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Energy & Fuels 2008, 22, 98–102
Hydrogen Fermentation and Methane Production from Sludge with Pretreatments† Tsai-Wu Jan,‡ Sunil S. Adav,‡ D. J. Lee,*,‡ R. M. Wu,§ Ay Su,| and Joo-Hwa Tay⊥ Department of Chemical Engineering, National Taiwan UniVersity, Taipei 10617, Taiwan, Department of Chemical and Materials Engineering, Tamkang UniVersity, Taipei County 10617, Taiwan, Fuel Cell Research Center, Department of Mechanical Engineering, Yuan-Ze UniVersity, Taoyuan 300, Taiwan, and Institute of EnVironmental Science and Engineering, Nanyang Technological UniVersity, Singapore ReceiVed May 27, 2007. ReVised Manuscript ReceiVed July 26, 2007
Excess wastewater sludge collected from the recycling stream of the wastewater treatment process of the food industry is biomass that has a high potential to produce energy. This work examined the anaerobic digestion of wastewater sludge using a Clostridium strain isolated from the sludge as the seed sludge. Four pretreatments (acidification, basification, freezing/thawing, and sterilization) were applied on wastewater sludge, and their effects on biogas yields were examined. The suspension pH and the presence of strains in seed sludge and sludge substrate significantly affected the fermentation process for wastewater sludge. The pretreatment using basification, freezing and thawing, and sterilization enhanced methane production. A critical pH of above 5.5 was needed to initiate the methanogenesis stage. Acidification could yield control of the fermentation process of wastewater sludge to separate into two sequential stages.
1. Introduction Bioconversion of biomass to produce hydrogen has been demonstrated, using the anaerobic fermentation of some welldefined compounds in water.1–4 Wang et al.5 conducted the first systematic study of the production of hydrogen from wastewater sludge and found a rather high hydrogen yield from wastewater sludge using a Clostridium strain isolated from the sludge sample. Later, Wang et al.6 claimed that applying the filtrate of the sludge could produce more hydrogen than could be obtained using all of the particles in the sludge. Although these studies successfully established the feasibility of producing hydrogen from wastewater sludge, the hydrogen formed during the first 16–24 h of fermentation was consumed in a later stage. Wang et al.5 blocked the methanogenetic pathway using a pretreatment. However, much of the produced hydrogen was still consumed. The pathway for hydrogen consumption remains unknown but is of academic and practical interest.7 Wang et al.8 identified the strain in hydrogen-producing liquor with wastewater sludge. * To whom correspondence should be addressed. Fax: +886-2-23623040. E-mail:
[email protected]. † Presented at the International Conference on Bioenergy Outlook 2007, Singapore, April 26–27, 2007. ‡ National Taiwan University. § Tamkang University. | Yuan-Ze University. ⊥ Nanyang Technological University. (1) Lin, C. Y.; Chang, R. C. J. Chem. Technol. Biotechnol. 1999, 74, 498–500. (2) Lay, J. J. Biotechnol. Bioeng. 2000, 68, 269–278. (3) Bai, M. D.; Cheng, S. S.; Tseng, T. C. Proceedings of the IWA Asia– Pacific Regional Conference (Water Quality 2001), Fukuoka, Japan, 2001. (4) Wu, K. J.; Chang, J. S.; Chang, C. F. J. Chin. Inst. Chem. Eng. 2006, 37, 545–550. (5) Wang, C. C.; Chang, C. W.; Chu, C. P.; Lee, D. J.; Chang, B. V.; Liao, C. S. J. Biotechnol. 2003, 102, 83–92. (6) Wang, C. C.; Chang, C. W.; Chu, C. P.; Lee, D. J.; Chang, B. V.; Liao, C. S.; Tay, J. H. Water Res. 2003, 37, 2789–2793.
