Anaerobic Mineralization of Toluene by Enriched Sediments with Quinones and Humus as Terminal Electron Acceptors

The anaerobic microbial oxidation of toluene to CO₂ coupled to humus respiration was demonstrated by use of enriched anaerobic sediments from the Amsterdam petroleum harbor (APH) and the Rhine River. Both highly purified soil humic acids (HPSHA) and the humic quinone moiety model compound anthraquinone-2,6-disulfonate (AQDS) were utilized as terminal electron acceptors. After 2 weeks of incubation, 50 and 85% of added uniformly labeled [¹³C]toluene were recovered as ¹³CO₂ in HPSHA- and AQDS-supplemented APH sediment enrichment cultures, respectively; negligible recovery occurred in unsupplemented cultures. The conversion of [¹³C]toluene agreed with the high level of recovery of electrons as reduced humus or as anthrahydroquinone-2,6-disulfonate. APH sediment was also able to use nitrate and amorphous manganese dioxide as terminal electron acceptors to support the anaerobic biodegradation of toluene. The addition of substoichiometric amounts of humic acids to bioassay reaction mixtures containing amorphous ferric oxyhydroxide as a terminal electron acceptor led to more than 65% conversion of toluene (1 mM) after 11 weeks of incubation, a result which paralleled the partial recovery of electron equivalents as acid-extractable Fe(II). Negligible conversion of toluene and reduction of Fe(III) occurred in these bioassay reaction mixtures when humic acids were omitted. The present study provides clear quantitative evidence for the mineralization of an aromatic hydrocarbon by humus-respiring microorganisms. The results indicate that humic substances may significantly contribute to the intrinsic bioremediation of anaerobic sites contaminated with priority pollutants by serving as terminal electron acceptors.     

Quinones as terminal electron acceptors for anaerobic microbial oxidation of phenolic compounds

The capacity of anaerobic granular sludge for oxidizing phenoland p-cresol under anaerobic conditions was studied. Phenol and p-cresolwere completely converted to methane when bicarbonate was the only terminal electron acceptor available. When the humic model compound, anthraquinone-2,6-disulfonate, was included as an alternative electron acceptor in the cultures, the oxidation of the phenolic compounds was coupled to the reduction of the model humic compound to its corresponding hydroquinone, anthrahydroquinone-2,6-disulfonate. These results demonstrate for the first time that the anaerobic degradation of phenolic compounds can be coupled to the reduction of quinones as terminal electron acceptor.     

Contribution of quinone-reducing microorganisms to the anaerobic biodegradation of organic compounds under different redox conditions

The capacity of two anaerobic consortia to oxidize different organic compounds, including acetate, propionate, lactate, phenol and p-cresol, in the presence of nitrate, sulfate and the humic model compound, anthraquinone-2,6-disulfonate (AQDS) as terminal electron acceptors, was evaluated. Denitrification showed the highest respiratory rates in both consortia studied and occurred exclusively during the first hours of incubation for most organic substrates degraded. Reduction of AQDS and sulfate generally started after complete denitrification, or even occurred at the same time during the biodegradation of p-cresol, in anaerobic sludge incubations; whereas methanogenesis did not significantly occur during the reduction of nitrate, sulfate, and AQDS. AQDS reduction was the preferred respiratory pathway over sulfate reduction and methanogenesis during the anaerobic oxidation of most organic substrates by the anaerobic sludge studied. In contrast, sulfate reduction out-competed AQDS reduction during incubations performed with anaerobic wetland sediment, which did not achieve any methanogenic activity. Propionate was a poor electron donor to achieve AQDS reduction; however, denitrifying and sulfate-reducing activities carried out by both consortia promoted the reduction of AQDS via acetate accumulated from propionate oxidation. Our results suggest that microbial reduction of humic substances (HS) may play an important role during the anaerobic oxidation of organic pollutants in anaerobic environments despite the presence of alternative electron acceptors, such as sulfate and nitrate. Methane inhibition, imposed by the inclusion of AQDS as terminal electron acceptor, suggests that microbial reduction of HS may also have important implications on the global climate preservation, considering the green-house effects of methane.     

