Bioresource Technology 169 (2014) 299–306

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Bioresource Technology

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Enhancement of anaerobic digestion of shredded grass by co-digestionwith sewage sludge and hyperthermophilic pretreatment

http://dx.doi.org/10.1016/j.biortech.2014.06.0530960-8524/� 2014 Elsevier Ltd. All rights reserved.

⇑ Corresponding author. Tel.: +81 29 879 6765; fax: +81 29 879 6797.E-mail address: wanfun55@pwri.go.jp (F. Wang).

1 Tel.: +81 29 879 6765; fax: +81 29 879 6797.

Feng Wang ⇑, Taira Hidaka 1, Jun Tsumori 1

Recycling Research Team, Materials and Resource Research Group, Public Works Research Institute, 1-6, Minamihara, Tsukuba, Ibaraki 305-8516, Japan

h i g h l i g h t s

� Anaerobic co-digestion of shredded grass with sewage sludge was conducted.� An average methane yield was 0.19 L/g VS-grass under thermophilic conditions.� Methane conversion from grass was unstable under mesophilic conditions.� A C/N ratio of around 10 obtained the highest synergistic methane production.� Hyperthermophilic treatment enhanced particulate dissolution and methane production.

a r t i c l e i n f o

Article history:Received 31 March 2014Received in revised form 5 June 2014Accepted 6 June 2014Available online 21 June 2014

Keywords:Anaerobic digestionHyperthermophilic pretreatmentShredded grassSewage sludgeC/N ratio

a b s t r a c t

Anaerobic co-digestion of shredded grass with sewage sludge was investigated under various tempera-ture conditions. The conversion of grass to methane was difficult to achieve under mesophilic conditions,while its methane yield was 0.19 NL/g VS-grass under thermophilic conditions. The mixture ratio of grassto sludge affected the methane yield, and the highest synergistic effect was obtained at a C/N ratio ofaround 10. In a continuous experiment, hyperthermophilic (80 �C) pretreatment promoted a methaneyield of 0.34 NL/g VS-mixture, higher than that under mesophilic and thermophilic conditions (0.20and 0.30 NL/g VS-mixture, respectively). A batch experiment with hyperthermophilic pretreatmentshowed that 3 days of treatment was sufficient for subsequent methane production, in which the highestdissolution of particulate COD, carbohydrate and protein was 25.6%, 33.6% and 25.0%, respectively.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Grass has long been used as principal livestock forage and isnow drawing attention for its high potential for biogas production(Prochnow et al., 2009). In Japan, where biomass materials arebeing used to generate energy, the amount of available bioenergyis estimated at between 5% and 15% of the national primary energysupply (Matsumura and Yokoyama, 2005). One potential source forenergy generation is grass waste collected from public spaces andone option for utilizing the biomass is anaerobic digestion (AD),which can recover biogas and reduce organic matter. The existingdigesters in wastewater treatment plants (WWTPs) can be usedfor these biomass substrates without having to invest in the con-struction of new treatment facilities. Grass can be co-digested with

the sewage sludge to increase biogas production (Hidaka et al.,2013).

Although AD is a promising technology for the utilization ofgrass, digestion efficiency needs to be improved due to the com-plex structure of grass; lignin and cellulose in grass are resistantto hydrolysis and microbial activity (Nizami et al., 2009). Fibrouscomponents are difficult to solubilize, resulting in the accumula-tion of floating undigested grass in the digester (Nizami et al.,2010). Various pretreatment methods including alkaline delignifi-cation, diluted acid hydrolysis, steam explosion, and aqueousammonia soaking have been used to optimize hydrolysis in meth-ane fermentation from grass (Eliana et al., 2014; Lee et al., 2010;Njoku et al., 2012; Rafique et al., 2010). Despite the improvementin biodegradability, chemical and physiochemical pretreatmentmethods cause a considerable increase in energy costs due to thesevere treatment conditions including high temperature, highpressure and addition of acidity or alkalinity.

