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

Bioresource Technology 169 (2014) 299–306

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Enhancement of anaerobic digestion of shredded grass by co-digestion with sewage sludge and hyperthermophilic pretreatment 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 2014 Received in revised form 5 June 2014 Accepted 6 June 2014 Available online 21 June 2014 Keywords: Anaerobic digestion Hyperthermophilic pretreatment Shredded grass Sewage sludge C/N ratio

a b s t r a c t Anaerobic co-digestion of shredded grass with sewage sludge was investigated under various temperature 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 grass to sludge affected the methane yield, and the highest synergistic effect was obtained at a C/N ratio of around 10. In a continuous experiment, hyperthermophilic (80 °C) pretreatment promoted a methane yield of 0.34 NL/g VS-mixture, higher than that under mesophilic and thermophilic conditions (0.20 and 0.30 NL/g VS-mixture, respectively). A batch experiment with hyperthermophilic pretreatment showed that 3 days of treatment was sufficient for subsequent methane production, in which the highest dissolution of particulate COD, carbohydrate and protein was 25.6%, 33.6% and 25.0%, respectively. Ó 2014 Elsevier Ltd. All rights reserved.

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

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

E-mail address: [email protected] (F. Wang). Tel.: +81 29 879 6765; fax: +81 29 879 6797.

http://dx.doi.org/10.1016/j.biortech.2014.06.053 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.

the sewage sludge to increase biogas production (Hidaka et al., 2013). Although AD is a promising technology for the utilization of grass, digestion efficiency needs to be improved due to the complex structure of grass; lignin and cellulose in grass are resistant to hydrolysis and microbial activity (Nizami et al., 2009). Fibrous components are difficult to solubilize, resulting in the accumulation of floating undigested grass in the digester (Nizami et al., 2010). Various pretreatment methods including alkaline delignification, diluted acid hydrolysis, steam explosion, and aqueous ammonia soaking have been used to optimize hydrolysis in methane fermentation from grass (Eliana et al., 2014; Lee et al., 2010; Njoku et al., 2012; Rafique et al., 2010). Despite the improvement in biodegradability, chemical and physiochemical pretreatment methods cause a considerable increase in energy costs due to the severe treatment conditions including high temperature, high pressure and addition of acidity or alkalinity. Biological treatment is a mild process for degradation of organic matter without high energy costs. Co-digestion of sewage sludge

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with other types of organic wastes is expected to improve the biochemical 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/g VS-grass under mesophilic batch conditions using sewage sludge and grass as co-substrates. However, comparative biodegradation of grass under mesophilic and thermophilic AD conditions has not been fully discussed. Furthermore, hyperthermophilic (70–80 °C) pretreatment has proven to be effective for increasing the dissolution of particulate components and the methane yield of sewage sludge and kitchen garbage (Lee et al., 2008, 2009). Hyperthermophilic (80 °C) pretreatment has also proven to enhance the hydrolysis of polylactide and the following methane production under co-digestion with kitchen garbage (Wang et al., 2011, 2012a). Under hyperthermophilic conditions, the hydrolytic activity was dominant and the methanogenic activity was decreased to minimum (Hartmann and Ahring, 2005). This temperature range is lower than that of other types of heat pretreatment including steam explosion. If waste heat can be used for this treatment, an additional energy source is not required. However, its improvement effect on the biodegradability of grass is uncertain. In this study, co-digestion of grass and sludge was conducted under mesophilic and thermophilic conditions to compare the biodegradability of the grass, and the effect of the mixing ratio on the methane yield was evaluated based on the results of batch experiments. Hyperthermophilic pretreatment was introduced and the enhancement of methane yield was assessed in continuous and batch experiments.

