Microbial Phosphite Oxidation and Its Potential Role in the Global Phosphorus and Carbon Cycles.

Phosphite [Formula: see text] is a highly soluble, reduced phosphorus compound that is often overlooked in biogeochemical analyses. Although the oxidation of phosphite to phosphate is a highly exergonic process (Eo'=-650mV), phosphite is kinetically stable and can account for 10-30% of the total dissolved P in various environments. There is also evidence that phosphite was more prevalent under the reducing conditions of the Archean period and may have been involved in the development of early life. Its role as a phosphorus source for a variety of extant microorganisms has been known since the 1950s, and the pathways involved in assimilatory phosphite oxidation have been well characterized. More recently, it was demonstrated that phosphite could also act as an electron donor for energy metabolism in a process known as dissimilatory phosphite oxidation (DPO). The bacterium described in this study, Desulfotignum phosphitoxidans strain FiPS-3, was isolated from brackish sediments and is capable of growing by coupling phosphite oxidation to the reduction of either sulfate or carbon dioxide. FiPS-3 remains the only isolated organism capable of DPO, and the prevalence of this metabolism in the environment is still unclear. Nonetheless, given the widespread presence of phosphite in the environment and the thermodynamic favorability of its oxidation, microbial phosphite oxidation may play an important and hitherto unrecognized role in the global phosphorus and carbon cycles.

Heterologous expression and purification of a multiheme cytochrome from a Gram-positive bacterium capable of performing extracellular respiration.

Microbial electrochemical technologies are emerging as environmentally friendly biotechnological processes. Recently, a thermophilic Gram-positive bacterium capable of electricity production in a microbial fuel cell was isolated. Thermincola potens JR contains several multiheme c-type cytochromes that were implicated in the process of electricity production. In order to understand the molecular basis by which Gram-positive bacteria perform extracellular electron transfer, the relevant proteins need to be characterized in detail. Towards this end, a chimeric gene containing the signal peptide from Shewanella oneidensis MR-1 small tetraheme cytochrome c (STC) and the gene sequence of the target protein TherJR_0333 was constructed. This manuscript reports the successful expression of this chimeric gene in the Gram-negative bacterium Escherichia coli and its subsequent purification and characterization. This methodology opens the possibility to study other multiheme cytochromes from Gram-positive bacteria, allowing the extracellular electron transfer mechanisms of this class of organisms to be unraveled.

Evidence for direct electron transfer by a gram-positive bacterium isolated from a microbial fuel cell.

Despite their importance in iron redox cycles and bioenergy production, the underlying physiological, genetic, and biochemical mechanisms of extracellular electron transfer by Gram-positive bacteria remain insufficiently understood. In this work, we investigated respiration by Thermincola potens strain JR, a Gram-positive isolate obtained from the anode surface of a microbial fuel cell, using insoluble electron acceptors. We found no evidence that soluble redox-active components were secreted into the surrounding medium on the basis of physiological experiments and cyclic voltammetry measurements. Confocal microscopy revealed highly stratified biofilms in which cells contacting the electrode surface were disproportionately viable relative to the rest of the biofilm. Furthermore, there was no correlation between biofilm thickness and power production, suggesting that cells in contact with the electrode were primarily responsible for current generation. These data, along with cryo-electron microscopy experiments, support contact-dependent electron transfer by T. potens strain JR from the cell membrane across the 37-nm cell envelope to the cell surface. Furthermore, we present physiological and genomic evidence that c-type cytochromes play a role in charge transfer across the Gram-positive bacterial cell envelope during metal reduction.

Mercury methylation by dissimilatory iron-reducing bacteria.

The Hg-methylating ability of dissimilatory iron-reducing bacteria in the genera Geobacter, Desulfuromonas, and Shewanella was examined. All of the Geobacter and Desulfuromonas strains tested methylated mercury while reducing Fe(III), nitrate, or fumarate. In contrast, none of the Shewanella strains produced methylmercury at higher levels than abiotic controls under similar culture conditions. Geobacter and Desulfuromonas are closely related to known Hg-methylating sulfate-reducing bacteria within the Deltaproteobacteria.

