Environ. Sci. Technol. 2004, 38, 2934-2939
Identification of Inhibitory Substances Affecting Bioleaching of Heavy Metals from Anaerobically Digested Sewage Sludge X I A N G Y A N G G U †,‡ A N D J O N A T H A N W . C . W O N G * ,† Department of Biology, Hong Kong Baptist University, Kowloon Tong, Hong Kong SAR, and Department of Microbiology, Nanjing Agricultural University, Nanjing 210095, People’s Republic of China
Significant inhibitory effects of the filtrate medium of anaerobically digested sewage sludge on iron oxidation by Acidithiobacillus ferrooxidans ANYL-1 were observed in our preliminary experiments, indicating the presence of inhibitory substances in anaerobically digested sewage sludge. The objectives of the present study were to identify the possible inhibitory substances and to evaluate their impacts on metal solubilization during bioleaching of sewage sludge. The results showed that the concentrations of total reducing sugars, all tested metal ions, and anions were too low to suppress iron oxidation, and only organic acids, especially acetic and propionic acids, were found at concentrations higher than their inhibitory levels. The presence of 10.8 mM acetic acid and 9.88 mM propionic acid in sewage sludge (sludge N) led to long lag periods of 6 and 7 days for solubilization of Cu and Cr, respectively, as compared to a lag period of only 1 day in the control and another sludge (sludge S) with a low level of organic acids. Meanwhile the leaching time for maximum solubilization of Zn also extended to 6 days in the presence of organic acids as compared to 3 days in the control. Acetic and propionic acids posed an unfavorable bioleaching condition for anaerobically digested sewage sludge; therefore, further studies are required to explore the means to remove the inhibitory effects to improve the heavy metal bioleaching efficiency.
sewage sludge before land application, and the bioleaching technique appears to be more attractive due to its lower chemical consumption (10). During the bioleaching process, metal solubilization can be achieved by acidification of the sludge through ferrous iron oxidation by Acidithiobacillus ferrooxidans (3, 10-15) or sulfur oxidation by Acidithiobacillus thiooxidans (16, 17). However, the sulfur-based bioleaching process is restricted by residual sulfur in decontaminated sludge, which might lead to secondary pollution such as sludge or soil reacidification (15). In the iron-based bioleaching process, acidification of the sludge may be brought about by ferrous iron oxidation (eq 1) followed by precipitation of ferric iron in the form of jarosite (eq 2). A. ferrooxidans
4Fe2+ + O2 + 2H+ 98 4Fe3+ + 2H2O
(1)
3Fe3+ + X+ + 2HSO4- + 6H2O f
XFe3(SO4)2(OH)6 + 8H+ (2)
Precipitation of Fe3+ as ferric hydroxide may also contribute to reduction of the sludge pH (13). This process could be initiated either by standard strains from the American Type Culture Collection Center (ATCC) (3, 10-12) or by indigenous strains of A. ferrooxidans (13, 14). However, these processes were initiated at pH 4.0, and preacidification of the sludge to pH 4.0 with sulfuric acid made this process more expensive. Recently a new bioleaching approach without preacidification was developed using indigenous iron-oxidizing bacteria, and the bioleaching time could be reduced to 2-4 days (15). However, A. ferrooxidans is well-known for its sensitivity to various kinds of organic compounds such as simple sugars, amino acids, and organic acids (18, 19). Sludge is rich in organic compounds, which may affect the growth of bioleaching bacteria and thus metal solubilization efficiencies. Significant inhibitory effects of anaerobically digested sludge or sludge extract on iron oxidation and metal solubilization have been reported and attributed generally to soluble organic matters present in sewage sludge (20, 21). Unfortunately, no specific inhibitory compounds have been identified in these bioleaching systems. Therefore, the objectives of the present study were to identify the possible inhibitory substance(s) present in anaerobically digested sludge, and to evaluate their impacts on iron oxidation and metal solubilization during bioleaching of sewage sludge.