2.2. Continuous fermentation experiment Operational conditions of the continuous experiment are summarized in Table 2. Co-digestion was conducted using two labscale continuous stirred tank reactors (CSTRs), namely RM and RT, operated under mesophilic and thermophilic conditions, respectively. The substrate was a mixture of grass and sewage sludge with a volatile solids (VS) ratio of 0.5. According to a survey of the amount of sewage sludge and green waste from public spaces available in one municipal area in Japan by Public Works Research Institute, the TS ratio of sewage sludge and green waste from public spaces in the area was around 10:1. This study used a higher ratio of grass to sludge to understand the methane production from grass preciously. To evaluate the biodegradation extent of grass under these two temperature conditions, two control reactors, namely RCM (under mesophilic conditions) and RCT (under thermophilic conditions), were fed sewage sludge as the sole substrate. The seed sludge for RM, RT, RCM, and RCT had been operated with a mixture of grass and sewage sludge from a different WWTP for 210 days under thermophilic conditions. To evaluate the effect of hyperthermophilic (80 °C) treatment on the biodegradation of substrate, another reactor, namely RHM, was operated under mesophilic conditions. The inoculum of RHM was taken from RM on days 133–167. The substrate was the same mixture as that fed to RM and RT, but was pretreated in a hyperthermophilic reactor (RH) operated at the hydraulic retention time (HRT) of 5 days. RH, with a working volume of 0.8 L, was heated in an oil bath and stirred at 200 rpm. The inoculum was taken from RT on days 154–167.

2. Methods

2.3. Batch co-digestion experiments

2.1. Characteristics of sewage sludge and grass

To evaluate the improvement of co-digestion on grass biodegradation, three series of batch experiments (C1, C2 and C3) were conducted (Table 3) under thermophilic conditions. The inoculum sludge was taken from RT on days 91–106. The amount of sewage sludge added in the three series was 0, 0.18 and 0.35 g VS, respectively. In each series, there were four sub-experiments with grass addition amounts of 0, 0.09, 0.18 and 0.26 g VS. The total volume of each vial was 130 mL and the working volume was set as 100 mL by adding deionized water. Prior to the experiment, the vials were flushed with nitrogen gas for 3 min. The vials were placed in a shaking incubator and gas production was monitored periodically. The experiment was performed in triplicate for each condition. The generated biogas was injected by a syringe into a glass full of 3 M NaOH solution to completely adsorb CO2, and the methane gas volume was measured by the volume of NaOH solution replaced with the methane gas.

Characteristics of the sewage sludge and grass are summarized in Table 1. The sewage sludge was taken from a municipal WWTP in Japan. Its original total solids (TS) content was around 2%. Prior to the experiments, the sludge was centrifuged at 3000 rpm for 10 min and the TS content was adjusted to around 4%. The grass used in this study was Eleusine indica, which is distributed throughout Japan. It was taken from the roadside at the Public Works Research Institute on April, 2013. After collection, the grass was air dried, shredded into small pieces of about 10 mm in length using a blender (ABS-V, Osaka Chemical Co., Ltd., Japan), and stored in a sealed bottle at room temperature prior to feeding into the reactors. The biodegradability for grass depends on several factors such as harvest time, age of the grass, and storage conditions. Herrmann et al. (2011) indicated that prolonged storage caused the increase in methane yield and losses of dry matter. During the present study, the grass collected at one time was used to minimize these effects.

Table 1 Characteristics of sewage sludge and shredded grass. Items

Unit

Sludge

Shredded grass

pH TS VS T-COD T-COD C H N Lipid Carbohydrate Protein

– % % g/L g/g-VS % (dry) % (dry) % (dry) % (dry) % (dry) % (dry)

5.64 ± 0.05 4.0 ± 0.7 3.4 ± 0.6 63.4 ± 9.7 – 44.40 ± 0.01 6.72 ± 0.38 5.34 ± 0.32 6.07 ± 0.65 39.2 ± 2.1 30.1 ± 0.1

– 29.1 ± 1.13 26.8 ± 1.05 – 1.36 ± 0.02 42.3 ± 0.4 6.10 ± 0.46 1.10 ± 0.57 2.21 ± 0.40 60.9 ± 8.7 7.4 ± 0.9

