Autotrophic nitrite removal in the cathode of microbial fuel cells

Nitrification to nitrite (nitritation process) followed by reduction to dinitrogen gas decreases the energy demand and the carbon requirements of the overall process of nitrogen removal. This work studies autotrophic nitrite removal in the cathode of microbial fuel cells (MFCs). Special attention was paid to determining whether nitrite is used as the electron acceptor by exoelectrogenic bacteria (biologic reaction) or by graphite electrodes (abiotic reaction). The results demonstrated that, after a nitrate pulse at the cathode, nitrite was initially accumulated; subsequently, nitrite was removed. Nitrite and nitrate can be used interchangeably as an electron acceptor by exoelectrogenic bacteria for nitrogen reduction from wastewater while producing bioelectricity. However, if oxygen is present in the cathode chamber, nitrite is oxidised via biological or electrochemical processes. The identification of a dominant bacterial member similar to Oligotropha carboxidovorans confirms that autotrophic denitrification is the main metabolism mechanism in the cathode of an MFC.     

Anaerobic degradation of phenol by pure cultures of newly isolated denitrifying pseudomonads

From various oxic or anoxic habitats several strains of bacteria were isolated which in the absence of molecular oxygen oxidized phenol to CO₂ with nitrate as the terminal electron acceptor. All strains grew in defined mineral salts medium; two of them were further characterized. The bacteria were facultatively anaerobic Gramnegative rods; metabolism was strictly oxidative with molecular oxygen, nitrate, or nitrite as electron acceptor. The isolates were tentatively identified as pseudomonads. Besides phenol many other benzene derivatives like cresols or aromatic acids were anaerobically oxidized in the presence of nitrate. While benzoate or 4-hydroxybenzoate was degraded both anaerobically and aerobically, phenol was oxidized under anaerobic conditions only. Reduced alicyclic compounds were not degraded. Preliminary evidence is presented that the first reaction in anaerobic phenol oxidation is phenol carboxylation to 4-hydroxybenzoate.     

Metabolic flexibility of SUP05 under low DO growth conditions

Chemoautotrophic bacteria from the SUP05 clade often dominate anoxic waters within marine oxygen minimum zones (OMZs) where they use energy gained from the oxidation of reduced sulfur to fuel carbon fixation. Some of these SUP05 bacteria are facultative aerobes that can use either nitrate or oxygen as a terminal electron acceptor making them ideally suited to thrive at the boundaries of OMZs where they experience fluctuations in dissolved oxygen (DO). SUP05 metabolism in these regions, and therefore the biogeochemical function of SUP05, depends largely on their sensitivity to oxygen. We evaluated growth and quantified differences in gene expression in Ca. T. autotrophicus strain EF1 from the SUP05 clade under high DO (22 μM), anoxic, and low DO (3.8 μM) concentrations. We show that strain EF1 cells respire oxygen and nitrate and that cells have higher growth rates, express more genes, and fix more carbon when oxygen becomes available for aerobic respiration. Evidence that facultatively aerobic SUP05 are more active and respire nitrate when oxygen becomes available at low concentrations suggests that they are an important source of nitrite across marine OMZ boundary layers.     

Dissimilatory nitrate reduction in Clostridium tertium.

Fermentation balance studies were carried out on Clostridium tertium grown with and without nitrate in the medium. Nitrate reduction increased the efficiency of energy produced from glucose by permitting the utilization of additional sites of substrate level phosphorylation. The effect was even more dramatic in C. tertium than in C. perfringens, with increased cell yields of about 30% being observed in the former compared with 20% in the latter. Unlike C. perfringens, C. tertium responded to the presence of nitrate in the medium with an increased growth rate. A slight increase in the Y ATP of these cultures was also observed, and quantitatively, this appeared to be consistent with the prediction of Stouthammer and Bettenhaussen that Y ATP will vary with the growth rate. Thus, C. tertium, like C. perfringens, was able to use nitrate as an electron acceptor in conjunction with its energy metabolism, suggesting that this may be widespread among the nitrate-reducing anaerobes.     

