Bioresource Technology 142 (2013) 663–671

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Bioresource Technology

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Psychrophilic anaerobic digestion of lignocellulosic biomass:A characterization study

0960-8524/$ - see front matter Crown Copyright � 2013 Published by Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.biortech.2013.05.089

⇑ Corresponding author. Tel.: +1 819 780 7128; fax: +1 819 564 5507.E-mail addresses: daniel.masse@agr.gc.ca, massed@agr.gc.ca (D.I. Massé).

Noori M. Cata Saady, Daniel I. Massé ⇑Dairy and Swine Research and Development Centre, Agriculture and Agri-Food Canada, Stn. Lennoxville, Sherbrooke, Quebec, Canada J1M 08C

h i g h l i g h t s

� Cow feces and wheat strawpsychrophilic anaerobic digestion isfeasible at <32 days.� Cow feces and wheat straw yielded

around 235 Nl CH4 kg�1 VS fed.� Cellulose, xylan, and their mixture

achieved 95% conversion into CH4 in35 days.� Less than 20 days are required to

achieve a conversion of 80%.� Methane yield (Nl CH4 kg�1 VS) was

338 (cellulose), 310 (xylan), and 305(mixture).

g r a p h i c a l a b s t r a c t

a r t i c l e i n f o

Article history:Received 25 April 2013Received in revised form 21 May 2013Accepted 23 May 2013Available online 29 May 2013

Keywords:Psychrophilic anaerobic digestionXylanWheat strawCow manureCellulose

a b s t r a c t

Psychrophilic (20 �C) specific methane (CH4) yield from cellulose (C), xylan (X), cellulose/xylan mixture(CX), cow feces (CF), and wheat straw (WS) achieved (Nl CH4 kg�1 VS) of 338.5 ± 14.3 (C), 310.5 ± 3.4(X), 305.5 ± 29.6 (CX mixture), and 235.3 ± 22.7 (WS) during 56 days, and 237.6 ± 17.7 (CF) during70 days. These yields corresponded to COD recovery of 73.3 ± 3.1% (C) = 69.1 ± 0.76% (X) = 67.3 ± 5.8%(CX mixture) > 52.9 ± 2.6% (CF) > 46.5 ± 2.7% (WS). Cellulose-fed culture had a lower and statistically dif-ferent initial CH4 production rate from those calculated for cultures fed X, CX mixture, CF and WS. Itseemed that the presence of hemicellulose in complex substrate such as wheat straw and cow feces sup-ported the higher initial CH4 rate compared to cellulose. Biomethanation of the pure and complex ligno-cellulosic substrates tested is feasible at psychrophilic conditions given that a well-adapted inoculum isused; however, hydrolysis was the rate limiting step.

Crown Copyright � 2013 Published by Elsevier Ltd. All rights reserved.

1. Introduction

Agriculture residue and farm waste are abundant and valuablerenewable natural resources which could support sustainable va-lue-added products such as biofuels and chemicals through low-cost microbial conversion processes (Lynd et al., 2008). Manureand farm lingocellulosic waste biomass can supply biodegradablereduced carbonic substrates for anaerobic digestion. However, thisprocess is affected by operational parameters such as temperature.Low temperature is a common stress factor in extreme environ-

ment and affects the performance of anaerobic digesters. Increas-ing number of reports indicated that psychrophilic anaerobicdigestion has the potential to become an economical and easy-to-use process to treat animal manure under northern climaticconditions (Massé et al., 2003; Massé et al., 2010; Safley and Wes-terman, 1994). The cold-adapted microorganisms (psychrophiles)are known to inhabit natural and man-made low temperatureenvironments and contribute to the ecosystem bioactivity in recy-cling carbon and other nutrients. However, their biotechnologicalpotential is still underutilized (Kasana and Gulati, 2011). Theycould be economically and ecologically advantageous comparedto thermophilic and mesophilic counterparts. Psychrophily is anevolutionary development from thermophily (Kasana and Gulati,

664 Noori M. Cata Saady, D.I. Massé / Bioresource Technology 142 (2013) 663–671

2011), thus adaptation and enrichment could be used to establishpsychrophilic anaerobic consortium specialized in manure derivedlignocellulosic substrates such as cellulose and hemicellulose.

Psychrophilic anaerobic digestion (PAD) substantially reducesthe energy input required for heating the bioreactor and could of-fer a solution in cold climate regions to treat farm wastes such asmanure and recover green energy in the form of biogas (Masséet al., 2003). Because cellulose and xylan are the most abundantpolymers in plants, their microbial conversion processes are cur-rently under active research. Dairy manure contains undigestedlignocellulosic biomass plus bedding materials such as straw com-posed mainly of polymers: cellulose (6C), hemicellulose (5C), andlignin (phenol polymers). Quantatively, manure fibers contain (as% of dry matter) cellulose (25%), hemicellulose (12–20%), and lignin(15%) (Yue et al., 2011). Cellulose and hemicellulose need to behydrolyzed to produce hexoses and pentoses, respectively, while,lignin is the limiting step in the degradation of lignocellulosic sub-strates because it is highly recalcitrant (Neves et al., 2006). Theheterogeneity and complex structure are a major challenge forthe anaerobic digestion of lignocellulosic biomass from plantmaterial (Weiß et al., 2010).