2.4. Biological methane potential (BMP) assay The effect of hyperthermophilic treatment time on the methane yield of the substrate was investigated by biological methane potential 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 inoculum was taken from RHM on days 217–237, and that of BT was taken from RT on days 217–237. The substrate used in the assay was a raw mixture of sewage sludge and grass, and a mixture pretreated under hyperthermophilic conditions. The pretreatment duration varied from 1 to 5 days. For the pretreatment, the substrate mixture was added to glass vials and sealed, and the vials were put in an oil bath at 80 °C. The amount of inoculum in experiment 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 g VS. The inoculum and substrate were added to a vial with a total

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2.5. Analytical methods

Table 2 Operation conditions of co-digestion experiment.

Period (d) Working volume HRT (d) Temperature (°C) VS loading rate (g/L d) Mixing ratio of grass to sludge (%, on VS basis) * **

RM

RCM

RT

RCT

RHM

0–258 4.5/2.1* 42 35 1.2 ± 0.2 50:100

0–258 1.8 42 35 0.7 ± 0.2 –

0–258 4.5 42 55 1.2 ± 0.1 50:100

0–258 10 42 55 0.7 ± 0.2 –

168–271 2.1 42 55 1.1 ± 0.1 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 3 Operational conditions of co-digestion batch experiments. Assay C1-1 (used as blank) C1-2 C1-3 C1-4 C2-1 C2-2 C2-3 C2-4 C3-1 C3-2 C3-3 C3-4 *

Inoculum (g VS)

Grass (g VS)

Sewage sludge (g VS)

I/S ratio*

1.14

0.00

0

–

1.14 1.14 1.14 1.14 1.14 1.14 1.14 1.14 1.14 1.14 1.14

0.09 0.18 0.26 0.00 0.09 0.18 0.26 0.00 0.09 0.18 0.26

0 0 0 0.18 0.18 0.18 0.18 0.35 0.35 0.35 0.35

13.0 6.5 4.3 6.5 4.3 3.2 2.6 3.2 2.6 2.2 1.9

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

The pH, total ammonia (TAN), TS and VS were analyzed according to the standard method (APHA, 1995). Total chemical oxygen demand (TCOD), soluble chemical oxygen demand (SCOD) and organic acids were analyzed with reagents TNTplus-HR and TNT 872 (Hach, USA), respectively, and the concentration was measured using a DR3900 spectrophotometer (Hach). Soluble samples were filtered through glass fiber filters with pore size of 1 lm (Whatman-GE Healthcare). Carbohydrate analysis was performed by the phenol–sulfuric acid method (Dubois et al., 1956); protein analysis by the Lowry method (Lowry et al., 1951); and lipid analysis by the Soxhlet extraction method (JSWA, 1997). The methane yield of grass under co-digestion conditions was calculated using the following equations, with the assumption that the methane yield from the sewage sludge and the grass could be summed:

Y grass ¼

M total  M sludge Mgrass ¼ VSgrass VSgrass

ð1Þ

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

Msludge ¼ VSsludge  ysludge

ð2Þ

where VSsludge (g) is the VS of sludge added to the reactors under codigestion conditions (g), and ysludge (NL/g VS) is the methane yield of sludge, which was calculated using the data from RCM and RCT. The degree of dissolution of COD, carbohydrate and protein was calculated using the following equation:

pin  pout  100 pin ðT in  Sin Þ  ðT out  Sout Þ  100 ¼ T in  Sin

Dissolution ratio ð%Þ ¼ Table 4 BMP assays conditions. Assay

Substrate description

Inoculum (g VS)

Substrate amount (g VS)

I/S ratio

BMblank BM0 BM1

Blank assay Raw mixture Hyperthermophilic treatment, 1 day Hyperthermophilic treatment, 2 day Hyperthermophilic treatment, 3 day Hyperthermophilic treatment, 4 day Hyperthermophilic treatment, 5 day Blank assay Raw mixture Hyperthermophilic treatment, 1 day Hyperthermophilic treatment, 2 day Hyperthermophilic treatment, 3 day Hyperthermophilic treatment, 4 day Hyperthermophilic treatment, 5 day

