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Effects of COD/SO42- Ratios on an Acidogenic Sulfate-Reducing Reactor Nan-Qi Ren,† Hong Chua,‡ Shing-Yan Chan,*,§ Yiu-Fai Tsang,§ and Ngai Sin‡ Research Centre of EnVironment and Biotechnology, Harbin UniVersity of CiVil Engineering and Architecture, Harbin 150008, People’s Republic of China, Joint Research Centre of Water and Wastewater Treatment Technology, The Hong Kong Polytechnic UniVersity, Hong Kong, and Department of CiVil and Structural Engineering, The Hong Kong Polytechnic UniVersity, Hung Hom, Kowloon, Hong Kong
Biotreatment process is widely used to treat high-strength sulfate-laden organic wastewater. By applying molasses wastewater as the sole organic carbon source and sodium sulfate as the electron acceptor in an acidogenic sulfate-reducing reactor, the electron flows of sulfate-reducing bacteria (SRB), the physiological metabolic pathways and products of SRB, the population dynamics of SRB, and the effect of sulfide production on hydrogen production were examined under different chemical oxygen demand/sulfate (COD/SO42-) ratios. The results showed that the number of electrons flowing to SRB was reduced when the COD/SO42- ratio increased and, hence, improved the sulfate removal rate. SRB was also the consumer of hydrogen and volatile fatty acids (VFAs) produced by acidogenic bacteria. The metabolic activities of SRB resulted in 45%-82% acetic acid in the terminal liquid products in the acidogenic reactor. The SRB also maintained a low-level hydrogen partial pressure by transferring hydrogen among microbial populations and guaranteed high performance stability in the acidogenic reactor. Introduction Four different trophic groups of bacteria are involved in the active decomposition of organic matters in an anaerobic environment. The first group consists the fermentative microorganisms, which hydrolyze higher-molecular-weight polymers and ferment their respective monomers to hydrogen, carbon dioxide, acetate, and other organic acids, and alcohols. The second group consists of acetogenic bacteria (AB), which cleave organic acids and alcohols into acetate, hydrogen, and carbon dioxide. The third group of microorganisms is formed by methogenic bacteria, which utilize hydrogen, carbon dioxide, acetate, and formate to produce methane. The fourth group is the sulfate-reducing bacteria, which compete with the methanogens and acetogens for available substrates.1 Sulfate-reducing bacteria (SRB) are of great importance in the biodegradation of organic matter in all anaerobic environments rich in sulfates.2 High-molecular-weight hydrocarbons are biodegradable and transform to low-molecular-weight polynuclear aromatic hydrocarbons (PAHs), under the sulfate-reducing conditions.3 Biological treatment process is widely used to treat highstrength sulfate-laden organic wastewater discharged from the production of sugar, alcohol, pharmaceutical, paper, and monosodium glutamate. However, sulfate reduction restrains the activities of other anaerobes via primary inhibition and subinhibition by SRB population, thus disturbing the normal operation of the anaerobic reactor.4-6 Hence, the interactions of different microbial populations are attributed to the stability and the efficiency of the anaerobic reactor. In an anaerobic reactor, SRB has many physiological and ecological similarities to other anaerobes, although it is a special * To whom correspondence should be addressed. E-mail: 03900310r@ polyu.edu.hk. † Harbin University of Civil Engineering and Architecture. ‡ Joint Research Centre of Water and Wastewater Treatment Technology, The Hong Kong Polytechnic University. § Department of Civil and Structural Engineering, The Hong Kong Polytechnic University.