The strain was identified as a Clostridium species using the polymerase chain reaction (PCR) and 16S DNA sequence analysis. The residual liquor after hydrogen fermentation is strong liquor containing a high level of organic substances. Ting et al.9 and Lee and Mueller10 demonstrated that the sequential production of hydrogen and methane could be an ideal solution to both extract energy and remove pollutants from waste streams. In their preliminary study, some pretreatments were shown to enhance, but the others may deteriorate the hydrogen yield from fermented sludge. However, the mechanisms corresponding to the noted difference were not fully comprehended. This work aims at monitoring the anaerobic fermentation of wastewater sludge, original and pretreated, by observing the yields of hydrogen and methane using the Clostridium strain isolated by Wang et al.8 The metabolites were tracked to provide insight into the digestion process. The original and pretreated wastewater sludges were tested. 2. Experimental Section 2.1. Samples and Pretreatments. Waste-activated sludge was extracted from a wastewater treatment plant of the Uni-President Oven Bakery Corp., Taiwan, which daily treats 250 tons of foodprocessing wastewater using primary, secondary, and tertiary treatments. The pH of the sludge was about 6.4. The chemical oxygen demand (COD) of the sludge and filtrate (through a 0.45 µm membrane) was 9600 mg L-1 (total COD), as determined by directly reading a spectrometer (DR/2000, HACH, Loveland, CO). (7) Wang, C. C.; Chang, C. W.; Chu, C. P.; Lee, D. J.; Chang, B. V.; Liao, C. S. J. EnViron. Sci. Health, Part A: Toxic/Hazard. Subst. EnViron. Eng. 2003, 38, 1867–1875. (8) Wang, C. C.; Chang, C. W.; Chu, C. P.; Lee, D. J.; Chang, B. V.; Liao, C. S. J. Chem. Technol. Biotechnol. 2004, 79, 426–427. (9) Ting, C. H.; Lin, K. R.; Lee, D. J.; Tay, J. H. Water Sci. Technol. 2004, 50, 223–228. (10) Lee, D. J.; Mueller, J. A. In From Sludge to Biosolids; Spinosa, L., Vesilind, P. A., Eds.; International Water Association (IWA): U.K., 2001.
10.1021/ef700278j CCC: $40.75 2008 American Chemical Society Published on Web 09/11/2007
Hydrogen Fermentation and Methane Production The elemental composition of the dried samples was C, 34.3%; H, 5.6%; and N, 5.5%, according to an elemental analyzer (PerkinElmer 2400 CHN). Four pretreatments (acidification, basification, freezing and thawing, and sterilization) were applied to the original sludge. These pretreatments resulted in the release of insoluble organic matter into water to enhance methane production.11 In acidification, perchloric acid (1 N HClO4) was mixed with the sludge sample for 10 min to adjust its pH to 3. Then, the sample was stored at 4 °C for 6 h. The final pH was 3.9. In basification, sodium hydroxide (NaOH) solution (3 M) was mixed with the sludge for 10 min to reach a pH of 11. Then, the sample was stored at 4 °C for 6 h. The final pH is 9.2. In freezing and thawing, the sludge was frozen at -17 °C for 24 h in a freezer and then thawed for another 12 h in a water bath at 25 °C. In sterilization, sludge samples were stored in an autoclave (Huxley, HL-360) at 121 °C and 1.2 atm for 30 min. 2.2. Inoculum Isolation. The inoculum was isolated from the collected wastewater sludge. The applied procedures include (i) sterilization at 121 °C and 1.2 atm for 30 min, (ii) addition of 100 mM bromoethane sulfonic acid (BESA) to the sterilized sludge from step i and incubation for 24 h under anaerobic conditions, and (iii) isolation and purification of the incubated strains. Step iii includes colonization of the incubated sludge on a reinforced clostridial medium for 72 h, 3× isolation of strains by removing and incubating colonies on the agar, and the final selection of the strains by preliminary hydrogen production tests. The strains with the highest hydrogen yields were used as the inoculum in this study. 