effects of feed to inoculum ratio, co-digestion, and pretreatment on biogas production from...

Effects of Feed to Inoculum Ratio, Co-digestion, and Pretreatment onBiogas Production from Anaerobic Digestion of Cotton StalkXi-Yu Cheng*,†,§ and Cheng Zhong‡,§

†College of Life Science and Bioengineering, School of Science, Beijing Jiaotong University, Beijing 100044, People’s Republic ofChina‡Key Laboratory of Industrial Fermentation Microbiology (Ministry of Education), Tianjin University of Science & Technology,Tianjin 300457, People’s Republic of China

ABSTRACT: Biogas yield from anaerobic digestion of cotton stalk (CS) at feed to inoculum (F/I) ratios of 2−4 reached 175−180 mL/(g of VSadded) against 113 mL/(g of VSadded) observed at the F/I ratio of 6. CS was proven to be a promising co-substrate in the digestion of swine manure (SM), and a CS/SM ratio of 50:50 with a C/N ratio of 25 was found to be the best interms of the biogas production rate and yield with increases up to 1.8- and 1.9-fold, respectively, as compared to the control. Thehighest biogas yield (449 mL/(g of VSadded)) and production rate (0.65 L/(L·day)) with comparable technical digestion timewere obtained in co-digestion of SM with CS pretreated by NaOH, which was 241−255% of those achieved in the control. Thisstudy indicates that co-digestion of SM with alkali-pretreated CS is a potential option for alleviating the insufficient substrateresource problems and improving the energy output.

1. INTRODUCTION

China produced the largest numbers of cotton with a cottonstalk yield of 20 million tons/year.1 Traditionally, cotton stalkwastes have been combusted or discarded directly, causing anumber of ecological and environmental problems. Anaerobicdigestion, which has been successfully applied for biogasproduction at both home scale and large scale for more than 1century and is considered as a promising method for renewableenergy production from varied organic wastes,2−5 provided aviable alternative for the disposal of cotton stalk wastes.6−9 EI-Shinnawi et al. studied biogas production from anaerobicdigestion of different kinds of crop residues (maize, rice, andcotton stalks) and observed that cotton stalks gave a lowervalue of methane yield than that obtained in the maize stalk.6 Ina further study, constant amounts of maize, rice, and cottonstalks were mixed with cow dung and predigested at varioustime intervals before fermentation.7 The results showed that thebiogas potential of the maize stalk mixture was also higher thanthat of cotton stalk. Methane yields of 65−86 mL of CH4/(g ofcotton wastes) were observed from anaerobic digestion ofcotton stalks and seed hulls in the presence of supplementednutrients and trace elements.8 Improved methane yields (80−242 mL of CH4/(g of VS)) were obtained in the digestion ofcotton stalk pretreated with different agents.9

When considering its high carbon content, cotton stalk mayalso be used as a potential co-substrate of anaerobic digestion ofswine manure. Substrates (e.g., swine manure) with too low ofa carbon/nitrogen ratio (C/N) ratio would increase the risk ofammonia inhibition, while a too high ratio would not provideenough nitrogen for the maintenance of cell biomass.10−13

Some studies showed that the optimal C/N ratio for anaerobicdigestion was between 20 and 35,12−14 and some otherresearchers revealed that it ranged from 16 to 25.15,16 Co-digestion of agricultural wastes with manure wastes providedbetter performance than their monodigestion, and its main

benefits were that it could not only increase buffering capacityto help maintain an optimal pH for methangenic bacteria andprovide a better C/N ratio in the feedstock but also dilutepotentially toxic compounds, utilize the nutrients and bacterialdiversities in various wastes, and so on.17−19 Although co-digestion of manure wastes with many crop residues such ascorn stalk, wheat straw, and rice straw was frequentlystudied,13,17−19 information on cotton stalk was still limited.Moreover, one of the major drawbacks when cotton stalks arebeing digested is its recalcitrant structure and higher lignincontent than common lingocellulosic wastes such as corn stalkand wheat straw,4,13,20 which may hinder anaerobic digestion.Different pretreatments, which can reduce the crystallinity of

cellulose and remove the lignin from lingocellulosic wastes,have been applied in attempts to improve the biodegrababilityof lingocellulosic wastes.4,5 Several pretreatment studies ofcotton stalk have also been reported such as saccharification byalkaline pretreatment and microwave assisted alkaline pretreat-ment, ethanol fermentation by pretreatment with variouschemical agents, and biogas production by hydrothermalpretreatment with hot water, ammonia solution, and recycledliquid from anaerobic digestion.9,21,22 Among different pretreat-ment methods, the dilute acid pretreatment was frequently usedbecause it is effective and inexpensive.23 And alkalinepretreatment, an efficient method for delignification oflignocellulose, has been proposed as a compatible chemicalpretreatment for enhanced biogas production, since anaerobicdigestion usually requires a pH adjustment by improvingalkalinity.24 Given its large potential for biogas production,cotton stalk certainly deserves more research attention forbeing used as a feedstock in co-digestion with manures and the

Received: December 29, 2013Revised: April 17, 2014Published: April 17, 2014

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© 2014 American Chemical Society 3157 dx.doi.org/10.1021/ef402562z | Energy Fuels 2014, 28, 3157−3166

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following process enhancement. However, there are currentlylimited publications reporting anaerobic co-digestion of swinemanure with cotton stalk and the effect of acid and alkalinepretreatments.In addition, the feed to inoculum (F/I) ratio was considered

as a key parameter affecting the efficiency of anaerobicdigestion and the accuracy of the BMP assay.25−27 Significantdecreases of methane yield were observed in digestion ofvarious wastes such as straw, food, and green wastes when F/Iratios were over a certain level.25,26 At the same time, it is veryimportant to find a suitable F/I ratio to minimize therequirement of active inoculum for the start-up of a biogasplant.In the present study, the biogas production performance in

anaerobic mono- and co-digestion of cotton stalk was studiedusing batch digestion tests. The purpose of this study was toinvestigate the (i) effect of F/I ratio on the degradation rate andbiogas production in anaerobic monodigestion of cotton stalk;(ii) effect of the mixing ratio of swine manure and cotton stalkand the C/N ratio on system performance of co-digestion ofthese wastes including biogas production, substrate degrada-tion, and process stability, etc.; and (iii) effect of differentpretreatments on the performance of co-digestion of swinemanure and cotton stalk.

