A New Process for Efficiently Producing Methane from Waste Activated

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Environ. Sci. Technol. 2011, 45, 803–808

A New Process for Efficiently Producing Methane from Waste Activated Sludge: Alkaline Pretreatment of Sludge Followed by Treatment of Fermentation Liquid in an EGSB Reactor DONG ZHANG, YINGUANG CHEN,* YUXIAO ZHAO, AND ZHENGXIANG YE State Key Laboratory of Pollution Control and Resources Reuse, School of Environmental Science and Engineering, Tongji University, 1239 Siping Road, Shanghai 200092, China

Received August 8, 2010. Revised manuscript received November 17, 2010. Accepted November 23, 2010.

In the literature the production of methane from waste activated sludge (WAS) was usually conducted in a continuous stirred tank reactor (CSTR) after sludge was pretreated. It was reported in our previous publication that compared with other pretreatment methods the methane production in CSTR could be significantly enhanced when sludge was pretreated by NaOH at pH 10 for 8 days. In order to further improve methane production, this study reported a new process for efficiently producing methane from sludge, that is, sludge was fermented at pH 10 for 8 days, which was adjusted by Ca(OH)2, and then the fermentation liquid was treated in an expanded granular sludge bed (EGSB) for methane generation. First, for comparing the methane production observed in this study with that reported in the literature, the conventional operational model was applied to produce methane from the pH 10 pretreated sludge, that is, directly using the pH 10 pretreated sludge to produce methane in a CSTR. It was observed that the maximal methane production was only 0.61 m3CH4/m3-reactor/ day. Then, the use of fermentation liquid of pH 10 pretreated sludge to produce methane in the reactors of up-flow anaerobic sludge bed (UASB), anaerobic sequencing batch reactor (ASBR) and EGSB was compared. The maximal methane production in UASB, ASBR, and EGSB reached 1.41, 3.01, and 12.43 m3CH4/m3-reactor/day, respectively. Finally, the mechanisms for EGSB exhibiting remarkably higher methane production were investigated by enzyme, adenosine-triphosphate (ATP), scanning electron microscope (SEM) and fluorescence in situ hybridization (FISH) analyses. It was found that the granular sludge in EGSB had the highest conversion efficiency of acetic acid to methane, and the greatest activity of hydrolysis and acidification enzymes and general physiology with much more Methanosarcinaceae.

Introduction

Materials and Methods

Biological treatment of wastes, such as wastewater and waste activated sludge, under anaerobic conditions is a highly * Corresponding author phone: 86-21-65981263; fax: 86-2165986313; e-mail: [email protected]. 10.1021/es102696d

sustainable waste treatment process because this technique can reduce the environmental pollution of wastes with simultaneous energy (biogas, a mixture of methane and carbon dioxide) recovery (1). The anaerobic treatment process of wastes, such as waste activated sludge usually includes: hydrolysis (the complex primary polymers of carbohydrates and proteins are converted to soluble organic compounds and further to soluble monomers by extracellular enzymes), acidogenesis (the hydrolysis products are fermented to various intermediate products such as volatile fatty acids (VFA)), acetogenesis (the VFA are converted to acetic acid, carbon dioxide, and hydrogen by acetogenic bacteria), and methanogenesis (acetic acid and hydrogen are converted to methane and carbon dioxide by methanogenic bacteria) (2). It has been recognized that hydrolysis of sludge organic components is the rate-limiting step in anaerobic methane production from waste activated sludge (3). For increasing methane yield from sludge most studies in the literature focused on the improvement of sludge hydrolysis by physical, chemical, or biological pretreatment methods (4-6). The pretreated sludge was then used directly for methane production, which was usually conducted in a continuous stirred tank reactor (CSTR) (7). Although several advanced anaerobic reactors, such as up-flow anaerobic sludge bed (UASB) (9, 10), anaerobic sequencing batch reactor (ASBR) (11) and expanded granular sludge bed (EGSB) (12, 13), have been developed for efficiently recovering methane from wastewater, these reactors have seldom been applied to produce methane from waste activated sludge, which might be due to high concentration of sludge. If sludge particle organic components could be hydrolyzed and acidified to soluble organic wastewater, especially high VFA-containing wastewater, which was then treated in the high rate anaerobic reactor, such as UASB, ASBR, or EGSB, the methane production might be improved. Our previous batch experiments have shown that after waste activated sludge was pretreated by NaOH at pH 10 for 8 days, and then fermented in a CSTR, the methane production was significantly higher compared with other pretreatments because this pretreatment method enhanced not only the hydrolysis of sludge protein and carbohydrate but the acidification of hydrolysis products, especially provided a suitable VFA composition for methane generation (8). The purpose of this paper was to report a new process for efficiently producing methane from sludge, that is, sludge was fermented for 8 days at pH 10, which was adjusted by Ca(OH)2 instead of NaOH, and then the fermentation liquid was treated in an EGSB for methane generation. First, for comparing the methane production observed in this study with that reported in the literature, the conventional operational model was applied to produce methane from the pH 10 pretreated sludge, that is, the pH 10 pretreated sludge was directly treated in a CSTR for producing methane. Then, the use of fermentation liquid of pH 10 pretreated sludge to produce methane in UASB, ASBR, and EGSB was compared. Finally, the mechanisms for EGSB exhibiting remarkably higher methane production were investigated from the aspects of sludge organic compounds conversion, enzymes, and microbiology.