Biological treatment is a mild process for degradation of organicmatter without high energy costs. Co-digestion of sewage sludge

300 F. Wang et al. / Bioresource Technology 169 (2014) 299–306

with other types of organic wastes is expected to improve thebiochemical conditions for different microorganisms in digesters,including a C/N ratio and nutrient balance (Sosnowski et al.,2003). Hidaka et al. (2013) obtained a methane yield of 0.2 NL/gVS-grass under mesophilic batch conditions using sewage sludgeand grass as co-substrates. However, comparative biodegradationof grass under mesophilic and thermophilic AD conditions has notbeen fully discussed. Furthermore, hyperthermophilic (70–80 �C)pretreatment has proven to be effective for increasing the dissolu-tion of particulate components and the methane yield of sewagesludge and kitchen garbage (Lee et al., 2008, 2009). Hyperthermo-philic (80 �C) pretreatment has also proven to enhance the hydro-lysis of polylactide and the following methane production underco-digestion with kitchen garbage (Wang et al., 2011, 2012a).Under hyperthermophilic conditions, the hydrolytic activity wasdominant and the methanogenic activity was decreased to mini-mum (Hartmann and Ahring, 2005). This temperature range islower than that of other types of heat pretreatment includingsteam explosion. If waste heat can be used for this treatment, anadditional energy source is not required. However, its improve-ment effect on the biodegradability of grass is uncertain.

In this study, co-digestion of grass and sludge was conductedunder mesophilic and thermophilic conditions to compare thebiodegradability of the grass, and the effect of the mixing ratioon the methane yield was evaluated based on the results of batchexperiments. Hyperthermophilic pretreatment was introduced andthe enhancement of methane yield was assessed in continuous andbatch experiments.

2. Methods

2.1. Characteristics of sewage sludge and grass

Characteristics of the sewage sludge and grass are summarizedin Table 1. The sewage sludge was taken from a municipal WWTPin Japan. Its original total solids (TS) content was around 2%. Priorto the experiments, the sludge was centrifuged at 3000 rpm for10 min and the TS content was adjusted to around 4%. The grassused in this study was Eleusine indica, which is distributedthroughout Japan. It was taken from the roadside at the PublicWorks Research Institute on April, 2013. After collection, the grasswas air dried, shredded into small pieces of about 10 mm in lengthusing a blender (ABS-V, Osaka Chemical Co., Ltd., Japan), and storedin a sealed bottle at room temperature prior to feeding into thereactors. The biodegradability for grass depends on several factorssuch as harvest time, age of the grass, and storage conditions.Herrmann et al. (2011) indicated that prolonged storage causedthe increase in methane yield and losses of dry matter. Duringthe present study, the grass collected at one time was used tominimize these effects.

Table 1Characteristics of sewage sludge and shredded grass.

Items Unit Sludge Shredded grass

pH – 5.64 ± 0.05 –TS % 4.0 ± 0.7 29.1 ± 1.13VS % 3.4 ± 0.6 26.8 ± 1.05T-COD g/L 63.4 ± 9.7 –T-COD g/g-VS – 1.36 ± 0.02C % (dry) 44.40 ± 0.01 42.3 ± 0.4H % (dry) 6.72 ± 0.38 6.10 ± 0.46N % (dry) 5.34 ± 0.32 1.10 ± 0.57Lipid % (dry) 6.07 ± 0.65 2.21 ± 0.40Carbohydrate % (dry) 39.2 ± 2.1 60.9 ± 8.7Protein % (dry) 30.1 ± 0.1 7.4 ± 0.9

2.2. Continuous fermentation experiment

Operational conditions of the continuous experiment are sum-marized in Table 2. Co-digestion was conducted using two lab-scale continuous stirred tank reactors (CSTRs), namely RM andRT, operated under mesophilic and thermophilic conditions,respectively. The substrate was a mixture of grass and sewagesludge with a volatile solids (VS) ratio of 0.5. According to a sur-vey of the amount of sewage sludge and green waste from publicspaces available in one municipal area in Japan by Public WorksResearch Institute, the TS ratio of sewage sludge and green wastefrom public spaces in the area was around 10:1. This study used ahigher ratio of grass to sludge to understand the methane produc-tion from grass preciously. To evaluate the biodegradation extentof grass under these two temperature conditions, two controlreactors, namely RCM (under mesophilic conditions) and RCT

(under thermophilic conditions), were fed sewage sludge as thesole substrate. The seed sludge for RM, RT, RCM, and RCT had beenoperated with a mixture of grass and sewage sludge from a differ-ent WWTP for 210 days under thermophilic conditions. To evalu-ate the effect of hyperthermophilic (80 �C) treatment on thebiodegradation of substrate, another reactor, namely RHM, wasoperated under mesophilic conditions. The inoculum of RHM wastaken from RM on days 133–167. The substrate was the samemixture as that fed to RM and RT, but was pretreated in a hyper-thermophilic reactor (RH) operated at the hydraulic retention time(HRT) of 5 days. RH, with a working volume of 0.8 L, was heated inan oil bath and stirred at 200 rpm. The inoculum was taken fromRT on days 154–167.