Anaerobic degradation of monoaromatic hydrocarbons.

Over the last two decades significant advances have been made in our understanding of the anaerobic biodegradability of monoaromatic hydrocarbons. It is now known that compounds such as benzene, toluene, ethylbenzene, and all three xylene isomers can be biodegraded in the absence of oxygen by a broad diversity of organisms. These compounds have been shown to serve as carbon and energy sources for bacteria growing phototrophically, or respiratorily with nitrate, manganese, ferric iron, sulfate, or carbon dioxide as the sole electron acceptor. In addition, it has also been recently shown that complete degradation of monoaromatic hydrocarbons can also be coupled to the respiration of oxyanions of chlorine such as perchlorate or chlorate, or to the reduction of the quinone moieties of humic substances. Many pure cultures of hydrocarbon-degrading anaerobes now exist and some novel biochemical and genetic pathways have been identified. In general, a fumarate addition reaction is used as the initial activation step of the catabolic process of the corresponding monoaromatic hydrocarbon compounds. However, other reactions may alternatively be involved depending on the electron acceptor utilized or the compound being degraded. In the case of toluene, fumarate addition to the methyl group mediated by benzylsuccinate synthase appears to be the universal mechanism of activation and is now known to be utilized by anoxygenic phototrophs, nitrate-reducing, Fe(III)-reducing, sulfate-reducing, and methanogenic cultures. Many of these biochemical pathways produce unique extracellular intermediates that can be utilized as biomarkers for the monitoring of hydrocarbon degradation in anaerobic natural environments.

Anaerobic biooxidation of Fe(II) by Dechlorosoma suillum.

Anaerobic microbial oxidation of Fe(II) was only recently discovered and very little is known about this metabolism. We recently demonstrated that several dissimilatory perchlorate-reducing bacteria could utilize Fe(II) as an electron donor under anaerobic conditions. Here we report on a more in-depth analysis of Fe(II) oxidation by one of these organisms, Dechlorosoma suillum. Similarly to most known nitrate-dependent Fe(II) oxidizers, D. suillum did not grow heterotrophically or lithoautotrophically by anaerobic Fe(II) oxidation. In the absence of a suitable organic carbon source, cells rapidly lysed even though nitrate-dependent Fe(II) oxidation was still occurring. The coupling of Fe(II) oxidation to a particular electron acceptor was dependent on the growth conditions of cells of D. suillum. As such, anaerobically grown cultures of D. suillum did not mediate Fe(II) oxidation with oxygen as the electron acceptor, while conversely, aerobically grown cultures did not mediate Fe(II) oxidation with nitrate as the electron acceptor. Anaerobic washed cell suspensions of D. suillum rapidly produced an orange/brown precipitate which X-ray diffraction analysis identified as amorphous ferric oxyhydroxide or ferrihydrite. This is similar to all other identified nitrate-dependent Fe(II) oxidizers but is in contrast to what is observed for growth cultures of D. suillum, which produced a mixed-valence Fe(II)-Fe(III) precipitate known as green rust. D. suillum rapidly oxidized the Fe(II) content of natural sediments. Although the form of ferrous iron in these sediments is unknown, it is probably a component of an insoluble mineral, as previous studies indicated that soluble Fe(II) is a relatively minor form of the total Fe(II) content of anoxic environments. The results of this study further enhance our knowledge of a poorly understood form of microbial metabolism and indicate that anaerobic Fe(II) oxidation by D. suillum is significantly different from previously described forms of nitrate-dependent microbial Fe(II) oxidation.

Anaerobic benzene oxidation coupled to nitrate reduction in pure culture by two strains of Dechloromonas.