 2011 American Chemical Society

Published on Web 12/03/2010

Sludges. The waste activated sludge (WAS) used for methane production was obtained from the secondary sedimentation tank of a municipal wastewater treatment plant in Shanghai, China. The anaerobic granular sludge (AGS), which was used as the inoculums of methane production was obtained from VOL. 45, NO. 2, 2011 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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the UASB reactor of a food wastewater treatment plant in Yixing, China. Both of these two sludges were concentrated by settling at 4 °C for 24 h, and their characteristics are shown in Table S1 (Supporting Information (SI)). Before the anaerobic granular sludge was inoculated into the methane production reactors, it was cultured in the laboratory according to the method described in our previous publication (8). Pretreating Waste Activated Sludge at pH 10 for 8 d in the Semicontinuously Operated Reactor. To get enough sludge fermentation liquid or pH 10 pretreated sludge for methane production, six identical reactors, each with working volume of 20 L (internal diameter of 240 mm and height of 450 mm) and made of Plexiglas were operated simultaneously. All reactors were maintained at 35 ( 2 °C, equipped with stainless-steel stirrers for mixing the contents, and operated semicontinuously, that is, every day morning 2.5 L of sludge was wasted from each reactor and the same amount of fresh sludge was added. The sludge retention time was 8 days and fermentation pH was maintained at 10 ( 0.2, which were the same as our previous study (8). Nevertheless, in this study the pH was adjusted by Ca(OH)2 (100 g/L). After pretreatment, the fermentation mixture was used either for the experiments of “Methane Directly Produced from pH 10 Pretreated Sludge in Semi-continuous-flow CSTRs” or left at 4 °C for 48 h to get the supernatant (VFA-containing sludge fermentation liquid) for the experiments of “Methane Production from Sludge Fermentation Liquid”. The pH of sludge fermentation liquid was adjusted to pH 7 ( 0.2 before used for methane production. The TSS and VSS of the alkaline pretreated sludge were, respectively, 13605 and 6765 mg/L. The main characteristics of sludge fermentation liquid are shown in SI Table S2. Methane Directly Produced from pH 10 Pretreated Sludge in Semicontinuous-flow CSTRs. Three semicontinuous-flow CSTRs with volume of 1 L each and made of Teflon were operated to study the effect of organic loading rate (OLR) on methane production directly from pH 10 pretreated sludge. The above pH 10 pretreated sludge of 1410 mL was divided equally into three reactors, and the pH in each reactor was adjusted to pH 7.0 ( 0.1 by 4 M HCl. Then 30 mL anaerobic granular sludge was added to each reactor and all reactors were sealed with rubber stoppers and mechanically stirred at 80 rpm for methane production. Every day morning, 37, 45, and 58 mL of sludge mixture was manually wasted from each reactor (the granular sludge was put back to reactors by sifting the wasted sludge through an aperture 0.2 mm sifter), and 37, 45, and 58 mL pretreated (pH 10 for 8 days) sludge was added to each reactor, respectively, which resulted in the corresponding organic loading rate of 1.54, 1.88, and 2.42 kgCOD/m3/day. After the methane production in all reactors reached a stable state, the data were reported. Methane Produced from Sludge Fermentation Liquid. In this study three anaerobic reactors, UASB, ASBR, and EGSB were operated to compare their methane production from sludge fermentation liquid. All reactors were maintained at temperature (35 ( 2 °C), and had a working volume of 4.5 L each. The internal diameter and height of three reactors are as follows: EGSB(60 mm × 1400 mm), UASB(90 mm × 700 mm) and ASBR(110 mm × 500 mm). The granular sludge was initially cultured in three laboratory reactors (UASB, ASBR, and EGSB) by synthetic wastewater with glucose (2500 mg/L) as the main carbon source for 30 days before used as the inoculums for methane production from sludge fermentation liquid. The synthetic wastewater consisted of (mg/L of tap water) 1000 NH4Cl, 500 KH2PO4, 200 CaCl2, 200 MgCl2 · 6H2O, 50 FeCl3, 0.5 H3BO3, 0.5 (NH4)6Mo7O24 · 4H2O, 0.5 ZnSO4 · 7H2O, 0.5 CuSO4 · 5H2O, 0.5 CoCl2 · 6H2O, 0.5 AlCl3 · 6H2O, 4 EDTA, 1 MnCl2 · 4H2O, and 1 NiCl2 · 6H2O. These 804