Previous studies have investigated biogas production from cel-lulose, hemicellulose, cow manure, and wheat straw in mesophilicand thermophilic processes. Reviewing published results on ther-mophilic, mesophilic, and psychrophilic celluloytic and hemicellu-lytic microorganisms, it appears that each microorganism has acertain degree of substrate specificity. Weiß et al. (2010) concludedthat augmenting batch anaerobic digester by enriched hemicellu-lytic bacteria increased the xylanase activity up to 162% and in-creased methane yield of xylan by 53% compared to controls.Similarly, treating fibers separated from cattle manure with ther-mophilic (70 �C) hemicellulose bacteria B14 for seven days in-creased methane potential in thermophilic (55 �C) anaerobicdigestion to 300 l CH4 kg�1 VS compared to 230 l CH4 kg�1 VS foruntreated control (Angelidaki and Ahring, 2000).

Biomethanation of lignocellulosic substrates have been re-ported in mesophilic and thermophilic anaerobic digestion, how-ever, very few studies have been published on psychrophilicanaerobic digestion of cellulose and hemicellulose. The objectiveof this study was to assess the extent of methane production fromthe degradation of hemicellulose (xylan; X), cellulose (C), celluloseand xylan (CX) mixture, cow feces (CF), and wheat straw (WS) inbatch by psychrophilic anaerobic mixed culture.

2. Methods

2.1. Experimental setup

Hemicellulose (xylan; X), cellulose (C), cellulose and xylan (CX)mixture, cow feces (CF), and wheat straw (WS) have been anaero-bically digested in duplicate 1.0 L batch bioreactors under con-trolled psychrophilic conditions (20 �C). The batch reactors have

Table 1Experimental design of psychrophilic batch reactors.

Condition Culture (mL) Substrate f

Xylan

Control 500 0Xylan 500 1.239Cellulose 500 01:2 Cellulose/xylan mixture (CX) 500 0.618Cow feces (VF) 500 0Wheat straw 500 0

Note: bioreactors’ working volume is 500 ml.

been intermittently mixed for 1 min once a week just before mixedliquor sampling. The reactors were flushed with nitrogen gasimmediately after feeding the substrate and when sampled formixed liquor to maintain the anaerobic condition, then were closedwith butyl rubber stoppers, and sealed with aluminum crimps. Theexperimental design is given in Table 1. The purpose of the exper-iments was to assess the degradation and the CH4 production fromX, C, CX mixture, CF and WS under psychrophilic anaerobic diges-tion conditions. A strategic objective was also to adapt the cultureto ferment these substrates in long term cycles. Physico-chemicalcharacteristics of the inoculum and substrates before feeding thebioreactors were analyzed and are given in Table 2.

2.2. Inoculum

The initial inoculum was obtained from a laboratory scale (40 L)psychrophilic (20 �C) dry anaerobic digester fed with fresh dairymanure and wheat straw (TS 27%), and operated as a sequencebatch reactor (SBR). The culture was prepared by diluting 1.2 kgof culture from dry anaerobic digestion reactors with deionizedwater and then sieving through 250 lm sieve to separate the largefibers. Only the mixed liquor passed through the sieve was used asan inoculum (TS 0.96%) in this study. The alkalinity of the diluteculture was 1700 mg L�1 as CaCO3 and was adjusted by adding4.9 g of CaCO3 into 7 L of dilute culture. The final measured alkalin-ity was 2595 mg L�1 as CaCO3.

The inoculum-to-substrate ratio (ISR) has been chosen to en-sure fast start-up of the anaerobic digestion based on results pub-lished previously (Chynoweth et al., 1993; Hansen et al., 2004;Hashimoto, 1989; Møller et al., 2004). The ISR used in the batchreactors are given in Table 2.

2.3. Substrates

Fresh feces from dairy cows (CF) was collected, at the experi-mental farm of the Dairy and Swine Research and DevelopmentCentre, Sherbrooke, Quebec (DSRDC), on wood boards, before get-ting in contact with urine and bedding, transferred into a plasticdrum, and stored at 4 �C prior being fed to the reactors.

The experiment employed wheat straw (Triticum aestivum)(WS) harvested at the DSRDC’s experimental farm during fall2012 and chopped (<250 lm) using a laboratory mill (ThomasWiley Laboratory Mill Model 4, Arthur H. Thomas Company,Philadelphia, PA). The wheat straw was stored in a controlled tem-perature (20 �C) room in closed plastic containers until further use.The moisture content of the straw was 11% (89% DM). All chemi-cals (pure substrates and standards) have been purchased fromSigma–Aldrich (Canada). Insoluble cellulose fibers (particle size40 lm) and insoluble powder of xylan from birchwood (Sigma–Aldrich (Canada)) were used for the pure substrates in this study.The total solids, volatile solids, and chemical oxygen demand ofthe substrates are given in Table 2.

ed (gram)

Cellulose Cow feces (VF) Wheat straw (WS)

0 0 00 0 00.685 0 00.341 0 00 10.264 00 0 1.367

Table 2Physico-chemical characteristics of the inoculum and substrates.