2. MATERIALS AND METHODS2.1. Materials. Cotton stalk was collected from Hunan province,

China. It was dried in the sun and then milled to 16-mesh powder byusing a plant muller. The sample powder was dried in an oven at 60 °Cfor at least 48 h before use. The main components of cotton stalk wereas follows (% (w/w), on a dry weight basis): cellulose, 38.3;hemicellulose, 19.5; lignin, 21.6. Swine manure used in the co-digestion experiment was obtained from the Institute of AnimalSciences, Chinese Academy of Agricultural Sciences and keptrefrigerated at 4 °C before use. The characteristics of cotton stalkand swine manure can be seen from Table 1.

2.2. Experimental Design and Method. Table 2 shows asummary of all of the experiments performed in this study.Experimental sets F (F1, F2, F3, and F4), C (C1, C2, and C3) andP (P1 and P2) were conducted to investigate the effect of F/I ratio, co-digestion, and pretreatments on biogas production from batchanaerobic mono- and co-digestion of cotton stalk, respectively.All batch digestion experiments were carried out in a 500 mL serum

bottle with a working volume of 300 mL. The mixtures of pond bedsludge and anaerobic sludge obtained from Gaobeidian WastewaterTreatment Plant of Beijing was used as seed, which contained totalsolid (TS) of 50 g/L and volatile solid (VS) 30 g/L. The TSconcentrations of substrates (cotton stalk alone or cotton stalktogether with swine manure) at the beginning of digestion of eachexperimental set were 5% (w/v, based on the original substrate).Sludge inoculum was added based on predetermined feed to inoculumratios. The F/I ratio was calculated based on the initial VSconcentrations of the substrate and the inoculum. Suitable distilled

water was added, and pH was adjusted to 7 by using 1 M HCl orNaOH solution. No extra nutrient solutions were added in all batchexperiments. After being flushed for 10 min with N2, all serum bottleswere sealed with butyl rubber stoppers and aluminum crimps at onceand then were incubated in a rotary shaker (110 rpm) at 35 °C. Theblank trials containing the sludge inocula only were carried out tocorrect for the biogas produced from the inocula. Batch experimentswere terminated on day 45 when a clear downward trend in dailybiogas production was observed. Triplicate bottles were used in allexperiments, and all values were the means of replicates of triplicate ±standard deviation (SD).

For the first batch experiment (sets F1−F4) of studying the effect ofthe F/I ratio on biogas production, cotton stalk was used as the solesubstrate and sludge inoculum was added at the F/I ratios of 2, 3, 4,and 6, respectively. The F/I ratios were chosen based on the previousstudies.25,26 For the second batch experiment (sets C1−C3) ofinvestigating the effect of co-digestion, all experimental conditionswere the same as those of experimental set F2 except for the substrate.In brief, the F/I ratio was fixed at 4, and cotton stalk was co-digestedwith swine manure according to predetermined mixing ratios (Table2) to reach a total TS concentration of substrate of 5% (w/v, based onthe original substrate). The mixing ratios of substrates (Table 2) in co-digestion were chosen to reach a different C/N ratio (18−35) basedon the previous results.12−15

For the third batch experiment (P1 and P2) of investigating theeffect of pretreatment, all experimental conditions were the same asthose of experimental set C2 except for the substrate cotton stalkwhich was pretreated by acid or alkaline solution before it was co-digested with swine manure at a mixing ratio of 50:50. Allpretreatments were carried out in a 500 mL round-bottom glassflask with a water condenser. With ice water running through thecondensation pipe of the system, 15 g of the grounded cotton stalksample was mixed with 150 mL of acid or alkaline solutions in theflask. The flasks containing the mixtures were then gradually heateduntil 100 °C and maintained at the boiling point for 60 min. For acidhydrolysis pretreatment (AHP), the cotton stalk sample was placed in0.9% (w/w) H2SO4 solution and then boiled at 100 °C for 60 min. Foralkaline pretreatment (AP), the cotton stalk sample was placed inNaOH solution and boiled at 100 °C for 60 min. The alkaliconcentration in the AP was 6% (WNaOH/WStalk). The operationalparameters in AHP and AP were chosen based on the previousstudies.4,5,28 It should be mentioned that the pretreated samples weredivided into two parts in each experimental set. One part of thesamples (the mixtures of CS and water) was used for the following

Table 1. Characteristics of Cotton Stalk and Swine Manure

param swine manure cotton stalk

TS (% fresh weight) 29.5 ± 0.2 93.1 ± 1.2VS (%TS) 78.9 ± 0.6 89.0 ± 0.9pH 7.5 ± 0.3 −TKN (%TS) 2.42 ± 0.2 1.15 ± 0.05C/N 13 50cellulose (%TS) 38.3 ± 1.0hemicellulose (%TS) 19.5 ± 1.0lignin (%TS) 21.6 ± 0.9

Table 2. Batch Experimental Conditions of AnaerobicDigestion

proportion of thevarious mixtures

(%TS)a

exptl set F/Icottonstalk

swinemanure pretreatment

first batch: effect of F/I ratioF1 6 100 0 noF2 4 100 0 noF3 3 100 0 noF4 2 100 0 no

second batch: effect of co-digestion

C1 4 25 75 noC2 4 50 50 noC3 4 75 25 no

third batch: effect ofpretreatment

P1 4 50 50 AHPP2 4 50 50 AP

aPercentages are expressed in total solid (TS). AHP, acid hydrolysispretreatment; AP, alkaline pretreatment.

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digestibility tests without solid separation, after their pH was adjustedto 7 by using 1 M HCl or NaOH solution. Another part of the sampleswas used for the analysis of the component change in the pretreatmentprocess.2.3. Analytical Methods. TS, VS, total Kjeldahl nitrogen,

ammonium nitrogen, and soluble chemical oxygen demand (COD)were determined according to the standard methods described by theAmerican Public Health Association (APHA) and the State Environ-mental Protection Administration of China (SEPAC).29,30 Totalorganic carbon was measured by following the Walkey−Blackmethod.31 The C/N ratio was obtained by dividing the total organiccarbon by the total Kjeldahl nitrogen.13 Weight loss after pretreat-ments and digestion was recorded at the end of batch digestion. Thefree ammonia concentrations at the end of the tests were calculatedbased on the previous method.11