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three reactors were all made of Teflon and operated at organic loading rate of 2.5 kgCOD/m3/day and AGS concentration of around 29 200 mg/L in each reactor. Then the synthetic wastewater was gradually replaced by sludge fermentation liquid (the OLR maintained at 2.5 kgCOD/m3/day) with an initial ratio of fermentation liquid to glucose synthetic wastewater being 10%, then 30%, 50%, 70%, and finally 100% sludge fermentation liquid before the effect of OLR on methane production was investigated. The operation parameters are shown in SI Tables S3 and S4. The data were not reported until the methane production at each organic loading rate reached a relatively stable point. Analytical Methods. Protease and R-glucosidase activity were investigated according to Goel et al. (14) with the F-nitrophenyl phosphate disodium salt as the standard. Then, the acid-forming enzymes, phosphotransacetylase (PTA), phosphotransbutyrylase (PTB), acetate kinase (AK), butyrate kinase (BK), oxaloacetate transcarboxylase (OAATC), and CoA transferase were assayed. For determining the activities of acid-forming enzymes, 25 mL of the granular sludge was taken out of the three anaerobic fermentation reactors, then washed and resuspended in 10 mL of 100 mM sodium phosphate buffer. The suspension was sonicated at 20 kHz and 4 °C for 30 min to break down the cells and then centrifuged at 10 000 rpm and 4 °C for 30 min. The assays for PTA and PTB were based on the method of Andersch et al. (15) with acetyl-CoA and butyryl-CoA as substrate, respectively. The AK and BK activities were analyzed using the method of Allen (16) with potassium acetate and sodium butyrate as the substrate, respectively. OAATC activity was determined according to Wood et al. (17) with methylmalonyl-CoA as substrate. The CoA transferase activity was analyzed using the method of Schulman and Wood (18) with succinyl CoA as substrate. One unit of enzyme activity was defined as the amount of enzyme that catalyzes the conversion of 1 µmol substrate per minute. The specific enzyme activity was defined as the unit of enzyme activity per milligram of volatile suspended solids (VSS). According to the literature the following 16S rRNA-targeted oligonucleotide probes were used in this study: cy-3-labeled EUB338, 5′-GCTGCCTCCCGTAGGAGT-3′ for Bacteria (19), flourescein iso-thiocyanate (FITC) labeled ARC915, 5′-GTGCTCCCCCGCCAATTCCT-3′ for Archaea (methanogens) (20), cy-3-labeled MS1414, 5′-CTCACCCATACCTCACTCGGG3′ for Methanosarcinaceae, FITC labeled MX825, 5′-TCGCACCGTGGCCGACACCTAGC-3′ for Methanosaetaceae (21). Hybridizations were performed at 46 °C for 10 h with hybridization buffer (0.9 M NaCl, 20 mM Tris-HCl [pH 7.2], 0.01% sodium dodecyl sulfate) containing 5 ng of each labeled probe/mL. The hybridization stringency was adjusted by adding formamide to the hybridization buffer (5% for EUB338, 35% for ARC915, 35% for MS1414 and 20% for MX825). The washing step was done at 48 °C for 30 min with washing buffer containing the same components as the hybridization buffer. After hybridization, the specimens were stained with 4′, 6′-diamidino-2-phenylindole (DAPI) (1 mg/mL). Then, the sections hybridized with the probes were observed with a confocal laser scanning microscope (CLSM, Leica TCS, SP2 AOBS). Ten at-random fields were analyzed to determine the average numbers of cells in the samples. All other analyses (TSS, VSS, SCOD, TCOD, NH3-N, PO43-P, adenosine triphosphate (ATP), carbohydrate, protein, VFA, methane, scanning electron microscopy (SEM), and fluorescence in situ hybridization (FISH)) were the same as those described previously (8, 22). In this study methane production was reported as the amount (m3) of methane generated per m3 of reactor per day (m3/m3-reactor/day) unless otherwise stated. The analysis of ATP of anaerobic granular sludge was conducted when the maximal methane production reached.