group with independence and diversity in a view of phylogenesis.7 By now, the ecological functions and the roles of SRB have been studied in single-phase anaerobic reactor as follows: (1) SRB can overcome methane-producing bacteria (MPB) when they compete for the same resources, such as hydrogen and acetate, and consequently form primary inhibition to MPB;8,9 (2) SRB converts sulfate into sulfides including H2S, HS-, and S2-, which poison MPB intensively and decrease the production of CH4 sharply;10 (3) SRB plays an important role in hydrogen transferrals among the anaerobic microbial species;11 (4) SRB can compete with hydrogen-producing acetogens (HPAs) for propionic acid, butyric acid, and benzophenone.12,13 On the other hand, Polo et al.14 found that when the organic loading rate (OLR) applied to an anaerobic reactor is doubled, the rate of sulfide production by SRB will be increased proportionately. Thus, the addition of external carbon was necessary for the growth of SRB. Although it has been recognized that the acidogenic-phase reactor of two-phase anaerobic treatment is practical to treat sulfate-laden wastewater,15 little is known about the behaviors and roles of SRB populations in such a reactor. The objective of this study is to investigate the behaviors of SRB populations under different COD/SO42- ratios in an acidogenic sulfatereducing reactor. The electron flows of SRB by applying molasses wastewater as the sole organic carbon source and sodium sulfate as the electron donor, the physiological metabolic pathways and metabolic products of SRB, and the population dynamics and the effects of sulfide production on hydrogen production were examined. Materials and Methods Experimental Apparatus and Operational Conditions. A continuous-flow reactor, shown in Figure 1, was adopted as the acidogenic sulfate-reducing reactor in this study. It was a continuous stirred tank reactor (CSTR) equipped with an internal gas-liquid-solid three-phase separator. The reactor had a total volume of 20.7 L and an effective volume of 9.63 L. Pure
10.1021/ie060589w CCC: $37.00 © 2007 American Chemical Society Published on Web 02/10/2007
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phase is 2.5 mM potassium hydrogen phthalate). Hydrogen sulfide generated from the reactor was passed into a liquid trap containing zinc acetate solution, where zinc sulfide was formed.13 The sulfide concentration was then determined using the spectrophotometric methylene blue method.16,18 The oxidation-reduction potential (ORP) in the reactor (Eh) was calculated using the equation
Eh (mV) ) Ec + 249.1
Figure 1. Schematic of the acidogenic sulfate-reducing reactor system.
nitrogen gas was used to purge the vessels to maintain anaerobic conditions. H2S gas was collected using a liquid trap and was measured using a gas flow meter.13 The temperature of the reactor was controlled at 34 ( 1 °C. Substrate. Sodium sulfate was applied as the electron acceptor, and molasses wastewater obtained from a beet sugar refinery was applied as the electron donor. The proportion of molasses (COD) and sodium sulfate (SO42-) in the influent was controlled at different levels and the components were fed into the reactor. An adequate amount of multiplex fertilizer contains 15% nitrogen, and phosphorus was added to maintain a COD: N:P ratio of 500:5:1. A high proportion of carbon to nutrients was maintained for better control of the different COD/SO42ratios. Reactor Startup and Selective Enrichment of SulfateReducing Bacteria (SRB). The acidogenic sulfate-reducing reactor was seeded with two different sources of anaerobic sludge at a volume ratio of 1:1. One was from an acidogenic reactor with an anaerobic bacteria (AB) concentration of 1012 cells/mL, and the other was from a reactor treating sulfate-laden wastewater that had a SRB concentration of ∼1011 cells/mL. The total biomass of the sludge, measured in terms of mixed liquor volatile suspended solids (MLVSS), was 8.2 g/L. The initial operating conditions were as follows: COD/SO42- ) 5.0, SO42- ) 600 mg/L, SRB ) 2.1 × 1010 cells/mL, and sulfateloading rate (Ns) ) 1.0 kg SO42- m-3 d-1. Ns was increased at a rate of 0.5 kg SO42- m-3 d-1 when an obvious sulfate reduction rate was observed. After 40 days, Ns had been increased up to 3.0 kg SO42- m-3 d-1 and the sulfate removal rate was over 70%, with an MLVSS value of 15.1 g/L and a SRB concentration as high as 4.3 × 1014 cells/mL. These data indicated the successful startup of the reactor. Afterward, the initial SO42- concentration was increased to 1000 mg/L in moderate stages. The operational conditions of the remaining experimental stages are summarized in Table 1. Analytical Methods. Standard methods were used to determine the COD, biochemical oxygen demand (BOD), mixed liquor suspended solids (MLSS), MLVSS, and pH values.16 Ethanol and volatile fatty acids (VFAs) were analyzed using a gas chromatograph (model GC-122) that was equipped with a hydrogen flame-ionization detector, using the procedure developed by Ren.17 The sulfate content was determined by an ion exchange chromatograph (model CDD-6A, Shimadzu, ShimpackIC-AI, where the column temperature is 40 °C and mobile