Reduction of humic substances by halorespiring, sulphate-reducing and methanogenic microorganisms

Physiologically distinct anaerobic microorganisms were explored for their ability to oxidize different substrates with humic acids or the humic analogue, anthraquinone-2,6-disulphonate (AQDS), as a terminal electron acceptor. Most of the microorganisms evaluated including, for example, the halorespiring bacterium, Desulfitobacterium PCE1, the sulphate-reducing bacterium, Desulfovibrio G11 and the methanogenic archaeon, Methanospirillum hungatei JF1, could oxidize hydrogen linked to the reduction of humic acids or AQDS. Desulfitobacterium dehalogenans and Desulfitobacterium PCE1 could also convert lactate to acetate linked to the reduction of humic substances. Humus served as a terminal electron acceptor supporting growth of Desulfitobacterium species, which may explain the recovery of these microorganisms from organic rich environments in which the presence of chlorinated pollutants or sulphite is not expected. The results suggest that the ubiquity of humus reduction found in many different environments may be as a result of the increasing number of anaerobic microorganisms, which are known to be able to reduce humic substances.     

Humic Acid Reduction by Propionibacterium freudenreichii and Other Fermenting Bacteria

Iron-reducing bacteria have been reported to reduce humic acids and low-molecular-weight quinones with electrons from acetate or hydrogen oxidation. Due to the rapid chemical reaction of amorphous ferric iron with the reduced reaction products, humic acids and low-molecular-weight redox mediators may play an important role in biological iron reduction. Since many anaerobic bacteria that are not able to reduce amorphous ferric iron directly are known to transfer electrons to other external acceptors, such as ferricyanide, 2,6-anthraquinone disulfonate (AQDS), or molecular oxygen, we tested several physiologically different species of fermenting bacteria to determine their abilities to reduce humic acids. Propionibacterium freudenreichii, Lactococcus lactis, and Enterococcus cecorum all shifted their fermentation patterns towards more oxidized products when humic acids were present; P. freudenreichii even oxidized propionate to acetate under these conditions. When amorphous ferric iron was added to reoxidize the electron acceptor, humic acids were found to be equally effective when they were added in substoichiometric amounts. These findings indicate that in addition to iron-reducing bacteria, fermenting bacteria are also capable of channeling electrons from anaerobic oxidations via humic acids towards iron reduction. This information needs to be considered in future studies of electron flow in soils and sediments.     

Electron transfer by Desulfobulbus propionicus to Fe(III) and graphite electrodes

Desulfobulbus propionicus was able to grow with Fe(III), the humic acids analog anthraquinone-2,6-disulfonate (AQDS), or a graphite electrode as an electron acceptor. These results provide an explanation for the enrichment of Desulfobulbaceae species on the surface of electrodes harvesting electricity from anaerobic marine sediments and further expand the diversity of microorganisms known to have the ability to use both sulfate and Fe(III) as an electron acceptor.     

Selective enrichment of Geobacter sulfurreducens from anaerobic granular sludge with quinones as terminal electron acceptors

A quinone-respiring, enrichment culture derived from methanogenic granular sludge was phylogenetically characterized by using a combined cloning-denaturing gradient gel electrophoresis (DGGE) method, which revealed that the consortium developed was dominated by a single microorganism: 97% related, in a sequence of 1520 base pairs, to Geobacter sulfurreducens. The enrichment culture could grow with acetate, formate or H₂ when humic acids, the humic model compound, anthraquinone-2,6-disulfonate (AQDS), or chelated Fe(III) was provided as a terminal electron acceptor. The occurrence of a humic acid- or quinone-respiring microorganism in the microbial community of a wastewater treatment system suggests that this type of microorganisms may play a potential role in anaerobic bioreactors treating humus-containing wastewaters.     

