic effects when it is higher than8.5 (Chandra et al., 2012b).The initial pH values in all reactors ran-ged from 7.4 to 7.7, which were all suitable for the anaerobic diges-tion process. Although the final pH values for most of the reactorswas still in a healthy range, the final pH of PS was 8.8, which mayhave inhibited the activity of the anaerobic microorganisms andlead to a low biogas production rate.

3.2.3. Buffer capacityThe buffer capacity of the reactors inoculated with different

inoculums was represented by the alkalinity. Alkalinity can be amore reliable parameter than pH to measure digester imbalancebecause it decreased significantly before the pH decreased whenshort chain fatty acids were accumulating (Guwy et al., 1997).No extra alkalinity was added in this study and the only sourceof alkalinity was the inoculum. The alkalinity in the different reac-tors before and after digestion is shown in Fig. 3. The alkalinity ofDM, SM, PS and AGS inoculated reactors increased after 40 days ofdegradation. The DM inoculated reactors had the highest initialand final alkalinity. The initial alkalinity in the DM inoculated

Fig. 2. pH for reactors inoculated with different inoculums.

reactors was 9.7, and it increased to 10.6 g CaCO3/L after 40 daysof digestion. The value in this study was similar to the alkalinityof the reactors inoculated with dairy waste effluent in the sameI/S ratio, as reported by Xu et al. (2013). The alkalinity providedby DM was high enough to prevent the reactor from acidificationand no extra bases or carbonate salts were needed in the digestionprocess. On the contrary, the initial alkalinity of MS was lower thanthe other inoculums, and the final alkalinity decreased to 3.9 gCaCO3/L. The low biogas production in MS inoculated reactorsmay be related to their low alkalinity.

3.3. Effect of enzyme on cellulose and hemicellulose degradation

3.3.1. Cellulose and hemicellulose degradationDuring the anaerobic digestion of lignocellulose, cellulose and

hemicellulose are the main components reduced and convertedto generate biogas. The cellulose and hemicellulose degradationrates during the digestion are shown in Fig. 4. The rice straw inoc-ulated with DM obtained the highest cellulose degradation rate,followed by SM, AGS, CM, MS and PS. The cellulose degradationrate in the DM inoculated reactors was 60.3% after 40 days ofdigestion, which showed a good adaptability of DM on lignocellu-lose degradation. The SM and AGS inoculated reactors also attaineda relatively high degradation rate of 53.7% and 51.8%, respectively.Hemicellulose showed a lower degradability than cellulose. Thehighest hemicellulose degradation rate was also observed in theDM inoculated reactors, and was 33.7–247.5% higher than theother reactors. The lignin content before and after digestion alsohad been determined and no obvious lignin degradation wasobserved.

In this study, for rice straw inoculated with digested manure,cellulose and hemicellulose degradation was higher compared tothose inoculated with sludge. When corn stover was applied asthe substrate, the dairy digestate also achieved higher celluloseand hemicellulose degradation rates than the other digested waste(Xu et al., 2013). This also suggests that digested manures had bet-ter adaptability in lignocellulose digestion than other inoculumsources. The cellulose and hemicellulose degradation rate was inagreement with the biogas production. Higher cellulose and hemi-cellulose degradation was observed in reactors with higher biogasproduction. It could be concluded that the biogas production wasrelated to cellulose and hemicellulose degradation.

3.3.2. The effect of enzyme activityThe difference in the cellulose and hemicellulose degradation

(Fig. 4) implies differences in inoculum activities. Enzyme playsan important role in lignocellulose degradation. Microorganisms

Fig. 4. Degradation of cellulose and hemicellulose in reactors.

Fig. 5. Comparisons on (a) cellulase activity and (b) xylanase activity in reactorsinoculated with different inoculums.

Y. Gu et al. / Bioresource Technology 158 (2014) 149–155 153

produce enzyme to hydrolyze the cellulose and hemicellulose intomonosaccharide. An enzyme activity experiment was applied toassess the cellulase and xylanase activities of the aforementionedinoculums. Fig. 5 shows that cellulase and xylanase activities wererelated to the cellulose and hemicellulose degradation. Higher cel-lulase and xylanase activities were observed in the reactors with ahigher cellulose and hemicellulose degradation rate. It was appar-ent that the enzyme activities increased in all reactors after diges-tion, but the increasement in CM and MS inoculated reactors wasmuch lower than others. Reactors inoculated with DM had thehighest cellulase activity before and after digestion. Compared tothe cellulase activity, the xylanase activity in the reactors variedmore different. The xylanase activity in the reactors ranged from1.2 to 2.0 U/mL before digestion. After 40 days digestion, the xylan-ase activity in all reactors increased and the reactors with higherinitial activity also increased more in the final measurements.

