John D. Coates

Microorganisms pumping iron: anaerobic microbial iron oxidation and reduction

Iron (Fe) has long been a recognized physiological requirement for life, yet for many microorganisms that persist in water, soils and sediments, its role extends well beyond that of a nutritional necessity. Fe(II) can function as an electron source for iron-oxidizing microorganisms under both oxic and anoxic conditions and Fe(III) can function as a terminal electron acceptor under anoxic conditions for iron-reducing microorganisms. Given that iron is the fourth most abundant element in the Earths crust, iron redox reactions have the potential to support substantial microbial populations in soil and sedimentary environments. As such, biological iron apportionment has been described as one of the most ancient forms of microbial metabolism on Earth, and as a conceivable extraterrestrial metabolism on other iron-mineral-rich planets such as Mars. Furthermore, the metabolic versatility of the microorganisms involved in these reactions has resulted in the development of biotechnological applications to remediate contaminated environments and harvest energy.

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.

Microbial perchlorate reduction: rocket-fuelled metabolism

It is less than 7 years since perchlorate, a predominantly man-made toxic anion, was first identified as a significant water contaminant throughout the United States. Owing to its solubility and non-reactivity, bioremediation was targeted as the most promising solution for the problem of perchlorate contamination. Since 1996, concerted efforts have resulted in significant advances in our understanding of the microbiology, biochemistry and genetics of the microorganisms that are capable of reductively transforming perchlorate into innocuous chloride. The recent completion of the whole-genome sequence of the perchlorate-reducing microorganism Dechloromonas aromatica offers further insight into the evolution and regulation of this unique metabolic pathway. Several in situ and ex situ bioremediative processes have been engineered, and many monitoring tools that are based on immunology, molecular biology and stable isotope content are now available. As such, the rapid scientific response to this emerging contaminant offers great hope for its successful elimination from contaminated environments in the future.

Humic substances as a mediator for microbially catalyzed metal reduction

The potential for humic substances to serve as a terminal electron acceptor in microbial respiration and to function as an electron shuttle between Fe(III)-reducing microorganisms and insoluble Fe(III) oxides was investigated. The Fe(III)-reducing microorganism Geobacter metallireducens conserved energy to support growth from electron transport to humics as evidenced by continued oxidation of acetate to carbon dioxide after as many as nine transfers in a medium with acetate as the electron donor and soil humic acids as the electron acceptor. Growth of G. metallireducens with poorly crystalline Fe(III) oxide as the electron acceptor was greatly stimulated by the addition of as little as 100 μM of the humics analog, anthraquinone-2,6-disulfonate. Other quinones investigated, including lawsone, menadione, and anthraquinone-2-sulfonate, also stimulated Fe(III) oxide reduction. A wide phylogenetic diversity of microorganisms capable of Fe(III) reduction were also able to transfer electrons to humics. Microorganisms which can not reduce Fe(III) could not reduce humics. Humics stimulated the reduction of structural Fe(III) in clay and the crystalline Fe(III) forms, goethite and hematite. These results demonstrate that electron shuttling between Fe(III)-reducing microorganisms and Fe(III) via humics not only accelerates the microbial reduction of poorly crystalline Fe(III) oxide, but also can facilitate the reduction of Fe(III) forms that are not typically reduced by microorganisms in the absence of humics. Addition of humic substances to enhance electron shuttling between Fe(III)-reducing microorganisms and Fe(III) oxides may be a useful strategy to stimulate the remediation of soils and sediments contaminated with organic or metal pollutants. Huminstoffe als Vermittler bei der mikrobiell katalysierten Metallreduktion Es wurde untersucht, inwieweit Huminstoffe als terminale Elektronenakzeptoren bei der mikrobiellen Atmung und als Vermittler bei der Elektronenubertragung zwischen Fe(III)-reduzierenden Mikroorganismen und unloslichen Fe(III)-oxiden fungieren konnen. Das Fe(III)-reduzierende Bakterium Geobacter metallireducens gewinnt Energie zum Wachstum aus der Elektronenubertragung auf Huminstoffe. Das wurde offensichtlich, als nach 9 aufeinanderfolgenden Transfers des Bakteriums auf frisches Medium mit Acetat als Elektronendonor und Boden-Huminstoff als Elektronenakzeptor seine Fahigkeit zur Oxidation von Acetat zu CO2 erhalten blieb. Das Wachstum von G. metallireducens mit niedrigkristallinem Fe(III)-oxid als Elektronenakzeptor konnte durch den Zusatz des Huminstoff-Analogen Anthrachinon-2,6-disulfonat bereits in Konzentrationen von 100 μmol/L deutlich stimuliert werden. Auch weitere untersuchte Chinone wie z.B. Lawson (2-Hydroxy-1,4-naphthochinon), Menadion (2-Methyl-1,4-naphthochinon) und Anthrachinon-2-sulfonat stimulierten die Fe(III)-oxid-Reduktion. Eine grose Anzahl phylogenetisch unterschiedlicher Mikroorganismen, die zur Fe(III)-Reduktion befahigt sind, zeigten gleichzeitig die Fahigkeit zum Elektronentransfer auf Huminstoffe. Zur Fe(III)-Reduktion nicht befahigte Mikroorganismen konnten auch Huminstoffe nicht reduzieren. Durch Huminstoffe konnte die Reduktion von Fe(III) stimuliert werden, das in die Struktur von Tonmineralen und in kristalline Formen des Fe(III)-oxids, Goethit und Hamatit, eingebaut ist. Diese Ergebnisse zeigen, das durch die vermittelnde Funktion der Huminstoffe bei der Elektronenubertragung zwischen Fe(III)-reduzierenden Mikroorganismen und Fe(III) nicht nur die mikrobielle Reduktion von niedrigkristallinem Fe(III)-oxid beschleunigt wird, sondern auch die Reduktion von solchen Formen des Fe(III) erleichtert wird, welche im allgemeinen in Abwesenheit von Huminstoffen durch Fe(III)-reduzierende Mikroorganismen nicht reduziert werden. Die Zugabe von Huminstoffen zur Verbesserung der Elektronenubertragung zwischen Fe(III)-reduzierenden Mikroorganismen und Fe(III)-oxiden konnte eine nutzliche Strategie zur Stimulierung der Sanierung von mit organischen oder metallischen Kontaminanten verunreinigten Boden und Sedimenten sein.