Chemotactic responses to metals and anaerobic electron acceptors in Shewanella oneidensis MR-1

Although a previous study indicated that the dissimilatory metal-reducing bacterium Shewanella oneidensis MR-1 lacks chemotactic responses to metals that can be used as anaerobic electron acceptors, new results show that this bacterium responds to both Mn(III) and Fe(III). Cells were also shown to respond to another unusual electron acceptor, the humic acid analog anthraquinone-2,6-disulfonate. These results indicate that S. oneidensis is capable of moving towards a number of unusual anaerobic electron acceptors, including some that would normally be insoluble in the environment. Additionally, S. oneidensis was shown to migrate in gradients of several divalent cations under anaerobic conditions. Although responses to the reduced forms of redox-active metals, such as Mn(II) and Fe(II), might indicate that S. oneidensis uses gradients of these metals to locate the insoluble electron acceptors Mn(III/IV) and Fe(III) for dissimilatory purposes, responses to non-redox-active metals, such as Zn(II), suggest that movement towards divalent cations might serve other, potentially assimilatory, purposes.     

Novel physiological features of Carboxydothermus hydrogenoformans and Thermoterrabacterium ferrireducens

Carboxydothermus hydrogenoformans is able to grow by conversion of CO to H2 and CO2. Besides CO, only pyruvate was described as serving as an energy source. Based on 16S rRNA gene sequence similarity, C. hydrogenoformans is closely related to Thermoterrabacterium ferrireducens. T. ferrireducens is like C. hydrogenoformans a gram-positive, thermophilic, strict anaerobic bacterium. However, it is capable of using various electron donors and acceptors for growth. Growth of C. hydrogenoformans with multiple electron donors and acceptors was tested. C. hydrogenoformans oxidized formate, lactate, glycerol, CO, and H2 with 9,10-anthraquinone-2,6-disulfonate as an electron acceptor. Sulfite, thiosulfate, sulfur, nitrate, and fumarate were reduced with lactate as an electron donor. T. ferrireducens oxidized CO with 9,10-anthraquinone-2,6-disulfonate as an electron acceptor but did not produce H2 from CO. In contrast to what was published before, T. ferrireducens was able to grow on lactate with sulfite, sulfur, and nitrate as electron acceptors.     

Electron Transfer by Desulfobulbus propionicus to Fe(III) and Graphite Electrodes

Desulfobulbus propionicus was able to grow with Fe(III), the humic acids analog anthraquinone-2,6-disulfonate (AQDS), or a graphite electrode as an electron acceptor. These results provide an explanation for the enrichment of Desulfobulbaceae species on the surface of electrodes harvesting electricity from anaerobic marine sediments and further expand the diversity of microorganisms known to have the ability to use both sulfate and Fe(III) as an electron acceptor.     

Humic analog AQDS and AQS as an electron mediator can enhance chromate reduction by Bacillus sp. strain 3C3

Humus as an electron mediator is recognized as an effective strategy to improve the biological transformation and degradation of toxic substances, yet the action of humus in microbial detoxification of chromate is still unknown. In this study, a humus-reducing strain 3C₃ was isolated from mangrove sediment. Based on the analyses of morphology, physiobiochemical characteristics, and 16S rRNA gene sequence, this strain was identified Bacillus sp. Strain 3C₃ can effectively reduce humic analog anthraquinone-2,6-disulfonate (AQDS) and anthraquinone-2-sulfonate (AQS) with lactate, formate, or glucose as electron donors. When the cells were killed by incubation at 95°C for 30 min or an electron donor was absent, the humic reduction did not occur, showing that the humic reduction was a biochemical process. However, strain 3C₃ had low capability of chromate reduction under anaerobic conditions, despite of having strong tolerance of the toxic metal. But in the presence of humic substances AQDS or AQS, we found that chromate reduction by strain 3C₃ was enhanced greatly. Because strain 3C₃ is an effective humus-reducing bacterium, it is proposed that humic substances could serve as electron mediator to interact with chromate and accelerate chromate reduction. Our results suggest that chromate contaminations can be detoxified by adding humic analog (low to 0.1 mM) as an electron mediator in the microbial incubation.     