As the inoculation ratio was same in every reactor, the enzymeactivity reflected the microorganism activity and degradation effi-ciency of different inoculums. The increase in enzyme activitymight have been caused by the growth of microorganisms andthe different amounts of activity increase indicated different adap-tations of the inoculums in hydrolyzing the lignocellulose materi-als. Compared to cellulase, initial and final xylanase activities weremuch lower and this implied that hemicellulose was harder formicroorganism than cellulose. The less increasement in xylanaseactivity implied a slower reproduce of hemicellulose degradablemicroorganisms. DM had the highest cellulase and xylanase activ-ity before fermentation. It also had better adaptability than other

Table 2Comparison of the macro and micronutrient levels in different reactors (mg/L).a

Elements DM SM CM

Na 433.8 ± 56.2 106.2 ± 24.6 92.5 ± 13.K 1560.3 ± 233.0 408.4 ± 38.1 515.0 ± 45Ca 192.9 ± 13.2 88.5 ± 10.7 113.8 ± 9.Mg 204.6 ± 24.6 263.5 ± 35.8 130.0 ± 14Fe 98.8 ± 12.4 79.9 ± 8.6 48.5 ± 9.4Co 0.3 ± 0.0 0.1 ± 0.0 <LDNi 0.9 ± 0.1 0.6 ± 0.0 0.1 ± 0.0Mo 0.8 ± 0.2 0.6 ± 0.1 0.3 ± 0.1Cu <LDb <LD <LDZn 9.8 ± 1.2 9.2 ± 0.9 5.2 ± 0.4Pb 2.5 ± 0.2 3.6 ± 0.6 2.6 ± 0.2

Data is expressed as means ± SD (n P 3).a All the original measured values were translated to the same operating conditions (b <LD lower than detection limit.

inoculums, which led to the highest cellulase and xylanase activityafter 40 days of digestion.

The hemicellulose structural barrier was one of the main mech-anisms that stopped cellulase from getting close to the cellulose(Taherzadeh and Karimi, 2008). The synergism that occurs be-tween cellulase and xylanase could break this barrier and help cel-lulase reach the surface of the cellulose molecules (Himmel et al.,2007), which means that the combined effects of cellulase andxylanase are larger than the sum of the individual effects (Huet al., 2011). Higher xylanase activity not only led to higher hemi-cellulose degradation but also affected the cellulose degradationprocess. The synergism of both enzymes had an important affectduring substrate degradation and biogas production. Compared

AGS MS PS

5 991.7 ± 116.4 659.8 ± 78.7 637.8 ± 54.3.3 685.3 ± 56.2 426.4 ± 32.3 576.3 ± 58.9

6 68.3 ± 5.4 89.6 ± 18.3 43.9 ± 8.9.6 95.2 ± 10.1 66.9 ± 7.9 73.3 ± 9.4

25.1 ± 3.2 18.2 ± 1.3 22.3 ± 2.9<LD 0.1 ± 0.0 <LD0.3 ± 0.0 0.2 ± 0.0 0.1 ± 0.00.4 ± 0.0 0.1 ± 0.0 <LD<LD 0.3 ± 0.0 0.2 ± 0.114.6 ± 2.3 11.7 ± 1.1 17.1 ± 1.81.1 ± 0.0 0.7 ± 0.0 1.0 ± 0.1

TS: 5%, I/S: 0.5).

Table 3The C/N of substrates after being mixed with inoculums.

DM SM CM AGS MS PS

C/N 24.7 33.8 38.4 19.4 24.5 22.1

154 Y. Gu et al. / Bioresource Technology 158 (2014) 149–155

with other reactors, the DM inoculated reactors had the highestvalues for both enzymes, and the synergism made this processmore efficient. With higher cellulase and xylanase activities, theDM inoculated reactors degraded more substrate and producedmore biogas than other inoculums.

The high cellulase and xylanase activity of the DM inoculatedreactors might benefit from the rumen microorganism containedin DM. Before the dairy manure was excreted, the digesta wasmixed with fermentation juice and inoculated with the microor-ganisms in cattle rumen (Bayane and Guiot, 2011). The rumenmicroorganisms are believed to have great digestibility of lignocel-lulose (Hu and Yu, 2006). There were abundant rumen microorgan-isms in DM (Wen et al., 2005), and they provided high cellulase andxylanase activities in the digestion process (Xu et al., 2013).

