鈴木 志野

Serpentinization, a geochemical process found on modern and ancient Earth, provides an ultra-reducing environment that can support microbial methanogenesis and acetogenesis. Several groups of archaea, such as the order Methanocellales, are characterized by their ability to produce methane. Here, we generate metagenomic sequences from serpentinized springs in The Cedars, California, and construct a circularized metagenome-assembled genome of a Methanocellales archaeon, termed Met12, that lacks essential methanogenesis genes. The genome includes genes for an acetyl-CoA pathway, but lacks genes encoding methanogenesis enzymes such as methyl-coenzyme M reductase, heterodisulfide reductases and hydrogenases. In situ transcriptomic analyses reveal high expression of a multi-heme c-type cytochrome, and heterologous expression of this protein in a model bacterium demonstrates that it is capable of accepting electrons. Our results suggest that Met12, within the order Methanocellales, is not a methanogen but a CO2-reducing, electron-fueled acetogen without electron bifurcation.

Abstract Serpentinization of ultramafic rocks provides molecular hydrogen (H₂) that can support lithotrophic metabolism of microorganisms, but also poses extremely challenging conditions, including hyperalkalinity and limited electron acceptor availability. Investigation of two serpentinization-active systems reveals that conventional H₂-/CO₂-dependent homoacetogenesis is thermodynamically unfavorable in situ due to picomolar CO₂ levels. Through metagenomics and thermodynamics, we discover unique taxa capable of metabolism adapted to the habitat. This included a novel deep-branching phylum, “Ca. Lithacetigenota”, that exclusively inhabits serpentinite-hosted systems and harbors genes encoding alternative modes of H₂-utilizing lithotrophy. Rather than CO₂, these putative metabolisms utilize reduced carbon compounds detected in situ presumably serpentinization-derived: formate and glycine. The former employs a partial homoacetogenesis pathway and the latter a distinct pathway mediated by a rare selenoprotein—the glycine reductase. A survey of microbiomes shows that glycine reductases are diverse and nearly ubiquitous in serpentinite-hosted environments. “Ca. Lithacetigenota” glycine reductases represent a basal lineage, suggesting that catabolic glycine reduction is an ancient bacterial innovation by Terrabacteria for gaining energy from geogenic H₂ even under hyperalkaline, CO₂-poor conditions. Unique non-CO₂-reducing metabolisms presented here shed light on potential strategies that extremophiles may employ for overcoming a crucial obstacle in serpentinization-associated environments, features potentially relevant to primordial lithotrophy in early Earth.

Abstract Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and poly(butylene succinate-co-adipate) (PBSA) are typical biodegradable polyesters; however, their biodegradability in the ocean differs substantially. Herein, we focused on functional genes correlated with biodegradation in ocean environments using multi-meta-omics approaches to identify the microbial groups and esterase enzymes correlated with biodegradation. Within the PHBV plastispheres, five Gammaproteobacteria were abundant, several of which encoded over 10 different types of extracellular poly(3-hydroxybutyrate (PHB) depolymerases that are highly expressed in the ocean. Within PBSA plastispheres, ecosystems of microbes formed on plastics, only two species of Gammaproteobacteria genomes were highly abundant and expressed: one for hydrolyzing PBSA and the other for consuming cleaved monomers. The high diversity of degrading microorganisms and enzymes could be related to the stable biodegradability of PHBV, while the low biodiversity of PBSA-degraders and necessity of symbiotic relationships likely characterize the instability of the marine biodegradability of PBSA. These results provide fundamental knowledge for the development of biodegradable marine plastics.

The Committee on Space Research (COSPAR) Sample Safety Assessment Framework (SSAF) has been developed by a COSPAR appointed Working Group. The objective of the sample safety assessment would be to evaluate whether samples returned from Mars could be harmful for Earth's systems (e.g., environment, biosphere, geochemical cycles). During the Working Group's deliberations, it became clear that a comprehensive assessment to predict the effects of introducing life in new environments or ecologies is difficult and practically impossible, even for terrestrial life and certainly more so for unknown extraterrestrial life. To manage expectations, the scope of the SSAF was adjusted to evaluate only whether the presence of martian life can be excluded in samples returned from Mars. If the presence of martian life cannot be excluded, a Hold & Critical Review must be established to evaluate the risk management measures and decide on the next steps. The SSAF starts from a positive hypothesis (there is martian life in the samples), which is complementary to the null-hypothesis (there is no martian life in the samples) typically used for science. Testing the positive hypothesis includes four elements: (1) Bayesian statistics, (2) subsampling strategy, (3) test sequence, and (4) decision criteria. The test sequence capability covers self-replicating and non-self-replicating biology and biologically active molecules. Most of the investigations associated with the SSAF would need to be carried out within biological containment. The SSAF is described in sufficient detail to support planning activities for a Sample Receiving Facility (SRF) and for preparing science announcements, while at the same time acknowledging that further work is required before a detailed Sample Safety Assessment Protocol (SSAP) can be developed. The three major open issues to be addressed to optimize and implement the SSAF are (1) setting a value for the level of assurance to effectively exclude the presence of martian life in the samples, (2) carrying out an analogue test program, and (3) acquiring relevant contamination knowledge from all Mars Sample Return (MSR) flight and ground elements. Although the SSAF was developed specifically for assessing samples from Mars in the context of the currently planned NASA-ESA MSR Campaign, this framework and the basic safety approach are applicable to any other Mars sample return mission concept, with minor adjustments in the execution part related to the specific nature of the samples to be returned. The SSAF is also considered a sound basis for other COSPAR Planetary Protection Category V, restricted Earth return missions beyond Mars. It is anticipated that the SSAF will be subject to future review by the various MSR stakeholders.