BIOREMEDIATION OF METAL CONTAMINATION

Recent studies have demonstrated that microbes might be used to remediate metal contamination by removing metals from contaminated water or waste streams, sequestering metals in soils and sediments or solubilizing metals to aid in their extraction. This is primarily accomplished either by biosorption of metals or enzymatically catalyzed changes in the metal redox state. Bioremediation of metals is still primarily a research problem with little large-scale application of this technology.

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

Abstract 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 ɛ-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.

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.

A novel ecological role of the Firmicutes identified in thermophilic microbial fuel cells.

Significant effort is currently focused on microbial fuel cells (MFCs) as a source of renewable energy. Most studies concentrate on operation at mesophilic temperatures. However, anaerobic digestion studies have reported on the superiority of thermophilic operation and demonstrated a net energy gain in terms of methane yield. As such, our studies focused on MFC operation and microbiology at 55 °C. Over a 100-day operation, these MFCs were stable and achieved a power density of 37 mW m−2 with a coulombic efficiency of 89%. To infer activity and taxonomic identity of dominant members of the electricity-producing community, we performed phylogenetic microarray and clone library analysis with small subunit ribosomal RNA (16S rRNA) and ribosomal RNA gene (16S rDNA). The results illustrated the dominance (80% of clone library sequences) of the Firmicutes in electricity production. Similarly, rRNA sequences from Firmicutes accounted for 50% of those taxa that increased in relative abundance from current-producing MFCs, implying their functional role in current production. We complemented these analyses by isolating the first organisms from a thermophilic MFC. One of the isolates, a Firmicutes Thermincola sp. strain JR, not only produced more current than known organisms (0.42 mA) in an H-cell system but also represented the first demonstration of direct anode reduction by a member of this phylum. Our research illustrates the importance of using a variety of molecular and culture-based methods to reliably characterize bacterial communities. Consequently, we revealed a previously unidentified functional role for Gram-positive bacteria in MFC current generation.

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.

Biogenic Magnetite Formation through Anaerobic Biooxidation of Fe(II)

ABSTRACT 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.