Cyanate as an energy source for nitrifiers.

Ammonia- and nitrite-oxidizing microorganisms are collectively responsible for the aerobic oxidation of ammonia via nitrite to nitrate and have essential roles in the global biogeochemical nitrogen cycle. The physiology of nitrifiers has been intensively studied, and urea and ammonia are the only recognized energy sources that promote the aerobic growth of ammonia-oxidizing bacteria and archaea. Here we report the aerobic growth of a pure culture of the ammonia-oxidizing thaumarchaeote Nitrososphaera gargensis using cyanate as the sole source of energy and reductant; to our knowledge, the first organism known to do so. Cyanate, a potentially important source of reduced nitrogen in aquatic and terrestrial ecosystems, is converted to ammonium and carbon dioxide in Nitrososphaera gargensis by a cyanase enzyme that is induced upon addition of this compound. Within the cyanase gene family, this cyanase is a member of a distinct clade also containing cyanases of nitrite-oxidizing bacteria of the genus Nitrospira. We demonstrate by co-culture experiments that these nitrite oxidizers supply cyanase-lacking ammonia oxidizers with ammonium from cyanate, which is fully nitrified by this microbial consortium through reciprocal feeding. By screening a comprehensive set of more than 3,000 publically available metagenomes from environmental samples, we reveal that cyanase-encoding genes clustering with the cyanases of these nitrifiers are widespread in the environment. Our results demonstrate an unexpected metabolic versatility of nitrifying microorganisms, and suggest a previously unrecognized importance of cyanate in cycling of nitrogen compounds in the environment.

Desulfoconvexum algidum gen. nov., sp. nov., a psychrophilic sulfate-reducing bacterium isolated from a permanently cold marine sediment.

A sulfate-reducing bacterium, designated JHA1(T), was isolated from a permanently cold marine sediment sampled in an Artic fjord on the north-west coast of Svalbard. The isolate was originally enriched at 4 °C in a highly diluted liquid culture amended with hydrogen and sulfate. Strain JHA1(T) was a psychrophile, growing fastest between 14 and 16 °C and not growing above 20 °C. Fastest growth was found at neutral pH (pH 7.2-7.4) and at marine concentrations of NaCl (20-30 g l(-1)). Phylogenetic analysis of 16S rRNA gene sequences revealed that strain JHA1(T) was a member of the family Desulfobacteraceae in the Deltaproteobacteria. The isolate shared 99 % 16S rRNA gene sequence similarity with an environmental sequence obtained from permanently cold Antarctic sediment. The closest recognized relatives were Desulfobacula phenolica DSM 3384(T) and Desulfobacula toluolica DSM 7467(T) (both <95 % sequence similarity). In contrast to its closest phylogenetic relatives, strain JHA1(T) grew chemolithoautotrophically with hydrogen as an electron donor. CO dehydrogenase activity indicated the operation of the reductive acetyl-CoA pathway for inorganic carbon assimilation. Beside differences in physiology and morphology, strain JHA1(T) could be distinguished chemotaxonomically from the genus Desulfobacula by the absence of the cellular fatty acid C16 : 0 10-methyl. Phylogenetic differentiation from other genera was further supported by DsrAB and AprBA sequence analysis. Based on the described phylogenetic and phenotypic differences between strain JHA1(T) and its closest relatives, the establishment of a novel genus and a novel species, Desulfoconvexum algidum gen. nov., sp. nov. is proposed. The type strain is JHA1(T) ( = DSM 21856(T)  = JCM 16085(T)).

The genome of the ammonia-oxidizing Candidatus Nitrososphaera gargensis: insights into metabolic versatility and environmental adaptations.