Three highly alkaliphilic bacterial strains designated as A1^T, H1^T and B1^T were isolated from two highly alkaline springs at The Cedars, a terrestrial serpentinizing site. Cells from all strains were motile, Gram-negative and rod-shaped. Strains A1^T, H1^T and B1^T were mesophilic (optimum, 30 °C), highly alkaliphilic (optimum, pH 11) and facultatively autotrophic. Major cellular fatty acids were saturated and monounsaturated hexadecenoic and octadecanoic acids. The genome size of strains A1^T, H1^T and B1^T was 2 574 013, 2 475 906 and 2 623 236 bp, and the G+C content was 66.0, 66.2 and 66.1 mol%, respectively. Analysis of the 16S rRNA genes showed the highest similarity to the genera <italic> <named-content content-type="genus"> <ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="http://doi.org/10.1601/nm.9349" xlink:type="simple">Malikia</ext-link> </named-content> </italic> (95.1–96.4 %), <italic> <named-content content-type="genus"> <ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="http://doi.org/10.1601/nm.1817" xlink:type="simple">Macromonas</ext-link> </named-content> </italic> (93.0–93.6 %) and <italic> <named-content content-type="genus"> <ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="http://doi.org/10.1601/nm.1807" xlink:type="simple">Hydrogenophaga</ext-link> </named-content> </italic> (93.0–96.6 %) in the family <italic> <named-content content-type="family"> <ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="http://doi.org/10.1601/nm.1773" xlink:type="simple">Comamonadaceae</ext-link> </named-content> </italic>. Phylogenetic analysis based on 16S rRNA gene and phylogenomic analysis based on core gene sequences revealed that the isolated strains diverged from the related species, forming a distinct branch. Average amino acid identity values of strains A1^T, H1^T and B1^T against the genomes of related members in this family were below 67 %, which is below the suggested threshold for genera boundaries. Average nucleotide identity by <sc>blast</sc> values and digital DNA–DNA hybridization among the three strains were below 92.0 and 46.6 % respectively, which are below the suggested thresholds for species boundaries. Based on phylogenetic, genomic and phenotypic characterization, we propose <italic>Serpentinimonas</italic> gen. nov., <italic>Serpentinimonas raichei</italic> sp. nov. (type strain A1^T=NBRC 111848^T=DSM 103917^T), <italic>Serpentinimonas barnesii</italic> sp. nov. (type strain H1^T= NBRC 111849^T=DSM 103920^T) and <italic>Serpentinimonas maccroryi</italic> sp. nov. (type strain B1^T=NBRC 111850^T=DSM 103919^T) belonging to the family <italic> <named-content content-type="family"> <ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="http://doi.org/10.1601/nm.1773" xlink:type="simple">Comamonadaceae</ext-link> </named-content> </italic>. We have designated <italic>Serpentinimonas raichei</italic> the type species for the genus because it is the dominant species in The Cedars springs.

Microbial involvement in the pathogenesis have been suggested in both antineutrophil cytoplasmic antibody-associated vasculitis (AAV) and sarcoidosis, both of which have lung involvement. However, exhaustive research to assess the bacteria in the lung in AAV and in sarcoidosis have not been performed. We sought to elucidate the distinct dysbiotic lung microbiota between AAV and sarcoidosis. We used 16S rRNA gene high-throughput sequencing to obtain the bacterial community composition of bronchoalveolar lavage fluid (BALF) in patients with AAV (n = 16) compared to patients with sarcoidosis (n = 21). The patients had not undergone therapy with immunosuppressive medication when their BALF was acquired. No difference was observed in α-diversity between patients with AAV and patients with sarcoidosis when using all the detected taxa. We defined the taxa of the oral cavity by using the data of oral microbiota of healthy individuals from the Human Microbiome Project (HMP). The analysis using only oral taxa made the difference in α-diversity between AAV and sarcoidosis clearer compared with those using all the detected taxa. Besides, the analysis using detected taxa except for oral taxa also made the difference in α-diversity between AAV and sarcoidosis clearer compared with those using all the detected taxa. A linear negative relationship between the α-diversity and Birmingham vasculitis activity score (BVAS) was detected in the AAV group. The observed p-value for the effect of the disease groups on the ß-diversity was small while the effect of other factors including sex and smoking status did not have small p-values. By excluding oral taxa from all the detected taxa, we found a cluster mainly consisted of sarcoidosis patients which was characterized with microbial community monopolized by Erythrobacteraceae family. Our results suggested the importance of considering the influence of oral microbiota in evaluating lung microbiota.

© 2020 The Authors. Published by the Royal Society under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/4.0/, which permits unrestricted use, provided the original author and source are credited. The subduction of seamounts and ridge features at convergent plate boundaries plays an important role in the deformation of the overriding plate and influences geochemical cycling and associated biological processes. Active serpentinization of forearc mantle and serpentinite mud volcanism on the Mariana forearc (between the trench and active volcanic arc) provides windows on subduction processes. Here, we present (1) the first observation of an extensive exposure of an undeformed Cretaceous seamount currently being subducted at the Mariana Trench inner slope; (2) vertical deformation of the forearc region related to subduction of Pacific Plate seamounts and thickened crust; (3) recovered Ocean Drilling Program and International Ocean Discovery Program cores of serpentinite mudflows that confirm exhumation of various Pacific Plate lithologies, including subducted reef limestone; (4) petrologic, geochemical and paleontological data from the cores that show that Pacific Plate seamount exhumation covers greater spatial and temporal extents; (5) the inference that microbial communities associated with serpentinite mud volcanism may also be exhumed from the subducted plate seafloor and/or seamounts; and (6) the implications for effects of these processes with regard to evolution of life. This article is part of a discussion meeting issue ‘Serpentine in the Earth system’.