2.3. Batch co-digestion experiments

To evaluate the improvement of co-digestion on grass biodegra-dation, three series of batch experiments (C1, C2 and C3) were con-ducted (Table 3) under thermophilic conditions. The inoculumsludge was taken from RT on days 91–106. The amount of sewagesludge added in the three series was 0, 0.18 and 0.35 g VS, respec-tively. In each series, there were four sub-experiments with grassaddition amounts of 0, 0.09, 0.18 and 0.26 g VS. The total volumeof each vial was 130 mL and the working volume was set as100 mL by adding deionized water. Prior to the experiment, thevials were flushed with nitrogen gas for 3 min. The vials wereplaced in a shaking incubator and gas production was monitoredperiodically. The experiment was performed in triplicate for eachcondition. The generated biogas was injected by a syringe into aglass full of 3 M NaOH solution to completely adsorb CO2, andthe methane gas volume was measured by the volume of NaOHsolution replaced with the methane gas.

2.4. Biological methane potential (BMP) assay

The effect of hyperthermophilic treatment time on the methaneyield of the substrate was investigated by biological methanepotential assay conducted under mesophilic (experiment BM)and thermophilic (experiment BT) conditions (Table 4). In all cases,the batch experiment was performed in triplicate. The BM inocu-lum was taken from RHM on days 217–237, and that of BT wastaken from RT on days 217–237. The substrate used in the assaywas a raw mixture of sewage sludge and grass, and a mixture pre-treated under hyperthermophilic conditions. The pretreatmentduration varied from 1 to 5 days. For the pretreatment, the sub-strate mixture was added to glass vials and sealed, and the vialswere put in an oil bath at 80 �C. The amount of inoculum in exper-iment BM and experiment BT was 1.89 and 1.87 g VS, respectively.The amount of substrate in both series of experiments was 0.52 gVS. The inoculum and substrate were added to a vial with a total

Table 2Operation conditions of co-digestion experiment.

RM RCM RT RCT RHM

Period (d) 0–258 0–258 0–258 0–258 168–271Working volume 4.5/2.1* 1.8 4.5 10 2.1HRT (d) 42 42 42 42 42Temperature (�C) 35 35 55 55 55VS loading rate (g/L d) 1.2 ± 0.2 0.7 ± 0.2 1.2 ± 0.1 0.7 ± 0.2 1.1 ± 0.1Mixing ratio of grass to

sludge (%, on VS basis)50:100 – 50:100 – 50:100**

* The working volume of RM was changed to 2.1 L on day 77.** The substrate of RHM is a mixture pretreated under hyperthermophilic condition.

Table 3Operational conditions of co-digestion batch experiments.

Assay Inoculum(g VS)

Grass(g VS)

Sewage sludge(g VS)

I/Sratio*

C1-1 (used asblank)

1.14 0.00 0 –

C1-2 1.14 0.09 0 13.0C1-3 1.14 0.18 0 6.5C1-4 1.14 0.26 0 4.3C2-1 1.14 0.00 0.18 6.5C2-2 1.14 0.09 0.18 4.3C2-3 1.14 0.18 0.18 3.2C2-4 1.14 0.26 0.18 2.6C3-1 1.14 0.00 0.35 3.2C3-2 1.14 0.09 0.35 2.6C3-3 1.14 0.18 0.35 2.2C3-4 1.14 0.26 0.35 1.9

* I/S ratio, the ratio of inoculum to substrate.

Table 4BMP assays conditions.