where Ec is the observed ORP, measured by a pHS-25 acidity voltmeter (AVM) and the number 249.1 was the potential value of a saturated calomel electrode. Enumeration and Identification of SRBs, Acetogenic Bacteria (AB), and Hydrogen-Producing Acetogens (HPAs). The SRB concentration was evaluated using a serial dilution technique.19 The culture medium contained 0.5 g of K2HPO4, 1.0 g of NH4Cl, 0.5 g of Na2SO4, 0.1 g of CaCl2‚2H2O, 2.0 g of MgSO4·7H2O, 1.0 g of yeast extract, and 4 mL of 70% lactic sodium in 1000 mL of distilled water with a final pH value of 7.4-7.6. The advanced Postgate culture medium was used to purify the SRB, and the advanced Hungate technique was adopted to meet the anaerobic conditions of evaluation, purification, and identification.20 The most probable number (MPN) technique was used to evaluate AB and HPA. The culture medium for AB consisted of 10.0 g of glucose, 5.0 g of peptone, 3.0 g of beef extract, 3.0 g of NaCl, 0.5 g of cysteine, and 0.002 g of resin lazuline in 1000 mL of distilled water with a final pH value of 7.2-7.4. The culture medium for HPA consisted of 30 mmol CH3CH2COONa, 2.0 g of yeast extract, 0.1 g of MgCl2, 1.0 g of NH4Cl, 0.4 g of K2HPO4, 0.5 g of cysteine, and 0.002 g of resin lazuline in 1000 mL of distilled water with a final pH value of 7.0-7.3. Results and Discussion Effect of the COD/SO42- Ratio on the Electron-Flow Distribution of the SRB Population. Electron flow was initially reported by Isa to indicate the competition for substrates between MPB and SRB and to explain the influence of sulfate on singlephase anaerobic treatment.21 In single-phase anaerobic reactors, MPB and SRB competed for the available electrons to produce methane and sulfide, respectively. Electron-flow distribution could be used to express the degree of competition for electrons between MPB and SRB. Such a relationship was shown in eq 1 by Isa.21
electron flow of SRB (%) )
∆SO42∆SO42- + ∆CH4
× 100 (1)
where ∆SO42- and ∆CH4 (each expressed in units of mol/L) represent sulfate removal and methane production, respectively. However, in this study, the acidogenic sulfate-reducing reactor was actually operated at the acidogenic phase of a two-phase anaerobic biosystem, and the biochemical reaction for organic
Table 1. Operational Conditions of Experiments Operational Conditions experimental stage
COD/SO42- ratio
COD (mg/L)
SO42- (mg/L)
Ns (kg SO42- m-3 d-1)
HRT (h)
quick startup moderate COD/SO42- ratio high COD/SO42- ratio low COD/SO42- ratio
5.0 3.0 4.0 2.0
3000 3000 4000 4000
600 1000 1000 2000
3.0 4.0 4.0 10.0
4.8 6.0 6.0 4.8
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Figure 2. Effect of the COD/SO42- ratio on the electron flows of sulfatereducing bacteria (SRB).
wastewater degradation remained at the acid-producing stage, which meant that the reaction of methane production shown in eq 1 could not happen. Therefore, electron flow generated by total COD consumption could account for the COD consumption by the SRB. Because COD was a parameter indicating the amount of electrons in the pollutants available for oxidation,22 the electron flow of SRB in an acidogenic sulfate-reducing reactor might be calculated from the following equation:
electron flow of SRB (%) )
∆SO42- × 0.67 × 100 (2) ∆COD
where 0.67 is a constant of theoretical value to indicate the amount of COD consumed by SRB for reducing 1 g of sulfate,21 ∆SO42- (expressed in units of mg/L) represents the amount of sulfate removal, and ∆COD (also given in units of mg/L) represents the amount of COD removal. In this study, the change of electron flow and sulfate removal rate with different COD/SO42- ratios (Figure 2) illustrates that the percentage of electrons flowing toward SRB was dependent on the COD/SO42- ratio. When the COD/SO42- ratio was 4.0 (SO42- ) 1000 mg/L, COD ) 4000 mg/L), 44% of the available electrons were utilized by SRB. The percentage increased to 68% when the COD/SO42- ratio was reduced to 3.0 (SO42- ) 1333 mg/L, COD ) 4000 mg/L), and it further increased to 79% when the COD/SO42- ratio decreased to 2.0 (SO42- ) 2000 mg/L, COD ) 4000 mg/L). This might be because the increase in the sulfate supply created a decrease in the COD/SO42- ratio, so the number of electrons utilized by the SRB would increase, according to eq 4. Figure 2 also shows that the sulfate removal rate presented a tendency that was opposite from the electron flow. When the COD/SO42- ratio was 4.0, the sulfate removal rate was 90% and decreased gradually as the COD/SO42- ratio decreased from 4.0 to 3.0. When the COD/SO42- ratio was further decreased to 2.0, the sulfate removal rate decreased sharply from 87% to 76%. Although the sulfate removal rate decreased from 90% to 87% to 76% as the COD/SO42- ratio decreased from 4.0 to
