Denitrifying sulfide removal and carbon methanogenesis in a mesophilic, methanogenic culture

The inhibitory effects of 90-189 mg l⁻¹ of sulfide and 25-75 mg-N l⁻¹ of nitrate on methanogenesis were investigated in a mixed methanogenic culture using butyrate as carbon source. In the initial phase of 90 mg l⁻¹ S²⁻ test, autotrophic denitrification of nitrate occurred with sulfide as the electron donor. Then the sulfate-reducing strains converted the produced sulfur back to sulfide via heterotrophic oxidation pathway. Methanogenesis was not markedly inhibited when 90 mg l⁻¹ of sulfide was dosed alone. When 25-75 mg-N l⁻¹ of nitrate was presented, initiation of methanogenesis was seriously delayed. Nitrogen oxides (NO_x), the intermediates for nitrate reduction via denitrification pathway, inhibited methanogenesis. The 90 mg l⁻¹ of sulfide favored heterotrophic dissimilatory nitrate reduction to ammonia (DNRA) pathway for nitrate reduction. Possible ways of maximizing methane production from an organic carbon-rich wastewater with high levels of sulfide and nitrate were discussed.     

Sulfate reduction in methanogenic bioreactors

Abstract: In the anaerobic treatment of sulfate-containing wastewater, sulfate reduction interferes with methanogenesis. Both mutualistic and competitive interactions between sulfate-reducing bacteria and methanogenic bacteria have been observed. Sulfate reducers will compete with methanogens for the common substrates hydrogen, formate and acetate. In general, sulfate reducers have better growth kinetic properties than methanogens, but additional factors which may be of importance in the competition are adherence properties, mixed substrate utilization, affinity for sulfate of sulfate reducers, relative numbers of bacteria, and reactor conditions such as pH, temperature and sulfide concentration. Sulfate reducers also compete with syntrophic methanogenic consortia involved in the degradation of substrates like propionate and butyrate. In the absence of sulfate these methanogenic consortia are very important, but in the presence of sulfate they are thought to be easily outcompeted by sulfate reducers. However, at relatively low sulfate concentrations, syntrophic degradation of propionate and butyrate coupled to HZ removal via sulfate reduction rather than via methanogenesis may become important. A remarkable feature of some sulfate reducers is their ability to grow fermentatively or to grow in syntrophic association with methanogens in the absence of sulfate.     

Effect of sulfide on nitrate reduction in mixed methanogenic cultures

The effect of sulfide on nitrate reduction and methanogenesis was investigated in two mixed, mesophilic (35 degrees C) methanogenic cultures: sulfide-free and sulfide-acclimated (67 mg S/L total sulfide). A mixture of dextrin/peptone served as the carbon/electron donor source for the two stock cultures, as well as in all assays reported here. The sulfide-free enriched culture was amended with both nitrate (75-350 mg N/L) and sulfide (10-100 mg S/L). Denitrification was the predominant pathway at all sulfide levels tested and methanogenesis did not recover in any of the sulfide- and nitrate-amended cultures, except in the 10 mg S/L culture. Accumulation of denitrification intermediates such as NO and N(2)O took place, which irreversibly inhibited the methanogens and resulted in the complete cessation of methane production. In contrast, conversion of nitrate to nitrite and then to ammonia via dissimilatory nitrate reduction to ammonia (DNRA) prevented the accumulation of denitrification intermediates and led to the recovery of methanogenesis in the nitrate-amended, sulfide-acclimated, mixed methanogenic culture. The effect of the COD/N value on nitrate reduction was assessed with the sulfide-acclimated, methanogenic culture at COD/N values of 10, 20, and 60. As the COD/N value increased, the fraction of nitrate reduced through DNRA also increased. The results of this study have significant implications relative to the combined anaerobic treatment of carbon-, nitrogen-, and/or sulfur-bearing wastes.     