Assay Substrate description Inoculum(g VS)

Substrateamount (g VS)

I/Sratio

BMblank Blank assay 1.89 – –BM0 Raw mixture 1.89 0.52 3.6BM1 Hyperthermophilic

treatment, 1 day1.89 0.52 3.6

BM2 Hyperthermophilictreatment, 2 day

1.89 0.52 3.6

BM3 Hyperthermophilictreatment, 3 day

1.89 0.52 3.6

BM4 Hyperthermophilictreatment, 4 day

1.89 0.52 3.6

BM5 Hyperthermophilictreatment, 5 day

1.89 0.52 3.6

BTblank Blank assay 1.87 0.52 –BT0 Raw mixture 1.87 0.52 3.6BT1 Hyperthermophilic

treatment, 1 day1.87 0.52 3.6

BT2 Hyperthermophilictreatment, 2 day

1.87 0.52 3.6

BT3 Hyperthermophilictreatment, 3 day

1.87 0.52 3.6

BT4 Hyperthermophilictreatment, 4 day

1.87 0.52 3.6

BT5 Hyperthermophilictreatment, 5 day

1.87 0.52 3.6

F. Wang et al. / Bioresource Technology 169 (2014) 299–306 301

volume of 130 mL, and the working volume was adjusted to100 mL by adding deionized water. Prior to the experiment, thevials were flushed with nitrogen gas for 3 min, and the gas mea-surement method was the same as that described in Section 2.3.

2.5. Analytical methods

The pH, total ammonia (TAN), TS and VS were analyzed accord-ing to the standard method (APHA, 1995). Total chemical oxygendemand (TCOD), soluble chemical oxygen demand (SCOD) andorganic acids were analyzed with reagents TNTplus-HR and TNT872 (Hach, USA), respectively, and the concentration was mea-sured using a DR3900 spectrophotometer (Hach). Soluble sampleswere filtered through glass fiber filters with pore size of 1 lm(Whatman-GE Healthcare). Carbohydrate analysis was performedby the phenol–sulfuric acid method (Dubois et al., 1956); proteinanalysis by the Lowry method (Lowry et al., 1951); and lipid anal-ysis by the Soxhlet extraction method (JSWA, 1997).

The methane yield of grass under co-digestion conditions wascalculated using the following equations, with the assumption thatthe methane yield from the sewage sludge and the grass could besummed:

Ygrass ¼Mgrass

VSgrass¼ Mtotal �Msludge

VSgrassð1Þ

where ygrass (NL/g VS) is the methane yield of grass, Mgrass (NL) isthe methane generated from grass, VSgrass (g) is the VS of addedgrass, Mtotal (NL) is the total methane measured, Msludge (NL) isthe methane generated from sludge, which was calculated by thefollowing equation:

Msludge ¼ VSsludge � ysludge ð2Þ

where VSsludge (g) is the VS of sludge added to the reactors under co-digestion conditions (g), and ysludge (NL/g VS) is the methane yield ofsludge, which was calculated using the data from RCM and RCT.

The degree of dissolution of COD, carbohydrate and protein wascalculated using the following equation:

Dissolution ratio ð%Þ ¼ pin � pout

pin� 100

¼ ðT in � SinÞ � ðTout � SoutÞT in � Sin

� 100 ð3Þ

where, Tin, Sin and Pin are the influent concentration of total, solubleand particulate components (g/L), and Tout, Sout and Pout are theeffluent concentration of total, soluble and particulate components(g/L).

3. Results and discussion

3.1. Performance comparison in continuous experiment: mesophilic vs.thermophilic

The performance of RM and RT is summarized in Fig. 1. RCM andRCT used as control reactors showed considerable stability indi-cated by the typical performance in terms of pH, VS concentrationof the digested sludge and methane production rate. The methaneyield of sludge as the sole substrate under mesophilic and thermo-philic conditions was 0.34 and 0.36 NL/g VS, respectively. Through-out the experiment, RT showed good stability, as indicated in Fig. 1.However, the methane production rate in RM was unstable com-pared with that in RT. Initially, methane production rate in RM

was 2.0 NL/(Lreactor�week) and then it decreased gradually to0.28 NL/(Lreactor�week) by day 112. This was followed by anincrease and then it stabilized at 1.6 ± 0.1 NL/(Lreactor�week) afterday 203. Methane production rate in RT was 2.4 ± 0.3 NL/(Lreactor�week), which was 50% higher than that during the stable periodin RM. The ammonia concentration was 1300 ± 212 mg N/L and1219 ± 133 mg N/L in RM and RT, respectively. Ammonia of2500 mg/L was considered as an inhibitor for both mesophilicand thermophilic anaerobic digestion (Hashimoto, 1986). In the

Fig. 3. Cumulative methane production from grass under thermophilic conditions.

Fig. 1. Comparison of performance under mesophilic and thermophilic conditions:(a) pH; (b) VS; (c) Methane production rate; (d) Ammonia; (e) Organic acids.