Materials and Methods Introduction Land application represents the most economical way for final disposal of residual sludge as it combines the recycling of plant nutrients and sludge disposal at the same time. Unfortunately, the presence of high levels of heavy metals in sewage sludge often limits its use as a fertilizer (1, 2). The heavy metal content in sewage sludge is about 0.5-2% on a dry weight basis (3), and in some cases, extremely high concentrations of up to 4% of the total metal have been reported (4). Uptake of heavy metals by plants and subsequent accumulation along the food chain is a potential threat to animal and human health (5). Chemical leaching (6, 7) and bacterial leaching (5, 8, 9) have been developed for decontamination of metal-laden * Corresponding author phone: 852-3411-7056; fax: 852-34115995; e-mail:
[email protected]. † Hong Kong Baptist University. ‡ Nanjing Agricultural University. 2934
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Microorganism and Inoculum Preparation. An indigenous iron-oxidizing bacterium (strain ANYL-1) isolated from the sludge in the Yuen Long Sewage Treatment Plant in Hong Kong was used in the present study. It was a Gram-negative, rod-shaped bacterium which utilized either ferrous iron or elemental sulfur as the sole energy source. The optimum growth conditions were 30-35 °C and pH 2-2.5 in mineral salt iron medium. It was identified as A. ferrooxidans according to Kelly and Wood (22). This bacterium was grown and maintained in liquid FeP medium (23) at pH 2.0 with 20 g L-1 FeSO4‚7H2O as the energy source. The inoculum was prepared by growing the bacterium in 500 mL conical flasks containing 150 mL of FeP medium. The stock culture was initially activated in fresh medium three consecutive times, after which the active growing culture of 36 h age was used in toxicity and bioleaching tests. Effects of the Sludge Filtrate on Ferrous Iron Oxidation by A. ferrooxidans ANYL-1. Sludge filtrate media were freshly prepared from the sludges collected in September (sludge S) 10.1021/es0347134 CCC: $27.50
2004 American Chemical Society Published on Web 04/10/2004
TABLE 1. Selected Properties of Anaerobically Digested Sludge Collected from the Yuen Long Sewage Treatment Plant
sludge pH S N
solids [Zn] [Cu] [Cr] ORP content total [N] total [P] (mg (mg (mg (mV) (%) (g kg-1) (g kg-1) kg-1) kg-1) kg-1)
7.15 -283 7.09 -243
1.66 2.99
55.5 41.3
25.8 23.0
13301 153 79.2 27609 111 128
and November (sludge N) 2002 from a sewage treatment plant at Yuen Long District, Hong Kong. Selected physical and chemical properties of the sludge are shown in Table 1. Each sludge was centrifuged at 10000 rpm for 10 min, and the supernatant was adjusted to pH 2.0 with 1:4 (v/v) H2SO4 before filtering through a 0.45 µm mixed cellulose ester membrane (ADVANTEC MFS Inc.) to obtain the sludge filtrate medium. In a 500 mL flask, 112.5 mL of sludge filtrate media, 15 mL of membrane-filtered 20% FeSO4‚7H2O (m/v, equivalent to 0.072 M Fe2+ in final concentration), and a 22.5 mL inoculum of active growing bacteria were added. All flasks were incubated on a gyratory shaker at 30 °C and 180 rpm, and 2 mL samples were withdrawn from the conical flask at 6 h intervals for determination of ferrous iron by using the 1,10-phenanthroline method (24). Another portion of the sludge filtrate (pH 7.09) was dialyzed (MWCO e 1000) against distilled water at 4 °C for 48 h to remove small molecules before the adjustment of the pH to 2.0. Iron oxidation in the medium of the dialyzed sludge filtrate was also determined for comparison. Liquid medium FeP was used as the control. Identification of Potential Inhibitory Substances in the Sludge Filtrate. It is almost impossible to identify all kinds of compounds present in the sludge, but it is reasonable to confine the scope of the present study to those small molecular weight soluble substances which may have a higher capability of inhibition. Insoluble particles or macromolecules, such as cellulose, could be considered nontoxic due to their inability to enter the microbial cells through the cell membrane. In the present study, the inhibitory concentrations of selected metal ions (Zn2+ and Cu2+), anions (Cl- and NO3-), mono- and