The cohort of the ammonia-oxidizing archaea (AOA) of the phylum Thaumarchaeota is a diverse, widespread and functionally important group of microorganisms in many ecosystems. However, our understanding of their biology is still very rudimentary in part because all available genome sequences of this phylum are from members of the Nitrosopumilus cluster. Here we report on the complete genome sequence of Candidatus Nitrososphaera gargensis obtained from an enrichment culture, representing a different evolutionary lineage of AOA frequently found in high numbers in many terrestrial environments. With its 2.83 Mb the genome is much larger than that of other AOA. The presence of a high number of (active) IS elements/transposases, genomic islands, gene duplications and a complete CRISPR/Cas defence system testifies to its dynamic evolution consistent with low degree of synteny with other thaumarchaeal genomes. As expected, the repertoire of conserved enzymes proposed to be required for archaeal ammonia oxidation is encoded by N. gargensis, but it can also use urea and possibly cyanate as alternative ammonia sources. Furthermore, its carbon metabolism is more flexible at the central pyruvate switch point, encompasses the ability to take up small organic compounds and might even include an oxidative pentose phosphate pathway. Furthermore, we show that thaumarchaeota produce cofactor F420 as well as polyhydroxyalkanoates. Lateral gene transfer from bacteria and euryarchaeota has contributed to the metabolic versatility of N. gargensis. This organisms is well adapted to its niche in a heavy metal-containing thermal spring by encoding a multitude of heavy metal resistance genes, chaperones and mannosylglycerate as compatible solute and has the genetic ability to respond to environmental changes by signal transduction via a large number of two-component systems, by chemotaxis and flagella-mediated motility and possibly even by gas vacuole formation. These findings extend our understanding of thaumarchaeal evolution and physiology and offer many testable hypotheses for future experimental research on these nitrifiers.

Anaerobic degradation of naphthalene and 2-methylnaphthalene by strains of marine sulfate-reducing bacteria.

The anaerobic biodegradation of naphthalene, an aromatic hydrocarbon in tar and petroleum, has been repeatedly observed in environments but scarcely in pure cultures. To further explore the relationships and physiology of anaerobic naphthalene-degrading microorganisms, sulfate-reducing bacteria (SRB) were enriched from a Mediterranean sediment with added naphthalene. Two strains (NaphS3, NaphS6) with oval cells were isolated which showed naphthalene-dependent sulfate reduction. According to 16S rRNA gene sequences, both strains were Deltaproteobacteria and closely related to each other and to a previously described naphthalene-degrading sulfate-reducing strain (NaphS2) from a North Sea habitat. Other close relatives were SRB able to degrade alkylbenzenes, and phylotypes enriched anaerobically with benzene. If in adaptation experiments the three naphthalene-grown strains were exposed to 2-methylnaphthalene, this compound was utilized after a pronounced lag phase, indicating that naphthalene did not induce the capacity for 2-methylnaphthalene degradation. Comparative denaturing gel electrophoresis of cells grown with naphthalene or 2-methylnaphthalene revealed a striking protein band which was only present upon growth with the latter substrate. Peptide sequences from this band perfectly matched those of a protein predicted from genomic libraries of the strains. Sequence similarity (50% identity) of the predicted protein to the large subunit of the toluene-activating enzyme (benzylsuccinate synthase) from other anaerobic bacteria indicated that the detected protein is part of an analogous 2-methylnaphthalene-activating enzyme. The absence of this protein in naphthalene-grown cells together with the adaptation experiments as well as isotopic metabolite differentiation upon growth with a mixture of d(8)-naphthalene and unlabelled 2-methylnaphthalene suggest that the marine strains do not metabolize naphthalene by initial methylation via 2-methylnaphthalene, a previously suggested mechanism. The inability to utilize 1-naphthol or 2-naphthol also excludes these compounds as free intermediates. Results leave open the possibility of naphthalene carboxylation, another previously suggested activation mechanism.

Recent Origin of the Methacrylate Redox System in Geobacter sulfurreducens AM-1 through Horizontal Gene Transfer.

The origin and evolution of novel biochemical functions remains one of the key questions in molecular evolution. We study recently emerged methacrylate reductase function that is thought to have emerged in the last century and reported in Geobacter sulfurreducens strain AM-1. We report the sequence and study the evolution of the operon coding for the flavin-containing methacrylate reductase (Mrd) and tetraheme cytochrome с (Mcc) in the genome of G. sulfurreducens AM-1. Different types of signal peptides in functionally interlinked proteins Mrd and Mcc suggest a possible complex mechanism of biogenesis for chromoproteids of the methacrylate redox system. The homologs of the Mrd and Mcc sequence found in δ-Proteobacteria and Deferribacteres are also organized into an operon and their phylogenetic distribution suggested that these two genes tend to be horizontally transferred together. Specifically, the mrd and mcc genes from G. sulfurreducens AM-1 are not monophyletic with any of the homologs found in other Geobacter genomes. The acquisition of methacrylate reductase function by G. sulfurreducens AM-1 appears linked to a horizontal gene transfer event. However, the new function of the products of mrd and mcc may have evolved either prior or subsequent to their acquisition by G. sulfurreducens AM-1.

Starting up microbial enhanced oil recovery.

