Denitrification is a distinct means of energy conservation, making use of N oxides as terminal electron acceptors for cellular bioenergetics under anaerobic, microaerophilic, and occasionally aerobic conditions. The process is an essential branch of the global N cycle, reversing dinitrogen fixation, and is associated with chemolithotrophic, phototrophic, diazotrophic, or organotrophic metabolism but generally not with obligately anaerobic life. Discovered more than a century ago and believed to be exclusively a bacterial trait, denitrification has now been found in halophilic and hyperthermophilic archaea and in the mitochondria of fungi, raising evolutionarily intriguing vistas. Important advances in the biochemical characterization of denitrification and the underlying genetics have been achieved with Pseudomonas stutzeri, Pseudomonas aeruginosa, Paracoccus denitrificans, Ralstonia eutropha, and Rhodobacter sphaeroides. Pseudomonads represent one of the largest assemblies of the denitrifying bacteria within a single genus, favoring their use as model organisms. Around 50 genes are required within a single bacterium to encode the core structures of the denitrification apparatus. Much of the denitrification process of gram-negative bacteria has been found confined to the periplasm, whereas the topology and enzymology of the gram-positive bacteria are less well established. The activation and enzymatic transformation of N oxides is based on the redox chemistry of Fe, Cu, and Mo. Biochemical breakthroughs have included the X-ray structures of the two types of respiratory nitrite reductases and the isolation of the novel enzymes nitric oxide reductase and nitrous oxide reductase, as well as their structural characterization by indirect spectroscopic means. This revealed unexpected relationships among denitrification enzymes and respiratory oxygen reductases. Denitrification is intimately related to fundamental cellular processes that include primary and secondary transport, protein translocation, cytochrome c biogenesis, anaerobic gene regulation, metalloprotein assembly, and the biosynthesis of the cofactors molybdopterin and heme D1. An important class of regulators for the anaerobic expression of the denitrification apparatus are transcription factors of the greater FNR family. Nitrate and nitric oxide, in addition to being respiratory substrates, have been identified as signaling molecules for the induction of distinct N oxide-metabolizing enzymes.

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Cell biology and molecular basis of denitrification.

Biofilms are complex communities of microorganisms attached to surfaces or associated with interfaces. Despite the focus of modern microbiology research on pure culture, planktonic (free-swimming) bacteria, it is now widely recognized that most bacteria found in natural, clinical, and industrial settings persist in association with surfaces. Furthermore, these microbial communities are often composed of multiple species that interact with each other and their environment. The determination of biofilm architecture, particularly the spatial arrangement of microcolonies (clusters of cells) relative to one another, has profound implications for the function of these complex communities. Numerous new experimental approaches and methodologies have been developed in order to explore metabolic interactions, phylogenetic groupings, and competition among members of the biofilm. To complement this broad view of biofilm ecology, individual organisms have been studied using molecular genetics in order to identify the genes required for biofilm development and to dissect the regulatory pathways that control the plankton-to-biofilm transition. These molecular genetic studies have led to the emergence of the concept of biofilm formation as a novel system for the study of bacterial development. The recent explosion in the field of biofilm research has led to exciting progress in the development of new technologies for studying these communities, advanced our understanding of the ecological significance of surface-attached bacteria, and provided new insights into the molecular genetic basis of biofilm development.

Microbial Biofilms: from Ecology to Molecular Genetics

A large fraction of globally produced methane is converted to CO2 by anaerobic oxidation in marine sediments. Strong geochemical evidence for net methane consumption in anoxic sediments is based on methane profiles, radiotracer experiments and stable carbon isotope data. But the elusive microorganisms mediating this reaction have not yet been isolated, and the pathway of anaerobic oxidation of methane is insufficiently understood. Recent data suggest that certain archaea reverse the process of methanogenesis by interaction with sulphate-reducing bacteria. Here we provide microscopic evidence for a structured consortium of archaea and sulphate-reducing bacteria, which we identified by fluorescence in situ hybridization using specific 16S rRNA-targeted oligonucleotide probes. In this example of a structured archaeal-bacterial symbiosis, the archaea grow in dense aggregates of about 100 cells and are surrounded by sulphate-reducing bacteria. These aggregates were abundant in gas-hydrate-rich sediments with extremely high rates of methane-based sulphate reduction, and apparently mediate anaerobic oxidation of methane.

A marine microbial consortium apparently mediating anaerobic oxidation of methane

Soil microorganisms as controllers of atmospheric trace gases (H2, CO, CH4, OCS, N2O, and NO).

The important life-supporting role of hydrogen sulfide (H2S) has evolved from bacteria to plants, invertebrates, vertebrates, and finally to mammals. Over the centuries, however, H2S had only been known for its toxicity and environmental hazard. Physiological importance of H2S has been appreciated for about a decade. It started by the discovery of endogenous H2S production in mammalian cells and gained momentum by typifying this gasotransmitter with a variety of physiological functions. The H2S-catalyzing enzymes are differentially expressed in cardiovascular, neuronal, immune, renal, respiratory, gastrointestinal, reproductive, liver, and endocrine systems and affect the functions of these systems through the production of H2S. The physiological functions of H2S are mediated by different molecular targets, such as different ion channels and signaling proteins. Alternations of H2S metabolism lead to an array of pathological disturbances in the form of hypertension, atherosclerosis, heart failure, diabetes...