1.89 1.89 1.89

– 0.52 0.52

– 3.6 3.6

1.89

0.52

3.6

1.89

0.52

3.6

1.89

0.52

3.6

1.89

0.52

3.6

1.87 1.87 1.87

0.52 0.52 0.52

– 3.6 3.6

1.87

0.52

3.6

1.87

0.52

3.6

1.87

0.52

3.6

1.87

0.52

3.6

BM2 BM3 BM4 BM5 BTblank BT0 BT1 BT2 BT3 BT4 BT5

volume of 130 mL, and the working volume was adjusted to 100 mL by adding deionized water. Prior to the experiment, the vials were flushed with nitrogen gas for 3 min, and the gas measurement method was the same as that described in Section 2.3.

ð3Þ

where, Tin, Sin and Pin are the influent concentration of total, soluble and particulate components (g/L), and Tout, Sout and Pout are the effluent 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 and RCT used as control reactors showed considerable stability indicated by the typical performance in terms of pH, VS concentration of the digested sludge and methane production rate. The methane yield of sludge as the sole substrate under mesophilic and thermophilic conditions was 0.34 and 0.36 NL/g VS, respectively. Throughout the experiment, RT showed good stability, as indicated in Fig. 1. However, the methane production rate in RM was unstable compared with that in RT. Initially, methane production rate in RM was 2.0 NL/(Lreactorweek) and then it decreased gradually to 0.28 NL/(Lreactorweek) by day 112. This was followed by an increase and then it stabilized at 1.6 ± 0.1 NL/(Lreactorweek) after day 203. Methane production rate in RT was 2.4 ± 0.3 NL/(Lreactor week), which was 50% higher than that during the stable period in RM. The ammonia concentration was 1300 ± 212 mg N/L and 1219 ± 133 mg N/L in RM and RT, respectively. Ammonia of 2500 mg/L was considered as an inhibitor for both mesophilic and thermophilic anaerobic digestion (Hashimoto, 1986). In the

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

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

present study, the ammonia concentration in the two reactors was much lower than this value. The lower methane production rate in RM might not have been caused by the ammonia. The organic acid concentration in RM was 1197 ± 106 mg HOAc/L, higher than that of 925 ± 65 mg HOAc/L in RT, which also indicated the inferior performance of RM. The results of the continuous experiment indicated that under mesophilic conditions, the addition of grass had a detrimental effect on the methane production rate while thermophilic conditions were favorable for the digestion of grass. To evaluate the digestion of grass under different temperature conditions, 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 the initial duration, the total methane production was almost the same as the calculated methane production from sludge, and the methane production from grass was near zero, as shown in Fig. 2(a). After 9 weeks, the total methane production was lower than the calculated methane production from sludge, and the methane production from grass was negative. A negative slope means that the

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

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 operation was negative, the cumulative methane production from grass during days 161–202 increased from 17.0 to 12.7 NL, which indicated that the methane production from grass during this period was positive (Fig. 2(b)). The methane yield of grass was 0.17 NL/g VS-grass in this period. After day 202, however, the cumulative methane production from grass decreased again, which indicated that the methane production from grass under mesophilic conditions was unstable. By contrast, a positive slope in RT means that the addition of grass enhanced the methane production, as shown in Fig. 3. The