Part of pretreated samples was centrifuged at 4500 rpm for 20 min,and the solid residues were then dried at 60 °C to constant weight forweight loss calculation and lignocellulosic component analysis.Cellulose, hemicellulose, and lignin were measured by the methoddescribed by Goering and Van Soest.32 In brief, neutral detergent fiber(NDF) and acid detergent fiber (ADF) are determined gravimetricallyas the solid residue remaining after neutral and acid detergentextraction, respectively. Lignin is determined gravimetrically on a freeash basis after the ADF residue is extracted with 72% H2SO4 solution.Ash content was determined in an oven at 550 °C over 6 h. Celluloseis calculated by subtracting the preash lignin value from the ADF value.Hemicellulose is calculated by subtracting the ADF value from theNDF value. Weight loss and cellulose, hemicellulose, and lignincontent are calculated on the basis of TS of cotton stalk samples.During anaerobic fermentation, the samples were collected at

predetermined time points and centrifuged at 15000 rpm for 10 min.The supernatants were filtered by using a micropore membrane of 0.45μm. The filtrates of 0.9 mL were treated by addition of 0.1 mL of 10%formic acid solution and then used for volatile fatty acids (VFAs)analysis. The concentrations of VFAs were determined using a gaschromatograph (Agilent GC 6890, Santa Clara, CA, USA) equippedwith a flame ionization detector and a 30 m × 0.25 mm × 0.25 μmfused-silica capillary column (DB-FFAP). The temperatures of theinjector and detector were 250 and 300 °C, respectively. The oventemperature was initially kept at 70 °C for 3 min, followed by a ramp-up of 20 °C/min for 5.5 min, and held at a final temperature of 180 °Cfor 3 min. Nitrogen was used as the carrier gas with a flow rate of 2.6mL/min.The biogas composition was determined using gas chromatography

(Agilent GC 6890) equipped with a thermal conductivity detector andtwo columns separated by a switching valve. The first column was aPlot Q polymer column, to separate CO2 and the compounds with ahigher molecular weight, and the second was a molecular sieve columnto separate the lower molecular weight gases such as H2, O2, N2, andCH4. Helium was used as the carrier gas at a flow rate of 23 mL/min.The oven, injector, and thermal conductivity detector temperatureswere 50 °C, 150 °C, and 250 °C, respectively. Calibration curves of theabove gas components were linear and reproducible. The volume ofthe biogas produced during anaerobic digestion of cotton stalk wasmeasured using a glass syringe.33 The daily biogas production wasexpressed as biogas production per gram of VS of the substrate addedin 1 day [mL/(day·(g of VSadded))]. The cumulative biogas productionwas expressed as the cumulative biogas production per gram of VS ofthe substrate added (mL/(g of VSadded)). Yields of biogas and methanewere expressed as milliliters of biogas or methane produced per gramof VS of the substrate added (mL/(g of VSadded) and milliters of CH4

per gram of VSadded. Technical digestion time (T80) was defined as thetime needed to produce 80% of the maximal digester gas production.2

In the present study, the final cumulative biogas production on day 45was considered as the maximal digester gas production and used tocalculate the technical digestion time.The synergistic effect of co-substrates was estimated based on the

previous method.3,34 The synergistic effect could be seen as anadditional methane yield for co-digestion substrates over the weighted

average of the individual substrate’s methane yield. Weighted methaneyield (weighted MY) was calculated as follows:

= + +R R R Rweighted MY (MY MY )/( )CS CS SM SM CS SM (1)

where weighted MY represents the weighted average of methane yieldfor co-substrates, MYCS and MYSM refer to the methane yields for CSand SM, and RCS and RSM are the VS fractions for CS and SM in themixtures of co-digestion, respectively. Methane yield of swine manure(MYSM) is 277 mL/(g of VSadded), which was obtained in thepreliminary experiment of this study.

2.4. Statistical Analysis. The experimental data were statisticallyanalyzed using one-way analysis of variance (ANOVA) followed byDuncan’s multiple range test by using SPSS statistics 17 (IBM,Armonk, NY, USA).

2.5. Kinetic Modeling. The biogas production process in theanaerobic bottles can be modeled by modified Gompertz equation,35

which can be written as follows:

λ= − − +⎧⎨⎩

⎡⎣⎢

⎤⎦⎥⎫⎬⎭M t P

R eP

t( ) exp exp ( ) 1m

(2)

The kinetics of biogas production from cotton stalk can be welldescribed using the above Gompertz equation, where M(t) representscumulative biogas volume per gram of VS substrate added (mL/(g ofVSadded)), P is the biogas production potential (mL/(g of VSadded)), Rmis the maximum biogas production rate [mL/(day·(g of VSadded))], λ isthe lag time (days), and e equals about 2.718. The cumulative biogasproduction curve was nonlinearly fitted by the equation with Origin8.0 pro.

3. RESULTS AND DISCUSSION3.1. Effect of F/I Ratios on Biogas Production in

Anaerobic Monodigestion of Cotton Stalk. Biogasproduction in anaerobic digestion of cotton stalk at four F/Iratios was investigated, and the results are shown in Figure 1.Rapid biogas production started from day 4−5 at the F/I ratiosof 2−4 (Figure 1A). Biogas production decreased to someextent with the increase of F/Is. The highest daily biogasproduction reached 17.1, 14.8, and 14.2 mL/(day·(g ofVSadded)) on days 5, 7, and 10 of digestion at the F/I ratiosof 2, 3, and 4, respectively. When compared with thoseobtained at these three F/I ratios, a significant decrease ofbiogas production was noticed at a higher F/I ratio of 6. Thedaily biogas production fluctuated during the first 20 days ofdigestion with a highest level of 8.5 mL/(day·(g of VSadded))and then dropped to a low level after day 21.It is to be noted that cumulative biogas production in the

digesters operated at F/I ratios of 2, 3, and 4 increased fromday 3 until about day 21 and then gradually leveled offthereafter (Figure 1B). In the case of the F/I ratio of 6, thecumulative biogas production began to increase slowly until day5 (Figure 1B). After 45 days of digestion, the biogas yields ofcotton stalk at the F/I ratios of 2−4 reached 175−180 mL/(gof VSadded) against 113 mL/(g of VSadded) achieved at the F/Iratio of 6 (Table 3). Biogas yield of raw cotton stalk was 65mL/(g cotton stalk).8 Another studies reported that biogasyields of cotton stalk reached 157 mL/(g of TS) (approx-imately 177 mL/(g of VSadded)) and 89.6 mL/(g of VSadded),respectively.9,20 The biogas yield obtained in this study iscomparable with those results.The average methane contents of the biogas at the four F/I

ratios of 2, 3, 4, and 6 were 58.5%, 58.2%, 57.8%, and 56.5%,respectively (Table 3). And the corresponding methane yieldsat these F/I ratios were calculated to be 106, 102, 102, and 64mL of CH4/(g of VS added), respectively. The lower methanecontent and yield at the F/I ratio of 6 indicated that there may



be an inhibition of methanogenic bacteria in this case. Thecorresponding weight loss of substrate at the end of the

digestion was also lower than those obtained at the F/I ratios of2−4 (30.8% vs 37.1−39.3%). Technical digestion time, which isdefined as the time needed to produce 80% of the maximaldigester gas production,2 is another indicator of biogasproduction performance. In the present study, the finalcumulative biogas production on day 45 was considered asmaximal digester gas production and used to calculate technicaldigestion time based on formula 1. As shown in Table 3,technical digestion times at the F/I ratios of 2−4 wereobviously shorter than that obtained in the case of the F/I ratioof 6 (16−20 days vs 23 days). The biogas and methane yieldsof cotton stalk achieved in this study are lower than those ofsome other crop residues such as corn stalk and wheat straw.8