不同抑制剂对乙酸降解产甲烷及产甲烷菌群结构的影响

摘要:【目的】研究不同温度条件下的石油烃降解产甲烷菌系中是否存在乙酸互营氧化产甲烷代谢途径。【方法】以3个不同温度条件的正十六烷烃降解产甲烷菌系Y15(15℃)、M82(35℃)和SK(55℃)作为接种物,通过乙酸喂养实验、并添加乙酸营养型产甲烷古菌的选择性抑制剂NH4Cl和CH3F,结合末端限制性片段长度多态性(terminal restriction fragment length polymorphism,T-RFLP)和克隆文库技术,分析乙酸产甲烷潜力及产甲烷古菌群落的演替趋势,推测产甲烷代谢途径的变化趋势。【结果】无论是否添加NH4Cl和CH3 F,这3个菌系都可以利用乙酸生长并产生甲烷,但是添加NH4Cl和CH3 F后产甲烷延滞期增加,最大比甲烷增长速率降低;只添加乙酸后,3个不同温度的菌系的古菌群落主要由乙酸营养型产甲烷古菌甲烷鬃毛菌属(Methanosaeta)组成,其丰度分别为92.8±1.4%、97.3±2.4%和82.8±9.0%;当添加选择性抑制剂NH4Cl,3 个菌系中的Methanosaeta的丰度分别变为98.5±0.7%、87.4±4.8%和6.1±8.6%,中温菌系M82中氢营养型产甲烷古菌甲烷袋装菌属(Methanoculleus)的相对丰度增加到12. 6±4.0%,高温菌系SK中另一类氢营养型产甲烷古菌甲烷热杆菌属(Methanothermobacter)增至84.3±1.5%;当添加选择性抑制剂CH3 F,Methanosaeta丰度分别降至77.1 ± 14.5%,86.4±6.1%和35.8±7.8%,低温菌系Y15中的甲烷八叠球菌属(Methanosarcina)增高(15.7±21%),这类产甲烷古菌具有多种产甲烷代谢途径,M82中Methanoculleus丰度上升到13.6±13.1%,SK中Methanothermobacter丰度增大到48.5±11.2%。【结论】在低温条件下,菌系Y15可能主要通过乙酸裂解完成产甲烷代谢,在中高温条件下,菌系M82和SK中可能存在乙酸互营氧化产甲烷代谢途径,并且甲烷的产生分别通过不同种群的氢营养型产甲烷古菌来完成。     

Interconnection of methanogenic and acetogenic pathways

Abstract Methanopterin displays a folate-like biochemistry: methenyl-, methylene- and methyltetrahydromethanopterin are intermediates in the process of methanogenesis. A corrinoid-enzyme is involved in methanol conversion and the possible function of such enzymes in cell-carbon synthesis and acetate conversion is discussed. Energy conservation is proposed to proceed via the establishment of a proton motive force through a vectorial reduction in the final step of methanogenesis and via substrate level phosphorylation in the oxidation of formyltetrahydromethanopterin. The biochemical reactions of methanogenesis and acetogenesis are compared. Methanogenic bacteria appear to use an aberrant set of coenzymes in almost all of the reactions involved in methanogenesis.     

Sulfide fluxes in a microbial mat from the Ebro Delta, Spain

The sulfur cycle of Ebro Delta microbial mats was studied in order to determine sulfide production and sulfide consumption. Vertical distribution of two major functional groups involved in the sulfur cycle, anoxygenic phototrophic bacteria and dissimilatory sulfate-reducing bacteria (SRB), was also studied. The former reached up to 2.2×10⁸ cfu cm^–3 sediment in the purple layer, and the latter reached about 1.8×10⁵ SRB cm^–3 sediment in the black layer. From the changes in sulfide concentrations under light-dark cycles it can be inferred that the rate of H₂S production was 6.2 μmol H₂S cm^–3 day^–1 at 2.6 mm, and 7.6 μmol H₂S cm^–3 day^–1 at 6 mm. Furthermore, sulfide consumption was also assessed, determining rates of 0.04, 0.13 and 0.005 mmol l^–1 of sulfide oxidized at depths of 2.6, 3 and 6 mm, respectively. Electronic Publication     

Thermodynamics of hydrogen metabolism in methanogenic cocultures degrading ethanol or lactate

Pure cultures of Desulfovibrio vulgaris or Pelobacter acetylenicus do not grow with lactate or ethanol, respectively, under obligately proton- reducing conditions. However, a small part of these substrates was oxidized and molecular hydrogen was produced up to 4.2 and 3.2 kPa, respectively. During growth in syntrophic methanogenic cocultures with Methanospirillum hungatei as partner, maximum hydrogen partial pressures were significantly lower (0.7 to 2.5 kPa) than in the corresponding pure cultures. Calculation of Gibbs free energies for the prevailing culture conditions showed that H₂ partial pressures were kept in a range at which both, H₂-producing and H₂-consuming reactions, were thermodynamically permissive in pure as well as in syntrophic mixed cultures.     

Anaerobic degradation of citrate under sulfate reducing and methanogenic conditions