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cumulative methane production was positive and the methane yield of grass was 0.19 NL/g VS-grass. These results show that thermophilic conditions were favorable for the biodegradation of grass, and it was difficult to convert the grass to methane under mesophilic conditions. The seed sludge for RM had been cultivated under thermophilic conditions, and the adaptation period for mesophilic conditions might have been required. However, the methane yield of the sewage sludge in RCM and RCT was around 0.34 and 0.36 NL/g VS, respectively, and these similar values mean the effect of temperature change for sludge degradation was not significant. Despite the potential for biodegradability, the biogas production of grass may be low because of the high lignocellulose biofiber content (Klimiuk et al., 2010). Higher temperature is favorable for the hydrolysis 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 and sludge is summarized in Table 5. The methane yield of grass and sludge under mono-digestion conditions was 213 Nml/g VS-grass and 352 Nml/g VS-sludge, respectively. Under co-digestion conditions, the expected methane yield was calculated using the methane yield of grass and sludge under mono-digestion conditions. The enhancement effect of co-digestion was evaluated based on the difference between measured and expected methane yields (enhanced methane yield). With all mixture ratios, the measured value was higher than the expected value. The carbon–nitrogen (C/N) ratio of grass was 38.5, higher than that of sewage sludge at 8.3. After mixing, the C/N ratio of the mixture varied in the range of 9.7–15.0. Previous research indicated that when sludge is co-digested with a substrate having a high content of easily biodegradable carbohydrates, the C/N ratio can be increased; consequently, the methane yield was increased (Feng et al., 2009; Yen and Brune, 2007). Wang et al. (2012b) investigated the effect of C/N ratio on methane potential under co-digestion conditions, and found that the methane potential initially increased and then declined, and the maximum methane potential was obtained at a C/N ratio of 27.1. In the present study, when the C/N ratio of the mixture was 9.7 or 10.9, the enhanced methane yield of the mixture was the highest (around 20 Nml/g VS). A C/N ratio of around 10 was considered the optimum value for improve-

ment of the methane potential of the mixture. The difference in optimum C/N ratio between the present study and previous research may be due to the various characteristics of the substrate. In the present study, the methane yield of grass was much lower than that of sludge. Although the carbohydrate content in grass was around 1.5 times higher than that in sludge, grass is less biodegradable compared with sludge due to its lignin–carbohydrate complex. The higher measured methane yield of the mixture compared to the expected yield confirmed the effectiveness of co-digestion.

3.3. Promotion of hyperthermophilic treatment on methane yield The purpose of introducing hyperthermophilic treatment is to enhance the dissolution of the particulate components. After hyperthermophilic treatment, VS and COD of the mixture showed no obvious change. During the operation, no methane gas generation was observed in the hyperthermophilic reactor. This confirmed that under these pretreatment conditions, solubilization of particulate components was enhanced rather than methane production. Although TCOD during the hyperthermophilic treatment process was constant, SCOD increased significantly (Fig. 4c). SCOD of the raw mixture prior to hyperthermophilic treatment was 9.1 ± 5.1 g/L and after treatment it increased to 19.4 ± 4.4 g/L. The dissolution ratio of COD during the operation was 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 methane yield. 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 0.30 NL/g VS-mixture (Fig. 3), respectively. Ferrer et al. (2010) investigated the effect of hyperthermophilic (80 °C) treatment on the biodegradation of water hyacinth, which is an aquatic plant, and found that there was no obvious positive effect, possibly because the concentration of soluble organic matter was lower than 6 g SCOD/kg after 3 h of pretreatment. However, the present results indicate that after hyperthermophilic treatment, the biodegradability of the organic substrate was improved and more methane can be generated from the pretreated substrate. The treatment time in the present study was 5 days, much longer than in the previous research, and the final COD dissolution ratio was higher than

Table 5 CH4 yield from co-digestion of various mixture ratios of grass and sludge. Temperature (°C)

Substrate amount (g VS)

Mono-digestion 55 Grass 0.088 0.175 0.263 0 0 Co-digestion 55

* ** ***

Grass 0.088 0.088 0.175 0.263 0.175 0.263

Mixture ratio of grass to sludge (–, on VS basis)

C/N (–)

Measured methane yield (Nml/g VS)

Expected methane yield (Nml/g VS)

Sludge 0 0 0 0.175 0.350

() () () 0 0

38.5 38.5 38.5 8.3 8.3

210 214 214 356 349

213* – – 352** –

Sludge 0.350 0.15 0.350 0.350 0.175 0.175

0.25 0.50 0.50 0.75 1.0 1.5

9.7 10.9 10.9 12.1 13.1 15.0

343 328 323 309 293 275

325 306 306 293 283 269

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.