Benzene contamination is a significant problem. It is used in a wide range of manufacturing processes and is a primary component of petroleum-based fuels. Benzene is a hydrocarbon that is soluble, mobile, toxic and stable, especially in ground and surface waters. It is poorly biodegraded in the absence of oxygen. However, anaerobic benzene biodegradation has been documented under various conditions. Although benzene biomineralization has been demonstrated with nitrate, Fe(III), sulphate or CO2 as alternative electron acceptors, these studies were based on sediments or microbial enrichments. Until now there were no organisms in pure culture that degraded benzene anaerobically. Here we report two Dechloromonas strains, RCB and JJ, that can completely mineralize various mono-aromatic compounds including benzene to CO2 in the absence of O2 with nitrate as the electron acceptor. This is the first example, to our knowledge, of an organism of any type that can oxidize benzene anaerobically, and we demonstrate the potential applicability of these organisms to the treatment of contaminated environments.

Biogenic magnetite formation through anaerobic biooxidation of Fe(II).

The presence of isotopically light carbonates in association with fine-grained magnetite is considered to be primarily due to the reduction of Fe(III) by Fe(III)-reducing bacteria in the environment. Here, we report on magnetite formation by biooxidation of Fe(II) coupled to denitrification. This metabolism offers an alternative environmental source of biogenic magnetite.

Dechloromonas agitata gen. nov., sp. nov. and Dechlorosoma suillum gen. nov., sp. nov., two novel environmentally dominant (per)chlorate-reducing bacteria and their phylogenetic position.

Previous studies on the ubiquity and diversity of microbial (per)chlorate reduction resulted in the isolation of 20 new strains of dissimilatory (per)chlorate-reducing bacteria. Phylogenetic analysis revealed that all of the isolates were members of the Proteobacteria with representatives in the alpha-, beta- and gamma-subclasses. The majority of the new isolates were located in the beta-subclass and were closely related to each other and to the phototrophic Rhodocyclus species. Here an in-depth analysis of these organisms which form two distinct monophyletic groups within the Rhodocyclus assemblage is presented. Two new genera, Dechloromonas and Dechlorosoma, are proposed for these beta-subclass lineages which represent the predominant (per)chlorate-reducing bacteria in the environment. The type species and strains for these new genera are Dechloromonas agitata strain CKBT and Dechlorosoma suillum strain PST, respectively.

Geobacter hydrogenophilus, Geobacter chapellei and Geobacter grbiciae, three new, strictly anaerobic, dissimilatory Fe(III)-reducers.

Recent studies on the diversity and ubiquity of Fe(III)-reducing organisms in different environments led to the isolation and identification of four new dissimilatory Fe(III)-reducers (strains H-2T, 172T, TACP-2T and TACP-5). All four isolates are non-motile, Gram-negative, freshwater, mesophilic, strict anaerobes with morphology identical to that of Geobacter metallireducens strain GS-15T. Analysis of the 16S rRNA sequences indicated that the new isolates belong to the genus Geobacter, in the delta-Proteobacteria. Significant differences in phenotypic characteristics, DNA-DNA homology and G+C content indicated that the four isolates represent three new species of the genus. The names Geobacter hydrogenophilus sp. nov. (strain H-2T), Geobacter chapellei sp. nov. (strain 172T) and Geobacter grbiciae sp. nov. (strains TACP-2T and TACP-5) are proposed. Geobacter hydrogenophilus and Geobacter chapellei were isolated from a petroleum-contaminated aquifer and a pristine, deep, subsurface aquifer, respectively. Geobacter grbiciae was isolated from aquatic sediments. All of the isolates can obtain energy for growth by coupling the oxidation of acetate to the reduction of Fe(III). The four isolates also coupled Fe(III) reduction to the oxidation of other simple, volatile fatty acids. In addition, Geobacter hydrogenophilus and Geobacter grbiciae were able to oxidize aromatic compounds such as benzoate, whilst Geobacter grbiciae was also able to use the monoaromatic hydrocarbon toluene.

Emerging techniques for anaerobic bioremediation of contaminated environments.

Over the past decade, it has been recognized that the diversity of anaerobic microbial metabolism is far greater than was previously assumed, and that many contaminants previously considered to be recalcitrant under anoxic conditions can in fact be biotransformed in the absence of molecular oxygen. Here, we summarize recent advances in the understanding of novel forms of anaerobic microbial metabolism and their potential application to bioremediative technologies.

Novel forms of anaerobic respiration of environmental relevance.