Material TS (%) VS (%) COD (g g�1) Organic loading rate (g COD l�1) Inoculum-to-substrate ratio (g VS g�1 VS)

Inoculum 0.96 0.73 0.012 NA NAXylan (X) 93 87 1.110 2.75 3.4Cellulose (C) 96 96 1.265 1.73 5.61:2 Cellulose/xylan mixture (CX) 95 90 1.210 2.24 4.2Cow feces (CF)a 13 11 0.148 3.04 3.1Wheat Straw (WS) 89 85 1.230 3.36 3.1

a Other physico-chemical characteristics are: alkalinity (5730 mg L�1 as CaCO3), pH 6.18, acetic acid (3365 mg L�1), propionic acid (877 mg L�1), butyric acid (1390 mg L�1).

Noori M. Cata Saady, D.I. Massé / Bioresource Technology 142 (2013) 663–671 665

2.4. Organic loading rate

The organic loading rate (OLR) of the substrates fed to the batchbioreactors (Table 2) has been calculated based on the masses oftotal chemical oxygen demand (TCOD) fed. OLR was expressed ingram of TCOD fed per liter of inoculum. The inoculum to substrateratio (ISR) based on VS ranged from 3.1 to 5.6 (Table 2).

2.5. Biogas measurement

Biogas production has been monitored daily while biogas com-position has been analyzed weekly using a gas chromatograph (GC)as described in section 2.6. Biogas volume produced was measureddaily using calibrated gas pressure meter (Ashcroft Digital Gauges,Model #30-2089SD-02L-5PSI, Ashcroft Inc., Connecticut, USA).Methane (CH4) production is reported in normalized liters (NlCH4), i.e., the CH4 volume produced was corrected to standard tem-perature and pressure (STP) (273 K; 1 atm) using Eq. (1):

VCH4STP¼ g Vm

Ts � Pm

Tm � Psð1Þ

where, Vm is the measured volume of biogas, g is the percentage ofCH4 in biogas, Tm and Pm which are the actual temperature andatmospheric pressure at the time of measurement, and Ts and Ps

are the standard temperature and pressure. VCH4STPis the volume

of methane at the standard temperature and atmospheric pressure.The CH4 volume produced was also corrected to the CH4 pro-

duced by the control bioreactors (CH4 produced in each bioreactorwas calculated by subtracting the amount of CH4 produced by thecontrols). Total specific methane yield (SMY) reported in this studyis the cumulative specific CH4 yield. Specific CH4 yield was calcu-lated as the ratio of CH4 produced (Nl) over the mass of the totalVS or COD fed to the bioreactor at the beginning of the experiment.The COD recovery in biogas was calculated as the percentage ofCH4 produced relative to the biochemical methane potential(350 l CH4 kg�1 COD).

2.6. Analytical methods

Mixed liquor samples have been collected and analyzed weeklyfor total chemical oxygen demand (TCOD), pH, alkalinity, and vol-atile and total solids (VS and TS, respectively) according to thestandard methods (APHA, 1992). The concentrations of individualvolatile fatty acids (VFAs) including acetic, propionic, butyric, iso-butyric, valeric and isovaleric acids have been measured using Per-kin–Elmer gas chromatograph (GC) model 8310 (Perkin–Elmer,Waltham, Mass) fitted with FID and, equipped with a J&W Scien-tific DB-FFAP high resolution column (30 m � 0.53 mm � 1.00 lm;Chromatographic Specialties Inc., Ontario) (Massé et al., 2003).

Biogas components (CH4 and CO2) were determined weeklyusing a Hach Carle 400 AGC gas chromatograph (Model 04131-C,Chandler Engineering, Houston, TX) configured for the application131-C. The application uses a column (1/8 inches) composed of1.8 m (805 porapak N + 205 porapak Q), 2.1 m (80% molecular

sieve 13X + 20% molecular Sieve 5A), and 1.8 m (80% OV-101 onchromosorb WHP). The column and thermal conductivity detectorwere operated at 85 �C with a helium gas flow rate of 30 mL min�1

(Massé et al., 2003). Calibration was performed weekly with a stan-dard gas (27.3% CO2, 1.01% N2, 71.69% CH4, 0.53% H2S). The percentof H2S in the biogas samples was less than 0.06%.

Methane production is reported in normalized litre (Nl CH4), i.e.,the volume of methane production is based on standard tempera-ture and pressure (STP) (273 K; 1 atm).

2.7. Elemental analysis

The pure substrates (xylan and cellulose) were subjected to anelemental analysis. Glucose was also analyzed as a control sample.Elemental analyses for CHN and O were carried out with the Carlo–Erba elemental analyzer, model 1106, according to standard proce-dure and the results are accurate to ±0.3%. The analysis for carbon(C), hydrogen (H), and nitrogen (N) has been performed based on amodification of the classical Pregl–Dumas method (Pella andColombo, 1973). Samples have been weighed accurately to0.0001 mg, tightly enclosed in tin capsules, and analyzed. The anal-ysis for oxygen (O) has been performed based on the Unterzauchermodified method (Pella and Colombo, 1972). Samples have beenweighed accurately to 0.0001 mg, tightly enclosed in silver cap-sules, and analyzed.