3.4. The effect of nutrients

An appropriate nutrient content in the reactors is very impor-tant for the anaerobic digestion process of rice straw. In this study,the inoculum was not only added to provide active microorgan-isms but also to serve as a source of nutrients. The demand formacronutrients, micronutrients and nitrogen for the anaerobicdigestion process can be satisfied by the inoculum without theaddition of chemicals.

3.4.1. Macro and micronutrientsMacro- and micronutrients are necessary for cell growth and

the methanogens require special micronutrients. The concentra-tions of macro- and micronutrients in different inoculums areshown in Table 2. The macronutrient (Na, K, Ca and Mg) contentin the inoculums was sufficient for the metabolic activities of themicroorganisms (Pobeheim et al., 2010). The concentrations of Kand Ca in DM were higher than in all other inoculums. The calciumcontent in the DM inoculated reactors was 192.9 mg/L, which wasnearly the optimal concentration (200.0 mg/L) for methane pro-duction (Kugelman and McCarty, 1965). In addition to themacronutrients, micronutrients play a more crucial role in themetabolism and growth of microorganisms.

The micronutrients contained in the reactors were the key fac-tors for improving anaerobic digestion (Zhang et al., 2011). Micro-nutrients, such as Fe, Co, Ni and Mo, contained in the digestate canimprove the enzyme activity and enhance the anaerobic digestionof substrates and the optimal range of Fe, Ni, Co and Mo was 0.28–50.40, 0.02–1.00, 0.05–0.46 and 5.08–50.12 mg/L, respectively(Demirel and Scherer, 2011; Pobeheim et al., 2010). The digestedmanures clearly had a higher concentration of micronutrients thanthe sludge (Table 2). The micronutrient content in DM was thehighest of the three digested manures. Although the Fe contentwas higher than the suggested value, but an addition concentrationof 100 mg/L also had positive effect on stimulation (Zhang et al.,2011). DM and SM inoculated reactors had the most suitablemicronutrients concentrations among all inoculums. The concen-tration of Mo in all reactors were much lower than the optimalrange, but the effect of Mo was limited compared with Fe, Ni andCo. Higher biogas production, lignocellulose degradation, and cel-lulase and xylanase activity were also observed in the reactors thatcontained higher micronutrients. Therefore, the higher concentra-tion of micronutrients contained in the DM inoculated reactors

contributed to a better performance than seen with the otherinoculums.

Many nutrients can also cause the inhibition of the anaerobicdigestion process. Luckily, the concentration of nutrients containedin all six inoculums was lower than the reported inhibitory con-centrations (Chen et al., 2008).

3.4.2. Carbon and nitrogen contentThe C/N ratio plays an important role in fermentative biogas

production. A C/N ratio that is either too high or too low will inhibitbiogas production (Chandra et al., 2012b). The C/N ratio after inoc-ulation is calculated and shown in Table 3. The suitable range ofthe C/N ratio was 20–30 for anaerobic digestion processes, andthe optimal C/N ratio suggested for gas production is 25 (Chandraet al., 2012b). The C/N ratio of the rice straw used in the reactorswas 58.6 with high carbon content (35.2%) and low nitrogen con-tent (0.6%). Because there were no chemicals added to adjust thecarbon to nitrogen ratio, the carbon and nitrogen balance wasindependent from the addition of inoculum. The C/N ratio of DM,SM, CM, AGS, MS, and PS was 6.7, 13.2, 17.2, 5.6, 7.7 and 6.9,respectively, and the C/N ratio changed to 24.7, 33.8, 38.4, 19.4,24.5 and 22.1, respectively, after being mixed with rice straw. Be-cause the carbon content was detected by elemental analysis, thelignin carbon which cannot be used by the microorganisms wasalso calculated in the results (Mussoline et al., 2012). The non-lig-nin C/N ratio for DM, SM, CM, AGS, MS and PS inoculated reactorswas 22.5, 30.9, 35.1, 17.7, 22.4 and 20.2, respectively. The C/N ratioof the reactors inoculated with DM, SM, MS and PS was in the opti-mal range, and the biogas production should not be affected by thecarbon and nitrogen imbalance. The C/N ratio in the AGS inocu-lated reactors was lower than the optimal range, and the C/N ratioin the CM inoculated reactors was higher than the optimal range.In both cases, this may have a negative effect on biogas production.

