New Sludge Pretreatment Method to Improve Methane Production in

May 24, 2010 - State Key Laboratory of Pollution Control and Resources Reuse, School of Environmental Science and Engineering, Tongji University, 1239...
0 downloads 5 Views 3MB Size
Download PDF
Environ. Sci. Technol. 2010, 44, 4802–4808

New Sludge Pretreatment Method to Improve Methane Production in Waste Activated Sludge Digestion DONG ZHANG, YINGUANG CHEN,* YUXIAO ZHAO, AND XIAOYU ZHU State Key Laboratory of Pollution Control and Resources Reuse, School of Environmental Science and Engineering, Tongji University, 1239 Siping Road, Shanghai 200092, China

Received January 4, 2010. Revised manuscript received May 11, 2010. Accepted May 12, 2010.

During two-phase sludge anaerobic digestion, sludge is usually hydrolyzed and acidified in the first phase, then methane is produced in the second stage. To get more methane from sludge, most studies in literature focused on the increase of sludge hydrolysis. In this paper a different sludge pretreatment method, i.e., pretreating sludge at pH 10 for 8 d is reported, by which both waste activated sludge hydrolysis and acidification were increased, and the methane production was significantly improved. First, the effect of different sludge pretreatment methods on methane yield was compared. The pH 10 pretreated sludge showed the highest accumulative methane yield (398 mL per g of volatile suspended solids), which was 4.4-, 3.5-, 3.1-, and 2.3-fold of the blank (unpretreated), ultrasonic, thermal, and thermal-alkaline pretreated sludge, respectively. Nevertheless, its total time involved in the first (hydrolysis and acidification) and second (methanogenesis) stages was 17 (8 + 9) d, which was almost the same as other pretreatments. Then, the mechanisms for pH 10 pretreatment significantly improving methane yield were investigated. It was found that pretreating sludge at pH 10 caused the greatest sludge hydrolysis, acidification, soluble C:N and C:P ratios, and Fe3+ concentration with a suitable short-chain fatty acids composition in the first stage, which resulted in the highest microorganism activity (ATP) and methane production in the second phase. Further investigation on the second phase microorganisms with fluorescence in situ hybridization (FISH) and scanning electron microscopy (SEM) indicated that there were much greater active methanogenesis Archaea when methane was produced with the pH 10 pretreated sludge, and the predominant morphology of the microcolonies suggest a shift to Methanosarcina sp. like.

lization of sludge as a useful resource (2). Ways to improve methane yield from sludge have become an interesting topic to many researchers (3, 4). Usually, methane production from waste solids undergoes three stages, i.e., hydrolysis, acidification, and methane generation. As hydrolysis has always been believed the ratelimited stage of methane production, most of the studies focused on accelerating sludge hydrolysis by the strategies of thermal (5), thermal-alkaline (6), ultrasonic (7), mechanical (8), or thermo-chemical pretreatment (9). After sludge was pretreated by these methods, more methane was produced. However, if both of the first two stages could be accelerated, the accumulation of methane in the last step should be further increased. Until now, very few publications discussed the increase of methane production by improving both sludge hydrolysis and acidification. It has been reported in our previous publication that when WAS was anaerobically treated under conditions of pH 10 for 8 d, both sludge hydrolysis and short-chain fatty acids (SCFA) accumulation were significantly enhanced, and there was very little methane generated (10). Thus, if we want to increase methane production during WAS digestion, the following two-stage anaerobic configuration might be considered (Figure 1), i.e., in the first stage sludge is pretreated at pH 10 for 8 d for efficient sludge hydrolysis and SCFA accumulation, and then in the second stage methane is produced with the pH 10 pretreated sludge at neutral pH in which methanogens have been reported to show a maximal activity (11, 12). In this study a new pretreatment method, i.e., pretreating sludge at pH 10 for 8 d, for remarkably enhancing methane generation was reported. The effect of different pretreatment methods (ultrasonic, thermophilic, thermophilic-alkaline, and pH 10 for 8 d) on methane yield in WAS digestion was compared, and the mechanisms for significantly higher methane yield with the pH 10 pretreatment method were explored. Also, microorganisms responsible for methane generation with the pH 10 pretreated sludge were investigated.