This might be due to the relatively higher lignin content ofcotton stalk (21.6%), which hinders attack of enzymes todegradable hemicellulose and cellulose components.As shown in Figure 1 and Table 3, both daily biogas

production and biogas yield obtained a significant increasewhen the F/I ratios were decreased from 6 to 2−4. Lowefficiency of biogas fermentation at a high F/I ratio might bedue to the insufficient methanogens and/or low methanogenicactivity of sludge, which could cause the accumulation of thevolatile fatty acids in the bioreactor and the subsequent drop ofpH.15 The optimal pH values for anaerobic digestion rangefrom 6.5 to 7.5, and methanogenic bacteria will be inhibited at alower level of pH.12 The changes of VFA and pH duringdigestion of cotton stalk were monitored, and the results areshown in Figure 1C,D. As observed in the digestion of cottonstalk at the F/I ratio of 6, the pH in the digester decreased to6.3 on day 8 and remained at a low level until day 12 (Figure1D), corresponding well with higher VFA concentrations(Figure 1C) of fermentation broth in this period than thoseobtained at the F/I ratios of 2−4. Acetic acid was the main VFApresent in all four batch tests (Figure 1C). The VFAsaccumulation leads to the decrease of pH, therefore affectingmethanogenic activity during the anaerobic digestion proc-ess.12,36 As a result, an inhibited steady state with a lowmethane yield will be observed.12,36 Previous studies indicatedthat the methane yield of wheat straw showed an obviousdecrease at F/I ratio over 4.25 Another study verified thatmarkedly lower methane yields were observed in anaerobicdigestion of food wastes and/or green wastes (grass clippings)at a F/I ratio of 5 than those obtained at F/I ratios of 2−4.26It is true that the peak of daily biogas production at F/I = 2

was slightly higher and technical digestion time was relativelyshorter (16 days vs 19 days, Table 3) than those obtained at F/I= 4, but differences in biogas and methane yield between thesetwo F/I ratios were not statistically significant (p < 0.01; Table3). Swine manure may also contain some active micro-organisms for biogas production, and sufficient inoculum wastherefore anticipated in the co-digestion at these two F/I ratios.On this condition, a relatively higher F/I ratio will allow onedigester to dispose of more wastes when an ideal total TSconcentration of substrate and inoculum is fixed. It will reallybring economical benefit for improving efficiency or treatmentcapacity of one existing digester and reducing disposal cost ofwastes. Also, given that it will be very difficult to get a hugeamount of active inoculum for start-up of a biogas plant in thefuture, the F/I of 4 with low requirement for the amounts ofinoculum was chosen for the following studies of co-digestionand the effect of pretreatment.

3.2. Effect of Mixing Ratios on Biogas Production inCo-digestion of Swine Manure with Cotton Stalk. In an

Figure 1. Effect of the F/I ratios on daily biogas production (A),cumulative biogas production (B), VFA accumulation (C), and pH(D) from anaerobic digestion of cotton stalk.



effort to enhance the performance of anaerobic digestion by co-digesting swine manure with cotton stalk, the mixing ratios ofcotton stalk and swine manure (CS/SM) were varied toproduce different C/N ratios while operating the digesters atthe F/I ratio of 4. The results of anaerobic monodigestion ofcotton stalk (experimental set F2) were used as a control.As in the anaerobic monodigestion of cotton stalk, biogas

production was observed after day 6 in the co-digestionexperiments. The biogas production rate (it was calculated bydividing the cumulative biogas volume by the working volumeof the reactor and the time needed to produce 90% of the finalcumulative biogas volume (day 45)) and cumulative biogasproduction observed in the operation of co-digestion weresignificantly higher than those in the anaerobic monodigestionof cotton stalk. As seen in Figure 2A, at a CS/SM ratio of75:25, daily biogas production reached a higher level from day8 of digestion than other treatments and peaked on day 11[33.1 mL/(day·(g of VSadded))] before decreasing to a low levelon day 19. At a CS/SM ratio of 50:50, daily biogas productionsurpassed the former to become the highest producer on day 13before reaching the highest level on day 14. At a CS/SM ratioof 25:75, biogas production gradually increased after a lag phaseof about 8 days and peaked until day 18 [34.8 mL/(day·(g ofVSadded))]. The highest daily biogas production of 33.1−36.5mL/(day·(g of VSadded)) obtained at all three CS/SM ratios washigher than the 14.2 mL/(day·(g of VSadded)) in the control.The average biogas production rate of 0.41−0.49 L/(L·day)was also higher than that obtained from the control (Table 4).Equally significant was the consequential increase in

cumulative biogas production: 276−341 mL/(g of VSadded)against 176 mL/(g of VSadded) in the control (Figure 2B andTable 4). There was a positive relation between biogasaccumulation in the first days of digestion and the ratio ofcotton stalk to swine manure. As shown in Figure 2A,cumulative biogas production maintained at a higher level atthe CS/SM ratio of 75:25 than other treatments before it wassurpassed by the digester at the CS/SM ratio of 50:50 on day13. The final cumulative biogas production on day 45 (i.e.,biogas yield) of digestion at the CS/SM ratios of 25:75, 50:50,and 75:25 reached 337, 341, and 276 mL/(g of VSadded),respectively. Differences in biogas yields between co-digestionand monodigestion of cotton stalk were statistically significant(p < 0.01).The average methane contents of the biogas in co-digestion

at the three CS/SM ratios were 63.5−65.7%, which wasobviously higher than the methane content of 57.8% in themonodigestion of cotton stalk (Table 4). The technicaldigestion time at the three CS/SM ratios was 23, 21, and 19days, respectively, which is the same or slightly longer than thatin the control (Table 4). The final methane yields at the threeCS/SM ratios were calculated to be 220, 224, and 175 mL of