Competition between methanogenesis and quinone respiration for ecologically important substrates in anaerobic consortia

Anaerobic consortia obtained from a wide variety of environments were tested for oxidizing several ecologically significant substrates with the humic model compound, anthraquinone-2,6-disulfonate (AQDS), as terminal electron acceptor. All the substrates, including hydrogen, acetate, propionate, methanol and lactate, were completely or partially converted to methane when bicarbonate was the only electron acceptor available. Addition of AQDS (20 mM) to the cultures prevented methanogenesis in most cases and AQDS reduction became the preferred pathway. AQDS was shown to be toxic for methanogenesis and this effect played an important role in enabling quinone-respiring bacteria to outcompete methanogens. Furthermore, AQDS respiration is thermodynamically more favorable than methanogenesis. All the consortia evaluated were capable of oxidizing hydrogen linked to the reduction of AQDS. Most inocula tested were also able to oxidize acetate and lactate in the same way. When methanol was provided as an electron donor competition between methanogenesis and acetogenesis occurred. Acetate accumulated from the latter process was responsible for quinone respiration. These results suggest that quinone-respiring bacteria are ubiquitous and that quinones in humus may significantly contribute to carbon cycling process by serving as a terminal electron acceptor for the anaerobic microbial oxidation of a wide variety of ecologically important substrates.     

Quinone-respiration improves dechlorination of carbon tetrachloride by anaerobic sludge

The impact of humic acids and the humic model compound, anthraquinone-2,6-disulfonate (AQDS), on the biodegradation of carbon tetrachloride (CT) by anaerobic granular sludge was studied. Addition of both humic acids and AQDS at sub-stoichiometric levels increased the first-order rate of conversion of CT up to 6-fold, leading to an increased production of inorganic chloride, which accounted for 40–50% of the CT initially added. Considerably less dechlorination occurred in sludge incubations lacking humic substances. By comparison, very limited dechlorination occurred in sterile controls with autoclaved sludge. Accumulation of chloroform (1–10%) and dichloromethane (traces) also accounted for the CT converted. The accumulation of a chlorinated ethene, perchloroethylene (up to 9% of added CT), is also reported for the first time as an end-product of CT degradation. A humus-respiring enrichment culture (composed primarily of a Geobacter sp.) derived from the granular sludge also dechlorinated CT, yielding products similar to the AQDS-supplemented granular sludge consortium. The dechlorination of CT by the Geobacter enrichment was dependent on the presence of AQDS or humic acids, which were reduced during the assays. The reduced form of AQDS, anthrahydroquinone-2,6-disulfonate, was shown to cause the chemical reduction of CT when incubated in sterile medium. The results taken as a whole indicate that the formation of reduced humic substances by quinone-respiring microorganisms can contribute to the reductive dechlorination of CT.     

Sequential application of electron donors and humic acids for the anaerobic bioremediation of chlorinated aliphatic hydrocarbons

In situ anaerobic bioremediation of chlorinated solvents such as perchloroethene (PCE) frequently faces the problem of accumulating toxic, lower chlorinated compounds such as dichloroethene (cis-DCE) and vinyl chloride (VC). In the present study, the efficacy of the sequential application of electron donors, supporting reductive dechlorination, and of humic acids, acting as extracellular electron shuttles facilitating the anaerobic oxidation of recalcitrant intermediates, was explored in microcosm studies. Upon one initial dose of lactose, supplied in a 1000-fold superstoichiometric electron equivalent ratio, PCE was completely converted into cis-DCE within 35 days. Repeated electron donor additions did not entail exhaustive cis-DCE degradation over incubation time (120 days). Although the electron donor was quickly converted into fatty acids, about 30% of added reducing equivalents were recovered as acetate after four months of operation, indicating the inhibition of acetoclastic methanogenesis. In the next step, the substoichiometric addition of anthraquinone-2,6-disulfonate, a humic acid model compound, effected the complete removal of the accumulated cis-DCE within 15 days, probably as a result of the participation of the quinone in the biotic or abiotic anaerobic oxidation of cis-DCE. Cis-DCE degradation was not connected to the accumulation of VC, rendering the proposed two-step treatment an efficient and environmentally compliant remedy for anaerobic groundwater bodies contaminated with chlorinated solvents.     