TABLE 1. Comparison of Main Organic Components of Sludge Fermentation Liquid Consumption at Different OLRs in Three Reactorsa consumption OLR reactors acetic acid other VFAb

FIGURE 1. Comparison of methane production in three reactors at different organic loading rates. Error bars represent standard deviations of three different measurements. The one way analysis of variance (ANOVA) at a 0.05 level was used to analyze the data.

Results and Discussion Methane Directly Produced from pH 10 Pretreated Sludge at Different OLRs in Semicontinuous-flow CSTRs. Traditionally, CSTR was used to produce methane from pretreated sludge. It was observed in our previous study that compared with other pretreatment methods the methane production in the CSTR could be significantly enhanced when waste activated sludge was pretreated at pH 10 for 8 days (8). Nevertheless, only one organic loading rate (1.88 kgCOD/ m3/day) was investigated in that paper. One may say that directly producing methane from pH 10 pretreated sludge in CSTR could be improved by the change of OLR. Thus, the methane production from pH 10 pretreated sludge at different OLRs was investigated first. As seen in SI Figure S1, the increase of OLR from 1.54 to 1.88 kgCOD/m3/day caused the increase of methane production from 0.53 to 0.61 m3CH4/ m3-reactor/day. However, the methane production was not further improved with increasing the OLR to 2.42 kgCOD/ m3/day. Apparently, when pH 10 pretreated sludge was used directly for methane generation, the maximal methane production was 0.61 m3CH4/m3-reactor/day. Comparison of Methane Produced from Sludge Fermentation Liquid in Three Anaerobic Reactors. The comparison of methane production from sludge fermentation liquid at different OLR in UASB, ASBR, and EGSB is shown in Figure 1. At a lower OLR (e.g., 5 kgCOD/m3/day), three reactors showed almost the same methane production (1.41, 1.49, 1.54 m3CH4/m3-reactor/day). However, different performances of methane production were observed when the OLR increased. In the reactor of EGSB the methane production increased with OLR in the range of 2.5-40 kgCOD/m3/ day, and the maximal methane production (12.43 m3CH4/ m3-reactor/day) was observed at OLR of 40 kgCOD/m3/day. However, the maximal methane production was 3.01 in ASBR and 1.41 in UASB, which was achieved at OLR of 10 and 5 kgCOD/m3/day, respectively. Clearly, the methane production in EGSB was the greatest one. It is well-known that at standard temperature and pressure the maximum possible methane yield is 350 mL/g COD during anaerobic treatment of wastes, which is equal to 395 mL/g COD at 35 °C and standard pressure. In this study the expected methane potential should be increased from 0.99 to 15.8 m3CH4/m3-reactor/day when the OLR is increased from 2.5 to 40 kgCOD/m3/day. In the literature the reported methane production could reach 0.38 and 0.59 m3CH4/m3-

soluble protein

soluble carbohydrate

5

UASB ASBR EGSB

1.12 ( 0.03 0.76 ( 0.02 1.08 ( 0.01 0.50 ( 0.02 1.11 ( 0.05 0.79 ( 0.02 1.09 ( 0.03 0.50 ( 0.02 1.08 ( 0.05 0.83 ( 0.02 1.09 ( 0.05 0.50 ( 0.02

10

UASB ASBR EGSB

1.12 ( 0.03 0.69 ( 0.01 0.89 ( 0.02 0.46 ( 0.01 2.26 ( 0.12 1.59 ( 0.06 2.20 ( 0.04 1.01 ( 0.04 2.22 ( 0.06 1.66 ( 0.05 2.22 ( 0.07 1.02 ( 0.02