Sulfate reduction and anaerobic glycerol degradation by a mixed microbial culture

Anaerobic glycerol degradation by a mixed microbial culture from a fermenter fed with industrial alcohol distillation waste water, was investigated in the absence or presence of sulfate, at 37°C and at a constant pH of 7.2. In the absence of sulfate, glycerol utilization was found to be characterized by the transient formation of 1,3-propanediol prior to propionate and acetate accumulation. In the presence of sulfate, 1,3-propanediol production was minor, and the carbon balance reflected a considerable accumulation of intermediate(s). A study of the role of sulfate reduction and methanogenesis on anaerobic 1,3-propanediol degradation showed that consumption of this substrate by the mixed microbial culture required a terminal electron acceptor. The number of fermentative and sulfate-reducing bacteria with glycerol or 1,3-propanediol as carbon and energy source revealed that sulfate-reducing bacteria outcompete fermentative bacteria for these substrates. The possible ecological role of sulfate-reducing bacteria in the metabolism of these reduced substrates is discussed.     

Anaerobic metabolism of cyclohexanol by denitrifying bacteria

Three strains of denitrifying bacteria were anaerobically enriched and isolated from oxic or anoxic habitats with cyclohexanol or cyclohexanone as sole electron donor and carbon source and with nitrate as electron acceptor. The bacteria were facultatively anaerobic, Gramnegative and metabolism was strictly oxidative with molecular oxygen, nitrate, or nitrite as terminal electron acceptor. Cyclohexanol and cyclohexanone were degraded both anaerobically and aerobically. Aromatic compounds were oxidized in the presence of molecular oxygen only. One of the bacterial strains was further characterized. During anaerobic cyclohexanol degradation approximately 40% of the substrate was oxidized to phenol, which accumulated as dead-endproduct in the growth medium; 60% of cyclohexanol was completely oxidized to CO₂ and assimilated, respectively. In addition to phenol formation, transient accumulation of cyclohexanone, 2-cyclohexenone and 1,3-cyclohexanedione was observed. Based on these findings we propose a pathway for anaerobic cyclohexanol degradation involving these intermediates.     

Chemolithotrophic perchlorate reduction linked to the oxidation of elemental sulfur

Perchlorate (ClO(4)(-)) contamination of ground and surface water has been recently recognized as a widespread environmental problem. Biological methods offer promising perspectives of perchlorate remediation. Facultative anaerobic bacteria couple the oxidation of organic and inorganic electron-donating substrates to the reduction of perchlorate as a terminal electron acceptor, converting it completely to the benign end-product, chloride. Insoluble inorganic substrates are of interest for low maintenance bioreactor or permeable reactive barrier systems because they can provide a long-term supply of electron donor without generating organic residuals. The main objective of this research was to investigate the feasibility of utilizing elemental sulfur (S(0)) as an insoluble electron donor for the biological reduction of perchlorate. A chemolithotrophic enrichment culture derived from aerobic activated sludge was obtained which effectively coupled the oxidation of elemental sulfur to sulfate with the reduction of perchlorate to chloride and gained energy from the process for cell growth. The enrichment culture grew at a rate of 0.41 or 0.81 1/d in the absence and presence of added organic carbon for cell growth, respectively. The enrichment culture was also shown to carry out sulfur disproportionation to a limited extent as evidenced by the formation of sulfide and sulfate in the absence of added electron acceptor. When nitrate and perchlorate were added together, the two electron acceptors were removed simultaneously after an initial partial decrease in the nitrate concentration.     

Microbial fuel cells: novel biotechnology for energy generation

Microbial fuel cells (MFCs) provide new opportunities for the sustainable production of energy from biodegradable, reduced compounds. MFCs function on different carbohydrates but also on complex substrates present in wastewaters. As yet there is limited information available about the energy metabolism and nature of the bacteria using the anode as electron acceptor; few electron transfer mechanisms have been established unequivocally. To optimize and develop energy production by MFCs fully this knowledge is essential. Depending on the operational parameters of the MFC, different metabolic pathways are used by the bacteria. This determines the selection and performance of specific organisms. Here we discuss how bacteria use an anode as an electron acceptor and to what extent they generate electrical output. The MFC technology is evaluated relative to current alternatives for energy generation.     