disaccharides (cellobiose, fructose, galactose, glucose, and sucrose), and organic acids (formic acid, acetic acid, propionic acid, and butyric acid) were determined using the synthetic medium FeP (23). The effects of potential inhibitory substances on iron oxidation by A. ferrooxidans ANTL-1 were studied in 250 mL flasks, each containing 100 mL of synthetic medium with 20 g L-1 FeSO4‚7H2O as the energy source and 15% active iron-oxidizing bacteria as the inoculum in the presence of different selected concentrations of the inhibitory substances. The concentration ranges of potential inhibitory substances were determined in preliminary experiments. The whole setup was incubated on a gyratory shaker at 30 °C and 180 rpm. After 36 h of incubation, the concentration of ferrous iron in the medium was determined by the 1,10-phenanthroline method (24). The iron oxidation rate was calculated from the initial and final concentrations of ferrous iron after 36 h of incubation according to eq 3, where [Fe2+] is the concentration of Fe2+ (mg L-1) and 4000 is the initial concentration of Fe2+ (mg L-1). The inhibitory effect was expressed as inhibition percentage as shown in eq 4:
iron oxidation rate (%) )
4000 - [Fe2+] × 100 4000
(3)
inhibition % ) 1 - iron oxidation rate (%) in the presence of inhibitory substance × 100 (4) iron oxidation rate (%) in the control
The EC50 value, which is the effective concentration causing a 50% reduction in the iron oxidation rate as compared with that of the control, was also calculated (25). To bring about significant inhibitory effects, any inhibitory substance present in sewage sludge should have a concentration significantly higher than its inhibitory concentration. Therefore, the major inhibitory substances could be easily identified by comparison of the concentrations of the compounds present in the original sludge with their inhibitory levels. Monitoring of Potential Inhibitory Compounds in Anaerobically Digested Sludge. Anaerobically digested sludge was periodically sampled from the Yuen Long Sewage Treatment Plant. The sludge was centrifuged at 10000 rpm for 10 min, and the supernatant was filtered through a 0.45 µm mixed cellulose ester membrane (ADVANTEC MFS Inc.) before chemical analysis. Heavy metals were determined by atomic absorption spectroscopy (24). Chloride, nitrate, and nitrite were determined by standard methods (24). Total reducing sugars in the sludge were determined by the dinitrosalicylic acid reagent method (26). Organic acids in the sludge filtrate were determined by ion chromatography (27). Bioleaching Assays. The bioleaching was conducted in 500 mL conical flasks, each containing 170 mL of sludge (sludge N) at a 1% solid content, 4 g of FeSO4‚7H2O, and a 30 mL active growing culture of A. ferrooxidans ANYL-1. The initial concentration of iron-oxidizing bacteria was about 1.2 × 107 cells mL-1. To prevent any possible biased bioleaching results caused by the difference in the physicochemical properties of both sludges, sludge N with its liquid portion replaced by the filtrate of sludge S was used as the control. Low organic acid contents observed in sludge S (0.09 mM acetic acid and 0.08 mM propionic acid) might be due to the low organic loading rate in the sampling period as indicated by the significantly lower solids content as compared with that of sludge N (Table 1). Sludge S and sludge N with its liquid phase being replaced by that of sludge S followed by artificial addition of organic acids at levels similar to those in sludge N were also included for comparison purposes. The whole setup was incubated in a gyratory incubator at 30 °C and 180 rpm. pH and ORP were measured at either 1 or 2 day intervals followed by a sampling of 8 mL of the sludge mixture from each of the flasks for chemical analyses. The sludge samples were centrifuged at 10000 rpm for 10 min to separate the solids from the liquid fraction. The supernatant was filtered, acidified to about pH 1.0 with concentrated HNO3, and then stored at 4 °C prior to determination of soluble Zn, Cr, or Cu by AAS. Ferrous and ferric iron were determined by the 1,10-phenanthroline method immediately before acidification (24). All treatments and controls were done in triplicate.