Novel Physiological Features of Carboxydothermus hydrogenoformans and Thermoterrabacterium ferrireducens

Carboxydothermus hydrogenoformans is able to grow by conversion of CO to H₂ and CO₂. Besides CO, only pyruvate was described as serving as an energy source. Based on 16S rRNA gene sequence similarity, C. hydrogenoformans is closely related to Thermoterrabacterium ferrireducens. T. ferrireducens is like C. hydrogenoformans a gram-positive, thermophilic, strict anaerobic bacterium. However, it is capable of using various electron donors and acceptors for growth. Growth of C. hydrogenoformans with multiple electron donors and acceptors was tested. C. hydrogenoformans oxidized formate, lactate, glycerol, CO, and H₂ with 9,10-anthraquinone-2,6-disulfonate as an electron acceptor. Sulfite, thiosulfate, sulfur, nitrate, and fumarate were reduced with lactate as an electron donor. T. ferrireducens oxidized CO with 9,10-anthraquinone-2,6-disulfonate as an electron acceptor but did not produce H₂ from CO. In contrast to what was published before, T. ferrireducens was able to grow on lactate with sulfite, sulfur, and nitrate as electron acceptors.     

中国希瓦氏菌D14^T的厌氧腐殖质呼吸

实验证明,希瓦氏菌新种(ShewanellacinicaD14T)在厌氧条件下可以利用多种有机酸盐和甲苯等环境有毒污染物作为电子供体,以腐殖质作为唯一末端电子受体进行厌氧呼吸(即醌呼吸)。电子在细胞膜呼吸链的传递过程中,偶联能量的产生来支持菌体的生长,1mmol/LAQDS可支持细胞增殖约60倍。电子供体的氧化和唯一电子受体腐殖质还原之间存在着动态的偶联过程,随着电子供体量的增加腐殖质还原的量也随之增加。典型呼吸链抑制剂诸如:抑制Fe-S中心的Cu2 ,甲基萘醌类似物标桩菌素,抑制甲基萘醌氧化型向还原型转化的双香豆素和细胞色素P450的专一抑制物甲吡酮等对腐殖质的还原有着极为显著的抑制作用,为进一步证明希瓦氏菌(Shewanellacinica)D14T可利用腐殖质进行厌氧呼吸提供了有力的佐证。而D14T在进行腐殖质呼吸的同时,对于甲苯,苯胺等环境有毒物质的有效降解则具有着重要的环境学意义。     

腐殖质在环境污染物生物降解中的作用研究进展

腐殖质物质在地球的生态环境中大量存在,它不仅可以在有毒化合物的生物降解和生物转化过程中起到氧化还原中间体的作用,加速有毒物质的降解和转化。也可以作为唯一末端电子受体,接受来自一些有机酸或者甲苯等环境中有毒物质提供的电子,偶联能量的产生,支持菌体的生长,形成一种新的细菌厌氧呼吸形式——腐殖质呼吸。因此,对腐殖质在环境有毒物质的生物降解和生物转化过程中的作用进行研究,不仪对于深入理解细菌呼吸的本质具有重要的理论意义,而且对于环境有毒物质的降解和转化以及元素的生物地球化学循环具有重要的生态学意义,同时对地球表面的有毒物质进行更有效的生物降解具有重要的现实意义。     

Unexpected role of electron-transfer hub in direct degradation of pollutants by exoelectrogenic bacteria