Copyright © 2019 Eickenbusch, Takai, Sissman, Suzuki, Menzies, Sakai, Sansjofre, Tasumi, Bernasconi, Glombitza, Jørgensen, Morono and Lever. Serpentinitic systems are potential habitats for microbial life due to frequently high concentrations of microbial energy substrates, such as hydrogen (H2), methane (CH4), and short-chain organic acids (SCOAs). Yet, many serpentinitic systems are also physiologically challenging environments due to highly alkaline conditions (pH > 10) and elevated temperatures (>80.C). To elucidate the possibility of microbial life in deep serpentinitic crustal environments, International Ocean Discovery Program (IODP) Expedition 366 drilled into the Yinazao, Fantangisna, and Asut Tesoru serpentinite mud volcanoes on the Mariana Forearc. These mud volcanoes differ in temperature (80, 150, 250.C, respectively) of the underlying subducting slab, and in the porewater pH (11.0, 11.2, 12.5, respectively) of the serpentinite mud. Increases in formate and acetate concentrations across the three mud volcanoes, which are positively correlated with temperature in the subducting slab and coincide with strong increases in H2 concentrations, indicate a serpentinization-related origin. Thermodynamic calculations suggest that formate is produced by equilibrium reactions with dissolved inorganic carbon (DIC) + H2, and that equilibration continues during fluid ascent at temperatures below 80.C. By contrast, the mechanism(s) of acetate production are not clear. Besides formate, acetate, and H2 data, we present concentrations of other SCOAs, methane, carbon monoxide, and sulfate, d13C-data on bulk carbon pools, and microbial cell counts. Even though calculations indicate a wide range of microbial catabolic reactions to be thermodynamically favorable, concentration profiles of potential energy substrates, and very low cell numbers suggest that microbial life is scarce or absent. We discuss the potential roles of temperature, pH, pressure, and dispersal in limiting the occurrence of microbial life in deep serpentinitic environments.

© 2018 Elsevier Ltd Continental active serpentinization of ultramafic rocks is today recognized as a key process triggering a sequence of phenomena involving the passage from inorganic, to organic and metabolic reactions. These may have a role in the origin of life, and may explain the occurrence of abiotic hydrocarbons on Earth and other planets. Production of hyperalkaline waters and abiotic methane (CH4) are two critical steps in this sequence. They were described independently by specific hydrogeological and geochemical models. Here, we update and combine these models into a unified scheme using and integrating geological, hydrogeological, hydrogeochemical, gas-geochemical and microbial analyses acquired from 2002 to 2014 in the Cabeço de Vide (CdV) study site, Portugal. The hyperalkaline (pH &gt; 10.5), Na-Cl/Ca-OH mineral water of CdV evolve from groundwater-peridotite interaction (serpentinization) generating hydrogen (H2), which, according to multiple theoretical, laboratory and field evidence, likely reacted with CO2 within metal- (catalyst) rich rocks, abiotically producing CH4 (up to 1.2 mg/L; -24.4°/oo &lt; δ13C-CH4 &lt; -14.0°/oo and -285°/oo &lt; δ2H-CH4 &lt; -218°/oo). The hyperalkaline water hosts hydrogen oxidizing bacteria “Serpentinomonas” which may explain the paucity of H2 observed in the dissolved gas. The CdV gas-rich mineral waters ascend along a fault at the boundary of the peridotite intrusion. Temporal changes of pH and CH4 concentration result from episodic mixing with shallower Mg-HCO3-type waters. Soil-gas analyses show that methane migrates to the surface along the fault, also independently from the water emergences, consistently with non-aqueous abiotic CH4 production. Our integrated model is generally compatible with observations from other gas-bearing continental serpentinization sites.

<p>約半世紀の歴史を持つ海洋掘削科学は，プレートテクトニクスの実証や過去の劇的な地球環境変動など，教科書にその名を刻む輝かしい科学的成果をもたらしてきた．中でも，「海底下生命圏」の発見による生命生息可能域の大幅な拡大は，それまでの地球生命科学の概念（パラダイム）を覆すマイルストーン的な科学成果の一つである．これまでに，世界各地の海洋底から掘削されたコアサンプルの多面的な分析研究により，水・エネルギー供給が極めて限られた海底下環境に，固有の進化を遂げた膨大な数の未知微生物が生息していることが明らかとなっている．その生態系機能は，極めて低活性な生命活動により支えられている静的なものであるが，地質学的時間スケールで，地球規模の元素循環に重要な役割を果たしていることが明らかとなってきた．</p>

Water from The Cedars springs that discharge from serpentinized ultramafic rocks feature highly basic (pH=similar to 12), highly reducing (E-h&lt;-550 mV) conditions with low ionic concentrations. These conditions make the springs exceptionally challenging for life. Here, we report the metagenomic data and recovered draft genomes from two different springs, GPS1 and BS5. GPS1, which was fed solely by a deep groundwater source within the serpentinizing system, was dominated by several bacterial taxa from the phyla OD1 ('Parcubacteria') and Chloroflexi. Members of the GPS1 community had, for the most part, the smallest genomes reported for their respective taxa, and encoded only archaeal (A-type) ATP synthases or no ATP synthases at all. Furthermore, none of the members encoded respiration-related genes and some of the members also did not encode key biosynthesis-related genes. In contrast, BS5, fed by shallow water, appears to have a community driven by hydrogen metabolism and was dominated by a diverse group of Proteobacteria similar to those seen in many terrestrial serpentinization sites. Our findings indicated that the harsh ultrabasic geological setting supported unexpectedly diverse microbial metabolic strategies and that the deep-water-fed springs supported a community that was remarkable in its unusual metagenomic and genomic constitution.

Microbial fuel cells (MFCs) are one of the bioelectrochemical systems that exploit microorganisms as biocatalysts to degrade organic matters and recover energy as electric power. Here, we explored how the established electrogenic microbial communities were influenced by three different inoculum sources; anaerobic sludge of the wastewater plant, rice paddy field soil, and coastal lagoon sediment. We periodically characterized both electricity generation with sucrose consumption and 16S rRNA-basis microbial community composition. The electrochemical features of MFC5 were slightly different among three inocula, and the lagoon sediment-inoculated MFC showed the highest performance in terms of the treatment time. Meanwhile, although the inoculated microbial communities were highly diverse and quite different, only twelve genera affiliated with delta-Proteobacteria, gamma-Proteobacteria, Bacilli, Clostridia/Negativicutes or Bacteroidetes were abundantly enriched in all MFC anode communities. Within them, several fermentative genera were clearly different due to the inocula, while the inocula-specific phylotypes were identified in an electrogenic genus Geobacter. The relative abundances of phylotypes closely-related to Geobacter metallireducens were increased in later stages of all the sucrose-fed MFC5. These results indicate that key microbial members for the functional electrogenic community widely exist in natural ecosystems, but the community members presenting in inoculum sources affected the MFC performances. (C) 2017 Elsevier B.V. All sights reserved.