Materials and Methods Sludges. WAS used as substrate for methane production was obtained from the secondary sedimentation tank of a municipal wastewater treatment plant in Shanghai, China. Its main characteristics are (all values are expressed in mg/L except pH): pH 6.8, TSS (total suspended solids) 1.75 × 104, VSS (volatile suspended solids) 1.38 × 104, SCOD (soluble chemical oxygen demand) 62, TCOD (total chemical oxygen demand) 1.69 × 104, total carbohydrate (as COD) 3.16 × 103, total protein (as COD) 9.96 × 103, lipid and oil (as COD) 159,

Introduction Activated sludge technology is the widely used biological method for wastewater (especially municipal wastewater) treatment, but large amounts of waste activated sludge (WAS) are produced in this process. WAS usually contains high organic matter, such as protein and carbohydrate (1). Sludge disposal by landfill and incineration may not be appropriate in the near future due to land scarcity and increasingly stringent environmental control regulations. Thus one strategy for sludge management is moving toward reuti* Corresponding author e-mail: [email protected]; tel: 86-2165981263; fax: 86-21-65986313. 4802

9

ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 44, NO. 12, 2010

FIGURE 1. Flowsheet for methane production with the pH 10 pretreated sludge. 10.1021/es1000209

 2010 American Chemical Society

Published on Web 05/24/2010

soluble carbohydrate (as COD) 4.5, soluble protein (as COD) 22, soluble phosphate (PO43--P) 45.2, and ammonium nitrogen (NH3-N) 17.1. The anaerobic granular sludge (AGS) used as the inoculums for methane production was obtained from the upward-flow anaerobic sludge blanket (UASB) reactor of a food wastewater treatment plant in Yixing, China. The main features of AGS are (with mg/L as the unit except pH) pH 7.0, TSS 2.92 × 104, VSS 2.23 × 104, SCOD 76, total carbohydrate (as COD) 4.07 × 103, and total protein (as COD) 1.29 × 104. Culture of Anaerobic Granular Sludge. The AGS was cultured in a laboratory UASB (Up-flow Anaerobic Sludge Bed) by synthetic wastewater with glucose (2500 mg/L) as the main carbon source for 30 d before use as the inoculums for methane yield. 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. The UASB (diameter 90 mm and height 700 mm) was made of Teflon and operated with a hydrolytic retention time of 6 h and AGS concentration of around 2.92 × 104 mg/L. Pretreating WAS at pH 10 for 8 d. The pretreatment of WAS at pH 10 was almost the same as that described in our previous publication (10). Two reactors, each with working volume of 12 L, internal diameter of 155 mm, and height of 636 mm were maintained at 34-36 °C. Eighteen liters of WAS was divided equally into 2 reactors. The pH in one reactor was not adjusted, which was set as a blank one, and in another reactor was controlled at pH 10.0 by automatic addition of 5 M NaOH or 4 M HCl with an automatic titrator. The two reactors were sealed air-tightly and mechanically stirred at 80 rpm for 8 d, and then the mixtures were used for the following tests. All the following experiments were duplicated and one-way analysis of variance (ANOVA) at 0.05 level was used to analyze the data.Control of pH was conducted by automatic addition of 5 M NaOH or 4 M HCl with an automatic titrator. Unless otherwise stated, all reactors were maintained at 35 ( 1 °C. Comparison among Different Sludge Pretreatment Strategies Affecting Methane Generation. The following experiments were conducted in 5 identical reactors with working volume of 1 L each. The raw (unfermented) WAS of 1.88 L was divided equally into reactors 1-4. The sludge in reactor 1 was not pretreated and set as the control, whereas the sludge in other three reactors was pretreated respectively by ultrasonic (frequency 41 kHz for 150 min), thermophilic (70 °C for 9 h), and thermophilic-alkaline (90 °C and pH 11 for 10 h) according to the methods and operational conditions reported in the literature (6, 7, 13). After pretreatment, the pH value in these 4 reactors and in reactor 5 which contained 470 mL of the above (pH 10 pretreated for 8 d) sludge was adjusted to pH 7.0 ( 0.1 since it is well-known that the suitable pH for methane fermentation is usually at this value, and then 30 mL anaerobic granular sludge was added to each reactor for methane generation. All reactors were sealed with rubber stoppers and mechanically stirred at 80 rpm without pH control. The gas produced was collected by the water displacement method. In this study methane yield was reported as the amount of methane generated per gram of VSS added (mL/g VSS) unless otherwise stated. The analysis of adenosine triphosphate (ATP) of anaerobic granular sludge was conducted when the maximal methane production reached. Supplemented Experiments of Stirring Time Affecting Sludge Hydrolysis, Acidification, and Methane Generation after Sludge Pretreated by Different Methods. After sludge (470 mL) was pretreated by different methods (ultrasonic, thermal, and thermal-alkaline), the mixture was mechanically stirred (80 rpm) at 35 ( 1 °C for 8 d, and then SCOD, SCFA,