CH4/(g of VSadded), respectively, corresponding to increases upto 1.7−2.2-fold.When compared with the control, the advantages of co-

digesting cotton stalk and swine manure are obvious withincreases up to 1.8, 1.9, and 2.2 times in biogas production rate,biogas yield, and methane yield, respectively. It is worth notingthat the extended lag phase of more than 10 days in thedigestion of swine manure alone observed in previous studieswas shortened.13 The earlier peak of daily biogas productionobserved from the co-digestion at higher CS/SM ratio of 75:25than those from other treatments is probably because parts ofthe VS in stalks may be more easily degradable than the VS inmanure.37 However, the highest biogas yield of 341 mL/(g ofVSadded) was observed from the co-digestion at the CS/SMratio of 50:50. When the CS/SM ratios were 25:75, 50:50, and75:25, the corresponding C/N ratios were 18, 25, and 35,respectively. These results suggested that the ideal C/N ratio is25 in the co-digestion of swine manure and cotton stalk, whichwas consistent with the optimum C/N range of 20−30reported before.13,14

Possible mechanisms behind the improved performance ofthe co-digestion process included not only providing a bettercarbon/nitrogen ratio in the feedstock but also dilutingpotentially toxic compounds, utilizing the nutrients andbacterial diversities in various wastes, and increasing bufferingcapacity to help maintain an optimal pH for methangenicbacteria, etc.17−19 Changes of the VFA concentration and pHwere therefore determined, and the results are given in Figure2C,D. As can be seen from Figure 2C, acetic acid was the mainVFA present in all tested conditions. In the case ofmonodigestion of cotton stalk (set F2), VFA concentrationon day 4 was about 320 mg/L and then decreased to anextremely low level (<50 mg/L) after day 12. When cottonstalk was co-digested with swine manure, the VFA accumu-lation showed a significant increase with the increase of theamount of swine manure in the mixtures (Figure 2C). This maybe attributed mainly to the acidification of swine manure. ThepH of fermentation broth at all mixing ratios was above 6.5through the digestion process with highest VFA concentrationsnear 3000 mg/L (Figure 2C,D). However, process disturbancesdue to acidification were not observed even at the CS/SM ratioof 25:75 in that the highest VFA accumulation was achieved,which may be due to the sufficient buffering capacity of the co-digestion system. The total VFA concentration at the end ofdigestion did not exceed the level of 50 mg/L and did not affectthe process balance. Concentrations of ammonium nitrogen(NH4

+−N) and free ammonia (NH3−N) (Table 4) observedin all three CS/SM ratios were below the methanogenic activityinhibitory level10,11,13 and were therefore unlikely to produce asignificant effect. When compared with monodigestion ofcotton stalk (F2), more drops of pH were not observed in co-digestion (C1−C3) in spite of their significantly higher VFAs

Table 3. Effect of F/I Ratios on Biogas Production from Anaerobic Monodigestion of Cotton Stalka

F/I ratios (exptl set)

param 6 (F1) 4 (F2) 3 (F3) 2 (F4)

biogas yield (mL/(g of VSadded)) 113 ± 4a 176 ± 9b 175 ± 9b 180 ± 9b

methane content (%) 56.5 ± 0.4 57.8 ± 0.5 58.2 ± 0.3 58.5 ± 0.4methane yield (mL of CH4/(g of VSadded)) 64 ± 2a 102 ± 5b 102 ± 5b 106 ± 5b

weight loss after digestion (%TS) 30.8 ± 0.4 37.9 ± 0.6 37.1 ± 0.5 39.3 ± 0.9technical digestion time (days) 23 ± 1.0 19 ± 0.6 20 ± 1.0 16 ± 0.6

aValues followed by different superscript lowercase letters are significantly different at p < 0.01 according to Duncan’s test.



levels in the bioreactors (Figure 2C). At the same time, biogasproduction rates of co-digestion and biogas yield wereobviously higher than that observed in monodigestion (Table

4). These results may be attributed to an improved bufferingcapacity and methanogenic activity in the co-digestion system.It is to be noted that the propionate concentration was alsoimproved in co-digestion of swine manure and cotton stalkcompared with monodigestion of cotton stalk. The previousstudy reported that propionate had stronger inhibitory effect onmethanogenesis than acetate and butyrate.38 Slow biogasproduction during the first 12 days and longer technicaldigestion time (Table 4) at the CS/SM ratio of 25:75 may bedue to the higher total VFAs and propionate levels accumulatedin the digester (Figure 2C), as compared to the other CS/SMratios.Biogas production performance in co-digestion was further

investigated by estimating the synergistic effect of co-substratesbased on the previous method.3,34 The synergistic effect couldbe seen as an additional methane yield for co-digestionsubstrates over the weighted average of the individualsubstrate’s methane yield. If the difference (methane yield −weighted methane yield) was higher than the standarddeviation of methane yield, the synergistic effect could beconfirmed.3 SM could provide additional nitrogen and essentialelements for the microorganism community in the co-digestionsystem, so that higher methane yield could be obtained. Asshown in Table 4, increasing the ratio of CS to 25−50% in themixture resulted in a very significant (p < 0.01) increase ofmethane yield compared to the digestion of CS alone. Based onthe comparison of corresponding methane yields, synergisticeffects were found in the CS/SM ratios of 50:50 and 75:25(Table 4). The difference (MY − weighted MY) in the case ofCS/SM ratio of 50:50 was bigger, which indicated a strongersynergistic effect at the mixing ratio. This might be due to theadjustment of the C/N ratio from 50 (CS alone) and 13 (SMalone) to 25, which was in the optimal C/N range of 20−30.13,14 Although the methane yield obtained at the CS/SMratio of 25:75 was comparable compared with the CS/SM ratioof 50:50, the synergistic effect was not clear and the technicaldigestion time was longer in this case (Table 4). In the case of aCS/SM ratio of 75:25, the methane yield was significant lowerthan in the other two cases in spite of the observed synergisticeffect and short technical digestion time (Table 4). Whenconsidering the high biogas yield, strong synergistic effect, andmedium technical digestion time, mixtures at the CS/SM ratioof 50:50 were chosen for examining the effect of pretreatmentson biogas production (Table 4). An addition of inexpensivecotton stalk wastes up to 50% TS of substrates in the existingdigesters with manure wastes as substrate will not only alleviatethe problem of insufficient manure source but also produceviable economic and ecological benefits.