2.3. PCR–Denaturing-Gradient Gel Electrophoresis (DGGE) Analysis. The V6–V8 regions of 16S rDNA contain information on the biodiversity in the collected samples. The variable region of V6–V8 therein collected 16S rDNA was amplified using primers F968 (5′-AAC GCG AAG AAC CTT AC-3′) and R1387 (5′-GGG CGG TGA GTA CCA GGC-3′). The 40 base pair GC clamp was attached to the forward primer. The PCR mixture (50 µL) consisted of 5 µL of PCR buffer (10×), 2.5 µL of MgCl2 (25 mM), 2.5 µL of dNTP (2.5 mM), 0.25 µL (100 mM) each of the primer solutions, 0.5 µL of Taq polymerase (Promega, Madison, WI), and 10 ng of template DNA. The DNA was amplified using an eppendorf mastercycler (Eppendorf AG, Hamburg, Germany) by denaturation at 94 °C for 3 min, 35 cycles consisting of 94 °C for 15 s, 58 °C for 30 s, 72 °C for 45 s, and final extension at 72 °C for 7 min. The PCR-amplified 16S rRNA was sequenced using the ABI Prism model 3730 (version 3.2) DNA sequencer. The sequence analysis of the 16S rDNA in the inoculum showed that it was Clostridium bifermentans. DGGE tests were conducted using the Bio-Rad universal mutation detection system with 6% (w/v) polyacrylamide gels. The range of denaturants [100% denaturant corresponds to 7 M urea and 40% (v/v) deionized formamide] was 35–65%. DGGE was performed at 60 °C for 10 h at 120 V. Gels were stained with ethidium bromide and photographed using a DigiGel digital image system. 2.4. Biogas Production Test. Substrate (45 mL; original or pretreated sludge) was mixed with 5 mL of inoculum suspension and anaerobically incubated at 35 °C in 125 mL serum bottles without stirring or further addition of nutrients. The bottles were capped with butyl rubber stoppers and wrapped in aluminum foil to prevent photolysis of the substrate. Gas and liquor samples were extracted at 8, 16, 24, 32, 40, 48, 72, 96, 120, 144, 168, 196, 216, 240, 264, 288, 336, 384, 432, and 480 h of fermentation. At each time interval and for each substrate (original or pretreated), three serum bottles were arbitrarily selected and their average of hydrogen concentrations was reported. After measurements, the selected samples were abandoned to prevent the introduction of possible errors as a result of sampling, including gas leakage. 2.5. Analytical Methods. A gas chromatography (GC)–thermal conductivity detector (TCD) (Shimadzu, GC-8A), equipped with a stainless column packed with Porapack Q (50/80 mesh) at 70 °C and a TCD, was used to measure the hydrogen and methane concentrations in the gas phase. The temperature of both the injector (11) Jean, D. S.; Chang, B. V.; Liao, G. S.; Tsou, G. W.; Lee, D. J. Water Sci. Technol. 2000, 42, 97–102.
Energy & Fuels, Vol. 22, No. 1, 2008 99 and detector of the GC was 100 °C. Nitrogen flowed at 20 mL min-1 as the carrying gas. An integrator (HP3396 series II) integrated the area under the peak of the effluent curve and quantified the gaseous concentrations. Repeated measurements revealed that the determined hydrogen and methane contents included a maximum relative error of 15 and 10%, respectively. The hydrogen content in the anaerobic glove box was also measured and subtracted from the hydrogen concentrations obtained in the serum bottles. The volatile fatty acids (VFAs) were measured by high-performance liquid chromatography (HPLC, Ecom LCP 4100 Pump, LCD 2083 detector) with the mobile phase as 0.1% of phosphoric acid at 0.5 mL min-1. The column used was C18, and the absorption peaks at UV 210 nm were recorded and analyzed by software Peak-ABC. The dry weight of sludge was measured according to standard methods.12 The ammonium nitrogen (NH3–N) concentration was measured by mixing a sample with Nessler reagents and scanned at 425 nm with a spectrophotometer (DR/2500, HACH, Loveland, CO). COD for the sludge and filtrate was determined by spectrometry (DR/2500, HACH, Loveland, CO). The protein content was measured by the Biuret method13 using bovine serum albumin as a standard. The carbohydrates were measured by the dinitrosalicylic acid (DNSA) method14 using glucose as the standard.