Results and Discussion Ferrous Iron Oxidation by A. ferrooxidans ANYL-1 in Sludge Filtrate Media. A. ferrooxidans ANYL-1 showed the same efficiency for iron oxidation in the filtrate medium of sludge S and the synthetic medium, and complete iron oxidation could be achieved within 36 h of incubation (Figure 1). However, iron oxidation was totally inhibited in the filtrate medium of sludge N, and less than 5% of the ferrous iron was oxidized during the whole incubation period, indicating the presence of inhibitory substances in sludge N. When the filtrate of sludge N was subjected to dialysis (MWCO e 1000), the iron oxidation rate increased to a level similar to that in the synthetic medium (Figure 1). This indicated that these inhibitory compounds were small molecules with molecular weight e 1000. Similar inhibitory effects were also observed by Fournier et al. (20) and Cho et al. (21) and were generally attributed to soluble organic matter present in sewage sludge, but the compounds responsible for this inhibition are still VOL. 38, NO. 10, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 2. Concentrations of Selected Inhibitory Substances Present in Sludge N Compared to Their EC50 Values potential inhibitory compound
concn in sludge N (mM)
EC50 (mM)
potential inhibitory compound
concn in sludge N EC50 (mM) (mM)
Zn2+
4.22a
398
Cations Cu2+
0.018a
24
Cl-
3.61
198
Anions NO3-
BDLb
61
cellobiose fructose galactose
0.54c
Simple Sugars 438 glucose 126 sucrose 281
formic acid acetic acid
BDL 10.8
Organic Acids 0.063 propionic acid 0.23 butyric acid
382 491
9.88 BDL
0.42 0.38
a The highest concentration of soluble metal ions in the liquid phase assuming a solubilization rate of 100%. b BDL, below detection limit. c Total reducing sugars.
FIGURE 1. Effects of the sludge filtrate on iron oxidation by A. ferrooxidans ANYL-1. Symbols: ×, synthetic medium (control); 4, filtrate medium of sludge S; 0, filtrate medium of sludge N; 2, dialyzed sludge filtrate N; 9, dialyzed sludge filtrate N plus 10.8 mM acetic acid and 9.88 mM propionic acid.
FIGURE 2. Effects of selected metal ions, anions, simple sugars, and organic acids on iron oxidation by A. ferrooxidans ANYL-1. unknown. Hence, it is essential to identify the potential inhibitory substances present in sludge N and to monitor their dynamics during the anaerobic digestion of sewage sludge since identifying and controlling the potential inhibitory substances may pave the way for further improving the performance of these sludge bioleaching systems. Identification of Inhibitory Substance(s) in Sludge N. To identify the potential inhibitory substance(s) present in sludge N, the inhibitory concentrations and EC50 values of selected metal ions, anions, mono- and disaccharides, and low molecular weight organic acids were first determined in the synthetic medium and then compared with their respective concentrations detected in aludge N. The results are shown in Figure 2 and Table 2. A. ferrooxidans was generally considered to be tolerant of various heavy metals such as As3+, Cd2+, Co2+, Cu2+, and Zn2+ (28). In the present study, Zn2+ and Cu2+ had EC50 values of 398 and 24 mM, respectively 2936
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(Table 2), and the inhibitory effect occurred when Zn2+ and Cu2+ were added at concentrations g300 and g24 mM, respectively. Although the concentrations of soluble Zn2+ and Cu2+ in sludge N before bioleaching were e0.8 µM, they were expected to increase significantly due to metal solubilization during the bioleaching process. At an assumption of 100% solubilizaton of heavy metals from sludge N with a solids content of 1%, the calculated highest concentrations of Zn2+ and Cu2+ would be 4.22 and 0.018 mM, respectively, which were far below their inhibitory concentrations. Chromium was not expected to inhibit bacterial growth and iron oxidation since higher removal rates of Cr had been observed even when the sludge contained Cr at concentrations much higher than those of the present study (15). Therefore, the expected amount of dissolved metals from sludge N even at the highest dissolution efficiency would not inhibit bacterial growth and iron oxidation during the bioleaching process. The inhibitory concentrations of Cl- and NO3- were above 100 and 20 mM, respectively (Figure 2). However, the concentration of Cl- was only 3.61 mM, and NO3- ion in sludge N was below the detection limit. Normally nitrate would not accumulate due to denitrification under anaerobic conditions. Therefore, these two anions could also be excluded from the list of inhibitory substances affecting iron oxidation in the filtrate medium of sludge N. The effective concentrations which led to a 50% reduction in the iron oxidation rate (EC50) for mono- and disaccharides tested ranged from 126 to 491 mM and were higher than those of any other compounds except Zn2+ and Cl- (Table 2), confirming that they were not very toxic to A. ferrooxidans under normal growth conditions (19). In the present study toxic effects were observed only at concentrations higher than 400, 120, 200, 300, and 400 mM for cellobiose, fructose, galactose, glucose, and sucrose, respectively (Figure 2), while the content of total reducing sugars in sludge N was only 0.54 mM, which was too low to inhibit the growth of the iron-oxidizing bacteria. Among the compounds tested, organic acids were the most toxic as reflected by the lowest EC50 values of 0.0630.42 mM (Table 2). Formic acid was the most toxic one with iron oxidation almost totally inhibited at an extremely low concentration of 0.08 mM (Figure 2). The inhibitory concentrations of acetic acid, propionic acid, and butyric acid were g0.15, g0.3 and g0.3 mM, respectively. The addition of these organic acids at a concentration of 0.6 mM caused a significant reduction in iron oxidation to less than 30% that of the control (Figure 2). Low molecular weight organic
FIGURE 3. Dynamics of the concentration of organic acids in anaerobically digested sewage sludge in the Yuen Long Sewage Treatment Plant, Hong Kong.