Exoelectrogenic bacteria (EEB) are capable of anaerobic respiration with diverse extracellular electron acceptors including insoluble minerals, electrodes and flavins, but the detailed electron transfer pathways and reaction mechanisms remain elusive. Here, we discover that CymA, which is usually considered to solely serve as an inner-membrane electron transfer hub in Shewanella oneidensis MR-1 (a model EEB), might also function as a reductase for direct reducing diverse nitroaromatic compounds (e.g. 2,4-dichloronitrobenzene) and azo dyes. Such a process can be accelerated by dosing anthraquinone-2,6-disulfonate. The CymA-based reduction pathways in S. oneidensis MR-1 for different contaminants could be functionally reconstructed and strengthened in Escherichia coli. The direct reduction of lowly polar contaminants by quinol oxidases like CymA homologues might be universal in diverse microbes. This work offers new insights into the pollutant reduction mechanisms of EEB and unveils a new function of CymA to act as a terminal reductase.     

Geoalkalibacter ferrihydriticus gen. nov., sp. nov., the first alkaliphilic representative of the family Geobacteraceae, isolated from a soda lake

Investigation of iron reduction in bottom sediments of alkaline soda lakes resulted in the isolation of a new obligately anaerobic iron-reducing bacterium, strain Z-0531, from Lake Khadyn (Tuva Republic, Russia) sediment samples. The cells of strain Z-0531 are short (1.0-1.5 by 0.3-0.5 microm), motile, non-spore-forming, gram-negative rods. The isolate is an obligate alkaliphile, developing in the pH range of 7.8-10.0, with an optimum at pH 8.6. It does not require NaCl but grows at NaCl concentrations of 0-50 g/1l. It can oxidize acetate with such electron acceptors as amorphous Fe(llI) hydroxide (AFH), EDTA-Fe(III), anthraquinone-2,6-disulfonate (quinone), Mn(IV), and S(0). On media with EDTA-Fe(III), the isolate can oxidize, apart from acetate, ethanol, pyruvate, oxalate, arginine, tartrate, lactate, propionate, and serine. H2 is not utilized. The reduced products formed during growth with AFH are siderite or magnetite, depending on the growth conditions. The isolate is incapable of fermenting sugars, peptides, and amino acids. Yeast extract or vitamins are required as growth factors. The organism is capable of dinitrogen fixation and harbors the nifH gene. The DNA G+C content is 55.3 mol %. 16S rRNA analysis places strain Z-0531 into the family Geobacteraceae. Its closest relative (93% similarity) is Desulfuromonas palmitatis. Based on phenotypic distinctions and phylogenetic position, it is proposed that strain Z-0531 be assigned to the new genus and species Geoalkalibacter ferrihydriticus gen. nov., sp. nov.     

Reduction of Fe(III), Cr(VI), U(VI), and Tc(VII) by Deinococcus radiodurans R1

Deinococcus radiodurans is an exceptionally radiation-resistant microorganism capable of surviving acute exposures to ionizing radiation doses of 15,000 Gy and previously described as having a strictly aerobic respiratory metabolism. Under strict anaerobic conditions, D. radiodurans R1 reduced Fe(III)-nitrilotriacetic acid coupled to the oxidation of lactate to CO(2) and acetate but was unable to link this process to growth. D. radiodurans reduced the humic acid analog anthraquinone-2,6-disulfonate (AQDS) to its dihydroquinone form, AH(2)DS, which subsequently transferred electrons to the Fe(III) oxides hydrous ferric oxide and goethite via a previously described electron shuttle mechanism. D. radiodurans reduced the solid-phase Fe(III) oxides in the presence of either 0.1 mM AQDS or leonardite humic acids (2 mg ml(-1)) but not in their absence. D. radiodurans also reduced U(VI) and Tc(VII) in the presence of AQDS. In contrast, Cr(VI) was directly reduced in anaerobic cultures with lactate although the rate of reduction was higher in the presence of AQDS. The results are the first evidence that D. radiodurans can reduce Fe(III) coupled to the oxidation of lactate or other organic compounds. Also, D. radiodurans, in combination with humic acids or synthetic electron shuttle agents, can reduce U and Tc and thus has potential applications for remediation of metal- and radionuclide-contaminated sites where ionizing radiation or other DNA-damaging agents may restrict the activity of more sensitive organisms.