and methane were measured to investigate the effect of stirring time on sludge hydrolysis, acidification, and methane yield. The blank test was also conducted, in which the sludge was not pretreated but mechanically stirred for 8 d. Then the pH in all reactors was adjusted to pH 7.0 ( 0.1, and 30 mL of anaerobic granular sludge was added to each reactor for methane production. Experiments of the Mass Ratio of C:N:P on Methane Generation. The pH 10 pretreated WAS of 2.35 L was centrifuged at 100g for 10 min, and the released NH3-N and PO43--P were removed from the supernatant according to the method described in our previous publication (14) before the pH of the supernatant was adjusted to pH 7.0 ( 0.1 by 4 M HCl. The liquid was then divided equally into five reactors, which had a working volume of 1 L each. By the addition of NH4Cl or KH2PO4, different C:N:P ratios in five reactors were obtained. All reactors were seeded with 30 mL of anaerobic granular sludge before being sealed with rubber stoppers and mechanically stirred at 80 rpm for methane generation. Experiments of Fe3+ Concentration on Methane Generation. Sludge (1.41 L) was divided equally into three reactors and was pretreated respectively by ultrasonic, thermal, and thermal-alkaline method. The mixture in each reactor was divided equally into 2 parallel reactors, and then FeCl3 · 6H2O (3.5uM) was added to one reactor to get its final concentration of 4.9 mg/L. The pH value in all reactors was adjusted to pH 7.0 ( 0.1 before 30 mL of anaerobic granular sludge was added to each reactor for methane production. Long-Term Reactors Operation for Methane Generation with the pH 10 Pretreated Sludge for Microbial Community Study. Two semicontinuous-flow reactors with working volume of 1.0 L each were operated for microbial community study. The two reactors received, respectively, the unpretreated and pH 10 pretreated sludge of 470 mL. The pH value in both reactors was adjusted to pH 7.0 ( 0.1, and then 30 mL of anaerobic granular sludge was added to each reactor for methane production. Every day, 52 mL of sludge mixture was manually wasted from each reactor (the anaerobic granular sludge was put back to reactors by sifting the wasted sludge through an aperture 0.2 mm sifter), and 52 mL of unpretreated and pretreated (pH 10 for 8 d) sludges was added to each reactor, respectively. After operation for almost 3 months, the methane generation remained stable, and then the analysis of microbial community was conducted. Analytical Methods. The analyses of TSS, VSS, SCOD, TCOD, NH3-N and PO43--P were conducted in accordance with Standard Methods (15). Sludge lipid was extracted by the Bligh-Dyer method from the acidification sample, and then measured gravimetrically after the solvent was evaporated at 80 °C (15). Carbohydrate was measured by the phenol-sulfuric method with glucose as standard (16). Soluble protein was determined by the Lowry-Folin method with BSA as standard (17). The total sludge protein was estimated from the corresponding TKN concentration by subtracting the inorganic nitrogen concentration and dividing the difference by 0.16, then multiplying the result by 1.5 (18). Trace elements were detected by plasma-optical spectrometry (Perkin-Elmer, Optima 2100DU). The measurement of SCFA was the same as that described in our previous publication (10). The ATP content of anaerobic granular sludge was determined by quantification of the luminescence released from the reaction of luciferase with ATP (19). A 10 mL portion of anaerobic granular sludge was collected from each reactor when the methane yield reached the maximal, and then washed three times (10 mL each) with PBS (0.1 M phosphate buffer solution, pH 7.2). Each sample was resuspended in 10 mL of PBS, and then sonicated (80 w, 20 kHz, 4 °C) for 5 min in an ultrasonic cell disruptor (VCX105) before being diluted with 20 mM Tris-EDTA (pH 7.75), boiled for 30 min, and VOL. 44, NO. 12, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