The elemental analysis revealed that the molecular formulae forcellulose and xylan are C5:928H10O4:965 and C5:148H8O4:418, respec-tively. The theoretical methane yield can be calculated using theresults of the elemental analysis and Buswell and Mueller (1952)formula.

2.8. Fiber analysis

The complex substrate (cow feces and wheat straw) were sub-jected to fiber analysis to determine their content of cellulose,hemicellulose, and lignin. Serial extractions have been conductedfor NDF (neutral detergent fiber), ADF (acid detergent fiber), andthen ADL (acid determined lignin), respectively, to determine thefiber content of cow feces and wheat straw. The NDF extractionwas conducted using a sample-containing bag in soapy water solu-tion. During NDF, soluble cell contents such as carbohydrates, lip-ids, pectin, starch, soluble proteins and non-protein nitrogen havebeen washed off while hemicellulose, proteins bound to the cellwalls, cellulose, lignin, and recalcitrant materials remained in thebag. During ADF, hemicellulose and bound proteins were washedoff using a 1.00 normal H2SO4 and detergent solution while cellu-lose, lignin, and recalcitrant materials were left. During ADL, cellu-lose has been washed off using 72% H2SO4 solution while onlylignin and recalcitrant materials were left. Hemicellulose has beenbe determined as the difference between NDF and ADF, cellulose asthe difference between ADF and ADL while lignin was consideredequivalent to ADL (Bauer et al., 2009).

0.00

0.10

0.20

0.30

0 14 28 42 56 70 84 98

Time (days)

Spec

ific

CH

4yi

eld

( Nl C

H4

g-1C

OD

)(A)

0.00

0.05

0.10

0.15

0.20

0 7 14

Time (days)

Spec

ific

CH

4yi

eld

( Nl C

H4

g-1C

OD

)

(B)

Xylan CelluloseXylane / Celulose Cow fecesWheat Straw

Fig. 1. Profile of specific methane yield during psychrophilic anaerobic digestion ofcellulose, xylan, cellulose/xylan mixture, cow feces and wheat straw. (A) During theentire fermentation period; (B) during the first 15 days of the fermentation period.(Values shown are average of duplicate bioreactors.)

666 Noori M. Cata Saady, D.I. Massé / Bioresource Technology 142 (2013) 663–671

2.9. Statistical methods

Data given in tables and shown in figures are the means andstandard deviations. Statistical analysis was carried out usingMinitab v. 14 statistical software (Minitab Inc., PA, USA). Data wereanalyzed by one-way ANOVA, followed by post hoc multiple com-parisons (Tukey’s test) with a confidence level of 95% (i.e., p < 0.05).

3. Results and discussion

3.1. Methane production

During batch anaerobic digestion, CH4 production curve patternreveals useful information such as the extent of substrate biode-gradability and the rate kinetic. The profiles of SMY in psychro-philic (20 �C) mixed cultures fed cellulose (C), xylan (X),cellulose/xylan (CX) mixture, cow feces (CF), and wheat straw(WS) are given in Fig. 1 (A). Examining CH4 production profilestwo segments can be recognized; an early fast phase when theCH4 production is increasing almost linearly with time followedby a second phase of decreasing CH4 production until a plateauhas been reached for each substrate. However, there were varia-tions in the CH4 profiles among the various substrates. The CH4

production profiles indicate that the degradation of all substratesfed started immediately with a relatively high rate (the straightportion of the curve), however, the duration of this phase for thecultures fed C, X, and CX mixture was longer (11 ± 1 day) than thatfor the complex (CF and WS) substrates fed cultures (7 ± 1 day).

Similarly, the rates of biomethanation for the pure substrates ortheir mixture were faster than those of complex substrates (noticethe slopes of the initial part of each curve (Fig. 1(A)). The enlargedsection (Fig. 1(B)) depicts three distinctive CH4 production patternsfor the substrates fermented. Careful examination of the curvesduring the first 15 days of the fermentation (Fig. 1(B)) reveals thatonly cultures fed with cellulose experienced a lag phase of 2 daysin CH4 production while in cultures fed other substrates (X, CXmixture, CF, and WS) the lag phase was shorter (1 day). Notice thatthe rate of CH4 generation was the fastest in the X and CX mixturefed cultures and the lowest in cellulose fed culture until betweenday 3 and 5 when cellulose-fed culture achieved the same rate asthose of X and CX mixture-fed cultures. The CH4 production ratesof CF and WS were almost the same and were lower than that ofpure substrate-fed cultures.