FIGURE 5. pH and ORP changes during the bioleaching of anaerobically digested sewage sludge. Symbols: 4, sludge N; 0, sludge N with the liquid phase replaced by the filtrate of sludge S (control); ×, sludge S; ], sludge N with the liquid phase replaced by the filtrate of sludge S plus 10.8 mM acetic acid and 9.88 mM propionic acid.
FIGURE 4. Ferrous iron oxidation during the bioleaching of anaerobically digested sewage sludge. Symbols: 4, sludge N; 0, sludge N with the liquid phase replaced by the filtrate of sludge S (control); ×, sludge S; ], sludge N with the liquid phase replaced by the filtrate of sludge S plus 10.8 mM acetic acid and 9.88 mM propionic acid. acids are common metabolites of anaerobic digestion of organic matter (29) and are highly toxic to A. ferrooxidans (18). In the present study, 10.8 mM acetic acid and 9.88 mM propionic acid were found in sludge N, while other organic acids were below the detection limit. Therefore, acetic and propionic acids would likely be the major factors responsible for inhibition of iron oxidation in sludge filtrate N. This was further confirmed by the results from an 8 month monitoring of sludge quality, in which six out of the eight samples randomly collected from the Yuen Long Sewage Treatment Plant from September 2002 to April 2003 contained acetic and propionic acids at concentrations higher than their respective toxic levels (Figure 3). Ferrous Iron Oxidation during Bioleaching of Sludge N. During the bioleaching process, ferrous iron oxidation in sludge N was relatively slower as compared to that in the control or sludge S in which complete iron oxidation was achieved at day 3 (Figure 4). The presence of 10.8 mM acetic acid and 9.88 mM propionic acid in sludge N resulted in a long bioleaching period of 8 days to achieve complete iron oxidation. About one-fourth of the added ferrous iron was precipitated as ferrous iron hydroxide. After that almost all ferrous iron was oxidized to ferric iron, which remained in the sludge mixture at a concentration of about 1500 mg
L-1 until the end of the bioleaching period, while the concentration of ferrous iron was e25 mg L-1. Residual ferric iron in the sludge liquid phase was far below the theoretical concentration of about 3000 mg L-1, and this could be attributed to precipitation of ferric iron in the form of Fe(OH)3 or jarosite that represented key reactions controlling sludge pH and ORP during the iron-based bioleaching process (13). Due to the inhibition of iron oxidation caused by acetic and propionic acids, the time required to reach a pH < 2.5 and an ORP > 550 mV increased to 8 days compared to 2 days in the control or sludge S (Figure 5). Despite a delay in iron oxidation, the pH and ORP of sludge N were almost identical to those of the control after 8 days of bioleaching. This may be attributed to the removal of organic acids by heterotrophic microorganisms under aerobic conditions. Chemical analysis confirmed that both acetic acid and propionic acid disappeared after 5 days of bioleaching. Metal Solubilization during Bioleaching of Sludge N. Solubilization of Zn, Cu, and Cr during the bioleaching process is shown in Figure 6. Zinc solubilized quickly following addition of ferrous sulfate and inoculation of iron-oxidizing bacteria; maximum solubilization could be achieved before the pH decreased to below 2.5 (Figure 6). Solubilization of Zn could be attributed to a purely chemical reaction, which could be initiated at pH e 3.5 (15, 30). In the control treatment, complete removal of Zn was achieved after 3 days of bioleaching compared to 6 days in sludge N. Nevertheless, the final magnitude of Zn removal was almost the same for all three treatments. On the other hand, organic acids showed