9

4803

equilibrated to room temperature. After centrifugation at 6000 rpm for 5 min the supernatant was collected for its ATP content analysis. The luminescence reaction of sample ATP with luciferase was measured as a Relative Luminescence Unit (RLU) in a Spectra Max L microplate luminometer (Melocular Devices Corp.) at 570 nm, after ATP samples added with luciferase (Bac Titer-Glo microbial cell viability assay, Promega Corp.). A calibration graph was established at varying ATP concentrations (Bac Titer-Glo Microbial Cell Viability Assay, Promega Corp.) with luciferase to quantify the sample ATP. The fixation and sectioning of anaerobic granular sludge were conducted as follows. The granule samples were washed with phosphate-buffered saline (PBS [0.13 M NaCl, 10 mM Na2HPO4, pH 7.2]), and settled for 2 h. The granular sludge was then fixed with 4% paraformaldehyde in PBS for 6 h at 4 °C, before being exposed to 50% ethanol in PBS for 12 h at 4 °C. Five freeze-thaw cycles (-80 to 60 °C) were done after fixation to allow probes to penetrate the cells in granule samples. The granules were then dehydrated by successively passaging through 50, 80, and 100% ethanol (three times), 50:50 (v/v) ethanol-xylene, and 100% xylene (three times), and embedded in melted paraffin wax. Serial sections (10-15 um) were cut with a rotary microtome and mounted on gelatin-coated glass slides. The sections were dewaxed through 100% xylene (two times) and 100% ethanol (twice), and dried at room temperature for fluorescence in situ hybridization (FISH). 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 (20), flourescein iso-thiocyanate (FITC) labeled ARC915, 5′-GTGCTCCCCCGCCAATTCCT-3′ for Archaea (methanogens) (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 and 35% for ARC915). 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. Before microbial observation of the anaerobic granular sludge by scanning electron microscopy (SEM) using a Hitachi S570 electron microscope, the samples were washed with PBS and fixed with 2.5% glutaraldehyde (pH 7.2-7.4) overnight at 4 °C, then washed with PBS for 2 h. Samples were fixed for 1.5 h by 1% osmic acid, then washed with double distilled water for 2 h. To obtain cleaved preparations of the granules, the fixed samples were quick-frozen in liquid nitrogen and cleaved with a rotary microtome. Then samples were dehydrated by successively passaging through 25, 50, 75, 80, 90, 95, and 100% ethanol followed by drying with a critical drier and platinizing with an ion coater (IB5). The particle size of anaerobic granular sludge was determined by image analysis system (Image-Pro Plus, V5.0, Media Cybernetics).

Results and Discussion Comparison among Sludge Different Pretreatment Methods Affecting Methane Yield. As shown in Figure 2 the cumulative methane yield with pH 10 pretreated sludge reached the maximal (398 mL/g VSS or 325 mL/g COD) on the ninth day, and there was no significantly increase after day 9 (Fobserved ) 2.08 × 10-16, Fsignificance ) 6.59, P(0.05) ) 0.99 4804

9

ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 44, NO. 12, 2010

FIGURE 2. Effect of sludge different pretreatment methods on methane yield. Error bars represent standard deviations of duplicate tests.

FIGURE 3. Effect of different pretreatment methods on the observed SCOD and SCFA. Error bars represent standard deviations of duplicate tests (before pretreatment the SCOD concentration was 62 mg/L, and the SCFA was nondetectable). > 0.05), which suggested that the suitable time for methane production was 9 d. It is well-known that the maximum possible methane yield is 350 mL/g COD during anaerobic treatment of wastes at standard temperature and pressure (22), which is equal to 395 mL/g COD at 35 °C and standard pressure. The methane yield in the blank test increased gradually with time from day 1 to day 17 (Fobserved ) 99.08, Fsignificance ) 2.29, P(0.05) ) 1.34 × 10-13 < 0.05), and very little increase was observed between time 17 and 20 d (Fobserved ) 0.01, Fsignificance ) 6.59, P(0.05) ) 0.99 > 0.05). When sludge was pretreated by other methods the methane yield was also observed to increase with time between 1 and 17 d, and no significant increase occurred in the range of time 17-20 d (Table S1, Supporting Information). During the first 17 d, the cumulative methane yield was 90.4, 115.4, 127.8, and 171.2 mL/g VSS with the unpretreated (blank), ultrasonic, thermal, and thermal-alkaline pretreated sludge, respectively. Obviously, the maximal methane yield of the pH 10 pretreated sludge was almost 4.4-, 3.5-, 3.1-, and 2.3-fold that of the unpretreated (blank), ultrasonic, thermal, and thermalalkaline pretreated sludge, respectively. Nevertheless, the total time involved in methane production with pH 10 pretreated sludge was 17 (8 + 9) d, which was no longer than that with other pretreatment methods. It should be emphasized that although the new pretreatment method showed higher methane yield than other documented methods, we can not give a definite conclusion about its cost because this study was conducted at a laboratory scale. It is well-known that the cost of a laboratory