Novel forms of anaerobic respiration continue to be discovered. Many of these are environmentally significant as they have important impacts on the fate of organic carbon and the cycling of many inorganic compounds. Furthermore, anaerobic respiration is becoming increasing recognized as a strategy for the remediation of organic and metal contaminants in the subsurface.

Ubiquity and diversity of dissimilatory (per)chlorate-reducing bacteria.

Environmental contamination with compounds containing oxyanions of chlorine, such as perchlorate or chlorate [(per)chlorate] or chlorine dioxide, has been a constantly growing problem over the last 100 years. Although the fact that microbes reduce these compounds has been recognized for more than 50 years, only six organisms which can obtain energy for growth by this metabolic process have been described. As part of a study to investigate the diversity and ubiquity of microorganisms involved in the microbial reduction of (per)chlorate, we enumerated the (per)chlorate-reducing bacteria (ClRB) in very diverse environments, including pristine and hydrocarbon-contaminated soils, aquatic sediments, paper mill waste sludges, and farm animal waste lagoons. In all of the environments tested, the acetate-oxidizing ClRB represented a significant population, whose size ranged from 2.31 x 10(3) to 2.4 x 10(6) cells per g of sample. In addition, we isolated 13 ClRB from these environments. All of these organisms could grow anaerobically by coupling complete oxidation of acetate to reduction of (per)chlorate. Chloride was the sole end product of this reductive metabolism. All of the isolates could also use oxygen as a sole electron acceptor, and most, but not all, could use nitrate. The alternative electron donors included simple volatile fatty acids, such as propionate, butyrate, or valerate, as well as simple organic acids, such as lactate or pyruvate. Oxidized-minus-reduced difference spectra of washed whole-cell suspensions of the isolates had absorbance maxima close to 425, 525, and 550 nm, which are characteristic of type c cytochromes. In addition, washed cell suspensions of all of the ClRB isolates could dismutate chlorite, an intermediate in the reductive metabolism of (per)chlorate, into chloride and molecular oxygen. Chlorite dismutation was a result of the activity of a single enzyme which in pure form had a specific activity of approximately 1,928 micromol of chlorite per mg of protein per min. Analyses of the 16S ribosomal DNA sequences of the organisms indicated that they all belonged to the alpha, beta, or gamma subclass of the Proteobacteria. Several were closely related to members of previously described genera that are not recognized for the ability to reduce (per)chlorate, such as the genera Pseudomonas and Azospirllum. However, many were not closely related to any previously described organism and represented new genera within the Proteobacteria. The results of this study significantly increase the limited number of microbial isolates that are known to be capable of dissimilatory (per)chlorate reduction and demonstrate the hitherto unrecognized phylogenetic diversity and ubiquity of the microorganisms that exhibit this type of metabolism.

Geothrix fermentans gen. nov., sp. nov., a novel Fe(III)-reducing bacterium from a hydrocarbon-contaminated aquifer.

In an attempt to understand better the micro-organisms involved in anaerobic degradation of aromatic hydrocarbons in the Fe(III)-reducing zone of petroleum-contaminated aquifers, Fe(III)-reducing micro-organisms were isolated from contaminated aquifer material that had been adapted for rapid oxidation of toluene coupled to Fe(III) reduction. One of these organisms, strain H-5T, was enriched and isolated on acetate/Fe(III) medium. Strain H-5T is a Gram-negative strict anaerobe that grows with various simple organic acids such as acetate, propionate, lactate and fumarate as alternative electron donors with Fe(III) as the electron acceptor. In addition, strain H-5T also oxidizes long-chain fatty acids such as palmitate with Fe(III) as the sole electron acceptor. Strain H-5T can also grow by fermentation of citrate or fumarate in the absence of an alternative electron acceptor. The primary end-products of citrate fermentation are acetate and succinate. In addition to various forms of soluble and insoluble Fe(III), strain H-5T grows with nitrate, Mn(IV), fumarate and the humic acid analogue 2,6-anthraquinone disulfonate as alternative electron acceptors. As with other organisms that can oxidize organic compounds completely with the reduction of Fe(III), cell suspensions of strain H-5T have absorbance maxima indicative of a c-type cytochrome(s). It is proposed that strain H-5T represents a novel genus in the Holophaga-Acidobacterium phylum and that it should be named Geothrix fermentans sp. nov., gen. nov.