3.3. Effect of Pretreatments on Biogas Production inCo-digestion of Swine Manure and Cotton Stalk. Cottonstalk wastes are mainly composed of cellulose, hemicellulose,and lignin. The lignin content (21.6%) of cotton stalk wasmuch higher than that observed in corn stalk and wheat strawsamples, while contents of degradable cellulose and hemi-cellulose components were comparable.4,13,20 It was anticipatedthat biogas production could be further improved by usingpretreatments to increase the accessibility of cotton stalksamples. To evaluate the possibilities, cotton stalk waspretreated by acid hydrolysis pretreatment and alkalinepretreatment before co-digestion with swine manure at amixing ratio of 50:50.As can be seen from Table 5, the cotton stalk pretreated by

AP had higher weight loss than that by AHP. The weight loss of

Figure 2. Effect of the CS/SM ratios on daily biogas production (A),cumulative biogas production (B), VFA accumulation (C), and pH(D) from co-digestion of swine manure and cotton stalk.



cotton stalk was correlated with solubilization of itscomponents, such as cellulose, hemicellulose, and lignin. Thehigher weight loss revealed that more cellulosic componentswere converted into soluble substances, which was furtherverified by a higher level of soluble COD in the solutionpretreated with AP than those pretreated with AHP. As can beseen from Table 5, the weight loss of cotton stalk was mainlydue to the hemicellulose hydrolysis in both AHP and AP.Shevchenko et al. also verified that the soluble sugar was mainlyoriginated from the hemicellulose fraction of softwood chips.39

Hemicellulose components can be effectively solubilized intomonomeric sugar and soluble oligomers by the dilute acidpretreatment,40 while much more lignin components wereremoved by AP (Table 5).Cotton stalk samples pretreated by AHP and AP were used

in anaerobic co-digestion for biogas production, and the resultsare shown in Figure 3. It is noteworthy that both daily biogasproduction (Figure 3A) and cumulative biogas production(Figure 3B) were markedly improved by using AHP and AP.The biogas produced from these pretreatment samples has65.6−65.8% methane. When compared with AHP, AP appeared

to be a relatively efficient pretreatment technique for obtainingthe highest cumulative biogas production after 45 days,although the lag phase was slightly longer in this case (Figure3). An average biogas production rate of 0.65 L/(L·day) wasalso higher than 0.49 L/(L·day) in the control. Equallysignificant was the resulting increase in biogas and methaneyields of 449 mL/(g of VSadded) and 296 mL of CH4/(g ofVSadded) against 341 mL/(g of VSadded) and 224 mL of CH4/(gof VSadded) in the control. The technical digestion time was keptat a constant level (Table 5). Differences in biogas yields in co-digestion of cotton stalk with and without pretreatments werestatistically significant (p < 0.01). As reported before, biogasyield of raw cotton stalk was not very high.8,9 Destroyedmicrostructure and increase in extractives’ contents wereobserved during mild pretreatments of cotton stalk withammonia solution or recycled liquid of anaerobic digestion,which provided more biodegradable components and madecellulosic components more exposed to microorganism.9 As aresult, a significantly higher biogas yield was carried out thanthat obtained in untreated cotton stalk (experimental set C2).9

When compared with untreated cotton stalk and cotton stalk

Table 4. Effect of the CS/SM Ratios on Biogas Production from Co-digestion of Swine Manure with Cotton Stalk

CS/SM ratios (exptl set)

param 25/75 (C1) 50/50 (C2) 75/25 (C3) 100/0 (F2)

biogas yield (mL/)g of VSadded)) 337 ± 23a 341 ± 17a 276 ± 6b 176 ± 9c

methane content (%) 65.5 ± 0.4 65.7 ± 0.6 63.5 ± 0.7 57.8 ± 0.4methane yield (mL of CH4/(g of VSadded)) 220 ± 15 a 224 ± 11 a 175 ± 4 b 102 ± 5 c

weighted MY (mL of CH4/(g of VSadded)) 229 184 142differential (MY − weighted MY) −9 40 33synergistic effect not clear synergistic synergisticbiogas production rate (L/(L·days))a 0.41 ± 0.03 0.49 ± 0.02 0.47 ± 0.01 0.27 ± 0.01initial NH4

+−N (mg/L) 1754 ± 85 1321 ± 65 881 ± 43 442 ± 23final NH4

+−N (mg/L) 1980 ± 132 1641 ± 124 1185 ± 89 691 ± 56final free ammonia (NH3−N, mg/L) 161 ± 12 108 ± 9 63 ± 5 15 ± 2weight loss after digestion (%TS) 49.9 ± 0.3 50.9 ± 0.8 45.6 ± 1.0 37.9 ± 0.6technical digestion time (days) 24 ± 1.0 21 ± 0.6 19 ± 0.6 19 ± 0.6

aThe biogas production rate was calculated by dividing the cumulative biogas volume by the working volume of the reactor and the time needed toproduce 90% of the final cumulative biogas volume (day 45). MY, methane yield. Values followed by different superscript lowercase letters aresignificantly different at p < 0.01 according to Duncan’s test.

Table 5. Effect of Pretreatments on Cotton Stalk Hydrolysis and Biogas Production from Co-digestion of Swine Manure withCotton Stalk

exptl set C2 exptl set P1 exptl set P2

param untreated CS CS by AHP CS by AP

cellulose (%TS) 38.3 ± 1.0 36.5 ± 0.5 37.3 ± 0.6hemicellulose (%TS) 19.5 ± 1.0 15.5 ± 0.6 16.7 ± 0.4lignin (%TS) 21.6 ± 0.9 17.3 ± 0.3 12.8 ± 0.2weight loss in pretreatments (%) 22.3 ± 0.3 27.6 ± 0.5soluble COD (mg/L) 15.6 ± 0.5 19.8 ± 0.3biogas yield (mL/(g of VSadded))

a 341 ± 17a 407 ± 29ab 449 ± 22b

methane content (%) 65.7 ± 0.6 65.6 ± 0.5 65.8 ± 0.6methane yield (mL of CH4/(g of VSadded))

a 224 ± 11a 267 ± 19ab 296 ± 14b

biogas production rate (L/(L·day))b 0.49 ± 0.02 0.61 ± 0.04 0.65 ± 0.03weight loss after digestion (%TS)c 50.9 ± 0.8 57.0 ± 0.4 59.8 ± 1.1technical digestion time (days) 21 ± 0.6 21 ± 0.6 21 ± 1.0

aBiogas and methane yield was calculated based on the VS of the substrate before pretreatments. bThe biogas production rate was calculated bydividing the cumulative biogas volume by the working volume of the reactor and the time needed to produce 90% of the final cumulative biogasvolume (day 45). cWeight loss after digestion was the total weight reduction in both pretreatment and the digestion process. AHP, acid hydrolysispretreatment; AP, alkaline pretreatment. Values followed by different superscript lowercase letters are significantly different at p < 0.01 according toDuncan’s test.