Citrate is an important component of metal processing effluents such as chemical mechanical planarization wastewaters of the semiconductor industry. Citrate can serve as an electron donor for sulfate reduction applied to promote the removal of metals, and it can also potentially be used by methanogens that coexist in anaerobic biofilms. The objective of this study was to evaluate the degradation of citrate with sulfate-reducing and methanogenic biofilms. During batch bioassays, the citrate, acetate, methane and sulfide concentrations were monitored. The results indicate that independent of the biofilm or incubation conditions used, citrate was rapidly fermented with specific rates ranging from 566 to 720 mg chemical oxygen demand (COD) consumed per gram volatile suspended solids per day. Acetate was found to be the main fermentation product of citrate degradation, which was later degraded completely under either methanogenic or sulfate reducing conditions. However, if either sulfate reduction or methanogenesis was infeasible due to specific inhibitors (2-bromoethane sulfonate), absence of sulfate or lack of adequate microorganisms in the biofilm, acetate accumulated to levels accounting for 90–100% of the citrate-COD consumed. Based on carbon balances measured in phosphate buffered bioassays, acetate, CO₂ and hydrogen are the main products of citrate fermentation, with a molar ratio of 2:2:1 per mol of citrate, respectively. In bicarbonate buffered bioassays, acetogenesis of H₂ and CO₂ increased the yield of acetate. The results taken as a whole suggest that in anaerobic biofilm systems, citrate is metabolized via the formation of acetate as the main metabolic intermediate prior to methanogenesis or sulfate reduction. Sulfate reducing consortia must be enriched to utilize acetate as an electron donor in order to utilize the majority of the electron-equivalents in citrate.     

不同pH缓冲液对由乙酸产甲烷菌群结构的影响

【目的】研究不同p H缓冲液对乙酸产甲烷过程及对细菌和古菌群落结构的影响。【方法】分别添加磷酸盐(PB)、4-羟乙基哌嗪乙磺酸(HEPES)、哌嗪-1,4-二乙磺酸(PIPES)和Na HCO3/CO2缓冲液到乙酸产甲烷菌系中,定期监测甲烷产生趋势,到稳定期后收集菌体,进行16S rRNA基因的末端限制性片段多态性分析(T-RFLP)。【结果】发现PB组的乙酸产甲烷菌系延滞期约为40d,显著高于其他组的20-24 d(P0.05);Na HCO3/CO2组乙酸转化为甲烷的比例为(88.3±0.5)%,显著高于其他组的77%-81%(P0.05);不同缓冲液组的最大甲烷比生长速率为0.46-0.57 d-1(P0.05);Na HCO3/CO2组的细菌群落变化最明显,主要是未培养细菌(unclassified bacteria)、螺旋菌科细菌(Spirochaetaceae)和未培养WWE1类群的丰度较其他组分别增加到(15.5±9.4)%、(7.3±4.6)%和(17.6±6.3)%,而互养菌科(Synergistaceae)的细菌丰度降低到(8.9±8.1)%。AC+PB组中的古菌类群发生了明显变化,以竹节状甲烷鬃毛菌(Methanosaeta harundinacea)相关的产甲烷古菌占主导(97±2%),而在HEPES、PIPES和Na HCO3/CO2组和不加缓冲液组中同时存在两类乙酸营养型产甲烷古菌M.harundinacea和联合鬃毛甲烷菌(Methanosaeta concilii),以及属于甲烷杆菌目(Methanobacteriales)的氢营养型产甲烷古菌。【结论】在乙酸产甲烷菌系中加入PB增加了甲烷产生的延滞期,加入Na HCO3/CO2增加了甲烷产量,但是添加p H缓冲液不会影响到菌系的最大甲烷比生长速率。加入PB和Na HCO3/CO2都会显著改变微生物的菌群结构。这些研究为设计适宜的产甲烷菌系生长条件提供了参考。     

The effect of sulfate reduction on the thermophilic (55°C) methane fermentation process

Summary The continuously operated suspended growth anaerobic contact system was utilized to estimate the effect of sulfate reduction on the thermophilic (55°C) methane fermentation process. Results indicated that reduction in methanogenesis in the presence of sulfate was due to two separate, but related, processes;i.e. competitive and sulfide inhibition. Although prevention of competitive inhibition would be difficult under normal fermenter operation, sulfide inhibition could be minimized by environmental selection of sulfide tolerant microbial populations through biomass recycle and pH control. Stable fermenter operation was achieved at soluble sulfide concentrations as high as 330 mg/l soluble sulfide. Using batch fermenters, a maximum thermophilic sulfate reduction rate of 3.7 mg SO₄ ^2––S/g volatile solids (VS)-day was estimated. The importance of reporting sulfate reduction rates on a biomass basis is demonstrated by a simple population adjustment kinetic model.This research study was conducted at the Department of Agricultural Engineering, Cornell University, Riley Robb Hall, Ithaca, NY 14853, U.S.A.     

Molecular characterization of surfactant-driven microbial community changes in anaerobic phenanthrene-degrading cultures under methanogenic conditions

SDS and Triton X-100 added at their critical micelle concentrations (CMCs), increased phenanthrene solubility in the presence of sediments and inhibited phenanthrene biodegradation. Triton X-100 caused more inhibition than SDS. 16S rDNA analyses revealed that both surfactants changed the microbial communities of phenanthrene-degrading cultures. Further, after the surfactant additions, parts of the microbial populations were not detected and methane production decreased. Surfactant applications, necessary to achieve actual CMCs, alter microbial community structure and diminish methanogenic activity under anaerobic conditions. We propose that this change may be related to the inhibitory effects of SDS and Triton X-100 on phenanthrene biodegradation under methanogenic conditions.     