40

UASB ASBR EGSB

0.95 ( 0.04 0.70 ( 0.02 0.85 ( 0.04 0.46 ( 0.01 2.23 ( 0.06 1.57 ( 0.05 2.11 ( 0.04 0.98 ( 0.03 8.52 ( 0.26 6.58 ( 0.20 8.65 ( 0.43 3.96 ( 0.16

a The data are the averages of three different measurements after methane production reached steady state at each organic loading rate. The unit is kgCOD/m3/ day. b Including propionic, iso-butyric, n-butyric, iso-valeric and n-valeric acids.

reactor/day after sludge was respectively pretreated under alkaline and thermophilic-alkaline conditions (23, 24). As seen from the above study it can be seen that the maximal methane production in EGSB at OLR of 40 kgCOD/m3/day was 12.43 m3CH4/m3-reactor/day (approximately 78% of the expected methane potential), which was much greater than that reported in the references. SI Table S5 shows the chemical costs and income when methane was produced respectively from raw (unpretreated) sludge, pH 10 pretreated sludge and sludge fermentation liquid at maximal methane production. It can be seen that although some chemicals (Ca(OH)2 and HCl) were used to adjust the pH first to 10 and then to 7, the net balance for the application of EGSB to produce methane reached 1478.3 RMB per ton of total suspended sludge solids per day, which was much greater than the use of traditional CSTR to produce methane from raw sludge (12.6 RMB/t-TSS/day). (the other costs, such as labor and power consumption were not compared in this study) In the coming text the reasons for EGSB showing significantly higher maximal methane production than ASBR and UASB were investigated. Consumption of Main Organic Components of Sludge Fermentation Liquid in Three Reactors. VFA and soluble protein and carbohydrate are the main organic components of sludge fermentation liquid, (SI Table S2). It is well-known that VFA is a main substance of methane production. As the content of acetic acid was much higher than any other VFA, in this study the consumption of acetic acid among UASB, ASBR, and EGSB was compared. Although three reactors received identical sludge fermentation liquid, different consumptions of acetic acid were observed (Table 1). In UASB and ASBR, the maximal acetic acid consumption occurred respectively at OLR of 5 and 10 kgCOD/m3/day, but in EGSB the consumption of acetic acid increased with OLR in the range of 5-40 kgCOD/m3/day. Thus, one reason for three reactors showing different methane production was due to their different conversion ability of acetic acid to methane. Also, it was found that consumptions of other VFA and soluble protein and carbohydrate were varied among three reactors (Table 1). The EGSB reactor exhibited the greatest conversion of soluble protein and carbohydrate and other VFA at OLR of 40 kgCOD/m3/day, which indicated that another reason for much higher methane produced in EGSB was its highest hydrolysis and acidification abilities. Observed Activities of Key Hydrolysis and Acidification Enzymes in Three Reactors. The above observations in three VOL. 45, NO. 2, 2011 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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TABLE 2. Comparison of Specific Activity of Key Enzymes Involved in Hydrolysis and Acidification at Different OLRs in Three Reactorsa OLRb

reactor

protease

r-glucosidase

PTA

AK

PTB

BK

OAATC

CoA transferase

5

UASB ASBR EGSB

0.0042 0.0043 0.0045

0.0137 0.0139 0.0143

0.1188 0.1231 0.1272

1.8769 1.7998 1.8343

0.0055 0.0056 0.0057

0.0328 0.0335 0.0336

0.6389 0.6499 0.6581

0.3107 0.3265 0.3362

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UASB ASBR EGSB

0.0041 0.0058 0.0061

0.0136 0.0148 0.0153

0.1147 0.1334 0.1397

1.7634 2.5540 2.5973

0.0053 0.0066 0.0067

0.0326 0.0523 0.0798

0.6373 0.7573 0.7592

0.2927 0.3952 0.4267

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UASB ASBR EGSB

0.0040 0.0057 0.0091

0.0136 0.0142 0.0178

0.1133 0.1304 0.1562

1.7358 2.5816 3.8984

0.0052 0.0066 0.0073

0.0323 0.0521 0.0904

0.6259 0.7405 0.8943

0.2816 0.3902 0.5637

a Data are the averages and their standard deviations in three different measurements. The unit of enzyme activity is U/ mg VSS. b The unit of OLR is kgCOD/m3/day.