Humics as an electron donor for anaerobic respiration.

The possibility that microorganisms might use reduced humic substances (humics) as an electron donor for the reduction of electron acceptors with a more positive redox potential was investigated. All of the Fe(III)- and humics-reducing microorganisms evaluated were capable of oxidizing reduced humics and/or the reduced humics analogue anthrahydroquinone-2,6,-disulphonate (AHODS), with nitrate and/or fumarate as the electron acceptor. These included Geobacter metallireducens, Geobacter sulphurreducens, Geothrix fermentans, Shewanella alga, Wolinella succinogenes and 'S. barnesii'. Several of the humics-oxidizing microorganisms grew in medium with AHQDS as the sole electron donor and fumarate as the electron acceptor. Even though it does not reduce Fe(III) or humics, Paracoccus denitrificans could use AHQDS and reduced humics as electron donors for denitrification. However, another denitrifier, Pseudomonas denitrificans, could not. AHODS could also serve as an electron donor for selenate and arsenate reduction by W. succinogenes. Electron spin resonance studies demonstrated that humics oxidation was associated with the oxidation of hydroquinone moieties in the humics. Studies with G. metallireducens and W. succinogenes demonstrated that the anthraquinone-2,6-disulphonate (AQDS)/AHQDS redox couple mediated an interspecies electron transfer between the two organisms. These results suggest that, as microbially reduced humics enter less reduced zones of soils and sediments, the reduced humics may serve as electron donors for microbial reduction of several environmentally significant electron acceptors.

Reduction of (per)chlorate by a novel organism isolated from paper mill waste.

As part of a study on the microbiology of chlorate reduction, several new dissimilatory chlorate-reducing bacteria were isolated from a broad diversity of environments. One of these, strain CKB, was selected for a more complete characterization. Strain CKB was enriched and isolated from paper mill waste with acetate as the sole electron donor and chlorate as the sole electron acceptor. Strain CKB is a completely oxidizing, non-fermentative, Gram-negative, facultative anaerobe. Cells of strain CKB are 0.5 x 2 microm and are highly motile, with a single polar flagellum. In addition to acetate, strain CKB can use propionate, butyrate, lactate, succinate, fumarate, malate or yeast extract as electron donors, with chlorate as the sole electron acceptor. Strain CKB can also couple chlorate reduction to the oxidation of ferrous iron, sulphide, or the reduced form of the humic substances analogue 2,6-anthrahydroquinone disulphonate. Fe(II) is oxidized to insoluble amorphous Fe(II) oxide, whereas sulphide is oxidized to elemental sulphur. Growth is not associated with this metabolism, even when small quantities of acetate are added as a potential carbon source. In addition to chlorate, strain CKB can also couple acetate oxidation to the reduction of oxygen or perchlorate. Chlorate is completely reduced to chloride. Strain CKB has an optimum temperature of 35 degrees C, a pH optimum of 7.5 and a salinity optimum of 1% NaCl. Strain CKB can grow in chlorate and perchlorate concentrations of 80 or 20 mM respectively. Under anaerobic conditions, strain CKB can dismutate chlorite into chloride and O2, and is only the second organism shown to be capable of this metabolism. Oxidized minus reduced spectra of whole-cell suspensions of strain CKB showed absorbance maxima at 423, 523 and 552nm, which are indicative of the presence of c-type cytochrome(s). Analysis of the complete sequence of the 16S rDNA indicates that strain CKB is a member of the beta subclass of the Proteobacteria. The phototroph Rhodocyclus tenuis is the closest known relative. When tested, strain CKB could not grow by phototrophy and did not contain bacteriochlorophyll. Phenotypically and phylogenetically, strain CKB differs from all other described bacteria and represents the type strain of a new genus and species.

Recovery of humic-reducing bacteria from a diversity of environments.