3. Results and Discussion 3.1. Pretreatments of Sludge. Table 1 lists the physiochemical characteristics of the six batches of tested samples. The soluble part of the original sludge had a COD of only 30 mg L-1, nearly neutral pH, and nondetectable VFAs. The NH3–N level was about 21 mg L-1. The seed sludge contained a high level of COD (2200 mg L-1), mainly in the form of VFAs. Acidification yielded a slight increase in soluble COD (SCOD, 300 mg L-1), by transferring some solids to formate, acetate, and lactate. Basification and freezing and thawing increased the soluble fraction of carbohydrates for increased SCOD. Their effects on the VFA distributions are different. Sterilization largely dissolved carbohydrates to suspension but not in the form of VFAs. 3.2. Fermentation Test. Figure 1 depicts the time evolution of solution pH, carbohydrates, NH3–N, and protein concentrations of the samples under anaerobic digestion tests. The pH of the samples, except for acidified sample, first decreased and then increased, eventually approached 6.5–7.0 (Figure 1a). The soluble carbohydrate contents of the sterilized sample slowly decreased from 300 to 230 mg L-1, while those of the acidified sample increased from 80 to 160 mg L-1 (Figure 1b). The quantities of NH3–N of all tested samples quickly approached certain plateau values and remained there (Figure 1c), indicating that the degradable nitrogen-containing compounds were mostly hydrolyzed in the first 100 h of fermentation. The concentrations of soluble proteins in all tested samples remained low during the test (Figure 1d). The removal ratios of TCOD for samples 1–6 following 600 h of fermentation were 18.0, 30.6, 8.5, 31.5, 29.8, and 28.9%, respectively. Restated, the acidified sludge with seed presented the worst case for organics degradation, only about 47% of the original sludge without seed. 3.3. Biogas Production and Metabolites. Figure 2 shows the hydrogen and methane yields during fermentation tests and the VFA levels in the suspensions for sample 1. This process is a hydrogen-consuming process, with acetate and propionate (12) American Public Health Association (APHA). Standard Methods for the Examination of Water and Wastewater, 20th ed.; APHA: Washington, D.C., 1998. (13) Gornall, A. G.; Bardawill, C. S.; David, M. M. J. Biol. Chem. 1949, 177, 751–756. (14) Miller, G. L. Anal. Chem. 1959, 31, 426–428.
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Table 1. Characteristics of Tested Samples before the Anaerobic Digestion Testa sample 1
sample 2
sample 3
sample 4
sample 5
sample 6
species and items (units)
sludge only
sludge plus seed
acidified sludge plus seed
basified sludge plus seed
F/T sludge plus seed
sterilized sludge plus seed
formate (mg L-1) acetate (mg L-1) propionate (mg L-1) butyrate (mg L-1) lactate (mg L-1) succinate (mg L-1) pH carbohydrates (mg L-1) NH3–N (mg L-1) proteins (mg L-1) SCOD (mg L-1) TCOD (mg L-1)
0 0 0 0 0 0 6.67 5.21 20.8 1.49 30 6050
228 356 267 22.9 200 160 6.01 79.7 82.8 3.44 2260 9010
305 428 262 27.4 335 167 4.72 82.0 77.8 3.91 2560 8720
73.4 199 267 26.8 213 165 6.87 115 133 16.8 2980 8880
241 376 262 22.4 260 167 5.71 100 95.4 15.9 2660 8940
218 376 230 47.4 197 146 6.01 291 87.9 13.6 3240 9040
a
All measurements, except for TCOD, were made on the supernatant following 0.45 µm filtration.
Figure 2. Hydrogen and methane yields and end products in liquid of the original sludge.