pretreated by AHP, higher biogas production rate and biogasyield (Table 5) in co-digestion of cotton stalk pretreated by APwere probably due to more soluble compounds released fromthe pretreated cotton stalk and higher biological accessibility ofthe pretreated cotton stalk. In the AP process, parts ofhemicellulose were converted into soluble components andmore lignin was removed than AHP (Table 5). The pretreatedcotton stalk by AP became more accessible to anaerobicmicroorganisms. In the digestion process, the use of celluloseand hemicellulose was therefore enhanced. As a result, themaximum weight loss of 59.8% was observed from the cottonstalk by AP in the digestion process, which is markedly higherthan those obtained in the case of untreated cotton stalk(50.9%) as well as with AHP (57.0%, Table 5). Previous study

on cotton stalk also verified that alkaline pretreatment resultedin the highest level of delignification (65.63% for 2% NaOH, 90min, 121 °C/15 psi) and cellulose conversion (60.8%), whilesulfuric acid pretreatment resulted in the highest xylanreduction (95.23% for 2% acid, 90 min, 121 °C/15 psi) butthe lowest cellulose to glucose conversion during hydrolysis(23.85%).22 Enhanced biogas production was also reported inanaerobic digestion of corn stalk pretreated with NaOH, whichis due to the breakdown of its recalcitrant structures.23 Whencompared to AP and AHP, less reduction of hemicelluloses andlignin was obtained in cotton stalk pretreated with hot water at100 °C for 60 min and the increase of biogas yield was alsolower (10% vs 19−32%; data not shown). However, it is stilldifficult to conclude the chemical pretreatment plays a moreimportant role for improving biogas yield than hydrothermalpretreatment in the present pretreatment process. A detailedstudy is ongoing to elucidate the relevant mechanism andoptimize the AP process.Reported biogas yield of raw cotton stalk was very low (65

mL/(g of cotton stalk)).8 Biogas yield in monodigestion ofcotton stalk pretreated for 7 days at 35 °C using a compositemicrobial system screened through the method of multi-generation selection reached 206 mL/(g of TS), which ismarkedly higher than 157 mL/(g of TS) achieved in the case ofuntreated cotton stalk (about 231 vs 177 mL/(g of VSadded),estimated based on the VS content (89% of TS) of cotton stalkin the present study).20 High biogas yield of 114−448 mL/(g ofVSadded) and methane yield of 80−242 mL of CH4/(g ofVSadded) was also obtained from digestion of cotton stalkpretreated using hot water, ammonia solution, and recycledliquid of anaerobic digestion.9 In the present study, biogas andmethane yields of raw cotton stalk were 176 mL/(g of VSadded)and 102 mL of CH4/(g of VSadded). The co-digestion of swinemanure and pretreated cotton stalk by AP significantlyenhanced biogas production from cotton stalk, and final biogasand methane yields reached 449 mL/(g of VSadded) and 296 mLof CH4/(g of VSadded). The present biogas and methane yieldsare also comparable with those observed in anaerobic digestionof other lignocellulosic biomass.13,19,41 Biogas and methaneyields of 410 mL/(g of TS) and 259 mL of CH4/(g of TS)were carried out in mesophilic co-digestion of vermicompostand corn stalk.19 And batch mesophilic co-digestion of cowmanure and pretreated Salix by steam explosion producedmethane yield of 230 mL of CH4/(g of VS).41

3.4. Kinetic Modeling of Biogas Production in Mono-and Co-digestion of Cotton Stalk. Table 6 summarizes the

Figure 3. Effect of pretreatments on daily biogas production (A) andcumulative biogas production (B) from co-digestion of swine manureand cotton stalk.

Table 6. Kinetic Parameters for Biogas Production from Anaerobic Mono- and Co-digestion of Cotton Stalk with or withoutPretreatments

MY (mL/(g of VSadded))a

exptl set Rm [mL/(day·(g of VSadded))] λ (days) R2 pred (P) measd diff (%)

F1 5.3 4 0.997 116 113 2.6F2 10.3 4 0.998 174 176 1.1F3 9.9 2 0.993 173 175 1.1F4 13.1 2 0.993 176 180 2.3C1 25.3 11 0.997 329 337 2.4C2 27.5 9 0.998 338 341 0.9C3 20.9 6 0.998 274 276 0.7P1 24.4 6 0.995 409 407 0.5P2 30.8 8 0.998 448 449 0.2

aMY, methane yield.



results of a kinetic study of biogas production. As indicated bythe R2 value of 0.993−0.998 and the difference of 0.2−2.6%between the measured and predicted methane yield, themodified Gompertz model was found to have very good fit tobiogas production from the substrates used. The lag phase inco-digestion of cotton stalk was longer than its monodigestion,but the biogas yield reached 194% of that in the latter. Theextended lag phase may be due to the acclimation ofmicroorganisms to introduced swine manure. Kinetic param-eters also revealed that the maximum biogas production ratewas significantly improved by co-digesting cotton stalk withswine manure at an optimal CS/SM ratio of 50:50 compared tothat in the monodigestion of cotton stalk (Table 6). Theproduction rate was further enhanced by using cotton stalkpretreated by AP in the co-digestion at this mixing ratio. Thecorresponding biogas yield in this case was also much higherthan that in the monodigestion of cotton stalk (Table 6).Together with existing engineering experience of biogastechnology, the current studies demonstrated that co-digestionof manure wastes with pretreated cotton stalk by AP provided apromising and viable alternative for renewable energyproduction from lignocellulosic biomass.

4. CONCLUSION

Biogas yields of 175−180 mL/(g of VSadded) with shortertechnical digestion times were observed from anaerobicdigestion of cotton stalk at the F/I ratios of 2−4 against 113mL/(g of VSadded) at the F/I ratio of 6. The performance wasimproved by co-digesting swine manure with cotton stalk. Thehighest biogas production rate and biogas yield were recordedin co-digestion of swine manure with cotton stalk at the CS/SM ratio of 50:50 with a C/N ratio of 25, which were 1.8- and1.9-fold of those achieved in monodigestion of cotton stalk,respectively. The higher biogas (methane) yields of 449 (296)mL/(g of VSadded) were obtained from co-digestion of swinemanure and cotton stalk pretreated by AP, which was 255% and132% of those achieved in anaerobic mono- and co-digestion ofraw cotton stalk, respectively. The maximum biogas productionrate was also significantly higher, while the technical digestiontime was comparable. It is interesting to note that the above co-digestion mixtures possessed a considerable amount ofpretreated cotton stalk (up to 50% of TS content). Cottonstalk available in the agricultural sector are then a good optionfor improving the energy output by co-digesting them withmanure wastes while giving a stable and viable digestionprocess.