The maximum SMY (corrected to that produced in the controlcultures) expressed per TCOD (Nl CH4 kg�1 TCOD) and VS(Nl CH4 kg�1 VS) fed are given in Table 3. The ranking of the SMY(Nl CH4 kg�1 VS) was 338.5 ± 14.3 (C) > 310.5 ± 3.42 (X) = 305.5 ±29.6 (CX mixture) > 237.6 ± 17.7 (CF) = 235.3 ± 22.7 (WS). UsingTukey’s multiple comparisons test at 95% confidence level, theSMYs from CX was statistically the same as those from C and X,however; the SMY’s from C and X were statistically different fromeach other. Moreover, the SMY’s from C, X, and CX were statisti-cally different from those from CF and WS, while the SMY’s fromCF was statistically the same as that from WS.

Theoretically, stoichiometrical methane potential of cellulose(C6H10O5) and hemicellulose (C5H8O4) can be calculated usingEqs. (2) and (3) (Buswell and Mueller, 1952). According to themolecular formula determined using the results of the elementalanalysis, theoretical methane yields of 414.5, 394, and 346.3 lCH4 kg�1 VS of C, X, and CX mixture were calculated, respectively.The theoretical SMY calculated at STP condition for the celluloseused in this study is the same as that reported in literature(415 Nl CH4 kg�1 of VS (Wang et al., 1994)). However, the theoret-ical SMY calculated at STP condition for the xylan used in thisstudy (394 Nl CH4 kg�1 VS) was lower than that reported forhemicellulose (424 l CH4 kg�1 (Wang et al., 1994)). Although thetheoretical methane yield of cellulose amounts to 414.5 Nl kg�1 VSaccording to Buswell and Mueller (1952) equation Eq. (2) methanepotential of 379 l CH4 kg�1 VS of cellulose has been reported byBauer et al. (2009) from mesophilic (35 �C) reactor at 28 daysretention time and by Hansen et al. (2004) from thermophilic(55 �C) reactor and 50 days retention time. The SMY of cellulosecalculated in this study (376.4 ± 14.0 Nl CH4 kg�1 VS) is close tothose reported for mesophilic and thermophilic reactors. Forexample, operating at 55 �C shortened the HRT to around 15 dayscompared to 94 days for psychrophilic operation.

CnHaOb þ n� a4� b

2

� �H2O! ½n� a

8� b

4�CO2 þ n� a

8� b

4

� �CH4

ð2Þ

Yu ¼n2þ a

8� b4

� ��22:4

12nþ aþ 16bLCH4kg�1VS ð3Þ

Although the theoretical methane yield of WS is 373.4 NlCH4 kg�1 DM (Bauer et al., 2009) yields between 189 Nl CH4 kg�1

untreated WS and 275 Nl CH4 kg�1 VS of untreated WS (<1 mmparticle size) have been reported (Bauer et al., 2009). The differ-ence in the reported yields can be attributed to difference in theparticle size of the fibers and the experimental conditions usedin each study. Although the theoretical methane yield of WS(373.4 l CH4 kg�1 VS) (Bauer et al., 2009) is lower than that ofcow feces (469 l CH4 kg�1 VS) (Møller et al., 2004) because of theprotein and lipids content of the cow feces the experimentally

Table 3Specific methane yield for xylan cellulose, cow feces and wheat straw.

Substrate Theoretical methane yield (l CH4 kg�1 VS) Maximum methane yielda Time (days) required to achieve the indicatedpercentage of the maximum SMY

Nl CH4 kg�1 TCOD Nl CH4 kg�1 VS 50% 80% 95% 99%

Control NA 46.5 ± 12.6 93.2 ± 78.1 NA NA NA NAXylan (C5.148H8O4.418) 394b 242.2 ± 3.0 310.5 ± 3.4 7 17 35 56Cellulose (C5.928H10O4.965) 414.5b 257 ± 11.0 338.5 ± 14.3 8 17 36 491:2 Cellulose/xylan mixture 346.3b 236 ± 23.0 305.5 ± 29.6 6 17.5 36 49Cow feces 469.0c 185 ± 14.0 237.6 ± 17.7 10.5 31.5 56 70Wheat straw 373.4–432d 163 ± 16.0 235.3 ± 22.7 8 22 42 56

a The yields are corrected for the volume of methane produced by the control culture (no substrate fed) shown in the first raw.b Calculated from Buswell and Mueller (1952) Eq. (2).c Møller et al. (2004).d Bauer et al. (2009) and Møller et al. (2004), respectively.

0

10

20

30

40

50

60

70

80

90

0 14 28 42 56 70 84 98

CO

D r

ecov

ery

in C

H4

(%)

Time (day)

Xylan CelluloseXylane / Celulose Cow fecesWheat Straw

Fig. 2. Percent COD recovered as CH4 during psychrophilic batch anaerobicdigestion.

Noori M. Cata Saady, D.I. Massé / Bioresource Technology 142 (2013) 663–671 667

calculated ultimate SMY (237.6 ± 17.7 and 235.3 ± 22.7 Nl CH4 -kg�1 VS for CF and WS, respectively) were close to each other; sim-ilar finding has been reported previously by Møller et al. (2004)(Table 3).

Table 5Initial methane production rate.

Substrate Time (day)

Control 12Xylan 12Cellulose 12Cellulose/xylan 12Cow feces 7Wheat straw 7

Note: numbers with the similar superscripts are statistically the same at 95% confidence

Table 4Average of percent COD recovery as methane for pure and complex substrates.