This study evaluated the influence of the membrane type on the performance of bioelectromethanogenesis reactors. The functional activities and taxonomic composition of bioelectrochemical systems (BES) with Nafion 117 or Ultrex CMI-7000 membranes were assessed. Functional activity was measured as methane production and current consumption rates throughout operation. Microbial biomass and phylogenetic diversity were characterized at strategic intervals related to the membrane type used. The Nafion-BES reactor showed the best performance in terms of current consumption and methane production in the early operational period and a strong selection for fermentative bacteria. However, the Nafion-BES was not able to sustain this activity over the course of 7 subpassages since methanogenic species were ultimately selected against and did not appear in the community composition for the last two subpassages. In contrast, the Ultrex-BES had a lower pH concentration gradient and lower overall current consumption activity; however, the methane production activity from the Ultrex-BES was equivalent or better than the Nafion-BES reactor and was sustained throughout the seven subpassages. The membrane type appeared to be responsible not only for differences in the electrochemical operation of the BESs but it also influenced microbial taxonomic composition and dynamics. (C) The Author(s) 2016. Published by ECS. All rights reserved.

The conceptual model of Cabeco de Vide mineral waters, issuing at the contact metamorphism zone developed by the local mafic/ultramafic pluton, is described. These mineral waters discharge with a temperature between 17 and 20 degrees C, show a very alkaline pH (up to 11.8), and have: Na-Cl/Ca-OH facies; dry residuum approximate to 200 mg/L; C-total &lt; 3.0 mg/L; and extremely low Mg2+ and silica concentrations (&lt; 0.3 and 6.5 mg/L, respectively). CH4 isotopic composition (-24.4 parts per thousand &lt; delta C-13-CH4 &lt; -14.0 parts per thousand and 285 parts per thousand &lt; delta H-2-CH4 &lt; -218 parts per thousand) falls in the range of typical abiotic gas. In the case of Cabeco de Vide mineral waters, the understanding of the resource will help to preserve the mineral water quality, creating public interest. (C) 2017 The Authors. Published by Elsevier B.V. 2017 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license.

Present-day serpentinization generates groundwaters with conditions (pH&gt; 11, Eh&lt; -550 mV) favorable for the microbial and abiotic production of organic compounds from inorganic precursors. Elevated concentrations of methane, C-2-C-6 alkanes, acetate, and formate have been detected at these sites, but the microbial or abiotic origin of these compounds remains unclear. While geochemical data indicate that methane at most sites of present-day serpentinization is abiogenic, the stable carbon, hydrogen, and clumped isotope data as well as the hydrocarbon gas composition from The Cedars, CA, USA, are consistent with a microbial origin for methane. However, there is no direct evidence of methanogenesis at this site of serpentinization. We report on laboratory experiments in which the microbial communities in fluids and sediments from The Cedars were incubated with C-13 labeled substrates. Increasing methane concentrations and the incorporation of C-13 into methane in live experiments, but not in killed controls, demonstrated that methanogens converted methanol, formate, acetate (methyl group), and bicarbonate to methane. The apparent fractionation between methane and potential substrates (alpha C-13(CH4-CO2(g)) = 1.059 to 1.105, alpha(13)(CCH4-acetate) = 1.042 to 1.119) indicated that methanogenesis was dominated by the carbonate reduction pathway. Increasing concentrations of volatile organic acid anions indicated microbial acetogenesis. alpha(13)(CCO2(g)-acetate) values (0.999 to 1.000), however, were inconsistent with autotrophic acetogenesis, thus suggesting that acetate was produced through fermentation. This is the first study to show direct evidence of microbial methanogenesis and acetogenesis by the native microbial community at a site of present-day serpentinization.

The functional and taxonomic microbial dynamics of duplicate electricity-consuming methanogenic communities were observed over a 6 months period to characterize the reproducibility, stability and recovery of electromethanogenic consortia. The highest rate of methanogenesis was 0.72 mg-CH4/L/day, which occurred during the third month of enrichment when multiple methanogenic phylotypes and associated Desulfovibrionaceae phylotypes were present in the electrode-associated microbial community. Results also suggest that electromethanogenic microbial communities are very sensitive to electron donor-limiting open-circuit conditions. A 45 min exposure to open-circuit conditions induced an 87% drop in volumetric methane production rates. Methanogenic performance recovered after 4 months to a maximum value of 0.30 mg-CH4/L/day under set potential operation (-700 mV vs Ag/AgCl); however, current consumption and biomass production was variable over time. Long-term functional and taxonomic analyses from experimental replicates provide new knowledge toward understanding how to enrich electromethanogenic communities and operate bioelectrochemical systems for stable and reproducible performance. (C) 2015 Elsevier Ltd. All rights reserved.

Microorganisms almost always exist as mixed communities in nature. While the significance of microbial community activities is well appreciated, a thorough understanding about how microbial communities respond to environmental perturbations has not yet been achieved. Here we have used a combination of metagenomic, genome binning, and stimulus-induced metatranscriptomic approaches to estimate the metabolic network and stimuli-induced metabolic switches existing in a complex microbial biofilm that was producing electrical current via extracellular electron transfer (EET) to a solid electrode surface. Two stimuli were employed: to increase EET and to stop EET. An analysis of cell activity marker genes after stimuli exposure revealed that only two strains within eleven binned genomes had strong transcriptional responses to increased EET rates, with one responding positively and the other responding negatively. Potential metabolic switches between eleven dominant members were mainly observed for acetate, hydrogen, and ethanol metabolisms. These results have enabled the estimation of a multi-species metabolic network and the associated short-term responses to EET stimuli that induce changes to metabolic flow and cooperative or competitive microbial interactions. This systematic meta-omics approach represents a next step towards understanding complex microbial roles within a community and how community members respond to specific environmental stimuli.