Fig. 2. Cumulative methane production from grass under mesophilic conditions: (a)entire duration; (b) day 161–202.

302 F. Wang et al. / Bioresource Technology 169 (2014) 299–306

present study, the ammonia concentration in the two reactors wasmuch lower than this value. The lower methane production rate inRM might not have been caused by the ammonia. The organic acidconcentration in RM was 1197 ± 106 mg HOAc/L, higher than thatof 925 ± 65 mg HOAc/L in RT, which also indicated the inferior per-formance of RM. The results of the continuous experiment indi-cated that under mesophilic conditions, the addition of grass hada detrimental effect on the methane production rate while thermo-philic conditions were favorable for the digestion of grass.

To evaluate the digestion of grass under different temperatureconditions, the methane yield of grass was calculated using Eqs.(1) and (2); and the result is shown in Figs. 2 and 3. In RM, at theinitial duration, the total methane production was almost the sameas the calculated methane production from sludge, and the meth-ane production from grass was near zero, as shown in Fig. 2(a).After 9 weeks, the total methane production was lower than thecalculated methane production from sludge, and the methane pro-duction from grass was negative. A negative slope means that the

addition of grass decreased the total methane production. In RM,the methane production from sewage sludge was inhibited.Although the total methane yield of grass during the operationwas negative, the cumulative methane production from grassduring days 161–202 increased from �17.0 to �12.7 NL, whichindicated that the methane production from grass during this per-iod was positive (Fig. 2(b)). The methane yield of grass was0.17 NL/g VS-grass in this period. After day 202, however, thecumulative methane production from grass decreased again, whichindicated that the methane production from grass under meso-philic conditions was unstable.

By contrast, a positive slope in RT means that the addition ofgrass enhanced the methane production, as shown in Fig. 3. The

F. Wang et al. / Bioresource Technology 169 (2014) 299–306 303

cumulative methane production was positive and the methaneyield of grass was 0.19 NL/g VS-grass. These results show that ther-mophilic conditions were favorable for the biodegradation of grass,and it was difficult to convert the grass to methane under meso-philic conditions. The seed sludge for RM had been cultivated underthermophilic conditions, and the adaptation period for mesophilicconditions might have been required. However, the methane yieldof the sewage sludge in RCM and RCT was around 0.34 and 0.36 NL/gVS, respectively, and these similar values mean the effect of tem-perature change for sludge degradation was not significant.Despite the potential for biodegradability, the biogas productionof grass may be low because of the high lignocellulose biofiber con-tent (Klimiuk et al., 2010). Higher temperature is favorable for thehydrolysis of complex lignocellulosic structures (Garba, 1996).

3.2. Effects of mixture ratio of grass and sludge on methane yield

Methane production with various mixing ratios of grass andsludge is summarized in Table 5. The methane yield of grass andsludge under mono-digestion conditions was 213 Nml/g VS-grassand 352 Nml/g VS-sludge, respectively. Under co-digestion condi-tions, the expected methane yield was calculated using the meth-ane yield of grass and sludge under mono-digestion conditions.The enhancement effect of co-digestion was evaluated based onthe difference between measured and expected methane yields(enhanced methane yield). With all mixture ratios, the measuredvalue was higher than the expected value.

The carbon–nitrogen (C/N) ratio of grass was 38.5, higher thanthat of sewage sludge at 8.3. After mixing, the C/N ratio of the mix-ture varied in the range of 9.7–15.0. Previous research indicatedthat when sludge is co-digested with a substrate having a highcontent of easily biodegradable carbohydrates, the C/N ratio canbe increased; consequently, the methane yield was increased(Feng et al., 2009; Yen and Brune, 2007). Wang et al. (2012b) inves-tigated the effect of C/N ratio on methane potential under co-diges-tion conditions, and found that the methane potential initiallyincreased and then declined, and the maximum methane potentialwas obtained at a C/N ratio of 27.1. In the present study, when theC/N ratio of the mixture was 9.7 or 10.9, the enhanced methaneyield of the mixture was the highest (around 20 Nml/g VS). A C/Nratio of around 10 was considered the optimum value for improve-

Table 5CH4 yield from co-digestion of various mixture ratios of grass and sludge.