3.0 to 2.0, respectively, the absolute sulfate removal still increased from 900 mg/L to 1160 mg/L to 1520 mg/L, respectively. VFA Productions and Metabolic Pathways. The effect of the SRB on the VFA distribution in terminal liquid products in an acidogenic sulfate-reducing reactor is shown in Table 2. Because acetic acid was dominant in the end products, the metabolism pattern could be defined as acetate-type metabolism, and the typical community in the reactor could be defined as an acetate-type climax community. The metabolic pathways of bacteria in an acidogenic sulfate-reducing reactor and in a typical acidogenic phase reactor of a two-phase anaerobic treatment system were compared (Figure 3). The comparatively simple metabolic pathways in a typical acidogenic reactor is shown in region I of Figure 3. The formation of acetate-type metabolism in the sulfate-reducing reactor illustrating the symbiotic relationship of interspecies is shown in region II of Figure 3. In an acidogenic sulfate-reducing reactor, four types of bacteria might coexist: AB, hydrogen-utilized SRB (HSRB), acetic acidutilized SRB (ASRB), and fatty acids-utilized SRB (FSRB) (including propionate-utilized SRB (p-SRB), lactic acid-utilized SRB (l-SRB), and butyric acid-utilized SRB (b-SRB)). The sulfate-reducing consortium was able to grow with formate, propionate, butyrate, and lactate, with lactate, which is described in the literature as the “classical” substrate for SRB.21 Following the metabolic pathways, H2 produced by AB could be utilized by HSRB, VFAs produced by AB could be utilized by FSRB and HPAs, acetic acid produced by FSRB and HPAs could be utilized by ASRB, and H2 produced by FSRB and HPAs could be utilized by HSRB. Therefore, AB, SRB (including FSRB, HSRB, and ASRB), and HPAs formed biochains via the relationships of substrate provisions and consumptions. AB was located at the first level of biochains, and FSRBs and HPAs were located at the second level, whereas HSRB and ASRB were located at the third level. In this ecosystem, the balance between biochains existed via diversifications of the acetatetype metabolisms of multipopulations. VFAs would accumulate in the terminal products in a typical acidogenic phase reactor, whereas, in the acidogenic sulfatereducing reactor, VFAs could be converted to acetic acid quickly by FSRB and HSRB. FSRB and HSRB were considered to be the consumers of VFAs produced by AB in the reactor, and, thereafter, more acetic acid formed in the terminal products. Acetic acid was one of the optimal substrates for methanogens in the subsequent treatment unit, thereby improving the efficiency and capacity of the entire treatment system. Hydrogen Consumers in the Acidogenic Sulfate-Reducing Reactor. Interspecies hydrogen transfer is the transfer of molecular hydrogen from a H2-evolving bacterium to a H2utilizing bacterium in mixed cultures, while maintaining a low H2 partial pressure.21 Figure 4 illustrates that the COD consumption for sulfide production was closely related to that of hydrogen production with controlled sulfate concentrations of 0, 1000, 1500, and 2000 mg/L. It has been well-documented that the energy yield for the reduction of bacterial SO42- is
Table 2. Effect of COD/SO42- Ratio on Volatile Fatty Acid (VFA) Distribution in Terminal Liquid Products VFA in Terminal Liquid Products (mmol/L) sulfate concentration
acetic acid
propionic acid
butyric acid
lactic acid
ethanol
[SO42-] ) 0 mg/L [SO42-] ) 600 mg/L (COD/SO42- ) 5.0) [SO42-] ) 1000 mg/L (COD/SO42- ) 3.0) [SO42-] ) 1000 mg/L (COD/SO42- ) 4.0) [SO42-] ) 2000 mg/L (COD/SO42- ) 2.0)
9.2 ( 1.53 17.6 ( 0.61 22.0 ( 3.38 22.6 ( 3.78 22.3 ( 1.41
3.8 ( 0.02 6.77 ( 0.12 2.36 ( 0.16 1.96 ( 0.04 4.41 ( 0.03
20.2 ( 3.23 7.01 ( 0.08 7.47 ( 0.07 4.91 ( 0.02 8.72 ( 0.03
3.5 ( 0.06 2.81 ( 0.1 1.41 ( 0.01 1.82 ( 0.01 4.25 ( 0.05
3.41 ( 0.03 0.56 ( 0.02 0.76 ( 0.01 2.01 ( 0.04
acidification rate (%)
proportion of acetic acid (%)
31.0 ( 10.55 62.9 ( 7.65 74.3 ( 9.10 76.5 ( 14.10 54.3 ( 7.55
21.9 ( 3.7 52.1 ( 1.8 67.2 ( 10.4 70.3 ( 11.8 58.5 ( 3.7
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Figure 3. Comparison of metabolic pathways between two types of reactors: (I) pathway in a typical acidogenic phase reactor and (II) pathway in an acidogenic sulfate-reducing reactor. (Legend: Ace-, acetic acid; Pro-, propionic acid; Lact-, lactic acid; and Buty-, butyric acid.)