Successful bioelectrochemical systems (BESs) applications and research efforts require a comprehensive understanding about the microbial activities associated with the bioelectrocatalytic conversion of chemical and electrical inputs. BES reactors utilize living microorganisms to drive catalytic activity, and these microbes will respond to system changes in different ways than abiotic catalysts. This chapter introduces methods and protocols for quantifying microbial biomass, extracting DNA from electrode surfaces, 16S rRNA gene sequencing, and sequence data analysis. Using these methods, researchers can quantify electron transfer activity per unit biomass and more accurately normalize electricity production results. In addition, 16S rRNA gene sequencing enables researchers to begin taxonomically describing the microbial populations that are present in anode-associated biofilms. This type of information is critical for gaining a basic understanding about the types of microbes that are interacting with electrode surfaces, estimating their function, and quantifying total biomass. These combined data sets can be used as a basis for more detailed analyses relative to microbial function and population dynamics in BESs.

Alkaliphilic bacteria typically grow well at pH 9, with the most extremophilic strains growing up to pH values as high as pH 12-13. Interest in extreme alkaliphiles arises because they are sources of useful, stable enzymes, and the cells themselves can be used for biotechnological and other applications at high pH. In addition, alkaline hydrothermal vents represent an early evolutionary niche for alkaliphiles and novel extreme alkaliphiles have also recently been found in alkaline serpentinizing sites. A third focus of interest in alkaliphiles is the challenge raised by the use of proton-coupled ATP synthases for oxidative phosphorylation by non-fermentative alkaliphiles. This creates a problem with respect to tenets of the chemiosmotic model that remains the core model for the bioenergetics of oxidative phosphorylation. Each of these facets of alkaliphilic bacteria will be discussed with a focus on extremely alkaliphilic Bacillus strains. These alkaliphilic bacteria have provided a cogent experimental system to probe adaptations that enable their growth and oxidative phosphorylation at high pH. Adaptations are clearly needed to enable secreted or partially exposed enzymes or protein complexes to function at the high external pH. Also, alkaliphiles must maintain a cytoplasmic pH that is significantly lower than the pH of the outside medium. This protects cytoplasmic components from an external pH that is alkaline enough to impair their stability or function. However, the pH gradient across the cytoplasmic membrane, with its orientation of more acidic inside than outside, is in the reverse of the productive orientation for bioenergetic work. The reversed gradient reduces the trans-membrane proton-motive force available to energize ATP synthesis. Multiple strategies are hypothesized to be involved in enabling alkaliphiles to circumvent the challenge of a low bulk proton-motive force energizing proton-coupled ATP synthesis at high pH.

Microbial extracellular electron transfer (EET) to solid surfaces is an important reaction for metal reduction occurring in various anoxic environments. However, it is challenging to accurately characterize EET-active microbial communities and each member's contribution to EET reactions because of changes in composition and concentrations of electron donors and solid-phase acceptors. Here, we used bioelectrochemical systems to systematically evaluate the synergistic effects of carbon source and surface redox potential on EET-active microbial community development, metabolic networks and overall electron transfer rates. The results indicate that faster biocatalytic rates were observed under electropositive electrode surface potential conditions, and under fatty acid-fed conditions. Temporal 16S rRNA-based microbial community analyses showed that Geobacter phylotypes were highly diverse and apparently dependent on surface potentials. The well-known electrogenic microbes affiliated with the Geobacter metallireducens clade were associated with lower surface potentials and less current generation, whereas Geobacter subsurface clades 1 and 2 were associated with higher surface potentials and greater current generation. An association was also observed between specific fermentative phylotypes and Geobacter phylotypes at specific surface potentials. When sugars were present, Tolumonas and Aeromonas phylotypes were preferentially associated with lower surface potentials, whereas Lactococcus phylotypes were found to be closely associated with Geobacter subsurface clades 1 and 2 phylotypes under higher surface potential conditions. Collectively, these results suggest that surface potentials provide a strong selective pressure, at the species and strain level, for both solid surface respirators and fermentative microbes throughout the EET-active community development.

Serpentinization, or the aqueous alteration of ultramafic rocks, results in challenging environments for life in continental sites due to the combination of extremely high pH, low salinity and lack of obvious electron acceptors and carbon sources. Nevertheless, certain Betaproteobacteria have been frequently observed in such environments. Here we describe physiological and genomic features of three related Betaproteobacterial strains isolated from highly alkaline (pH 11.6) serpentinizing springs at The Cedars, California. All three strains are obligate alkaliphiles with an optimum for growth at pH 11 and are capable of autotrophic growth with hydrogen, calcium carbonate and oxygen. The three strains exhibit differences, however, regarding the utilization of organic carbon and electron acceptors. Their global distribution and physiological, genomic and transcriptomic characteristics indicate that the strains are adapted to the alkaline and calcium-rich environments represented by the terrestrial serpentinizing ecosystems. We propose placing these strains in a new genus 'Serpentinomonas'.

This study explores the photosynthetic microbial colonization of rhyolitic ignimbrites in Lomas de Tilocalar, a hyper-arid region of the Atacama Desert, Chile. Colonization appeared in the form of a green layer a few millimeters beneath the ignimbrite surface. Some ignimbrite rocks revealed two distinct micromorphological areas of identical mineralogical and chemical composition but different textural properties. According to texture, colonization patterns varied in terms of the extension and depth of colonization. The diversity of photosynthetic microorganisms was assessed by denaturing gradient gel electrophoresis (DGGE) of the 23S rRNA gene and by generating clone libraries of the 16S rRNA gene. We observed a low diversity of photosynthetic microorganisms colonizing the ignimbrite microhabitat. Most rRNA gene sequences recovered greatly resembled those of Chroococcidiopsis hypolith clones from arid deserts. These results point to highly restrictive conditions of the hyper-arid Atacama Desert conditioning the diversity of cyanobacteria, and suggest that microbial colonization and composition patterns might be determined by the microscale physico-chemical properties of the ignimbrite rocks.