Temperature(�C)

Substrateamount (g VS)

Mixture ratio of grass tosludge (–, on VS basis)

C/N(–)

Measuredyield (Nml

Mono-digestion55 Grass Sludge

0.088 0 (�) 38.5 2100.175 0 (�) 38.5 2140.263 0 (�) 38.5 2140 0.175 0 8.3 3560 0.350 0 8.3 349

Co-digestion55 Grass Sludge

0.088 0.350 0.25 9.7 3430.088 0.15 0.50 10.9 3280.175 0.350 0.50 10.9 3230.263 0.350 0.75 12.1 3090.175 0.175 1.0 13.1 2930.263 0.175 1.5 15.0 275

* An average value calculated by the data from exp. C1-2 to C1-4.** An average value calculated by the data from exp. C2-1 and C3-1.

*** Measured minus expected methane yield.

ment of the methane potential of the mixture. The difference inoptimum C/N ratio between the present study and previousresearch may be due to the various characteristics of the substrate.In the present study, the methane yield of grass was much lowerthan that of sludge. Although the carbohydrate content in grasswas around 1.5 times higher than that in sludge, grass is less bio-degradable compared with sludge due to its lignin–carbohydratecomplex. The higher measured methane yield of the mixturecompared to the expected yield confirmed the effectiveness ofco-digestion.

3.3. Promotion of hyperthermophilic treatment on methane yield

The purpose of introducing hyperthermophilic treatment is toenhance the dissolution of the particulate components. Afterhyperthermophilic treatment, VS and COD of the mixture showedno obvious change. During the operation, no methane gas gener-ation was observed in the hyperthermophilic reactor. This con-firmed that under these pretreatment conditions, solubilizationof particulate components was enhanced rather than methaneproduction. Although TCOD during the hyperthermophilic treat-ment process was constant, SCOD increased significantly(Fig. 4c). SCOD of the raw mixture prior to hyperthermophilictreatment was 9.1 ± 5.1 g/L and after treatment it increased to19.4 ± 4.4 g/L. The dissolution ratio of COD during the operationwas 14.3 ± 2.7%.

As shown in Fig. 4, the performance of RHM was kept stable,which is indicated by the pH, ammonia, organic acids and methaneyield. The methane yield of the mixture was 0.34 NL/g VS-mixture(Fig. 4d), higher than that in RM and RT at 0.20 (Fig. 2a) and 0.30NL/g VS-mixture (Fig. 3), respectively. Ferrer et al. (2010) investi-gated the effect of hyperthermophilic (80 �C) treatment on thebiodegradation of water hyacinth, which is an aquatic plant, andfound that there was no obvious positive effect, possibly becausethe concentration of soluble organic matter was lower than6 g SCOD/kg after 3 h of pretreatment. However, the presentresults indicate that after hyperthermophilic treatment, the biode-gradability of the organic substrate was improved and more meth-ane can be generated from the pretreated substrate. The treatmenttime in the present study was 5 days, much longer than in the pre-vious research, and the final COD dissolution ratio was higher than

methane/g VS)

Expected methaneyield (Nml/g VS)

Enhanced methaneyield*** (Nml/g VS)

Sub-experimentNo.

213* C1-2

– C1-3

– C1-4

352** C2-1

– C3-1

325 19 C3-2

306 22 C2-2

306 17 C3-3

293 16 C3-4

283 10 C2-3

269 6 C2-4

Fig. 4. Time course of RHM: (a) pH, ammonia and organic acids; (b) VS profile: VSraw

is the VS of the mixture prior to hyperthermophilic treatment; (c) COD profile:TCODraw and SCODraw are the TCOD and SCOD of the mixture prior to hypertherm-ophilic treatment; (d) Cumulative methane production from mixture pretreatedunder hyperthermophilic conditions.

304 F. Wang et al. / Bioresource Technology 169 (2014) 299–306

10%. This possibly caused an obvious enhancement of methaneproduction. Also, the results in the present study agree with previ-ous research that hyperthermophilic treatment (70–80 �C)improved the biodegradability of organic substrates (Bonmatíet al., 2001; Ferrer et al., 2008).