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Physiological Implications of Hydrogen Sulfide: A Whiff Exploration That Blossomed

A procedure which includes the Total Reduced Inorganic Sulfur (TRIS) in a single distillation step is described for the radiotracer measurement of sulfate reduction in sediments. The TRIS includes both Acid Volatile Sulfide (AVS: H2S + FeS) and the remaining Chromium Reducible Sulfur (CRS: S0, FeS2). The single-step distillation was simpler and faster than the consecutive distillations of AVS and CRS. It also resulted in higher (4–50%) sulfate reduction rates than those obtained from the sum of35S in AVS and CRS. The difference was largest when the sediment had been dried after AVS but before CRS distillation. Relative to the35S-AVS distillation alone, the35S-TRIS single-step distallation yielded 8–87% higher reduction rates. The separation and recovery of FeS, S0 and FeS2 was studied under three distillation conditions: 1) cold acid, 2) cold acid with Cr2+, and 3) hot acid with Cr2+. The FeS was recovered by cold acid alone while pyrite was recovered by cold acid with Cr2+. A smaller S0 fraction, presumably of the finer crystal sizes, was recovered also in the cold acid with Cr2+ while most of the S0 required hot acid with Cr2+ for reduction to H2S.

Measurement of bacterial sulfate reduction in sediments: Evaluation of a single-step chromium reduction method

Pathways of organic carbon oxidation in three continental margin sediments

Manganese, iron and sulfur cycling in a coastal marine sediment, Aarhus bay, Denmark

Some microbes are capable of extracellular electron transport through so-called bacterial nanowires or electron shuttles. It is now shown that this may be a significant process in the marine sediment, allowing oxygen to oxidize compounds located centimetres away. The remains of dead algae and faeces buried in marine sediment are a good food resource for microbes, but at sediment depths greater than a few millimetres the lack of oxygen limits utilization of this resource. A study of sediment samples from the seabed off Aarhus in Denmark reveals that microbes can overcome this obstacle by making intercellular electric connections and establishing a division of labour. Cells at the surface utilize sufficient oxygen for all cells in the community, and those at depth acquire nutrients for all. It has been previously demonstrated that some microbes are capable of extracellular electron transport through so–called nanowires or electron shuttles. Here it is demonstrated that this may be a significant process in the marine sediment. Some bacteria are capable of extracellular electron transfer, thereby enabling them to use electron acceptors and donors without direct cell contact1,2,3,4. Beyond the micrometre scale, however, no firm evidence has previously existed that spatially segregated biogeochemical processes can be coupled by electric currents in nature. Here we provide evidence that electric currents running through defaunated sediment couple oxygen consumption at the sediment surface to oxidation of hydrogen sulphide and organic carbon deep within the sediment. Altering the oxygen concentration in the sea water overlying the sediment resulted in a rapid (<1-h) change in the hydrogen sulphide concentration within the sediment more than 12 mm below the oxic zone, a change explicable by transmission of electrons but not by diffusion of molecules. Mass balances indicated that more than 40% of total oxygen consumption in the sediment was driven by electrons conducted from the anoxic zone. A distinct pH peak in the oxic zone could be explained by electrochemical oxygen reduction, but not by any conventional sets of aerobic sediment processes. We suggest that the electric current was conducted by bacterial nanowires combined with pyrite, soluble electron shuttles and outer-membrane cytochromes. Electrical communication between distant chemical and biological processes in nature adds a new dimension to our understanding of biogeochemistry and microbial ecology.

Electric currents couple spatially separated biogeochemical processes in marine sediment

MARINE species of Thioploca occur over 3,000 km along the continental shelf off Southern Peru and North and Central Chile1–4. These filamentous bacteria live in bundles surrounded by a common sheath and form thick mats on the sea floor under the oxygen-minimum zone in the upwelling region, at between 40 and 280 m water depth. The metabolism of this marine bacterium5,6 remained a mystery until long after its discovery1,7. We report here that Thioploca cells are able to concentrate nitrate to up to 500 mM in a liquid vacuole that occupies >80% of the cell volume. Gliding filaments transport this nitrate 5–10 cm down into the sediment and reduce it, with concomitant oxidation of hydrogen sulphide, thereby coupling the nitrogen and sulphur cycles in the sediment.

Henrik Fossing

Papers

Measurement of bacterial sulfate reduction in sediments: Evaluation of a single-step chromium reduction method

Pathways of organic carbon oxidation in three continental margin sediments

Manganese, iron and sulfur cycling in a coastal marine sediment, Aarhus bay, Denmark

Electric currents couple spatially separated biogeochemical processes in marine sediment

Concentration and transport of nitrate by the mat-forming sulphur bacterium Thioploca