granules cultured with ph10 pretreated sludge granules cultured with unpretreated sludge

a Data are the averages and their standard deviations in duplicate tests. b Total number of Bacteria and Archaea accounting for the total cells. the total number of Bacteria and Archaea. d Number of Archaea accounting for the total number of Bacteria and Archaea.

c

Number of Bacteria accounting for

71.8 ( 0.3 51.4 ( 0.2 28.2 ( 0.1 48.6 ( 0.2 33.8 ( 0.3 19.4 ( 0.2 2.0 ( 0.1 × 1010 1.8 ( 0.1 × 1010 2.1 ( 0.1 × 1011 1.9 ( 0.1 × 1011

granules type

5.1 ( 0.2 × 1010 1.9 ( 0.1 × 1010

active Archaead (%) active Bacteriac (%) total active cellsb (%) Archaea (no. of ARC915 cells/ g of sludge) Bacteria (no. of EUB338 cells/ g of sludge)

work is different from that of full-scale. As the main purpose of this study was to improve methane yield from sludge, in the following text the reasons for pH 10 pretreatment method showing higher methane yield than others were investigated. Effect of Different Pretreatments on WAS Hydrolysis, Acidification, and the Composition of SCFA. In this study sludge hydrolysis and acidification were expressed, respectively, by the changes of observed soluble COD and SCFA. Figure 3 describes the influence of different sludge pretreatment methods on WAS hydrolysis and acidification. The observed SCOD and SCFA was 1.18 × 104 mg/L, and 4.93 × 103 mg COD/L when sludge was treated at pH 10 for 8 d. To all other pretreatments, the SCOD was less than 4000 mg/L, and the SCFA was below 200 mg COD/L. It can be concluded that pretreating sludge at pH 10 for 8 d caused much greater of both sludge hydrolysis and acidification than any other pretreatment methods. Different pretreatments also influenced the composition of generated SCFA (Figure 4), which might affect the methane production because several researchers reported that different SCFA showed different methane production (23-25), i.e., acetic > butyric > propionic acid. It seems that higher acetic with lower propionic acid fraction would cause greater methane generation. As seen in Figure 4, although acetic acid was the greatest SCFA in all tests, its percentage reached almost 60% with the pH 10 pretreatment method, whereas

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

FIGURE 5. Effects of concentrations of NH3-N and PO43--P on methane yield. In the supernatant of pH 10 pretreated sludge the SCOD, NH3-N and PO43--P were 1.18 × 104, 173.4, and 58.5 mg/L, respectively. Error bars represent standard deviations of duplicate tests.

TABLE 1. Quantification of the Total (DAPI) and Hybridized (Active) Cells with Universal Bacterial (EUB338) and Archaeal (ARC915) Probes in the Granules Cultured Respectively with Un-Pretreated and pH 10 Pretreated Sludgea

FIGURE 4. Effect of different pretreatment methods on SCFA composition (in the blank test the SCFA was nondetectable). Acetic (A), propionic (P), iso-butyric (iso-B), n-butyric (n-B), iso-valeric (iso-V), and n-valeric (n-V). Error bars represent standard deviations of duplicate tests.

VOL. 44, NO. 12, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

9

4805

FIGURE 6. Effect of Fe3+ addition on the 9 d (a) and 18 d (b) cumulative methane production after sludge pretreated respectively by ultrasonic, thermal, and thermal-alkaline method. Error bars represent standard deviations of duplicate tests.