4. Conclusion

The results of this study demonstrated that there were signifi-cant differences between different inoculums. The reactors inocu-lated with digested manures have shorter starting time andachieved higher biogas production than reactors inoculated withsludge. Sludge such as MS and PS failed to create a suitable envi-ronmental for rice straw digestion. Biogas productions was unsta-ble and kept in a low level in reactors inoculated with MS and PS.DM, with the highest enzyme activities and most suitable nutrientscontent, achieved the highest biogas production and showed thebest adaptability among all inoculums.

Acknowledgements

This research was funded by the National Natural ScienceFoundation of China (Nos. 20976139, 21246001, 51138009), theNational Key Technologies R&D Program of China (No.2012BAJ25B02), New Century Excellent Talents in University(NCET-11-0391) and the Project of Shanghai Science and Technol-ogy Commission (No. 11QH1402600).

References

APHA, 2005. Standard Methods for the Examination of Water and Wastewater, 21sted. American Public Health Association (APHA), Washington, DC.

Bayane, A., Guiot, S.R., 2011. Animal digestive strategies versus anaerobic digestionbioprocesses for biogas production from lignocellulosic biomass. Rev. Environ.Sci. Bio-Technol. 10 (1), 43–62.

Bi, Y., Gao, C., Wang, Y., Li, B., 2009. Estimation of straw resources in China. NongyeGongcheng Xuebao/Trans. Chin. Soc. Agric. Eng. 25 (12), 211–217.

Chandra, R., Takeuchi, H., Hasegawa, T., 2012a. Hydrothermal pretreatment of ricestraw biomass: a potential and promising method for enhanced methaneproduction. Appl. Energy 94, 129–140.

Y. Gu et al. / Bioresource Technology 158 (2014) 149–155 155

Chandra, R., Takeuchi, H., Hasegawa, T., 2012b. Methane production fromlignocellulosic agricultural crop wastes: a review in context to secondgeneration of biofuel production. Renew. Sustain. Energy Rev. 16 (3), 1462–1476.

Chen, Y., Cheng, J.J., Creamer, K.S., 2008. Inhibition of anaerobic digestion process: areview. Bioresour. Technol. 99 (10), 4044–4064.

Cheng, Y.S., Zheng, Y., Yu, C.W., Dooley, T.M., Jenkins, B.M., VanderGheynst, J.S.,2010. Evaluation of high solids alkaline pretreatment of rice straw. Appl.Biochem. Biotechnol. 162 (6), 1768–1784.

Demirel, B., Scherer, P., 2011. Trace element requirements of agricultural biogasdigesters during biological conversion of renewable biomass to methane.Biomass Bioenergy 35 (3), 992–998.

Elbeshbishy, E., Nakhla, G., Hafez, H., 2012. Biochemical methane potential (BMP) offood waste and primary sludge: influence of inoculum pre-incubation andinoculum source. Bioresour. Technol. 110, 18–25.

Guwy, A., Hawkes, F., Wilcox, S., Hawkes, D., 1997. Neural network and on-offcontrol of bicarbonate alkalinity in a fluidised-bed anaerobic digester. WaterRes. 31 (8), 2019–2025.

Himmel, M.E., Ding, S.Y., Johnson, D.K., Adney, W.S., Nimlos, M.R., Brady, J.W., Foust,T.D., 2007. Biomass recalcitrance: engineering plants and enzymes for biofuelsproduction. Science 315 (5813), 804–807.

Hu, Z.H., Yu, H.Q., 2006. Anaerobic digestion of cattail by rumen cultures. WasteManage. (Oxford) 26 (11), 1222–1228.

Hu, J., Arantes, V., Saddler, J.N., 2011. The enhancement of enzymatic hydrolysis oflignocellulosic substrates by the addition of accessory enzymes such asxylanase: is it an additive or synergistic effect? Biotechnol. Biofuels 4 (1), 1–14.

Jing, D.U., Zhizhou, C., Xiaomei, Y.E., Yuting, Q., 2011. Effect of substrateconcentration and inoculums ratio on the start-up of anaerobic digestion ofrice straw. J. Agro-Environ. Sci. 30 (7), 1456–1459.

Kugelman, I.J., McCarty, P.L., 1965. Cation toxicity and stimulation in anaerobicwaste treatment. J. Water Pollut. Control Fed. 37 (1), 97–116.