reactors could be explained from the aspect of enzyme. According to the proposed metabolic pathway for methane production from soluble protein and carbohydrate (SI Figure S2), polysaccharide and protein are first hydrolyzed respectively into monosaccharide and amino acids, and then into pyruvate and other intermediates. In the metabolic pathway, acetyl-CoA and butyryl-CoA are first converted to acetyl and butyryl phosphate by PTA and PTB, respectively. These acyl phosphates are then converted to acetic and butyric acid by AK and BK, respectively. OAATC, a key enzyme for propionic acid synthesis catalyzing pyruvate to oxaloacrtate and methylmalonyl CoA to propionyl CoA, is responsible for supplying the carbon flux from the central carbon metabolism to propionic acid. CoA transferase also catalyzes the reactions of succinic acid to succinyl CoA and propionyl CoA to propionic acid. As seen in Table 2, the protein and polysaccharide hydrolysis enzymes (protease andR-glucosidase) and key enzymes relevant to VFA generation (PTA, AK, PTB, BK, OAATC, and CoA transferase) showed the highest activity at organic loading rate of 40 kgCOD/m3/day in EGSB, which was consistent with the consumption of fermentation liquid organic components observed in Table 1. General Physiological Activity of Anaerobic Granular Sludge in Three Reactors. It was reported in literatures that the measurement of ATP could be used to assess the general physiological activity of anaerobic cells (25, 26), and the decrease or increase of methane production during anaerobic digestion was observed to be in correspondence with that of ATP (27). In this study the relationships among ATP content, organic loading rate and reactor configuration are illustrated in Figure 2. The maximal ATP in UASB, ASBR, and EGSB was observed at OLR of 5, 10, and 40 kgCOD/m3/day, respectively, and the ATP at OLR of 40 kgCOD/m3/day in EGSB was the greatest one, which was in correspondence with the methane production in three reactors. It seems that the general physiological activity of anaerobic cells in EGSB at OLR of 40 kgCOD/m3/day was the highest, which resulted in its significantly higher methane production. SEM and FISH Analyses of Anaerobic Granular Sludge Cultured in Three Reactors. After the methane production in three reactors reached stable, the granules were observed with SEM, and the results are shown in SI Figure S3. The rods, filaments and cocci could be observed in the surface of all three granular sludges, but there were more rods in UASB (SI Figure S3U-1) and more filaments in ASBR (SI Figure S3A-1). The internal Archaeal morphotypes study indicated that the large microcolonies in UASB were composed almost exclusively of angular-shaped rods (SI Figure S3U-2), which possessed a structure similar to those of Methanosaeta sp. In the granules of EGSB (SI Figure S3E-2), however, the microorganisms looking like large Archaeal cocci in groups of four individuals were predominant, which might be the 806

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FIGURE 2. Comparison of ATP content in UASB, ASBR, and EGSB at different organic loading rates. Error bars represent standard deviations in three different measurements. Methanosarcina sp-like. Both of these Archaeal morphotypes could be observed in the internal of granular sludge cultured in ASBR, but the large Archaeal cocci were less than that in EGSB (Figure S3A-2). The microorganisms in three reactors were further examined by FISH. To visualize the Bacteria and Archaea (methanogens), Cy-3-labeled EUB338 probe and FITClabeled ARC915 probe were used simultaneously in sections of three granular sludges, and the results are shown in Figure 3. The brighter green fluorescent signal of the FITC-labeled Archaea could be observed in EGSB granules than in UASB and ASBR at high (×60) magnifications (Figure 3U-4, A-4, and E-4), and the same observation could be made at other magnifications (×10 and ×20, data not shown), which indicated that more active Archaea (methanogens) were in EGSB. Similarly, the brighter red fluorescent signal of the Cy-3-labeled Bacteria was observed in granules cultured in EGSB, whereas lower red fluorescent signal was seen in UASB and ASBR (Figure 3U-5, A-5, and E-5), suggesting that the EGSB granules had greater active bacteria. From the Cy-3labeled Methanosarcinaceae-domain probe (MS1414) (red) (Figure 3U-3, A-3, and E-3), it can be seen that the EGSB granules were brighter (with red fluorescent signal of Cy3-labeled Methanosarcinaceae), whereas the granules in ASBR and UASB were less bright (with red fluorescent signal of Cy-3-labeled Methanosarcinaceae), which indicated that more active Methanosarcinaceae appeared in EGSB. As seen from the FITC -labeled Methanosaetaceae -domain probe (MX825) (green) (Figure 3U-2, A-2, and E-2), the UASB granules were brighter (with green fluorescent signal of FITClabeled Methanosaetaceae), whereas the granules in both