Figure 4. Chemical oxygen demand (COD) consumption for hydrogen production and sulfide production.
greater than that of methanogenesis in anaerobic mixedpopulation systems.22,23 With increasing sulfate concentration, sulfide production had a tendency to increase and COD consumption for H2 production decreased correspondingly. While the sulfate concentration was at a level of 2000 mg/L, the partial pressure of H2 was low, down to 0 Pa, after the hydraulic retention time was >6 h. This was one of the most important features indicative of a typical acidogenic sulfatereducing reactor, because HSRB acts as a consumer of hydrogen. The end products of AB were H2, acetic acid, and VFAs. H2 was the substrate of HSRB. Similarly, VFAs produced by AB were converted by HPAs to H2 and acetic acid, and H2 was also utilized by HSRB. Thus, at the third level of the biochains, HSRB transferred H2 between species via co-metabolism and
consequently accelerated the biochemical reactions of AB and HPAs. Therefore, the conclusion could be drawn that the symbiotic relationship between HSRB, AB, and HPAs was able to maintain the balance of hydrogen in the ecosystem and thereby made the partial pressure of hydrogen reasonably low. The HSRB isolated in this study were DesulfoVibrio, Desulfonema, Desulfobacter, and Desulfococcus. SRB Population Dynamics. The pH and ORP values, the alkalinity (ALK) under different COD/SO42- ratios, and the dominant microbial populations observed in the study are shown in Table 3. The quantity distributions of SRB, AB, and HPAs under different COD/SO42- ratios also are shown. At a COD/ SO42- ratio of 3.0, the cell number of SRB was 7.5 × 1015 cells/mL, which was lower than that of AB by 1 order of magnitude and higher than that of HPA by 3 orders of magnitudes. When the COD/SO42- ratio increased to 4.0, the quantities of SRB, AB, and HPA increased to 5.4 × 1016, 2.1 × 1017, and 4.7 × 1013 cells/mL, respectively. When the COD/ SO42- ratio decreased to 2.0, the quantities of the three populations reduced sharply (2.0 × 1014, 3.2 × 1015, and 4.5 × 1011 cells/mL, respectively). SRB grow better under slightly alkaline conditions over a relatively restricted pH range (pH 7-8) and tolerate pH values in the range of 5.5-9.24,25 In the acidogenic sulfate-reducing reactor, the population dynamics of SRB changed with the operating conditions in different experiment stages. After the quick startup, the COD/SO42- ratio decreased from 5.0 to 3.0, and the operating conditions changed to pH 6.1, ORP ) -380 mV, and ALK ) 1500 mg/L, which indicated that the anabolism was dominated by the metabolic pathways of AB, SRB, and HPAs. The quantities of the three groups of bacteria had a tendency to increase: SRB increased from 4.3 × 1014 cells/mL to 7.5 × 1015 cells/mL. When the COD/SO42- ratio increased from 3.0 to 4.0, the operating conditions were pH 6.2, ORP )
Table 3. Ecological Characters and Dominant Populations in Different Experimental Stages COD/SO42- ) 3.0 pH value ORP ALK amount SRB AB HPA dominant population
COD/SO42- ) 4.0
COD/SO42- ) 2.0
6.1 -380 mV 1500 mg/L
6.2 -430 mV 1700 mg/L
5.7 -280 mV 2000 mg/L
7.5 × 1015 cells/mL 6.8 × 1016 cells/mL 5.5 × 1012 cells/mL Clostridium, Sporosarcina, Bacteroides, Aeromonas, Fosobucterium, Streoptococus, DesulfoVibrio, Desulfococcus, Desulfotomaculum, Desulfobacter
5.4 × 1016 cells/mL 2.1 × 1017 cells/mL 4.7 × 1013 cells/mL Clostridium, Bacteroides, Fusobacterium, Leptotrichia, Streoptococus, Aeromonas, Aerobacter, Desulfonema DesulfoVibrio, Desulfobacter, Desulfotomaculum, Desulfococcus
2.0 × 1014 cells/mL 3.2 × 1015 cells/mL 4.5 × 1011 cells/mL Dialister, Aeromonas, acteroides, Zymomonas, Desulfonema, Desulfobacter, Desulfococcus
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transferring the hydrogen among populations and guaranteed high performance stability. Acknowledgment The authors would like to thank the National Natural Science Foundation of China, the Hong Kong Polytechnic University Research Grant, and the Hong Kong Research Grants Council for financial support. Literature Cited Figure 5. Schematic diagram of the realized niche of the SRB populations.