This chapter gives the reader a practical introduction into microbial enhanced oil recovery (MEOR) including the microbial production of natural gas from oil. Decision makers who consider the use of one of these technologies are provided with the required scientific background as well as with practical advice for upgrading an existing laboratory in order to conduct microbiological experiments. We believe that the conversion of residual oil into natural gas (methane) and the in situ production of biosurfactants are the most promising approaches for MEOR and therefore focus on these topics. Moreover, we give an introduction to the microbiology of oilfields and demonstrate that in situ microorganisms as well as injected cultures can help displace unrecoverable oil in place (OIP). After an initial research phase, the enhanced oil recovery (EOR) manager must decide whether MEOR would be economical. MEOR generally improves oil production but the increment may not justify the investment. Therefore, we provide a brief economical assessment at the end of this chapter. We describe the necessary state-of-the-art scientific equipment to guide EOR managers towards an appropriate MEOR strategy. Because it is inevitable to characterize the microbial community of an oilfield that should be treated using MEOR techniques, we describe three complementary start-up approaches. These are: (i) culturing methods, (ii) the characterization of microbial communities and possible bio-geochemical pathways by using molecular biology methods, and (iii) interfacial tension measurements. In conclusion, we hope that this chapter will facilitate a decision on whether to launch MEOR activities. We also provide an update on relevant literature for experienced MEOR researchers and oilfield operators. Microbiologists will learn about basic principles of interface physics needed to study the impact of microorganisms living on oil droplets. Last but not least, students and technicians trying to understand processes in oilfields and the techniques to examine them will, we hope, find a valuable source of information in this review.

Growth of nitrite-oxidizing bacteria by aerobic hydrogen oxidation.

The bacterial oxidation of nitrite to nitrate is a key process of the biogeochemical nitrogen cycle. Nitrite-oxidizing bacteria are considered a highly specialized functional group, which depends on the supply of nitrite from other microorganisms and whose distribution strictly correlates with nitrification in the environment and in wastewater treatment plants. On the basis of genomics, physiological experiments, and single-cell analyses, we show that Nitrospira moscoviensis, which represents a widely distributed lineage of nitrite-oxidizing bacteria, has the genetic inventory to utilize hydrogen (H2) as an alternative energy source for aerobic respiration and grows on H2 without nitrite. CO2 fixation occurred with H2 as the sole electron donor. Our results demonstrate a chemolithoautotrophic lifestyle of nitrite-oxidizing bacteria outside the nitrogen cycle, suggesting greater ecological flexibility than previously assumed.

Enrichment and genome sequence of the group I.1a ammonia-oxidizing Archaeon "Ca. Nitrosotenuis uzonensis" representing a clade globally distributed in thermal habitats.

The discovery of ammonia-oxidizing archaea (AOA) of the phylum Thaumarchaeota and the high abundance of archaeal ammonia monooxygenase subunit A encoding gene sequences in many environments have extended our perception of nitrifying microbial communities. Moreover, AOA are the only aerobic ammonia oxidizers known to be active in geothermal environments. Molecular data indicate that in many globally distributed terrestrial high-temperature habits a thaumarchaeotal lineage within the Nitrosopumilus cluster (also called "marine" group I.1a) thrives, but these microbes have neither been isolated from these systems nor functionally characterized in situ yet. In this study, we report on the enrichment and genomic characterization of a representative of this lineage from a thermal spring in Kamchatka. This thaumarchaeote, provisionally classified as "Candidatus Nitrosotenuis uzonensis", is a moderately thermophilic, non-halophilic, chemolithoautotrophic ammonia oxidizer. The nearly complete genome sequence (assembled into a single scaffold) of this AOA confirmed the presence of the typical thaumarchaeotal pathways for ammonia oxidation and carbon fixation, and indicated its ability to produce coenzyme F420 and to chemotactically react to its environment. Interestingly, like members of the genus Nitrosoarchaeum, "Candidatus N. uzonensis" also possesses a putative artubulin-encoding gene. Genome comparisons to related AOA with available genome sequences confirmed that the newly cultured AOA has an average nucleotide identity far below the species threshold and revealed a substantial degree of genomic plasticity with unique genomic regions in "Ca. N. uzonensis", which potentially include genetic determinants of ecological niche differentiation.

Desulfopila inferna sp. nov., a sulfate-reducing bacterium isolated from the subsurface of a tidal sand-flat.