FIGURE 7. SEM of anaerobic granule sludge long-term cultured respectively with pH10 pretreated sludge (P1-P3) and unpretreated sludge (UP1-UP3). P-1 and UP-1, P-2 and UP-2, and P-3 and UP-3 represent the appearance, surface, and interior of two anaerobic granule sludges. it was only 38-48% with others. In addition, the pH 10 pretreatment showed the lowest propionic acid percentage. Some might say that the pretreatment time for pH 10 (8 d) was longer than any other methods, which resulted in its greater SCOD and SCFA concentration and acetic acid percentage. Thus, supplemental experiments of stirring time affecting sludge hydrolysis, acidification, and methane generation after sludge pretreatment by different methods were conducted. It was observed that even the sludge stirred for 8 d after pretreatment, the concentrations of SCOD and SCFA with the ultrasonic, thermal, and thermal-alkaline pretreated sludge and in the blank test were still much lower than those with the pH10 pretreated sludge (Table S2, Supporting Information). Also, during the 8-d-stirring time the percentages of individual SCFA did not change significantly (Table S3, Supporting Information). In addition, there was very small amount of methane produced during the 8-day stirring time (Table S4, Supporting Information), which might be due to the pH (Figure S1, Supporting Information) not being suitable for methane generation. No matter which pretreatment method was applied the total methane produced during the 8-day stirring time and the followed 9-day 4806

9

ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 44, NO. 12, 2010

methanogenesis stage was still much lower than that with the pH 10 method (Table S4, Supporting Information). Thus, pretreating sludge at pH 10 not only improved sludge hydrolysis and SCFA accumulation, but it provided a suitable substrate (SCFA) composition for methanogens, which resulted in its significantly high methane yield. Effect of Different Pretreatments on the Released C:N:P Ratio. After sludge was pretreated by different methods, the released NH3-N and PO43--P and the ratios of C:N:P were different (Table S5, Supporting Information). The C:N:P has been reported to give impact on methane accumulation during anaerobic fermentation (26). As the pH 10 pretreatment showed the greatest released C:N and C:P ratios, its liquid was used to study the effect of C:N:P on methane generation in this study. With an identical SCOD concentration (1.18 × 104 mg/L), the effect of C:N:P on methane yield is shown in Figure 5. Obviously, when the C:N and C:P ratio increased or decreased, an improved or declined methane yield occurred. Although more investigations are required in the future about the optimal C:N:P ratio for methane production, it can be concluded that one reason for pH 10

pretreated sludge showing the highest methane yield was that it had the greatest C:N and C:P ratios. Effect of Different Pretreatments on the Release of Trace Metals. It was observed that pH 10 pretreatment caused the greatest Fe3+ release among the four pretreatment methods (Figure S2, Supporting Information). Since several cases were reported in the literature about the increase of trace metal elements, such as Fe3+, resulting in the improvement of methane generation (27-29), in this study the effect of Fe3+ addition on methane yield with sludge pretreated by different methods was investigated. As seen in Figure 6, no matter which pretreatment method was used, the increase (addition) of Fe3+ caused the enhancement of methane yield. It seems that one reason for pH 10 pretreated sludge showing the greatest methane yield was due to the highest Fe3+ release. Effect of Different Pretreatments on the General Physiological Activity of Anaerobic Granular Sludge during Methane Production. The measurement of ATP has been used in literatures to assess the general physiological activity of anaerobic cells (19, 30), and the decrease or increase of methane generation during anaerobic digestion is observed to be in correspondence with that of ATP (31). In this study different pretreatment methods showed different effects on ATP content of granular sludge with the following sequence: pH 10 (2.15 ug/L) > thermal-alkaline (0.66) > thermal (0.45) > ultrasonic (0.39), and the blank test had the lowest ATP content (0.32 ug/L). Further investigation showed that the increase of ATP was in correspondence with that of methane yield (Figure S3, Supporting Information). Fluorescence In Situ Hybridization of Anaerobic Granular Sludge Long-Term Cultured with pH 10 Pretreated Sludge. The FISH analysis (Figure S4, Supporting Information) indicated that compared with unpretreated sludge there were more active Archaea (methanogens) and Bacteria in the reactor fed with pH 10 pretreated sludge. Further investigation using DAPI staining showed that the anaerobic granular sludge cultured with pH 10 pretreated sludge had significantly higher percentages of total active cells and active Archaea than those with unpretreated sludge (Table 1). Scanning Electron Microscopy. As shown in Figures 7P-1 and UP-1, there were no significant differences in the appearance and diameter of these two granular sludges cultured respectively with unpretreated and pH 10 pretreated sludge. However, in the surface of granular sludge cultured with pH 10 pretreated sludge there were more cocci (Figure 7P-2), whereas large numbers of rods appeared in the surface of granular sludge fed with raw sludge (Figure 7UP-2). Further study indicated that the microorganisms looking like large Archaeal cocci in groups of four individuals were predominant in the granules fed with pH 10 pretreated sludge (Figure 7P-3), which might be the Methanosarcina sp., but to the granules cultured with unpretreated sludge the large microcolonies were composed almost exclusively of angularshaped rods (Figure 7UP-3), which possessed a structure similar to those of Methanosaeta sp. Both Methanosarcina sp. and Methanosaeta sp. have been reported to be able to convert SCFA, especially acetic acid, to methane and these two microorganisms belong to the domain Archaea (32). Nevertheless, Methanosarcina sp. can grow on higher acetate concentration, whereas lower acetate concentration benefits the predominance of Methanosaeta sp. (33), which might be a reason to explain that the predominant microcolonies were Methanosarcina sp. in granules using pH 10 pretreated sludge as substrates (high acetate concentration), whereas predominant microcolonies were Methanosaeta sp. in granules using raw sludge as substrates (low acetate concentration).