Microbial fuel cells (MFCs) are devices that exploit microorganisms as "biocatalysts" to recover energy from organic matter in the form of electricity. MFCs have been explored as possible energy neutral wastewater treatment systems; however, fundamental knowledge is still required about how MFC-associated microbial communities are affected by different operational conditions and can be optimized for accelerated wastewater treatment rates. In this study, we explored how electricity-generating microbial biofilms were established at MFC anodes and responded to three different operational conditions during wastewater treatment: 1) MFC operation using a 750 Omega external resistor (0.3 mA current production); 2) set-potential (SP) operation with the anode electrode potentiostatically controlled to +100 mV vs SHE (4.0 mA current production); and 3) open circuit (OC) operation (zero current generation). For all reactors, primary clarifier effluent collected from a municipal wastewater plant was used as the sole carbon and microbial source. Batch operation demonstrated nearly complete organic matter consumption after a residence time of 8-12 days for the MFC condition, 4-6 days for the SP condition, and 15-20 days for the OC condition. These results indicate that higher current generation accelerates organic matter degradation during MFC wastewater treatment. The microbial community analysis was conducted for the three reactors using 16S rRNA gene sequencing. Although the inoculated wastewater was dominated by members of Epsilonproteobacteria, Gammaproteobacteria, and Bacteroidetes species, the electricity-generating biofilms in MFC and SP reactors were dominated by Deltaproteobacteria and Bacteroidetes. Within Deltaproteobacteria, phylotypes classified to family Desulfobulbaceae and Geobacteraceae increased significantly under the SP condition with higher current generation; however those phylotypes were not found in the OC reactor. These analyses suggest that species related to family Desulfobulbaceae and Geobacteraceae are correlated with the electricity generation in the biofilm and may be key players for optimizing wastewater treatment rates and energy recovery in applied MFC systems. (C) 2013 Elsevier Ltd. All rights reserved.

Ultra-basic (pH 11-12) reducing (-656 to -585 mV) groundwater springs discharging from serpentinized peridotite of The Cedars, CA, were investigated for their geochemistry and geobiology. The spring waters investigated were of meteoric origin; however, geochemical modeling suggests that there were two sources of groundwater, a shallow source with sufficient contact with The Cedars' peridotite body to be altered geochemically by serpentinization, and a deeper groundwater source that not only flows through the peridotite body but was also in contact with the marine sediments of the Franciscan Subduction Complex (FSC) below the peridotite body. We propose that the groundwater discharging from lower elevations (GPS1 and CS1) reflect the geochemistry of the deeper groundwater in contact with FSC, while groundwaters discharging from springs at higher elevations (NS1 and BSC) were a mixture of the shallow peridotite-only groundwater and the deeper groundwater that has been in contact with the FSC. Cell densities of suspended microbes within these waters were extremely low. In the NS1 and BSC spring fluids, cell densities ranged from 10(2) to 10(3) cells/ml, while suspended cells at GPS were lower than 10 cells/mL. However, glass slides incubated in the BSC and GPS1 springs for 2-3 weeks were colonized by cells with densities ranging from 10(6) to 10(7) cells/cm(2) attached to their surfaces. All of the springs were very low (&lt;= 1 mu M) in several essential elements and electron acceptors (e. g. nitrate/ammonium, sulfate, and phosphate) required for (microbial) growth, which is not uncommon at sites of continental serpentinization. Gases rich in N-2, H-2, and CH4 were exsolving from the springs. The stable carbon isotope value (delta C-13(CH4) = -68 +/- 0.6 parts per thousand) and the CH4/C2+ (&gt;10(3)) of methane and other gaseous hydrocarbons exsolving from NS1 were typical of microbially sourced methane, whereas the isotope values and the CH4/C2+ of BSC and CS1 springs were more enriched in C-13 and had CH4/C2+ &lt; 10(3), suggesting a mixture of microbial and non-microbial methane. The concentrations of aromatic compounds, and ethane, propane, iso- and n-butane were well described by simple physical mixing between the aromatic- and alkane-poor, shallow groundwater and the relatively aromatic, and alkane-rich groundwater that flows through both the peridotite and the FSC suggesting that these aromatic and alkane compounds originated in the deeper FSC groundwater and are not produced in the shallow peridotite-only groundwater. The aromatic compounds most probably originated from the diagenesis/degradation of organic matter in the marine sediments below the peridotite body, while the gaseous alkanes may have multiple sources including thermal degradation of the organic matter in the marine sediments below the peridotite body and possibly by abiogenic reactions occurring within the peridotite body. This geochemical study demonstrates the complexity of The Cedars, and the possible sources of hydrocarbons at continental sites of serpentinization. (C) 2013 Elsevier Ltd. All rights reserved.

Microbial respiration via extracellular electron transfer (EET) is a ubiquitous reaction that occurs throughout anoxic environments and is a driving force behind global biogeochemical cycling of metals. Here we identify specific EET-active microbes and genes in a diverse biofilm using an innovative approach to analyse the dynamic community-wide response to changing EET rates. We find that the most significant gene expression responses to applied EET stimuli occur in only two microbial groups, Desulfobulbaceae and Desulfuromonadales. Metagenomic analyses reveal high coverage draft genomes of these abundant and active microbes. Our metatranscriptomic results show known and unknown genes that are highly responsive to EET stimuli and associated with our identified draft genomes. This new approach yields a comprehensive image of functional microbes and genes related to EET activity in a diverse community, representing the next step towards unravelling complex microbial roles within a community and how microbes adapt to specific environmental stimuli.