To evaluate which microorganisms might be responsible for microbial reduction of humic substances in sedimentary environments, humic-reducing bacteria were isolated from a variety of sediment types. These included lake sediments, pristine and contaminated wetland sediments, and marine sediments. In each of the sediment types, all of the humic reducers recovered with acetate as the electron donor and the humic substance analog, 2,6-anthraquinone disulfonate (AQDS), as the electron acceptor were members of the family Geobacteraceae. This was true whether the AQDS-reducing bacteria were enriched prior to isolation on solid media or were recovered from the highest positive dilutions of sediments in liquid media. All of the isolates tested not only conserved energy to support growth from acetate oxidation coupled to AQDS reduction but also could oxidize acetate with highly purified soil humic acids as the sole electron acceptor. All of the isolates tested were also able to grow with Fe(III) serving as the sole electron acceptor. This is consistent with previous studies that have suggested that the capacity for Fe(III) reduction is a common feature of all members of the Geobacteraceae. These studies demonstrate that the potential for microbial humic substance reduction can be found in a wide variety of sediment types and suggest that Geobacteraceae species might be important humic-reducing organisms in sediments.

Carbohydrate oxidation coupled to Fe(III) reduction, a novel form of anaerobic metabolism.

An isolate, designated GC-29, that could incompletely oxidize glucose to acetate and carbon dioxide with Fe(III) serving as the electron acceptor was recovered from freshwater sediments of the Potomac River, Maryland. This metabolism yielded energy to support cell growth. Strain GC-29 is a facultatively anaerobic, gram-negative motile rod which, in addition to glucose, also used sucrose, lactate, pyruvate, yeast extract, casamino acids or H2 as alternative electron donors for Fe(III) reduction. Stain GC-29 could reduce NO3(-), Mn(IV), U(VI), fumarate, malate, S2O3(2-), and colloidal S0 as well as the humics analog, 2,6-anthraquinone disulfonate. Analysis of the almost complete 16S rRNA sequence indicated that strain GC-29 belongs in the Shewanella genus in the epsilon subdivision of the Proteobacteria. The name Shewanella saccharophilia is proposed. Shewanella saccharophilia differs from previously described fermentative microorganisms that metabolize glucose with the reduction of Fe(III) because it transfers significantly more electron equivalents to Fe(III); acetate and carbon dioxide are the only products of glucose metabolism; energy is conserved from Fe(III) reduction; and glucose is not metabolized in the absence of Fe(III). The metabolism of organisms like S. saccharophilia may account for the fact that glucose is metabolized primarily to acetate and carbon dioxide in a variety of sediments in which Fe(III) reduction is the terminal electron accepting process.

Dissimilatory arsenate and sulfate reduction in Desulfotomaculum auripigmentum sp. nov.

A newly discovered arsenate-reducing bacterium, strain OREX-4, differed significantly from strains MIT-13 and SES-3, the previously described arsenate-reducing isolates, which grew on nitrate but not on sulfate. In contrast, strain OREX-4 did not respire nitrate but grew on lactate, with either arsenate or sulfate serving as the electron acceptor, and even preferred arsenate. Both arsenate and sulfate reduction were inhibited by molybdate. Strain OREX-4, a gram-positive bacterium with a hexagonal S-layer on its cell wall, metabolized compounds commonly used by sulfate reducers. Scorodite (FeAsO42. H2O) an arsenate-containing mineral, provided micromolar concentrations of arsenate that supported cell growth. Physiologically and phylogenetically, strain OREX-4 was far-removed from strains MIT-13 and SES-3: strain OREX-4 grew on different electron donors and electron acceptors, and fell within the gram-positive group of the Bacteria, whereas MIT-13 and SES-3 fell together in the epsilon-subdivision of the Proteobacteria. Together, these results suggest that organisms spread among diverse bacterial phyla can use arsenate as a terminal electron acceptor, and that dissimilatory arsenate reduction might occur in the sulfidogenic zone at arsenate concentrations of environmental interest. 16S rRNA sequence analysis indicated that strain OREX-4 is a new species of the genus Desulfotomaculum, and accordingly, the name Desulfotomaculum auripigmentum is proposed.