Acknowledgments This work was financially supported by the Foundation of State Key Laboratory of Pollution Control and Resources Reuse (PCRRK09002) and the program for NCET in university (06-0373).

Supporting Information Available Supplementary Tables S1-S5 and Figures S1-S4. This information is available free of charge via the Internet at http://pubs.acs.org.

Literature Cited (1) Elefsiniotis, P.; Wareham, D. G.; Oldham, W. K. Particulate organic carbon solubilization in an acid-phase upflow anaerobic sludge blanket system. Environ. Sci. Technol. 1996, 30, 1508– 1514. (2) Mossakowska, A.; Hellstro¨m, B. G.; Hultman, B. Strategies for sludge handling in the Stockholm region. Water Sci. Technol. 1998, 38, 111–118. (3) Cui, R.; Jahng, D. Enhanced methane production from anaerobic digestion of disintegrated and deproteinized excess sludge. Biotechnol. Lett. 2006, 28, 531–538. (4) Ting, C. H.; Lee, D. J. Production of hydrogen and methane from wastewater sludge using anaerobic fermentation. Int. J. Hydrogen Energy 2007, 32, 677–682. (5) Li, Y. Y.; Noike, T. Upgrading of anaerobic digestion of waste activated sludge by thermal pretreatment. Water Sci. Technol. 1992, 26, 857–866. (6) Vlyssides, A. G.; Karlis, P. K. Thermal alkaline solubilization of waste activated sludge as a pretreatment stage for anaerobic digestion. Bioresour. Technol. 2004, 91, 201–206. (7) Tiehm, A.; Nickel, K.; Zellhorn, M.; Neis, U. Ultrasonic waste activated sludge disintegration for improving anaerobic stabilization. Water Res. 2001, 35, 2003–2009. (8) Nah, I. W.; Kang, Y. W.; Hwang, K. Y.; Song, W. K. Mechanical pretreatment of waste activated sludge for anaerobic digestion process. Water Res. 2000, 34, 2362–2368. (9) Tanaka, S.; Kobayashi, T.; Kamiyama, K. L; Bildan, M. L. Effects of thermochemical pretreatment on the anaerobic digestion of waste activated sludge. Water Sci. Technol. 1997, 35, 209–215. (10) Yuan, H.; Chen, Y.; Zhang, H.; Jiang, S.; Zhou, Q.; Gu, G. Improved bioproduction of short-chain fatty acids (SCFAs) from excess sludge under alkaline condition. Environ. Sci. Technol. 2006, 40, 2025–2029. (11) Jan, T. W.; Adav, S. S.; Lee, D. J.; Wu, R. M.; Su, A.; Tay, J. H. Hydrogen fermentation and methane production from sludge with pretreatments. Energy Fuels 2008, 22, 98–102. (12) Eekert, M. H.; Ras, N. J.; Mentink, G. H.; Rijnaarts, H. H.; Stams, A. J.; Field, J. A.; Schraa, G. Anaerobic transformation of β-hexachlorocyclohexane by methanogenic granular sludge and soil microflora. Environ. Sci. Technol. 1998, 32, 3299– 3304. (13) Ferrer, I.; Ponsa´b, S.; Va´zquezc, F.; Font, X. Increasing biogas production by thermal (70°C) sludge pretreatment prior to thermophilic anaerobic digestion. Biochem. Eng. J. 2008, 42, 186–192. (14) Tong, J.; Chen, Y. Recovery of nitrogen and phosphorus from alkaline fermentation liquid of waste activated sludge and application of the fermentation liquid to promote biological municipal wastewater treatment. Water Res. 2009, 43, 2969– 2976. (15) Standard Methods for the Examination of Water and Wastewater, 20th ed.; American Public Health Association (APHA), American Water Works Association, and Water Environment Federation: Washington, DC, 1998. (16) Herbert, D.; Philipps, P. J.; Strange, R. E. Methods Enzymol. 1971, 5B, 265–277. (17) Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 1951, 193, 265–275. (18) Miron, Y.; Zeeman, G.; Vanlier, J. B.; Lettinga, G. The role of sludge retention time in the hydrolysis and acidification of lipids, carbohydrates and proteins during digestion of primary sludge in CSTR systems. Water Res. 2000, 34, 1705–1713. (19) Chung, Y. C.; Neetheling, J. B. ATP as a measure of anaerobic sludge digester activity. Water Pollut. Control Fed. 1988, 60, 107–112. VOL. 44, NO. 12, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