-430 mV, and ALK ) 1700 mg/L. The increasing carbon source (COD) made the AB population increase significantly and, thus, produced more VFAs. When the COD/SO42- ratio was decreased from 4.0 to 2.0 by increasing the sulfate concentration, the SRB populations were enhanced. Consequently, when the operating conditions were pH 5.7, ORP ) -280 mV, and ALK ) 2000 mg/L, the optimum ranges of the anabolism of the three populations were exceeded and their quantities decreased. A three-dimensional diagram showing the niche of the acetate-type climax community with restrictive ORP, pH, and ALK levels is presented in Figure 5. It was obvious that Fusobacterium, Bacteroides, Aeromonas, and Desulfobacter had an overlapped niche. The niche of the SRB population was dependent on the restriction and adjustment between its overlapped niche and its detached niche. The competition for substrates between FSRB and HPAs formed the separation of their nutritional niches, and the specialization of the nutritional niche also occurred within the SRB population. To form a stable climax community, ASRB, HSRB, and FSRB (including p-SRB, b-SRB, and l-SRB) inhabited different spatial niches, which could afford to utilize the substrate as much as possible and, therefore, widened the ecological niche of the SRB populations. On the other hand, even though different bacterial populations that had similar niches could coexist when the available substrate was sufficient, the change of operating conditions would lead to different metabolic activities of the microorganisms. Therefore, each population in the acidogenic sulfate-reducing reactor had different metabolic activities and finally led to different COD and sulfate removal rates and different sulfide production rates. Conclusions In an acidogenic sulfate-reducing reactor, the COD/SO42ratio could significantly affect the compositions of the SBR populations and their metabolic pathways. The electron flow to the sulfate-reducing bacteria (SRB) population decreased as the COD/SO42- ratio increased, which resulted in improvement of the sulfate removal rate. It was also observed that the SRB population was the consumer of terminal liquid products in the acidogenic sulfate-reducing reactor and was involved in acetatetype metabolism, which resulted in the presence of 45%-82% acetic acid in the terminal liquid products. Acetic acid was one of the optimal substrates for methanogens in the subsequent treatment unit, thereby improving the efficiency and capacity of the entire treatment system. Moreover, the SRB population was the hydrogen consumer in the acidogenic sulfate-reducing reactor, which maintained a low hydrogen partial pressure by
(1) Fauque, G. D. Ecology of sulfate-reducing bacteria. In SulfateReducing Bacteria; Barton, L. L., Ed.; Biotechnology Handbooks, Vol. 8; Plenum Press: New York, 1995; p 217. (2) Azabou, S.; Mechichi, T.; Sayadi, S. Sulfate reduction from phosphogypsum using mixed culture of sulfate-reducing bacteria. Int. Biodeterior. Biodegrad. 2005, 56, 236-242. (3) Kuwano, Y.; Shimizu, Y. Bioremediation of coal contaminated soil under sulphate-reducing conditions. EnViron. Technol. 2006, 27, 95-102. (4) Anderson, G. K. Identification and control of inhibition in the anaerobic treatment of industrial wastewater. Process Biochem. 1982, 17 (4), 28-32. (5) Anderson, G. K.; Sau, C. B. State of Art of Anaerobic Digestion for Industrial Applications in the United Kingdom. Proceedings of the 39th Industrial Waste Conference, Purdue University, West Lafayette, IN, 1984; pp 783-793. (6) Anderson, G. K. Fate of COD in an Anaerobic System Treating High Sulfate Bearing Wastewater. Presented at the International Conference on Toxic Waste Treatment, Washington, DC, 1986. (7) Zhou, G. M.; Fang, H. H. P. Competition between methanogenesis and sulfidogenesis in anaerobic wastewater treatment. Water Sci. Technol. 