Microbial fuel cells (MFCs) are devices that exploit microorganisms as biocatalysts to recover energy from organic matter in the form of electricity. One of the goals of MFC research is to develop the technology for cost-effective wastewater treatment. However, before practical MFC applications are implemented it is important to gain fundamental knowledge about long-term system performance, reproducibility, and the formation and maintenance of functionally-stable microbial communities. Here we report findings from a MFC operated for over 300 days using only primary clarifier effluent collected from a municipal wastewater treatment plant as the microbial resource and substrate. The system was operated in a repeat-batch mode, where the reactor solution was replaced once every two weeks with new primary effluent that consisted of different microbial and chemical compositions with every batch exchange. The turbidity of the primary clarifier effluent solution notably decreased, and 97% of biological oxygen demand (BOD) was removed after an 8-13 day residence time for each batch cycle. On average, the limiting current density was 1000 mA/m(2), the maximum power density was 13 mW/m(2), and coulombic efficiency was 25%. Interestingly, the electrochemical performance and BOD removal rates were very reproducible throughout MFC operation regardless of the sample variability associated with each wastewater exchange. While MFC performance was very reproducible, the phylogenetic analyses of anode-associated electricity-generating biofilms showed that the microbial populations temporally fluctuated and maintained a high biodiversity throughout the year-long experiment. These results suggest that MFC communities are both self-selecting and self-optimizing, thereby able to develop and maintain functional stability regardless of fluctuations in carbon source(s) and regular introduction of microbial competitors. These results contribute significantly toward the practical application of MFC systems for long-term wastewater treatment as well as demonstrating MFC technology as a useful device to enrich for functionally stable microbial populations.

A parA gene in-frame deletion mutant of Azorhizobium caulinodans ORS571 (ORS571-Delta parA) was constructed to evaluate the roles of the chromosome-partitioning gene on various bacterial traits and on the development of stem-positioned nodules. The Delta parA mutant showed a pleiomorphic cell shape phenotype and was polyploid, with differences in nucleoid sizes due to dramatic defects in chromosome partitioning. Upon inoculation of the Delta parA mutant onto the stem of Sesbania rostrata, three types of immature nodule-like structures with impaired nitrogen-fixing activity were generated. Most showed signs of bacteroid early senescence. Moreover, the Delta parA cells within the nodule-like structures exhibited multiple developmental-stage phenotypes. Since the bacA gene has been considered an indicator for bacteroid formation, we applied the expression pattern of bacA as a nodule maturity index in this study. Our data indicate that the bacA gene expression is parA dependent in symbiosis. The presence of the parA gene transcript was inversely correlated with the maturity of nodule; the transcript was switched off in fully mature bacteroids. In summary, our experimental evidence demonstrates that the parA gene not only plays crucial roles in cellular development when the microbe is free-living but also negatively regulates bacteroid formation in S. rostrata stem nodules.

Here we report the phenotypic characteristics of a novel hypernodulation mutant, Ljrdh1 (root-determined hypernodulation 1) of Lotus japonicus. At 12 weeks after rhizobial inoculation, there were no differences between the growth of Ljrdh1 and, wild-type. However, Ljrdh1 showed 2 to 3 times higher nitrogen-fixing activity, and seed and pod yields, were approximately 50% higher than the wild-type. This is the first report of a legume hypernodulation mutant showing normal growth and a high-yielding characteristic under optimal cultivation conditions.

Two strains of marine, heterotrophic, alkaliphilic bacteria, designated A3F-7(T) and ssthio04PA2-7c, were isolated from hard coral and marine sand, respectively, collected in the Republic of Palau. A phylogenetic analysis based on 16S rRNA gene sequences showed that the isolates were related to members of the genus Moritella. However, the sequence similarities between the isolates and the type strains of the Moritella species were less than 93%. The G + C contents of the isolates were around 57 mol%, the major respiratory quinone was Q-8 and the predominant cellular fatty acids were 16 : 1 omega 7c, 16: 0, 18: 1 omega 7c and 14: 0. On the basis of the phylogenetic data, phenotypic characteristics and DNA-DNA hybridization results, strains A3F-7(T) and ssthio04PA2-7c represent a novel species of a novel genus, for which the name Paramoritella alkaliphila gen. nov., sp. nov. is proposed. The type strain of Paramoritella alkaliphila is A3F-7(T) (=MBIC06429(T) -DSM 19956(T)).

Two strains of marine, heterotrophic, alkaliphilic bacteria, designated A3F-7(T) and ssthio04PA2-7c, were isolated from hard coral and marine sand, respectively, collected in the Republic of Palau. A phylogenetic analysis based on 16S rRNA gene sequences showed that the isolates were related to members of the genus Moritella. However, the sequence similarities between the isolates and the type strains of the Moritella species were less than 93%. The G + C contents of the isolates were around 57 mol%, the major respiratory quinone was Q-8 and the predominant cellular fatty acids were 16 : 1 omega 7c, 16: 0, 18: 1 omega 7c and 14: 0. On the basis of the phylogenetic data, phenotypic characteristics and DNA-DNA hybridization results, strains A3F-7(T) and ssthio04PA2-7c represent a novel species of a novel genus, for which the name Paramoritella alkaliphila gen. nov., sp. nov. is proposed. The type strain of Paramoritella alkaliphila is A3F-7(T) (=MBIC06429(T) -DSM 19956(T)).

In this study, we investigated the function of a putative high-molecular-weight outer membrane protein, azorhizobial outer membrane autotransporter A (AoaA), of Azorhizobium caulinodans ORS571. Sequence analysis revealed that AoaA was an autotransporter protein belonging to the type V protein secretion system. Azorhizobium caulinodans forms N(2)-fixing nodules on the stems and roots of Sesbania rostrata. The sizes of stem nodules formed by an aoaA mutant having transposon insertion within this ORF were as large as those in the wild-type strain, but the N(2)-fixing activity of the nodules by the aoaA mutant was lower than that of wild-type nodules. cDNA-amplified fragment length polymorphism and reverse transcriptase-PCR analysis revealed that the expressions of several pathogen-related genes of host plants were induced in the aoaA mutant nodules. Furthermore, exopolysaccharide production was defective in the aoaA mutant under free-living conditions. These results indicate that AoaA may have an important role in sustaining the symbiosis by suppressing plant defense responses. The exopolysaccharide production controlled by AoaA might mediate this suppression mechanism.