9

4807

(20) Amann, R. I.; Gish, W.; Miller, W.; Myers, E. W.; Lipman, D. J. Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations. Appl. Environ. Microbiol. 1990, 56, 1919–1925. (21) Stahl, D. A.; Flesher, B.; Mansfield, H. R.; Montgomery, L. Use of phylogenetically based hybridization probes for studies of ruminal microbial ecology. Appl. Environ. Microbiol. 1988, 54, 1079–1084. (22) Rittmann, B. E.; McCarty, P. L. Environmental Biotechnology: Principles and Applications; McGraw-Hill Inc.: New York, 2001. (23) Wang, Q. H.; Kuninobu, M.; Ogawa, H. I.; Kato, Y. Degradation of volatile fatty acids in highly efficient anaerobic digestion. Biomass Bioenergy 1999, 16, 407–416. (24) Ozturk, M. Conversion of acetate, propionate and butyrate to methane under thermophilic conditions in batch reactors. Water Res. 1991, 25, 1509–1513. (25) Fukuzaki, S.; Nishio, N.; Shobayashi, M.; Nagai, S. Inhibition of the fermentation of propionate to methane by hydrogen, acetate and propionate. Appl. Environ. Microbiol. 1990, 56, 719–723. (26) Salminen, E. A.; Rintala, J. A. Semi-continuous anaerobic digestion of solid poultry slaughterhouse waste: effects of hydraulic retention time and loading. Water Res. 2002, 36, 3175– 3182.

4808

9

ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 44, NO. 12, 2010

(27) Speece, R. E. Anaerobic biotechnology for industrial wastewater treatment. Environ. Sci. Technol. 1983, 17, 416–427. (28) Speece, R. E.; Gallagher, D.; Parkin, G. F. Nickel stimulation of anaerobic digestion. Water Res. 1983, 17, 677–683. (29) Zehnder, A. J. B.; Brock, T. D. Anaerobic methane oxidation: occurrence and ecology. Appl. Environ. Microbiol. 1980, 39, 194–204. (30) Yu, Y.; Hansen, C. L.; Hwang, S. Biokinetics in acidogenesis of highly suspended organic wastewater by adenosine 5′ triphosphate analysis. Biotechnol. Bioeng. 2002, 78, 147–156. (31) Perle, M.; Kimchie, S.; Shelef, G. Some biochemical aspects of the anaerobic degradation of dairy wastewater. Water Res. 1995, 29, 1549–1554. (32) Le, M. J.; Roger, P. Production, oxidation, emission and consumption of methane by soils: a review. Eur. J. Soil Biol. 2001, 37, 25–50. (33) Groβkopf, R.; Janssen, P.; Liesack, W. Diversity and structure of the methanogenic community in anoxic rice paddy soil microcosms as examined by cultivation and direct 16S rRNA gene sequence retrieval. Appl. Environ. Microbiol. 1998, 64, 960–969.

ES1000209