1998, 38 (8-9), 317-324. (8) Nielsen, P. H. Bio-film dynamics and kinetics during high-rate sulfate reduction under anaerobic conditions. Appl. EnViron. Microbiol. 1987, 53, 27-32. (9) Zuo, J.; Hu, J. Anaerobic treatment of sulfate organic wastewater. EnViron. Sci. 2001, 3, 69 (in Chin.). (10) Kang, F. The process and mechanism of two-phase anaerobic treatment of sulfate-reduction and methanation. Ph.D. Dissertation paper, Wu Xi Light Industrial Institute, 1994. (11) Li, Y. Y.; Fang, H. H. P. Interactions between methanogenic, sulfate-reducing and syntrophic acetogenic bacteria in the anaerobic degradation of benzoate. Water Res. 1996, 30, 1555-1562. (12) Mizuno, O.; Li, Y. Y.; Noike, T. Effects of sulfate concentration and sludge retention time on the interaction between methane production and sulfate reduction for buturate. Water Sci. Technol. 1994, 30 (8), 4554. (13) Mizuno, O.; Li, Y.; Noike, T. The behavior of sulfate-reducing bacteria in acidogenic phase of anaerobic digestion. Water Res. 1999, 32 (5), 1626-1634. (14) Polo, B. C.; Bewtra, J. K.; Biswas, N. Effect of hydraulic retention time and attachment media on sulfide production by sulfate reducing bacteria. J. EnViron. Eng. Sci. 2006, 5 (1), 47-57. (15) Seger, O. M. The behavior of sulfate-reducing bacteria in acidogenic phase of anaerobic digestion. Water Res. 1998, 32 (5), 1626-1634. (16) Standard Methods for the Examination of Water and Wastewater, 20th Edition; American Public Health Association (APHA): Washington, DC, 1998. (17) Ren, N. Q. Hydrogen Production Biotechnology from Wastewater FermentationsTheory and Method; Heilongjiang Science and Technology Press: Harbin, PRC, 1994. (18) Truper, H. G.; Schlegel, H. G. Sulphur metabolism in Thiorhodaceae1. quantitative measurements on growing cells of Chromatium okenii. Antonie Van Leeuwenhoek 1964, 30, 225-238. (19) Disappearing dilution technique, an oil field water-infusing bacteria analytical method of petroleum and natural gas standard in China, Report No. SY/T0532-93, China National Environmental Protection Bureau, Beijing, PRC, 1993. (20) Postgate, J. R. The Sulphate-Reducing Bacteria; Cambridge University Press: Cambridge, U.K., 1984. (21) Isa, Z. Sulfate reduction relative to methane production in high rate anaerobic digestion: microbiological aspects. Appl. EnViron. Microbiol. 1986, 51, 580-587.
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(22) Wang, A. The ecology of SRB in acidogenic de-sulfate reactors forming of climax community and controlling of ecological factors. Ph.D. Dissertation, Harbin Institute of Technology, Harbin, PRC, 2000. (23) Kuivila, K. M.; Murray, J. W.; Devol, A. H.; Novelli, P. C. Methane production, sulfate reduction, and competition for substrates in the sediments of Lake Washington. Geochim. Cosmochim. Acta 1989, 53, 409-416. (24) Zobell, C. E. Ecology of sulphate-reducing bacteria. Prod. Mon. Penn. Oil Prod. Assoc. 1996, 22, 12-29. (25) Devereux, R.; Stahl, D. A. Phylogeny of sulfate-reducing bacteria and a perspective for analyzing their natural communities. In The Sulfate-
Reducing Bacteria: Contemporary PerspectiVes; Odom, J. M., Singleton, R., Eds.; Brock/Springer Series in Contemporary Bioscience; SpringerVerlag: New York, 1993; Chapter 6.
ReceiVed for reView May 12, 2006 ReVised manuscript receiVed January 1, 2007 Accepted January 9, 2007 IE060589W