Figure 1. pH and soluble compounds in fermentation tests.
mainly accumulated in the suspension up to 100 h. Afterward, butyrate started to form. Methane was noted to be yielded since 100 h and reached 22 g kg-1 dried solids (DSs) at 600 h. The acetate and butyrate were consumed accordingly. As Figure 1 shows, the pH of the suspension continuously dropped to around 6.0 on day 120, corresponding to the production of VFA as shown in Figure 2. However, the development of the subsequent methanogenesis stage raised the pH to around 6.8 by producing alkalinity as a byproduct. Figure 3 shows the hydrogen and methane concentrations in the head spaces of serum bottles during fermentation tests and the VFA levels in the suspensions when the freezing and thawing sludge was used (sample 5). The patterns noted for basified and sterilized samples (samples 5 and 6) resembled that depicted in Figure 3 and were not shown here for brevity sake. Hydrogen was formed and peaked at hour 16 and then dropped to a low level at >60 h. The formate and succinate in the suspension were consumed rapidly. Acetate, propionate, and
Figure 3. Hydrogen and methane yields and end products in liquid of the freeze-and-thaw sludge.
butyrate were formed in this period (Figure 3). In this period, the pH of the suspension dropped to 5.6 for the freezing and thawing sludge (Figure 1). Accordingly, the pH of suspensions was increased to around 6.6 with a stable methanogenesis process (Figure 1).
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Figure 4. Hydrogen and methane yields and end products in liquid of the acidified sludge.
Figure 4 shows the tests with acidified sludge. Unlike the other tests, hydrogen was formed and consumed over 0–70 h, while methane was produced until t > 400 h. Restated, the establishment of stable methanogenesis was significantly delayed for the acidified sludge system. Over an idle period of 300 h, no noticeable changes occurred in the reactor. The formate was first consumed at t > 350 h to produce methane. While hydrogen was produced, lactate was consumed at the first stage t < 40 h, resulting in a peak level of hydrogen production. The decrease in the hydrogen production rate resulted an increase in lactate content (t < 40–80 h) that stabilized the reactor pH. The changes in suspension pH for the acidified sludge test were worthy of further discussion. Researchers have hypothesized that the methane produced in bogs and peatlands occurs in localized, higher pH microniches and within the neutral pH niche inside the microorganism itself.15 While these environments are the primary source of atmospheric methane generated from H2/CO2,16 it has proven difficult to isolate methanogenic organisms capable of growing at low pH.17 Williams and Crawford successfully18 enriched cultures that generate methane at pH 3.0–4.0 as well as isolates of Methanobacterium capable of growth, and methane productions at pH values of 4.68 were isolated by Patel et al.19 Maestrojuan and Boon20 also noted that substrate use was affected by pH, because their isolates preferred acidic pH conditions when grown using H2/methanol and H2/CO2 and exhibited a significantly slower growth using acetate under acidic conditions. Taconi et al.21 reported a 30% increase in methane production when the initial pH was decreased from 7.0 to 4.5 during methanogenic digestion of a synthetic acetic acid wastewater inoculated using a mixed culture. Over 0–24 h, the microbes in seed were in a stationary phase. Then, hydrogen was produced together with an accumulation (15) Duval, B.; Goodwin, S. Int. Microbiol. 2000, 3, 89–95. (16) Goodwin, S.; Zeikus, G. Appl. EnViron. Microb. 1987, 53, 57–64. (17) Horn, M.; Matthies, C.; Kusel, K.; Schramm, A.; Drake, H. Appl. EnViron. Microb. 2003, 69, 74–83. (18) Williams, R.; Crawford, R. Appl. EnViron. Microb. 1985, 50, 1542– 1544. (19) Patel, G.; Sprott, G.; Fein, J. Int. J. Syst. Bacteriol. 1990, 40, 12– 18. (20) Maestrojuan, G.; Boone, D. Int. J. Syst. Bacteriol. 1991, 41, 267– 274. (21) Taconi, K. A.; Zappi, M. E.; French, T. W.; Brown, L. R. Bioresour. Technol. 2007, 98, 1579–1585.