■ AUTHOR INFORMATION

Corresponding Author*Tel.: +86-10-51684351-210. Fax: +86-10-51683887. E-mail:[email protected].

Author Contributions§X.-Y.C. and C.Z. contributed equally to this work.

NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTS

This work is financially supported by National Natural ScienceFoundation of China (Grant No. 21306009) and the ScienceFoundation of Beijing Jiaotong University (Grant 2011RC030).

■ ABBREVIATIONS

CS = cotton stalkSM = swine manureAHP = acid hydrolysis pretreatmentAP = alkaline pretreatmentTS = total solidVS = volatile solidF/I = feed to inoculumsCOD = chemical oxygen demandVFAs = volatile fatty acidsNDF = neutral detergent fiberADF = acid detergent fiber

■ REFERENCES(1) Li, Y. B.; Yang, G. H.; Chu, L. L.; Chen, Y. Resour. Sci. 2009, 31,231−237 (in Chinese).(2) Palmowski, L. M.; Muller, J. A. Water Sci. Technol. 2000, 41,155−162.(3) Labatut, R. A.; Angenent, L. T.; Scott, N. R. Bioresour. Technol.2011, 102, 2255−2264.(4) Luo, Q.; Li, X. J. Trans. Chin. Soc. Agric. Eng. 2005, 21, 111−115.(5) Liu, C. Z.; Cheng, X. Y. Energy Fuels 2009, 23, 6152−6155.(6) El-Shinnawi, M. M.; Alaa El-Din, M. N.; El-Shimi, S. A.; Badawi,M. A. Resour., Conserv. Recycl. 1989, 3, 33−45.(7) El-Shinnawi, M. M.; El-Houssieni, M.; Aboel-Naga, S. A.; Fahmy,S. Resour., Conserv. Recycl. 1990, 3, 217−230.(8) Isci, A.; Demirer, G. N. Renewable Energy 2007, 32, 750−757.(9) Adl, M.; Sheng, K.; Gharibi, A. Appl. Energy 2012, 93, 251−260.(10) Angelidaki, I.; Ahring, B. K. Water Res. 1994, 28, 727−731.(11) Hansen, K. H.; Angelidaki, I.; Ahring, B. K. Water Res. 1998, 32,5−12.(12) Zhang, T.; Liu, L. L.; Song, Z. L.; Ren, G. X.; Feng, Y. Z.; Han,X. H.; Yang, G. H. PLoS One 2013, 8, 1−7.(13) Cuetos, M. J.; Fernandez, C.; Gomez, X.; Moran, A. Biotechnol.Bioprocess Eng. 2011, 16, 1044−1052.(14) Wu, X.; Yao, W. Y.; Zhu, J.; Miller, C. Bioresour. Technol. 2010,101, 4042−4047.(15) Sievers, D. M.; Brune, D. E. Trans. ASAE 1978, 21, 537−549.(16) Hills, D. J. Agric. Wastes 1979, 1, 267−278.(17) Xie, S.; Wu, G.; Lawlor, P. G.; Frost, J. P.; Zhan, X. Bioresour.Technol. 2012, 104, 289−297.(18) Dechrugsa, S.; Kantachote, D.; Chaiprapat, S. Bioresour. Technol.2013, 146, 101−108.(19) Chen, G. Y.; Zheng, Z.; Yang, S. G. Waste Manage. 2010, 30,1834−1840.(20) Bai, Y.; Li, W.; Chen, C.; Liao, P. Biological Pretreatment ofCotton Stalks and Domestication of Inocula in Biogas Fermentation.Microbiol. China 2010, 37, 513−519 (in Chinese).(21) Deng, H.; Li, C.; Li, F.; Chen, J. Trans. Chin. Soc. Agric. Eng.2009, 3, 212−216 (in Chinese).(22) Silverstein, R. A.; Chen, Y.; Sharma-Shivappa, R. R.; Boyette, M.D.; Osborne, J. Bioresour. Technol. 2007, 98, 3000−3011.(23) Keikhosro, K.; Shauker, K.; Mohammad, J. T. Biomass Bioenergy2006, 30, 247−253.(24) Pavlostathis, S. G.; Gosset, J. M. Biotechnol. Bioeng. 1985, 27,334−344.(25) Hashimoto, A. G. Biol. Wastes 1989, 28, 247−255.(26) Liu, G. Q.; Zhang, R. H.; El-Mashad, H. M.; Dong, R. J.Bioresour. Technol. 2009, 100, 5103−5108.(27) Dechrugsa, S.; Kantachote, D.; Chaiprapat, S. Bioresour. Technol.2013, 146, 101−108.(28) Cheng, X. Y.; Liu, C. Z. J. Chem. Technol. Biotechnol. 2010, 85,127−131.(29) American Public Health Association (APHA). Standard methodsfor the examination of water and wastewater, 20th ed.; APHA:Washington, D.C, USA, 2005.



mailto:[email protected]

(30) State Environmental Protection Administration of China(SEPAC). The methods for water and wastewater monitoring & analysis,4th ed.; China Environmental Science Press: Beijing, 2002.(31) Walkey, A.; Black, I. A. Soil Sci. 1934, 37, 29−38.(32) Goering, H. K.; Van Soest, P. J. Forage fiber analyses, (apparatus,reagents, procedures and some applications); Agricultural ResearchService (ARS), United States Department of Agriculture (USDA):Washington, DC, USA, 1970; Agricultural Handbook No. 379.(33) Owen, W. F.; Stuckey, D. C.; Healy, J. B.; Young, L. Y.;McCarty, P. L. Water Res. 1979, 13, 485−493.(34) Li, Y. Q.; Zhang, R. H.; Chen, C.; Liu, G. Q.; He, Y. F.; Liu, X.Y. Bioresour. Technol. 2013, 149, 406−412.(35) Kafle, G. K.; Kim, S. H. Appl. Energy 2013, 103, 61−72.(36) Kayhanian, M. Environ. Technol. 1999, 20, 355−365.(37) Lehtomaki, A.; Huttunen, S.; Rintala, J. A. Resour., Conserv.Recycl. 2007, 51, 591−609.(38) Siegert, I.; Banks, C. Process Biochem. 2005, 40, 3412−3418.(39) Shevchenko, S. M.; Chang, K.; Robinson, J.; Saddler, J. N.Bioresour. Technol. 2000, 72, 207−211.(40) Sun, Y.; Cheng, J. J. Bioresour. Technol. 2005, 96, 1599−1606.(41) Maria, M.; Linjordet, R.; Morken, J. Bioresour. Technol. 2012,104, 749−756.



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