FIGURE 3. FISH analysis of granular sludge long-term cultured respectively in UASB (U1-U5), ASBR (A1-A5), and EGSB (E1-E5) viewed by CLSM and photographed at magnification of 60×. The 4′,6′diamidino-2-phenylindole (DAPI)-labeled total cells (blue) are shown in U-1, A-1, and E-1. The sections were simultaneously hybridized with FITC -labeled Methanosaetaceae-domain probe (MX825) (green U-2, A-2, and E-2) and Cy-3-labeled Methanosarcinaceae-domain probe (MS1414) (red U-3, A-3, and E-3) after hybridization. FITC-labeled Archaeal-domain probe (ARC915) (green) and Cy-3-labeled Bacterial-domain probe (EUB338) (red) are shown in U-4, A-4, E-4, U-5, A-5, and E-5,

TABLE 3. Quantification of the Total (DAPI) and Hybridized (Active) Cells with Universal Bacterial (EUB338) and Archaeal (ARC915) Probes in the Granules Cultured in Three Reactorsa granules source

total cells (no. of DAPI cells/g of sludge)

Bacteria (no. of EUB338 cells/g of sludge)

Archaea (no. of ARC915 cells/g of sludge)

active Bacteriab (%)

active Archaeac (%)

UASB ASBR EGSB

(2.7 ( 0.1) × 1011 (3.1 ( 0.2) × 1011 (3.5 ( 0.2) × 1011

(1.8 ( 0.1) × 1010 (1.9 ( 0.1) × 1010 (2.0 ( 0.2) × 1010

(5.2 ( 0.1) × 1010 (5.5 ( 0.1) × 1010 (6.8 ( 0.1) × 1010

25.7 25.7 22.7

74.3 74.3 77.3

UASB ASBR EGSB

Methanosaetaceae (no. of MX825 cells/g of sludge)

Methanosarcinaceae (no. of MS1414 cells/g of sludge)

active Methanosaetaceaed (%)

active Methanosarcinaceaee (%)

(2.5 ( 0.1) × 1010 (2.1 ( 0.2) × 1010 (0.9 ( 0.1) × 1010

(1.4 ( 0.1) × 1010 (1.9 ( 0.2) × 1010 (3.2 ( 0.2) × 1010

64.1 52.5 21.9

35.9 47.5 78.1

a The data are the averages and their standard deviations in two different measurements. b The number Bacteria accounting for the total number of Bacteria and Archaea. c The number Archaea accounting for the total number of Bacteria and Archaea. d The number Methanosaetaceae accounting for the total number of Methanosaetaceae and Methanosarcinaceae. e The number Methanosarcinaceae accounting for the total number of Methanosaetaceae and Methanosarcinaceae.

ASBR and EGSB were less bright (with green fluorescent signal of FITC-labeled Methanosaetaceae), which indicated that more active Methanosaetaceae appeared in UASB. Using DAPI staining, the number of microorganisms in the granules of three reactors was counted (Table 3). The total cells number and active Archaea (Methanogens) in the granules of EGSB was higher than that in ASBR and UASB. Furthermore, higher number of Methanosarcinaceae cells was also observed in EGSB granules than in ASBR and UASB. This indicated that more methanogenesis Archaea and Methanosarcinaceae were cultured in EGSB. Both Methanosarcinaceae and Methanosaetaceae have the ability to convert VFA especially acetic acid to methane, and these

two microorganisms belong to the domain Archaea (28). Nevertheless, the maximum specific utilization rate of acetic acid to methane with Methanosarcinaceae has been reported to be higher than that with Methanosaetaceae (29). As there was more Methanosarcinaceae in EGSB than in other two reactors, it might be a reason to explain that the methane production in EGSB was the highest among three reactors.

Acknowledgments This work was financially supported by the Foundation of State Key Laboratory of Pollution Control and Resources Reuse (PCRRK09002). VOL. 45, NO. 2, 2011 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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Supporting Information Available This file contains Tables S1-S5 and Figures S1-S3. This material is available free of charge via the Internet at http:// pubs.acs.org.

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