Nitrate respiration and diel migration patterns of diatoms are linked in sediments underneath a microbial mat

Diatoms are among the few eukaryotes known to store nitrate (NO₃⁻) and to use it as an electron acceptor for respiration in the absence of light and O₂. Using microscopy and ¹⁵N stable isotope incubations, we studied the relationship between dissimilatory nitrate/nitrite reduction to ammonium (DNRA) and diel vertical migration of diatoms in phototrophic microbial mats and the underlying sediment of a sinkhole in Lake Huron (USA). We found that the diatoms rapidly accumulated NO₃⁻ at the mat-water interface in the afternoon and 40% of the population migrated deep into the sediment, where they were exposed to dark and anoxic conditions for ~75% of the day. The vertical distribution of DNRA rates and diatom abundance maxima coincided, suggesting that DNRA was the main energy generating metabolism of the diatom population. We conclude that the illuminated redox-dynamic ecosystem selects for migratory diatoms that can store nitrate for respiration in the absence of light. A major implication of this study is that the dominance of DNRA over denitrification is not explained by kinetics or thermodynamics. Rather, the dynamic conditions select for migratory diatoms that perform DNRA and can outcompete sessile denitrifiers.     

Aerobic nitrate respiration in a nitrite-oxidising bioreactor

The ability of heterotrophic bacteria in a nitrite-oxidising bioreactor to respire with nitrate as an electron acceptor was examined. Approximately 70% of 1000 heterotrophic isolates were able to express a nitrate reductase. A detailed survey of 15 isolates showed that five expressed the azide-insensitive nitrate reductase encoded by the napA gene. A two-round PCR amplification of the napA gene using degenerate PCR primers and DNA sequence analysis of these products confirmed the presence of this gene in the positive isolates. Partial 16S rDNA products and napA products were amplified from the biomass in the bioreactor and denaturing gradient gel electrophoresis of these products identified 21 distinct ribotypes and 12 distinct napA sequences. The results show that the ability to respire with nitrate as an electron acceptor under aerobic conditions is widespread among the heterotrophic population of this bioreactor.     

Nitrate,fumarate, and oxygen as electron acceptors for a late step in microbial heme synthesis

Nitrate can serve as anaerobic electron acceptor for the oxidation of protoporphyrinogen to protoporphyrin in cell-free extracts of Escherichia coli grown anaerobically in the presence of nitrate. Two kinds of experiments indicated this: anaerobic protoporphyrin formation from protoporphyrinogen, followed spectrophotometrically, was markedly stimulated by addition of nitrate; and anaerobic protoheme formation from protoporphyrinogen, determined by extraction procedures, was markedly stimulated by addition of nitrate. In contrast, anaerobic protoheme formation from protoporphyrin was not dependent upon addition of nitrate. This was the first demonstration of the ability of nitrate to serve as electron acceptor for this late step of heme synthesis. Previous studies with mammalian and yeast mitochondria had indicated an obligatory requirement for molecular oxygen at this step.In confirmation of our previous preliminary report, fumarate was also shown to be an electron acceptor for anaerobic protoporphyrinogen oxidation in extracts of E. coli grown anaerobically on fumarate. For the first time, anaerobic protoheme formation from protoporphyrinogen, but not from protoporphyrin, was shown to be dependent upon the addition of fumarate.The importance of these findings is 2-fold. First, they establish that enzymatic protoporphyrinogen oxidation can occur in the absence of molecular oxygen, in contrast to previous observations using mammalian and yeast mitochondria. Secondly, these findings help explain the ability of some facultative and anaerobic bacteria to form very large amounts of heme compounds, such as cytochrome pigments, when grown anaerobically in the presence of nitrate or fumarate. In fact, denitrifying bacteria are known to form more cytochromes when grown anaerobically than during aerobic growth.An unexpected finding was that extracts of another bacterium, Staphylococcus epidermidis, exhibited very little ability to oxidize protoporphyrinogen to protoporphyrin as compared to E. coli extracts. This finding suggests some fundamental differences in these two organisms in this key step in heme synthesis. It is known that these two facultative organisms also differ in that E. coli synthesizes cytochrome during both aerobic and anaerobic growth, while Staphylococcus only synthesizes cytochromes when grown aerobically.     