A Gram-negative, rod-shaped, sulfate-reducing bacterium (strain JS_SRB250Lac(T)) was isolated from a tidal sand-flat in the German Wadden Sea. 16S rRNA gene sequence analysis showed that strain JS_SRB250Lac(T) belonged to the Desulfobulbaceae (Deltaproteobacteria), with Desulfopila aestuarii MSL86(T) being the closest recognized relative (94.2 % similarity). Higher similarity (96.6 %) was shared with 'Desulfobacterium corrodens' IS4, but this name has not been validly published. The affiliation of strain JS_SRB250Lac(T) to the genus Desulfopila was further supported by analysis of aprBA gene sequences and shared physiological characteristics, in particular the broad range of organic electron donors used for sulfate reduction. Compared with Desulfopila aestuarii MSL86(T), strain JS_SRB250Lac(T) additionally utilized butyrate and succinate and grew chemolithoautotrophically with hydrogen as an electron donor. CO dehydrogenase activity was demonstrated, indicating that the reductive acetyl-CoA pathway (Wood-Ljungdahl pathway) was used for CO(2) fixation. Results of cellular fatty acid analysis allowed chemotaxonomic differentiation of strain JS_SRB250Lac(T) from Desulfopila aestuarii MSL86(T) by the presence of C(17 : 0) cyclo and the absence of hydroxy and unsaturated branched-chain fatty acids. Based on phylogenetic, physiological and chemotaxonomic characteristics, strain JS_SRB250Lac(T) represents a novel species of the genus Desulfopila, for which the name Desulfopila inferna sp. nov. is proposed. The type strain is JS_SRB250Lac(T) (=DSM 19738(T) =NBRC 103921(T)).

Operation of the CO dehydrogenase/acetyl coenzyme A pathway in both acetate oxidation and acetate formation by the syntrophically acetate-oxidizing bacterium Thermacetogenium phaeum.

Thermacetogenium phaeum is a homoacetogenic bacterium that can grow on various substrates, such as pyruvate, methanol, or H2/CO2. It can also grow on acetate if cocultured with the hydrogen-consuming methanogenic partner Methanothermobacter thermautotrophicus. Enzyme activities of the CO dehydrogenase/acetyl coenzyme A (CoA) pathway (CO dehydrogenase, formate dehydrogenase, formyl tetrahydrofolate synthase, methylene tetrahydrofolate dehydrogenase) were detected in cell extracts of pure cultures and of syntrophic cocultures. Mixed cell suspensions of T. phaeum and M. thermautotrophicus oxidized acetate rapidly and produced acetate after addition of H2/CO2 after a short time lag. CO dehydrogenase activity staining after native polyacrylamide gel electrophoresis exhibited three oxygen-labile bands which were identical in pure culture and coculture. Protein profiles of T. phaeum cells after sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the strain exhibited basically the same protein patterns in both pure and syntrophic culture. These results indicate that T. phaeum operates the CO dehydrogenase/acetyl-CoA pathway reversibly both in acetate oxidation and in reductive acetogenesis by using the same biochemical apparatus, although it has to couple this pathway to ATP synthesis in different ways.

Cysteine-mediated electron transfer in syntrophic acetate oxidation by cocultures of Geobacter sulfurreducens and Wolinella succinogenes.

Syntrophic cocultures of Geobacter sulfurreducens and Wolinella succinogenes oxidize acetate with nitrate as terminal electron acceptor. It has been postulated earlier that electrons are transferred in these cocultures not via hydrogen, but via a different carrier, e.g., a small c-type cytochrome that is detected in the supernatant of growing cultures. In the present study, L -cysteine, which was provided as a reducing agent, was found to mediate the electron transfer between the two partners. Low concentrations of L -cysteine or L -cystine (10-100 microM) supported syntrophic growth, and no acetate oxidation was observed in the absence of cysteine or cystine. Cell suspensions of G. sulfurreducens or coculture cell suspensions reduced cystine to cysteine, and suspensions of W. succinogenes or coculture suspensions oxidized cysteine with nitrate, as measured by the formation or depletion of free thiol groups. Added cysteine was rapidly oxidized by the coculture during growth, but the formed cystine was not entirely rereduced even under acceptor-limited conditions. The redox potential prevailing in acetate-oxidizing cocultures was -160 to -230 mV. Sulfide at low concentrations supported syntrophic growth as well and could replace cysteine. Neither growth nor acetate degradation was found with D-cysteine, homocysteine, cysteamine, 3-mercaptopropionate, dithiothreithol, thioglycolate, glutathione, coenzyme M, dimethylsulfoxide, trimethylamine- N-oxide, anthraquinone-2,6-disulfonate, or ascorbate.