Substrate Percent C

31 days

Xylan, cellulose, and cellulose/xylan mixture 65 ± 2Cow feces, wheat straw 41 ± 1

Based on theoretical CH4 yield of 415 and 425 Nl kg�1 VS ofcellulose and xylan, respectively, a theoretical yield of 267.1Nl kg�1 VS of WS is estimated which is close to the experimentallyobtained CH4 yield (273.3 Nl kg�1 VS). However, the estimated the-oretical CH4 yield contributed by the cellulose and hemicellulosecomponents (as determined by the fiber analysis) for cow feces is(177.5 Nl kg�1 VS) which is far below the experimentally obtainedCH4 yield (278.1 Nl kg�1 VS). This difference might be explained bythe contribution of protein, fats, and other degradable organic mat-ter contained in the cow feces.

Based on the experimentally obtained CH4 yield of 338.5 and310.5 Nl kg�1 VS for cellulose and xyaln, and using the celluloseand hemicellulose percentage in the cow feces, the contributionof cellulose and xylan is estimated as 154.3 Nl kg�1 VS or 55.5% ofthe yield obtained experimentally and 44.5% was contributed bythe protein, fats, and other biodegradable organics in the cow feces.Using the same calculation for the wheat straw, cellulose and xylanhave contributed around 85.7% of the CH4 yield obtainedexperimentally.

Within 31 days of incubation the SMYs (Nl CH4 kg�1 TCOD) were(Fig. 1(A)): X (226.7 ± 2.0) = C (223.4 ± 10.3) = CX mixture(218.9 ± 24.4) > CF (145.2 ± 12.0) > WS (141.8 ± 13.5). These yieldsrepresent a COD recovery (%) in CH4 of around 65 ± 2 for the puresubstrates (X, C, and CX mixture) and around 41 ± 1 for the com-plex substrates (CF and WS). Extending the incubation period to63 days increased the percent COD recovered in CH4, on average,by 5% and 8% for pure and complex substrates, respectively(Fig. 2). Interestingly, no increase in the percent of COD recovery

Methane initial production rate (Nl CH4 kg�1 TCOD day�1)

5 ± 420 ± 2a

13 ± 1b

16 ± 3a

16 ± 2a

18 ± 3a

level.

OD recovery in CH4 at

63 days 94 days

70 ± 3 70 ± 349 ± 4 50 ± 5

30

40

50

60

70

80

0 14 28 42 56 70 84 98

Time (day)

CH

4in

bio

gas

(%)

Xyaln CelluloseXylan:Cellulose mixture Cow fecesWheat straw

Fig. 3. Profile of methane content in biogas.

668 Noori M. Cata Saady, D.I. Massé / Bioresource Technology 142 (2013) 663–671

in CH4 was observed during the period from day 63 to day 94 (Ta-ble 4). The COD recovery from X, C, and CX mixture were statisti-cally the same. The COD recovery from WS was the lowest andsignificantly different from that of other substrates tested.

Cellulose biodegradability observed in this study is comparableto those reported in previous studies at higher temperatures; Laba-tut et al. (2011) reported 80% and 87% cellulose biodegradability inmesophilic and thermophilic operations, respectively. Moreover,biodegradability of 84% (Wang et al., 1994), 88% (Stinson andHam, 1995) and 89% (Tong et al., 1990), has been reported for cel-lulose under mesophilic condition. Little information is availableon xylan biodegradability during anaerobic digestion; Labatutet al. (2011) reported 53% and 66% xylan (from beechwood) biode-gradability under mesophilic and thermophilic conditions, respec-tively, which is comparable to the 64.8 ± 0.54% observed in thisstudy at retention time of 31 days (Table 4). Given the differencein the thermal range of the operation, the xylan biodegradabilityobserved in this study reflects the quality of the psychrophilic cul-ture which is well adapted to lingocellulosic substrates.

The biodegradability (55%) of cow manure reported under mes-ophilic conditions (Labatut et al., 2011) is comparable to that ob-served in this study (51.5 ± 2.7%) at HRT (63 days) which isconsistent with the kinetic limitation imposed by the lowtemperature.

The initial CH4 production rate (Table 5) is another parameterwhich can be used to compare the effect of the substrate qualityon the CH4 production. Cellulose-fed culture had a lower and sta-tistically different initial methane production rate (13 ± 1 NlCH4 kg�1 TCOD day�1) from those calculated for cultures fed X,CX mixture, CF and WS (average of 17.5 ± 1.9 Nl CH4 kg�1

TCOD day�1). It seems that the presence of hemicellulose in com-plex substrate such as wheat straw and cow feces supported thehigher initial CH4 rate compared to cellulose.