Background: Biological nitrogen fixation is a prokaryotic process that plays an essential role in the global nitrogen cycle. Azorhizobium caulinodans ORS571 has the dual capacity to fix nitrogen both as free-living organism and in a symbiotic interaction with Sesbania rostrata. The host is a fast-growing, submergence-tolerant tropical legume on which A. caulinodans can efficiently induce nodule formation on the root system and on adventitious rootlets located on the stem. Results: The 5.37-Mb genome consists of a single circular chromosome with an overall average GC of 67% and numerous islands with varying GC contents. Most nodulation functions as well as a putative type-IV secretion system are found in a distinct symbiosis region. The genome contains a plethora of regulatory and transporter genes and many functions possibly involved in contacting a host. It potentially encodes 4717 proteins of which 96.3% have homologs and 3.7% are unique for A. caulinodans. Phylogenetic analyses show that the diazotroph Xanthobacter autotrophicus is the closest relative among the sequenced genomes, but the synteny between both genomes is very poor. Conclusion: The genome analysis reveals that A. caulinodans is a diazotroph that acquired the capacity to nodulate most probably through horizontal gene transfer of a complex symbiosis island. The genome contains numerous genes that reflect a strong adaptive and metabolic potential. These combined features and the availability of the annotated genome make A. caulinodans an attractive organism to explore symbiotic biological nitrogen fixation beyond leguminous plants.

A mutant line that develops an excess number of small nodules was found in Lotus japonicus Miyakojima MG20 during a screening for mutants defective in nodule development and nitrogen fixation. Genetic analysis revealed that the phenotype is inherited in a monogenic, recessive manner. The gene's locus was mapped on chromosome 1 between 53.7 and 61.4 cM. This mutant formed 5 - 10-fold more nodules than the wild-type plant, and a grafting experiment revealed that the root regulated the hypernodulation. Except for the nodulation's phenotype no other differences were found between the mutant and the wild-type plant with respect to growth and morphological characteristics. In the mapped locus for the mutant no nodulation genes were reported, and this fact strongly suggests that the gene's locus is a new one. The gene was named root-determined hypernodulation (rdh) 1.

The molecular and physiological mechanisms behind the maturation and maintenance of N-2-fixing nodules during development of symbiosis between rhizobia and legumes still remain unclear, although the early events of symbiosis are relatively well understood. Azorhizobium caulinodans ORS571 is a microsymbiont of the tropical legume Sesbania rostrata, forming N2-fixing nodules not only on the roots but also on the stems. In this study, 10,080 transposon-inserted mutants of A. caulinodans ORS571 were individually inoculated onto the stems of S. rostrata, and those mutants that induced ineffective stem nodules, as displayed by halted development at various stages, were selected. From repeated observations on stem nodulation, 108 Tn5 mutants were selected and categorized into seven nodulation types based on size and N-2 fixation activity. Tn5 insertions of some mutants were found in the well-known nodulation, nitrogen fixation, and symbiosis-related genes, such as nod, nif, and fix, respectively, lipopolysaccharide synthesis-related genes, C, metabolism-related genes, and so on. However, other genes have not been reported to have roles in legume-rhizobium symbiosis. The list of newly identified symbiosis-related genes will present clues to aid in understanding the maturation and maintenance mechanisms of nodules.

The Pseudomonas fluorescens strain HP72 used as biocontrol agent was isolated from the roots of creeping bentgrass on brown patch-suppressive soil. This strain can suppress brown patch disease caused by Rhizoetonia solani. The analysis of secondary metabolites from strain HP72 revealed that it produced known antifungal compounds, 2,4-diacetylphloroglucinol (2,4-DAPG), HCN, and a fluorescent siderophore. In the present study, the Tn5-inserted mutants of strain HP72, which did not show any antifungal activity, were selected. None of the mutants produced 2,4-DA-PG but they produced a fluorescent siderophore, while some strains produced HCN. Therefore, it is suggested that 2,4-DA-PG plays a major role in the biological control of brown patch disease caused by R. solani. In the genomic region where Tn5 was inserted, two open reading frames (ORFs A and B), which are not included in the 2,4-DAPG gene cluster of HP72, were detected. It was demonstrated that ORFs A and B are involved in the regulation of 2,4-DA-PG biosynthesis.

Three dibenzofuran (DF)-degrading strains were newly isolated from roots of white clover (Trifolium repens L.) and poplar trees grown in DF-contaminated soil samples. These strains, designated KD2, KD7, and PD1, were characterized as Comamonas sp. on the basis of their 16S rDNA sequences and physiological characteristics. The metabolites produced when strain KD7 was incubated with DF were identified by gas chromatography-mass spectrometry (GC-MS) analysis. Interestingly, strain KD7 was found to have two pathways for DF degradation, beginning with angular dioxygenation at carbons 4 and 4a, and lateral dioxygenation at carbons 1 and 2, respectively. Furthermore, strains KD2 and KD7 not only achieved efficient root colonization in clover but also promoted clover growth. They are the first reported Comamonas sp. strains capable of utilizing DF as a sole carbon source. This provides additional information on the diversity of DF-degrading bacteria.

Pseudomonas fluorescens HP72, which suppresses the brown patch disease on bentgrass, produces several secondary metabolites, 2,4-diacetylphloroglucinol (2,4-DAPG), HCN, siderophore, and indole-3-acetic acid (IAA). In this study, IAA biosynthesis in strain HP72 was investigated. After several repeated subcultures, the spontaneous IAA low-producing mutant HP72LI was isolated. The IAA low production of the strain HP72LI was due to the low tryptophan side chain oxidase (TSO) activity. Colonization of strain HP72 on the bentgrass root induced root growth reduction, while strain HP72LI did not induce such growth reduction. The colonization ability of strain HP72 on the bentgrass root is higher than that of strain HP72LI. However, as for biocontrol ability, a significant difference in both strains was not detected. IAA production by strain HP72 may play a role in the construction of short root systems and take advantage of root colonization, but does not contribute to the biocontrol properties of P. fluorescens HP72.