The dissolution of COD, carbohydrate and protein with differenthyperthermophilic treatment time for the BMP assay is summa-rized in Fig. 5. The TCOD concentration is included in this figure.The initial TCOD value of the raw mixture was 75.8 g/L and after5 days of treatment, there was a slight decrease to 75.3 g/L, which

was in accordance with the fact that no obvious gas productionwas detected during the hyperthermophilic treatment process.Retention time is a key parameter determining the solubilizationefficiency of solid components and acid generation of soluble com-ponents (Guerrero et al., 1999). The COD dissolution ratio was inthe range of 16.4–25.6%. Carbohydrate and protein dissolutionratios were in the range of 20.9–33.6% and 11.4–25.0%, respec-tively. The highest dissolution of COD, carbohydrate and proteinwas obtained with a retention time of 3 days. The dissolution ofcarbohydrate was higher than that of protein, which is similar tothe results obtained in previous research (Fang and Yu, 2002; Leeet al., 2009).

The mixture treated at different retention times was fermentedunder batch mesophilic and thermophilic conditions to evaluatethe effect of hyperthermophilic pretreatment on biodegradabilityof the substrate. Methane production increased quickly in the ini-tial period (before day 10) and then slowed down. As shown inFig. 6, after 50 days of fermentation, the final methane yield ofthe raw mixture and pretreated mixture under mesophilic condi-tions was 212.6 ± 4.7 Nml/g VS (raw), 225.3 ± 14.5 Nml/g VS(1 day), 243.0 ± 6.8 Nml/g VS (2 days), 309.7 ± 24.2 Nml/g VS(3 days), 287.3 ± 38.4 Nml/g VS (4 days) and 291.8 ± 28.3 Nml/gVS (5 days), while each mixture under thermophilic conditionsresulted in a higher methane yield of 279.5 ± 14.6 Nml/g VS(raw), 290.9 ± 32.3 Nml/g VS (1 day), 286.7 ± 30.4 Nml/g VS (2days), 335.0 ± 36.6 Nml/g VS (3 days), 339.9 ± 37.5 Nml/g VS(4 days) and 334.1 ± 30.4 Nml/g VS (5 days). Two important con-clusions were drawn from this experiment. First, after hypertherm-ophilic treatment, biodegradability of the grass and sludge wasimproved as indicated by the higher methane yield. The methaneyield increased with the retention time but when the retentiontime was more than 3 days, the methane yield showed no obviousincrease. Second, thermophilic conditions were more favorable formethane production compared with mesophilic conditions. Ther-mophilic AD has the proven advantage of high hydrolysis rateand methane yield over mesophilic AD (Coelho et al., 2011;Ramakrishnan and Surampalli, 2013). With the same hypertherm-ophilic treatment, the methane yield increased under thermophilicconditions compared with mesophilic conditions at 31% (raw), 29%(1 day), 18% (2 days), 8% (3 days), 18% (4 days) and 14% (5 days).Considering the particulate component dissolution ratio and meth-ane yield of the pretreated mixture, a retention time of 3 days wasa reasonable treatment time with the highest pretreatmentperformance.

In the batch experiment, no inoculum sludge was added, andthe hyperthermophilic treatment was mainly thermal. Lee et al.(2008) reported some acidogens adapted to the hyperthermophilicreactor continuously operated at the HRT of 4–5 days. As RH wascontinuously operated at the HRT of 5 days, the hyperthermophilictreatment in the continuous experiment was possibly both thermaland biological, and effective for improving the methane yield.

4. Conclusions

Grass was co-digested with sewage sludge under mesophilicand thermophilic conditions. Thermophilic conditions were morefavorable than mesophilic conditions for the biodegradation ofgrass, and the methane yield of grass was 0.19 NL/g VS-grass inthe continuous experiment. Under mesophilic conditions, the addi-tion of grass negatively affected the methane production. The high-est synergistic methane yield was obtained at a C/N ratio of around10 under thermophilic conditions. Hyperthermophilic pretreat-ment enhanced the dissolution of particulate components andmethane production in the subsequent fermentation.

Fig. 5. COD, carbohydrate, and protein dissolution ratios and organic acid concentration with various hyperthermophilic treatment times for the BMP assay.

Fig. 6. Methane yield of mixture treated under hyperthermophilic conditions: (a)fermented under mesophilic conditions; (b) fermented under thermophilicconditions.

F. Wang et al. / Bioresource Technology 169 (2014) 299–306 305

Acknowledgements

We express our appreciation to the staff of the local govern-ment and related offices, and to those at the sewage treatmentplants. This work was partially supported by JSPS KAKENHI GrantNumber 25289168.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.biortech.2014.06.053.

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