Anaerobic electron acceptor chemotaxis in Shewanella putrefaciens.

Shewanella putrefaciens MR-1 can grow either aerobically or anaerobically at the expense of many different electron acceptors and is often found in abundance at redox interfaces in nature. Such redox interfaces are often characterized by very strong gradients of electron acceptors resulting from rapid microbial metabolism. The coincidence of S. putrefaciens abundance with environmental gradients prompted an examination of the ability of MR-1 to sense and respond to electron acceptor gradients in the laboratory. In these experiments, taxis to the majority of the electron acceptors that S. putrefaciens utilizes for anaerobic growth was seen. All anaerobic electron acceptor taxis was eliminated by the presence of oxygen, nitrate, nitrite, elemental sulfur, or dimethyl sulfoxide, even though taxis to the latter was very weak and nitrate and nitrite respiration was normal in the presence of dimethyl sulfoxide. Studies with respiratory mutants of MR-1 revealed that several electron acceptors that could not be used for anaerobic growth nevertheless elicited normal anaerobic taxis. Mutant M56, which was unable to respire nitrite, showed normal taxis to nitrite, as well as the inhibition of taxis to other electron acceptors by nitrite. These results indicate that electron acceptor taxis in S. putrefaciens does not conform to the paradigm established for Escherichia coli and several other bacteria. Carbon chemo-taxis was also unusual in this organism: of all carbon compounds tested, the only positive response observed was to formate under anaerobic conditions.     

Anaerobic electron acceptor chemotaxis in Shewanella putrefaciens

Shewanella putrefaciens MR-1 can grow either aerobically or anaerobically at the expense of many different electron acceptors and is often found in abundance at redox interfaces in nature. Such redox interfaces are often characterized by very strong gradients of electron acceptors resulting from rapid microbial metabolism. The coincidence of S. putrefaciens abundance with environmental gradients prompted an examination of the ability of MR-1 to sense and respond to electron acceptor gradients in the laboratory. In these experiments, taxis to the majority of the electron acceptors that S. putrefaciens utilizes for anaerobic growth was seen. All anaerobic electron acceptor taxis was eliminated by the presence of oxygen, nitrate, nitrite, elemental sulfur, or dimethyl sulfoxide, even though taxis to the latter was very weak and nitrate and nitrite respiration was normal in the presence of dimethyl sulfoxide. Studies with respiratory mutants of MR-1 revealed that several electron acceptors that could not be used for anaerobic growth nevertheless elicited normal anaerobic taxis. Mutant M56, which was unable to respire nitrite, showed normal taxis to nitrite, as well as the inhibition of taxis to other electron acceptors by nitrite. These results indicate that electron acceptor taxis in S. putrefaciens does not conform to the paradigm established for Escherichia coli and several other bacteria. Carbon chemo-taxis was also unusual in this organism: of all carbon compounds tested, the only positive response observed was to formate under anaerobic conditions.     

厌氧氨氧化菌的中心代谢研究进展

摘要: 厌氧氨氧化是以NH +4为电子供体,以NO－2为电子受体产生N2的生物反应。厌氧氨氧化菌是厌氧氨氧化过程的执行者,在废水生物脱氮和地球氮素循环中扮演着重要角色。研究厌氧氨氧化菌的代谢特性,将有助于理解厌氧氨氧化过程,开发厌氧氨氧化工艺。厌氧氨氧化菌是化能自养型细菌,以CO2或HCO－3为碳源,并通过偶联NH+4氧化和NO －2还原的生物反应获得能量。在NH+4/NO－2的生物氧化还原反应过程中,检出了中间产物N2H4,但未检出其他中间产物(如NH2OH、NO)。此外,由基因组信息推断,厌氧氨氧化菌     