An extensive comparison of the SMY for CF and WS measured inthis study with literature reported values at different tempera-tures, organic loading rates, and hydraulic retention times (HRT)is presented in Table 6. Generally, the SMY of dairy manure rangesbetween 204–296 Nl CH4 kg�1 VS (Vedrenne et al., 2008). The SMY(145 ± 12 Nl CH4 kg�1 VS) from psychrophically (20 �C) digested CFat HRT of 31 days is higher than that reported by (Somayaji andKhanna, 1994) (135 l CH4 kg�1 VS) for cattle manure digestedmesophilically (30 �C) at HRT of 40 days (Table 6). Moreover, theultimate SMY achieved in this study (278 ± 14 Nl CH4 kg�1 VS CFat HRT of 118 days) is greater than 230, 148, and 260 l CH4 kg�1 VScow manure reported by Shyam (2002) at 30 �C and HRT 90 days,Møller et al. (2004) at 35 �C and HRT 100 days, and Güngör-Demi-rci and Demirer (2004) at 35 �C and HRT 90 days, respectively.

The SMY (142 ± 14 Nl CH4 kg�1 VS) from WS digested psycho-physically at HRT 31 days achieved in this study is greater than100 l CH4 kg�1 VS at HRT 25 days reported by Møller et al. (2004)at 35 �C. Moreover, the ultimate SMY achieved in this study(273 ± 18 Nl CH4 kg�1 VS) is similar to that reported by Mølleret al. (2004) (250 l CH4 kg�1 VS) at 35 �C and HRT 100 days, andsimilar to the 276 ± 20.5 l CH4 kg�1 VS reported by Bauer et al.(2009) for chopped WS (0.5–1.0 mm particle size) and digestedat 35 �C and HRT 28 days. Notice that the particle size of the WSused in this study was less than 0.25 mm. Wheat straw biodegrad-ability (54%) observed in this study compares reasonably to the66% biodegradability of wheat straw by mixed culture at 37 �C re-ported by de la Torre and Campillo (1984).

Interestingly, the specific methane yield (260 l CH4 kg�1 VS at35 �C) from cattle manure during mesophilic (Güngör-Demirciand Demirer, 2004) operation is almost twice the SMY (135 lCH4 kg�1 VS at 25 �C) during the psychrophilic operation (Somayajiand Khanna, 1994), however, latter required almost twice theretention time (40 days) compared to that of mesophilic (18 days).

The inoculum-to-substrate ratio (ISR) affects the rate and theyield of CH4 production. For example, Chynoweth et al. (1993)and Hashimoto (1989) concluded that ISR greater than two re-sulted in the maximum methane production rate from wheatstraw (expressed as l CH4 per VS added per unit time). Mølleret al. (2004) used ISR of 1.4 to digest cattle manure and wheatstraw while Labatut et al. (2011) used ISR of 1.0 to maximize thedegradation rate and achieve the methane potential of cellulose,xylan, and dairy manure. Chamchoi et al. (2011) reported that atISR of five, mixed culture produced 400 l CH4 kg�1 VS cellulose in17 days at 53 �C. The ISRs used in this study ranged between 3.1and 5.6 which were well above those used in the previous studiesto compensate for the low temperature stress. The initial rates ofCH4 production indicate that the ISR used was sufficient.

3.2. Quality of the biogas produced

The quality of the biogas is indicated by its CH4 content (Fig. 3).Generally, three segments can be recognized in Fig. 3. The first seg-ment is characterized by CH4 percent in biogas greater than 60%during the first two weeks (where almost 70% of the maximumCH4 specific yield was achieved in X, C and CX mixture fed bioreac-tors and 60% of the maximum CH4 specific yield was achieved in CFand WS fed bioreactors). The second segment where the CH4 per-cent decreased to between 45% and 60% (from day 27 to day 63);and the third segment starts from day 82 forward where the CH4

percent increased to between 64% and 69%. Interestingly, the CH4

percent from CF fed bioreactors were always above 50%. Torres-Castillo et al. (1995) observed 46% and 52% CH4 in biogas fromWS digestion at 25 and 35 �C, respectively.

3.3. Volatile fatty acids

The major VFAs produced by all cultures were acetate and pro-pionate particularly during the first two weeks of the incubation(Fig. 4). Acetate (mg L�1) peaked during the first two weeks in cul-tures fed X to (3500 ± 900), C (310 ± 90), CX mixture (640 ± 25), CF(310 ± 75), and WS (370 ± 20) while the levels detected during therest of the incubation were within the instrument‘s detection limit(<30 mg L�1). Propionate (mg L�1) peaked in cultures fed X(1500 ± 150), CX mixture (190 ± 7), CF (50 ± 7) during the firsttwo weeks and during the rest of the incubation was within theinstrument’s detection limit. No propionate was detected inC- and WS-fed cultures. Given that the control culture producedpropionate (240 ± 59) during the first two weeks then the propio-

Table 6Methane yield (l CH4 kg�1 VS) of cow manure and wheat straw at different temperatures.

Substrate OLR (kg VS m�3 d�1) unless indicated otherwise HRT (day) Temperature (�C) Refs.