Enhanced methane yield*** (Nml/g VS)

Subexperiment No.

C1-2 C1-3 C1-4 C2-1 C3-1

19 22 17 16 10 6

C3-2 C2-2 C3-3 C3-4 C2-3 C2-4

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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 hyperthermophilic treatment; (d) Cumulative methane production from mixture pretreated under hyperthermophilic conditions.

was in accordance with the fact that no obvious gas production was detected during the hyperthermophilic treatment process. Retention time is a key parameter determining the solubilization efficiency of solid components and acid generation of soluble components (Guerrero et al., 1999). The COD dissolution ratio was in the range of 16.4–25.6%. Carbohydrate and protein dissolution ratios were in the range of 20.9–33.6% and 11.4–25.0%, respectively. The highest dissolution of COD, carbohydrate and protein was obtained with a retention time of 3 days. The dissolution of carbohydrate was higher than that of protein, which is similar to the results obtained in previous research (Fang and Yu, 2002; Lee et al., 2009). The mixture treated at different retention times was fermented under batch mesophilic and thermophilic conditions to evaluate the effect of hyperthermophilic pretreatment on biodegradability of the substrate. Methane production increased quickly in the initial period (before day 10) and then slowed down. As shown in Fig. 6, after 50 days of fermentation, the final methane yield of the raw mixture and pretreated mixture under mesophilic conditions 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/g VS (5 days), while each mixture under thermophilic conditions resulted 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 (2 days), 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 conclusions were drawn from this experiment. First, after hyperthermophilic treatment, biodegradability of the grass and sludge was improved as indicated by the higher methane yield. The methane yield increased with the retention time but when the retention time was more than 3 days, the methane yield showed no obvious increase. Second, thermophilic conditions were more favorable for methane production compared with mesophilic conditions. Thermophilic AD has the proven advantage of high hydrolysis rate and methane yield over mesophilic AD (Coelho et al., 2011; Ramakrishnan and Surampalli, 2013). With the same hyperthermophilic treatment, the methane yield increased under thermophilic conditions 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 methane yield of the pretreated mixture, a retention time of 3 days was a reasonable treatment time with the highest pretreatment performance. In the batch experiment, no inoculum sludge was added, and the hyperthermophilic treatment was mainly thermal. Lee et al. (2008) reported some acidogens adapted to the hyperthermophilic reactor continuously operated at the HRT of 4–5 days. As RH was continuously operated at the HRT of 5 days, the hyperthermophilic treatment in the continuous experiment was possibly both thermal and biological, and effective for improving the methane yield. 4. Conclusions

10%. This possibly caused an obvious enhancement of methane production. Also, the results in the present study agree with previous research that hyperthermophilic treatment (70–80 °C) improved the biodegradability of organic substrates (Bonmatí et al., 2001; Ferrer et al., 2008). The dissolution of COD, carbohydrate and protein with different hyperthermophilic treatment time for the BMP assay is summarized 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 after 5 days of treatment, there was a slight decrease to 75.3 g/L, which

Grass was co-digested with sewage sludge under mesophilic and thermophilic conditions. Thermophilic conditions were more favorable than mesophilic conditions for the biodegradation of grass, and the methane yield of grass was 0.19 NL/g VS-grass in the continuous experiment. Under mesophilic conditions, the addition of grass negatively affected the methane production. The highest synergistic methane yield was obtained at a C/N ratio of around 10 under thermophilic conditions. Hyperthermophilic pretreatment enhanced the dissolution of particulate components and methane production in the subsequent fermentation.

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

305

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 thermophilic conditions.

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