利用硝酸盐和亚硝酸盐同步富集厌氧甲烷氧化微生物的比较实验

【背景】反硝化厌氧甲烷氧化(Denitrifying anaerobic methane oxidation,DAMO)是以硝酸盐或亚硝酸盐为电子受体以甲烷为电子供体的厌氧氧化过程,对认识全球碳氮循环、削减温室气体排放和开发废水脱氮新技术等方面具有重要意义。【目的】认识以硝酸盐和亚硝酸盐为电子受体的DAMO微生物富集过程和结果的差异性。【方法】在序批式反应器(Sequencing batch reaetor,SBR)内接种混合物,分别以硝酸盐和亚硝酸盐为电子受体连续培养800 d,定期检测反应器基质浓度变化、计算转化速率;利用16S rRNA基因系统发育分析研究功能微生物的多样性,利用实时荧光定量PCR技术定量测定功能微生物。【结果】以亚硝酸盐为电子受体的1、3号反应器富集到了DAMO细菌,未检测到DAMO古菌;以硝酸盐为电子受体的2号反应器富集到了DAMO细菌和古菌的混合物;3个反应器的脱氮速率经过初始低速期、快速提升期,最终达到稳定,但2号快速提升期开始时间比1、3号晚了80 d左右,达到稳定的时间更长,稳定最大速率为1、3号的44.7%、40.3%。【结论】硝酸盐和亚硝酸盐对富集产物有决定性影响;以硝酸盐为电子受体富集得到的DAMO古菌和细菌协同体系可以长期稳定共存,DAMO古菌可能是协同体系中脱氮速率的限制性因素。     

Chemolithotrophic growth ofDesulfovibrio desulfuricans with hydrogen coupled to ammonification of nitrate or nitrite

Two of nine sulfate reducing bacteria tested,Desulfobulbus propionicus andDesulfovibrio desulfuricans (strain Essex 6), were able to grow with nitrate as terminal electron acceptor, which was reduced to ammonia. Desulfovibrio desulfuricans was grown in chemostat culture with hydrogen plus limiting concentrations of nitrate, nitrite or sulfate as sole energy source. Growth yields up to 13.1, 8.8 or 9.7 g cell dry mass were obtained per mol nitrate, nitrite or sulfate reduced, respectively. The apparent half saturation constants (K _s) were below the detection limits of 200, 3 or 100 mol/l for nitrate, nitrite of sulfate, respectively. The maximum growth rates {ie63-1} raised from 0.124 h^-1 with sulfate and 0.150 h^-1 with nitrate to 0.193 h^-1 with nitrite as electron acceptor. Regardless of the electron acceptor in the culture medium, cell extracts exhibited absorption maxima corresponding to cytochromec and desulfoviridin. Nitrate reductase was found to be inducible by nitrate or nitrite, whereas nitrite reductase was synthesized constitutively. The activities of nitrate and nitrite reductases with hydrogen as electron donor were 0.2 and 0.3 mol/min·mg protein, respectively. If limiting amounts of hydrogen were added to culture bottles with nitrate as electron acceptor, part of the nitrate was only reduced to the level of nitrite. In media containing nitrate plus sulfate or nitrite plus sulfate, sulfate reduction was suppressed.The results demonstrate that the ammonification of nitrate or nitrite can function as sole energy conserving process in some sulfate-reducing bacteria.     

Growth of Geobacter sulfurreducens with Acetate in Syntrophic Cooperation with Hydrogen-Oxidizing Anaerobic Partners

Pure cultures of Geobacter sulfurreducens and other Fe(III)-reducing bacteria accumulated hydrogen to partial pressures of 5 to 70 Pa with acetate, butyrate, benzoate, ethanol, lactate, or glucose as the electron donor if electron release to an acceptor was limiting. G. sulfurreducens coupled acetate oxidation with electron transfer to an anaerobic partner bacterium in the absence of ferric iron or other electron acceptors. Cocultures of G. sulfurreducens and Wolinella succinogenes with nitrate as the electron acceptor degraded acetate efficiently and grew with doubling times of 6 to 8 h. The hydrogen partial pressures in these acetate-degrading cocultures were considerably lower, in the range of 0.02 to 0.04 Pa. From these values and the concentrations of the other reactants, it was calculated that in this cooperation the free energy change available to G. sulfurreducens should be about −53 kJ per mol of acetate oxidized, assuming complete conversion of acetate to CO₂ and H₂. However, growth yields (18.5 g of dry mass per mol of acetate for the coculture, about 14 g for G. sulfurreducens) indicated considerably higher energy gains. These yield data, measurement of hydrogen production rates, and calculation of the diffusive hydrogen flux indicated that electron transfer in these cocultures may not proceed exclusively via interspecies hydrogen transfer but may also proceed through an alternative carrier system with higher redox potential, e.g., a c-type cytochrome that was found to be excreted by G. sulfurreducens into the culture fluid. Syntrophic acetate degradation was also possible with G. sulfurreducens and Desulfovibrio desulfuricans CSN but only with nitrate as electron acceptor. These cultures produced cell yields of 4.5 g of dry mass per mol of acetate, to which both partners contributed at about equal rates. These results demonstrate that some Fe(III)-reducing bacteria can oxidize organic compounds under Fe(III) limitation with the production of hydrogen, and they provide the first example of rapid acetate oxidation via interspecies electron transfer at moderate temperature.     