severe inhibitory effects on solubilization of Cu and Cr. There was a lag period of 6 and 7 days for Cu and Cr, respectively, before their solubilization took place in the sludge containing organic acids as compared with just 1 day in the control and sludge S. After the initial lag period, solubilization of Cu in the sludge without organic acids increased until it reached its highest level at day 4, and then stabilized more or less at this level during the rest of the bioleaching period. Meanwhile 10 days were required to obtain maximum removal of Cu from sludge N containing 10.8 mM acetic acid and 9.88 mM propionic VOL. 38, NO. 10, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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rather than acclimation of iron-oxidizing bacteria alone. Therefore, the bioleaching was actually initiated by both ironoxidizing bacteria and heterotrophic microorganisms, and an inhibitory effect might still exist in this system because the bioleaching time still required about 6-10 days for some of the sludge samples. In fact, the role of heterotrophic microorganisms in removing the inhibitory acetic and propionic acids has been confirmed in our subsequent experiments (data not shown). Until now there has been no report on the capability of A. ferrooxidans in decomposing or tolerating high concentrations of small molecular weight organic acids. The presence of organic acids represented an unfavorable bioleaching condition for iron oxidation, which would lead to a longer bioleaching time especially for solubilization of Cu and Cr from anaerobically digested sludge. Although removing organic acids by replacing the liquid phase with wastewater containing no organic acids can overcome this problem, separation of liquid from the solids followed by addition of water and possibly nutrient would increase the operational difficulties and also make this process more expensive. Therefore, further studies will be needed to improve the metal bioleaching efficiency in the presence of organic acids.
Acknowledgments The work described in this paper was fully supported by a research grant from the Research Grant Council of the Hong Kong Special Administrative Region, People’s Republic of China (Grant HKBU2010/99M). FIGURE 6. Metal solubilization during the bioleaching of anaerobically digested sewage sludge. Symbols: 4, sludge N; 0, sludge N with the liquid phase replaced by the filtrate of sludge S (control); ×, sludge S; ], sludge N with the liquid phase replaced by the filtrate of sludge S plus 10.8 mM acetic acid and 9.88 mM propionic acid. acid. A similar inhibitory effect on solubilization of Cr by organic acids was observed, but it showed a different bioleaching behavior. After an initial lag period, the removal of Cr increased steadily from day 2 to day 4 in the control and sludge S but showed only a slight increase in the rest of the bioleaching period. Unlike that in the control and sludge S, the removal rate of Cr increased steadily until the end of the bioleaching period in sludge N with organic acids (Figure 6). The maximum removal rates of Cu and Cr for sludge N were 86.4% and 56.1%, respectively, which were only slightly lower than those of the control, i.e., 97.5% and 63.3%, respectively. This might be due to the instability of the organic acids, which could be easily decomposed by heterotrophic microorganisms under aerobic conditions. In the present study there is no evidence for the presence of any other inhibitory substances in sludge N since artificial addition of similar amounts of organic acids into sludge N with the liquid fraction replaced by the filtrate of sludge S showed similar inhibitory effects on iron oxidation and metal solubilization (Figure 