Li, L., Yang, X., Li, X., Zheng, M., Chen, J., Zhang, Z., 2011. The influence of inoculumsources on anaerobic biogasification of NaOH-treated corn stover. EnergySources Part A 33 (2), 138–144.

Lin, L.-L., Thomson, J.A., 1991. An analysis of the extracellular xylanases andcellulases of< i> Butyrivibrio fibrisolvens</i> H17c. FEMS Microbiol. Lett. 84 (2),197–203.

Lin, Y., Wang, D., Wu, S., Wang, C., 2009. Alkali pretreatment enhances biogasproduction in the anaerobic digestion of pulp and paper sludge. J. Hazard.Mater. 170 (1), 366–373.

Lopes, W.S., Leite, V.D., Prasad, S., 2004. Influence of inoculum on performance ofanaerobic reactors for treating municipal solid waste. Bioresour. Technol. 94 (3),261–266.

Mussoline, W., Esposito, G., Lens, P., Garuti, G., Giordano, A., 2012. Designconsiderations for a farm-scale biogas plant based on pilot-scale anaerobicdigesters loaded with rice straw and piggery wastewater. Biomass Bioenergy46, 469–478.

O’keefe, D., Chynoweth, D., 2000. Influence of phase separation, leachate recycle andaeration on treatment of municipal solid waste in simulated landfill cells.Bioresour. Technol. 72 (1), 55–66.

Pang, Y.Z., Liu, Y.P., Li, X.J., Wang, K.S., Yuan, H.R., 2008. Improving biodegradabilityand biogas production of corn stover through sodium hydroxide solid statepretreatment. Energy Fuels 22 (4), 2761–2766.

Pobeheim, H., Munk, B., Johansson, J., Guebitz, G.M., 2010. Influence of traceelements on methane formation from a synthetic model substrate for maizesilage. Bioresour. Technol. 101 (2), 836–839.

Prochazka, J., Mrazek, J., Strosova, L., Fliegerova, K., Zabranska, J., Dohanyos, M.,2012. Enhanced biogas yield from energy crops with rumen anaerobic fungi.Eng. Life Sci. 12 (3), 343–351.

Quintero, M., Castro, L., Ortiz, C., Guzman, C., Escalante, H., 2012. Enhancement ofstarting up anaerobic digestion of lignocellulosic substrate: fique’s bagasse asan example. Bioresour. Technol. 108, 8–13.

Suwannoppadol, S., Ho, G., Cord-Ruwisch, R., 2011. Rapid start-up of thermophilicanaerobic digestion with the turf fraction of MSW as inoculum. Bioresour.Technol. 102 (17), 7762–7767.

Taherzadeh, M.J., Karimi, K., 2008. Pretreatment of lignocellulosic wastes toimprove ethanol and biogas production: a review. Int. J. Mol. Sci. 9 (9), 1621–1651.

Wen, Z., Liao, W., Chen, S., 2005. Production of cellulase by Trichoderma reesei fromdairy manure. Bioresour. Technol. 96 (4), 491–499.

Xu, F.Q., Shi, J., Lv, W., Yu, Z.T., Li, Y.B., 2013. Comparison of different liquidanaerobic digestion effluents as inocula and nitrogen sources for solid-statebatch anaerobic digestion of corn stover. Waste Manage. (Oxford) 33 (1), 26–32.

Yang, Y., Li, G., Zhang, P., 2013. Simulation and prediction of CO2 emissionreductions of biogas industry in China. Nongye Gongcheng Xuebao/Trans. Chin.Soc. Agric. Eng. 29 (15), 1–9.

Zhang, L., Lee, Y.W., Jahng, D., 2011. Anaerobic co-digestion of food waste andpiggery wastewater: focusing on the role of trace elements. Bioresour. Technol.102 (8), 5048–5059.

Zheng, M.X., Li, X.J., Li, L.Q., Yang, X.J., He, Y.F., 2009. Enhancing anaerobicbiogasification of corn stover through wet state NaOH pretreatment.Bioresour. Technol. 100 (21), 5140–5145.

Zhou, Y., Zhang, Z., Nakamoto, T., Li, Y., Yang, Y., Utsumi, M., Sugiura, N., 2011.Influence of substrate-to-inoculum ratio on the batch anaerobic digestion ofbean curd refuse-okara under mesophilic conditions. Biomass Bioenergy 35 (7),3251–3256.