20 25 30 35 40 50 55 60 65

Dairy cow manure 3.0 g TCOD L�1 31 145 ± 12 This studyDairy cow manure 3.0 g TCOD L�1 63 179 ± 14 This studyDairy cow manure 3.0 g TCOD L�1 94 237.6 ± 17.7 This studyWheat straw 3.4 g TCOD L�1 31 142 ± 14 This studyWheat straw 3.4 g TCOD L�1 63 162 ± 15 This studyWheat straw 3.4 g TCOD L�1 94 235.3 ± 22.7 This studyXylan 2.8 g TCOD L�1 94 310.5 ± 3.42 This studyCellulose 1.7 g TCOD L�1 94 338.5 ± 14.3 This studyCellulose/xylan mixture 2.2 g TCOD L�1 94 305.5 ± 29.6 This studyCellulose 2 g VS L�1 50 379 Hansen et al. (2004)Cellulose 2 g VS�1 28 356 Cho et al. (1995)a-Cellulose 9 g VS L�1 57 259 Lay et al. (2011)Avicel cellulose 2 g VS L�1 8 370 Chynoweth et al. (1993)Avicel cellulose 2 g VS L�1 17 400 Chamchoi et al. (2011)Cellulose 5.7 g VS L�1 14 200 Smiti et al. (1986)Cattle manure 3.3 18 260 270 250 280 270 240 Varel et al. (1980)Cattle manure 3.0 15 202 165 Ahring et al. (2001)Cattle manure 2.0 20 260 230 El-Mashad et al. (2004)Dairy manure NR 30 242.7 Labatut et al. (2011)Cattle manure NR 40 135 Somayaji and Khanna (1994)Cattle manure NR 50 164 Shyam (2002)

100 230 Shyam (2002)Dairy cattle manure NRa 100 148 ± 41 Møller et al. (2004)Cow manure NR 16 128 Preeti Rao and Seenayya (1994)Cow manure NR 90 260b Güngör-Demirci and Demirer (2004)Cow manure 3.0 15 215235c Nielsen et al. (2004)Wheat straw 1.5 155 Jewell et al. (1987)Wheat straw 3.5 212 Jewell et al. (1987)Wheat straw NRa 100 195 ± 5.9 Møller et al. (2004)Wheat straw (0.088 mm) 3.6 g VS L�1 56 249 ± 0�97 Sharma et al. (1988)Wheat straw (1 mm particles) 3.6 g VS L�1 56 241 ± 0�29 Sharma et al. (1988)Wheat straw (6 mm particles) 3.6 g VS L�1 56 227 ± 1�17 Sharma et al. (1988)Wheat straw (30 mm particles) 3.6 g VS L�1 56 162 ± 2.9 Sharma et al. (1988)Wheat straw (<1 mm particles) NR 60 161 ± 10 Møller et al. (2004)Wheat straw (30 mm particles) NR 60 145 ± 3 Møller et al. (2004)Wheat straw NR 25 100 Møller (2005)

170d

Wheat straw NR 50 190 Møller and Andersen (2006)100 250

Wheat straw (0.5–1.0 mm) NR 276 ± 20.5 Bauer et al. (2009)Cattle manure and 30% wheat straw 40 g L�1) 182e Müller and Trösch (1986)

233e

Cattle manure and 40% wheat straw NR 40 140 Somayaji and Khanna (1994)Cattle manure and 33% wheat straw NR 28 200 Demirbas (2006)Cattle manure and wheat straw NR 11 110 Oosterkamp (2011)Cattle manure and wheat straw NR 27 210 Oosterkamp (2011)Cattle manure and 100% rice straw NR 40 230 Somayaji and Khanna (1994)

NR = not reported.a The test was carried out as per ISO 11734.b With basal medium.c With thermal pre-treatment at 68 �C.d With thermal pre-treatment at 120 �C.e Pretreated with white rot fungi.

Noori

M.Cata

Saady,D.I.M

assé/Bioresource

Technology142

(2013)663–

671669

Fig. 4. Profiles of volatile fatty acids.

670 Noori M. Cata Saady, D.I. Massé / Bioresource Technology 142 (2013) 663–671

nate concentrations detected in CX mixture and CF-fed cultureswere probably from the inoculum itself rather than from the sub-strates fed. However, no explanation can be provided for why pro-pionate peaked in cultures fed X, to levels almost 6.5 times thatobserved in the control, but not in C- or CF-fed cultures. Obviously,the concentrations of VFAs detected after the second week of theincubation indicate that the fermentation was hydrolysis limited.Notice that the methanogenic component of the microbial consor-tia consumed the VFAs (Acetate 3500 ± 900 and propionate1500 ± 150 mg l�1) accumulated in X-fed samples during the sec-ond week efficiently.

4. Conclusion

Methane production from cellulose (C), xylan (X), cellulose andxylan mixture (CX), cow feces (CF), and wheat straw (WS) is feasi-ble at psychrophilic condition (20 �C) using a well-adapted inocu-lum; however, hydrolysis is a rate limiting step. 99% of thecumulative methane production from cultures fed C, X, CX, orWS was produced within 56 days compared to 70 days in CF-fedcultures. Cellulose-fed culture had a lower and statistically differ-ent initial methane production rate compared to cultures fed X,CX, CF, and WS. The presence of hemicellulose in WS and CF sup-ported higher initial CH4 rate compared to cellulose.

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