Figure 5. Microbial community change during fermentation tests at t ) 0, 8, 16, 24, 32, 40, 48, 72, 96, 120, 144, 168, 192, 240, 288, 360, 432, 504, and 600 h, respectively (from left to right) of the acidified sludge.
Figure 6. Microbial community change during fermentation tests at t ) 0, 8, 16, 24, 32, 40, 48, 72, 96, 120, 144, 168, 192, 240, 288, 360, 432, 504, and 600 h, respectively (from left to right) of the freeze– thawed sludge.
of acetate and butyrate in suspension (Figure 4). However, the pH of the suspension was increased to around 6.6 around hour 40. The hydrolysis of proteins, yielding NH3–N in suspension, may be attributable to the noted pH changes. However, the pH declined again and reached around 5.4 at t > 96 h. This pH value, although only slightly lower than that reached in the freezing and thawing sludge (5.6 in Figure 3), significantly retarded the activity of methanogenic bacteria. The methane yield at hour 600 was only around 9 g kg-1 DSs. The low yield of methane may be attributable to the long time needed for microorganisms required to adapt to the acidic environment. 3.4. Microbes. The strains isolated from the original sludge included Clostridium butyricum, Vellonella ratti, Methanobac
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-terium formicium, and Methanobacterium subterraneum. These four strains and the Clostridium bifermentans from the seed sludge were the dominating species in the experiments. Figures 5 and 6 show the microbial community changes in acidified and freeze–thawed sludge tests. Other tests revealed a similar pattern but occurred at different characteristic times. The bands for C. bifermentans and V. ratti were noted over the entire fermentation test. The strain C. butyricum was present up to hour 144. Restated, in the initial stage, the Clostridium strains produced VFAs and protons, while V. ratti may convert sulfate to sulfide with VFA degradation. The strains M. formicium and M. subterraneum started to be present since hour 150, corresponding to the methane production at >400 h. As noted above, suspension pH and the presence of C. bifermentans in seed sludge and sludge substrate (C. butyricum, V. ratti, M. formicium, and M. subterraneum) significantly affected the fermentation process for wastewater sludge, including the biogas production and metabolite distribution aspects. A critical pH of around 5.5 determined whether the methanogenesis stage could be initiated. The pretreatment using basification, freezing and thawing, and sterilization enhanced methane production. However, the hydrogen yield and methane production process cannot be controlled. On the other hand, acidification yields a chance to precisely control the fermentation process of wastewater sludge, making possible the combined production of hydrogen and methane from two sequential reactors.
Jan et al.
4. Conclusions This work examined the anaerobic digestion of wastewater sludge with four pretreatments (acidification, basification, freezing/thawing, and sterilization). The gaseous products, metabolites, and microbes in tested samples were probed over time. The adopted pretreatments released a small quantity of insoluble organic matter into water but could enhance biogas production. Over 600 h of fermentation, the removal ratios of TCOD were 18.0, 8.5, 31.5, 29.8, and 28.9% for tests with original sludge, acidified sludge, basified sludge, freezing and thawing sludge, and sterilized sludge, respectively. Different pretreatments had distinct impacts on the biogas production potentials. The suspension pH and the presence of strains in seed sludge (C. bifermentans) and sludge substrate (C. butyricum, V. ratti, M. formicium, and M. subterraneum) were noted to significantly affect the fermentation process. A critical pH of above 5.5 was noted to initiate the methanogenesis stage. Restated, because the pH of suspensions from basified, freezing and thawing, and sterilized sludge samples would not drop to lower than 5.5, methanogenesis occurs right after the consumption of hydrogen gas. On the other hand, acidification yielded a suspension pH of 5.4, hence leading to a chance of precise control of the fermentation process of wastewater sludge. Microbial strains corresponding to each stage of the fermentation reaction were identified. EF700278J