6). The presence of organic acids can also explain the low metal solubilization efficiencies reported by Fournier et al. (20) and Cho et al. (21) since autoclave-sterilized sewage sludge had been used in these two bioleaching systems, which might lead to an increased soluble fraction of organic matter (26). However, inhibitory substance(s) other than organic acids could not be ruled out. Blais et al. (13) reported that acclimation of the ironoxidizing microflora reduced the bioleaching time from 11-28 to 2-10 days for 23 sludges. However, repeated the adaptation procedure used in this study might also involve an enrichment of organic acid-degrading microorganism(s) 2938
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Literature Cited (1) Beckett, P. H. T.; Davis, R. D. Water Pollut. Control (Maidstone, Engl.) 1982, 81, 112-119. (2) Scheltinga, H. M. J. Water Sci. Technol. 1987, 19, 9-18. (3) Wong, L.; Henry, J. G. Water Sci. Technol. 1984, 17, 575-586. (4) Lester, J. N.; Sterrit, R. M.; Kirk, P. W. W. Sci. Total Environ. 1983, 30, 45-83. (5) Tyagi, R. D.; Couillard, D. In Encyclopedia of Environmental Control Technology, Water Treatment Technology; Chereminsinoff, P. E., Ed.; Gulf Publishing Co.: Houston, 1989; Vol. 3, pp 557-591. (6) Wozniak, D. J.; Heng, J. Y. C. J. Water Pollut. Control Fed. 1992, 54, 1574-1580. (7) Jenkins, R. L.; Scheybeler, B. J.; Smith, M. L.; Baird, R.; Lo, M. P.; Haug, R. T. J. Water Pollut. Control Fed. 1981, 53, 25-32. (8) Wong, L.; Henry, J. G. In Biotreatment System; Wise, D. L., Ed.; CRC Press: Boca Raton, FL, 1988; Vol. 2, pp 125-169. (9) Tyagi, R. D.; Couillard, D. In Biological Degradation of Wastes; Martin, A. M., Ed.; Elsevier Science Pulishers: New York, 1991; pp 307-322. (10) Tyagi, R. D.; Couillard, D.; Tran, F. Environ. Pollut. 1988, 50, 295-316. (11) Tyagi, R. D.; Couillard, D.; Grenier, Y. Environ. Pollut. 1991, 71, 57-67. (12) Couillard, D.; Zhu, S. Water, Air, Soil Pollut. 1992, 63, 67-80. (13) Blais, J. F.; Tyagi, R. D.; Auclair, J. C. J. Environ. Sci. Health 1993, A28, 443-467. (14) Tyagi, R. D.; Blais, J. F.; Auclair, J. C. Environ. Pollut. 1993, 82, 9-12. (15) Wong, J. W. C.; Xiang, L.; Chan, L. C. Water, Air, Soil Pollut. 2002, 138, 25-35. (16) Blais, J. F.; Tyagi, R. D.; Auclair, J. C. Water Sci. Technol. 1992, 26, 197-206. (17) Jain, D. K.; Tyagi, R. D. Enzyme Microb. Technol. 1992, 14, 376383. (18) Tuttle, J. H.; Dugan, P. D. Can. J. Microbiol. 1976, 22, 719-730. (19) Frattini, C. J.; Leduc, L. G.; Ferroni, G. D. Antonie van Leeuwenhoek 2000, 77, 57-64. (20) Fournier, D.; Lemieux, R.; Couillard, D. Environ. Pollut. 1998, 101, 303-309. (21) Cho, K. S.; Ryu, H. W.; Lee, I. S.; Choi, H. M. J. Air Waste Manage. Assoc. 2002, 52, 237-243.
(22) Kelly, D. P.; Wood, A. P. Int. J. Syst. Evol. Microbiol. 2000, 50, 511-516. (23) Johnson, D. B. J. Microbiol. Methods 1995, 23, 205-218. (24) APHA. Standard methods for the determination of waste and wastewater, 17th ed.; American Public Health Association: Washington, DC, 1985. (25) Wong, M. H.; Wong, J. W. C. Environ. Pollut. 1986, 40, 127-144. (26) Nelson, N. J. Biol. Chem. 1944, 153, 375-380. (27) Yang, H.; Wong, J. W. C.; Zhou, L. X.; Yang, Z. M. J. Instrum. Anal. 2001, 20, 19-21. (28) Leduc, L. G.; Ferroni, G. D. FEMS Microbiol. Rev. 1994, 14, 103120.
(29) Malina, J. F., Jr. In Design of anaerobic process for the treatment of industrial and municipal waste; Malina, J. F., Jr., Pohland, F. G., Eds.; Technomic Publishing: Lancaster, PA, 1992; pp 167212. (30) Blais, J. F.; Meunier, N.; Tyagi, R. D. Environ. Pollut. 1997, 18, 499-508.
Received for review July 4, 2003. Revised manuscript received November 27, 2003. Accepted February 23, 2004. ES0347134
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