Isolation and characterization of a sulfur-oxidizing chemolithotroph growing on crude oil under anaerobic conditions

Molecular approaches have shown that a group of bacteria (called cluster 1 bacteria) affiliated with the epsilon subclass of the class Proteobacteria constituted major populations in underground crude-oil storage cavities. In order to unveil their physiology and ecological niche, this study isolated bacterial strains (exemplified by strain YK-1) affiliated with the cluster 1 bacteria from an oil storage cavity at Kuji in Iwate, Japan. 16S rRNA gene sequence analysis indicated that its closest relative was Thiomicrospira denitrificans (90% identity). Growth experiments under anaerobic conditions showed that strain YK-1 was a sulfur-oxidizing obligate chemolithotroph utilizing sulfide, elemental sulfur, thiosulfate, and hydrogen as electron donors and nitrate as an electron acceptor. Oxygen also supported its growth only under microaerobic conditions. Strain YK-1 could not grow on nitrite, and nitrite was the final product of nitrate reduction. Neither sugars, organic acids (including acetate), nor hydrocarbons could serve as carbon and energy sources. A typical stoichiometry of its energy metabolism followed an equation: S(2-) + 4NO(3)(-) --> SO(4)(2-) + 4NO(2)(-) (Delta G(0) = -534 kJ mol(-1)). In a difference from other anaerobic sulfur-oxidizing bacteria, this bacterium was sensitive to NaCl; growth in medium containing more than 1% NaCl was negligible. When YK-1 was grown anaerobically in a sulfur-depleted inorganic medium overlaid with crude oil, sulfate was produced, corresponding to its growth. On the contrary, YK-1 could not utilize crude oil as a carbon source. These results suggest that the cluster 1 bacteria yielded energy for growth in oil storage cavities by oxidizing petroleum sulfur compounds. Based on its physiology, ecological interactions with other members of the groundwater community are discussed.     

Electric coupling between distant nitrate reduction and sulfide oxidation in marine sediment

Filamentous bacteria of the Desulfobulbaceae family can conduct electrons over centimeter-long distances thereby coupling oxygen reduction at the surface of marine sediment to sulfide oxidation in deeper anoxic layers. The ability of these cable bacteria to use alternative electron acceptors is currently unknown. Here we show that these organisms can use also nitrate or nitrite as an electron acceptor thereby coupling the reduction of nitrate to distant oxidation of sulfide. Sulfidic marine sediment was incubated with overlying nitrate-amended anoxic seawater. Within 2 months, electric coupling of spatially segregated nitrate reduction and sulfide oxidation was evident from: (1) the formation of a 4–6-mm-deep zone separating sulfide oxidation from the associated nitrate reduction, and (2) the presence of pH signatures consistent with proton consumption by cathodic nitrate reduction, and proton production by anodic sulfide oxidation. Filamentous Desulfobulbaceae with the longitudinal structures characteristic of cable bacteria were detected in anoxic, nitrate-amended incubations but not in anoxic, nitrate-free controls. Nitrate reduction by cable bacteria using long-distance electron transport to get privileged access to distant electron donors is a hitherto unknown mechanism in nitrogen and sulfur transformations, and the quantitative importance for elements cycling remains to be addressed.