Dynamics of sulfidogenesis associated to methanogenesis in horizontal-flow anaerobic immobilized biomass reactor

Two bench-scale horizontal anaerobic fixed bed reactors were tested to remove both sulfate and organic matter from wastewater. First, the reactors (R1 and R2) were supplied with synthetic wastewater containing sulfate and a solution of ethanol and volatile fatty acids. Subsequently, R1 and R2 were fed with only ethanol or acetate, respectively. The substitution to ethanol in R1 increased the sulfate reduction efficiency from 83% to nearly 100% for a chemical oxygen demand to sulfate (COD/sulfate) ratio of 3.0. In contrast, in R2, the switch in carbon source to acetate strongly decreased sulfidogenesis and the maximum sulfate reduction achieved was 47%. Process stability in long-term experiments and high removal efficiencies of both organic matter and sulfate were achieved with ethanol as the sole carbon source. The results allow concluding that syntrophism instead of competition between the sulfate reducing bacteria and acetoclastic methanogenic archaeal populations prevailed in the reactor.     

The effect of pH on ruminal methanogenesis

Abstract: When a fistulated cow was fed an all forage diet, ruminal pH remained more or less constant (6.7 to 6.9). The ruminal pH of a concentrate-fed cow decreased dramatically in the period soon after feeding, and the pH was as low as 5.45. Mixed ruminal bacteria from the forage-fed cow converted CO₂ and H₂ to methane, but the ruminal fluid from the concentrate-fed cow did not produce methane. When the pH of the ruminal fluid from the concentrate-fed cow was adjusted to pH 7.0, methane was eventually detected, and the absolute rate constant of methane production was as high as the one observed with ruminal fluid from the forage fed cow (0.32 h⁻¹). Based on the zero-time intercepts of methane production, it appeared that the concentrate-fed cow had fewer methanogens than the forage-fed cow. When the mixed ruminal bacteria were incubated in a basal medium containing 100 mM acetate, methanogenesis was pH-dependent, and no methane was detected at pH values less than 6.0. Because the removal of acetic acid completely reversed the inhibition of methanogenesis, it appeared that volatile fatty acids were causing the pH-dependent inhibition. Based on these results, concentrate diets that lower ruminal pH may provide a practical means of decreasing ruminal methane production.     

5,6,7,8-Tetrahydromethanopterin-dependent enzymes involved in methanogenesis

Abstract In the process of methanogenesis, 5,6,7,8-tetrahydromethanopterin (H₄MPT) is the carrier of the C₁ unit at the formyl through methyl state of reduction. By the transfer of a formyl group from formylmethanofuran, 5-formyl- and 10-formyl-H₄MPT are formed in hydrogenotrophic and methylotrophic organisms, respectively. Cyclohydrolysis of the 5- and 10-formyl derivatives then yields 5,10-methenyl-H₄MPT, which is reduced in two subsequent coenzyme F₄₂₀-dependent reactions to 5-methyl-H₄MPT. Following the transfer of the methyl group to coenzyme M, the substrate of the terminal step in methanogenesis, methylcoenzyme M, is produced. In this paper properties of the enzymes catalyzing the individual H₄MPT-dependent reactions are discussed.     

Effect of 2-bromo-ethane sulfonate, molybdate and chloroform on acetate consumption by methanogenic and sulfate-reducing populations in freshwater sediment

The relative importance of methanogenesis and sulfate reduction in freshwater sediment supplemented with acetate was investigated. Addition of acetate stimulated both methane formation and sulfate reduction, indicating that an active aceticlastic population of methanogens and sulfate reducers was present in the sediment. Sulfate reducers were most important in the consumption of acetate. However, when sulfate reducers were inhibited, acetate was metabolised at a similar rate by methanogens. Acetate, propionate and valerate accumulated only when both processes were inhibited by the combined addition of 2-bromo-ethane sulfonate and molybdate. The relative amounts of acetate, propionate and valerate were 93, 6 and 1 mol%, respectively. These results demonstrate the role of acetate as a key intermediate in the terminal step of organic matter mineralisation in the sediment. Addition of chloroform inhibited both methanogenesis and sulfate reduction. We studied the inhibitory effect of CHCl(3) on homoacetogenic bacteria, sulfate-reducing bacteria and methanogens. The results showed that inhibition by CHCl(3) correlates with microorganisms, which operate the acetyl-CoA cleavage pathway. We propose that chloroform can be used to elucidate the role of different metabolic types of sulfate reducers to sulfate reduction in natural environments.