Serpentinimonas gen. nov., Serpentinimonas raichei sp. nov., Serpentinimonas barnesii sp. nov. and Serpentinimonas maccroryi sp. nov., hyperalkaliphilic and facultative autotrophic bacteria isolated from terrestrial serpentinizing springs.

Three highly alkaliphilic bacterial strains designated as A1T, H1T and B1T 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 A1T, H1T and B1T 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 A1T, H1T and B1T 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 Malikia (95.1-96.4 %), Macromonas (93.0-93.6 %) and Hydrogenophaga (93.0-96.6 %) in the family Comamonadaceae. 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 A1T, H1T and B1T 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 blast 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 Serpentinimonas gen. nov., Serpentinimonas raichei sp. nov. (type strain A1T=NBRC 111848T=DSM 103917T), Serpentinimonas barnesii sp. nov. (type strain H1T= NBRC 111849T=DSM 103920T) and Serpentinimonas maccroryi sp. nov. (type strain B1T=NBRC 111850T=DSM 103919T) belonging to the family Comamonadaceae. We have designated Serpentinimonas raichei the type species for the genus because it is the dominant species in The Cedars springs.

Mariana serpentinite mud volcanism exhumes subducted seamount materials: implications for the origin of life.

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'.

Microbial diversity in The Cedars, an ultrabasic, ultrareducing, and low salinity serpentinizing ecosystem.

The Cedars, in coastal northern California, is an active site of peridotite serpentinization. The spring waters that emerge from this system feature very high pH, low redox potential, and low ionic concentrations, making it an exceptionally challenging environment for life. We report a multiyear, culture-independent geomicrobiological study of three springs at The Cedars that differ with respect to the nature of the groundwater feeding them. Within each spring, both geochemical properties and microbial diversity in all three domains of life remained stable over a 3-y period, with multiple samples each year. Between the three springs, however, the microbial communities showed considerable differences that were strongly correlated with the source of the serpentinizing groundwater. In the spring fed solely by deep groundwater, phylum Chloroflexi, class Clostridia, and candidate division OD1 were the major taxa with one phylotype in Euryarchaeota. Less-abundant phylotypes include several minor members from other candidate divisions and one phylotype that was an outlier of candidate division OP3. In the springs fed by the mixture of deep and shallow groundwater, organisms close to the Hydrogenophaga within Betaproteobacteria dominated and coexisted with the deep groundwater community members. The shallow groundwater community thus appears to be similar to those described in other terrestrial serpentinizing sites, whereas the deep community is distinctly different from any other previously described terrestrial serpentinizing community. These unique communities have the potential to yield important insights into the development and survival of life in these early-earth analog environments.

A non-methanogenic archaeon within the order Methanocellales.

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.

Bacterial and fungal bioburden reduction on material surfaces using various sterilization techniques suitable for spacecraft decontamination.

Planetary protection is a guiding principle aiming to prevent microbial contamination of the solar system by spacecraft (forward contamination) and extraterrestrial contamination of the Earth (backward contamination). Bioburden reduction on spacecraft, including cruise and landing systems, is required to prevent microbial contamination from Earth during space exploration missions. Several sterilization methods are available; however, selecting appropriate methods is essential to eliminate a broad spectrum of microorganisms without damaging spacecraft components during manufacturing and assembly. Here, we compared the effects of different bioburden reduction techniques, including dry heat, UV light, isopropyl alcohol (IPA), hydrogen peroxide (H2O2), vaporized hydrogen peroxide (VHP), and oxygen and argon plasma on microorganisms with different resistance capacities. These microorganisms included Bacillus atrophaeus spores and Aspergillus niger spores, Deinococcus radiodurans, and Brevundimonas diminuta, all important microorganisms for considering planetary protection. Bacillus atrophaeus spores showed the highest resistance to dry heat but could be reliably sterilized (i.e., under detection limit) through extended time or increased temperature. Aspergillus niger spores and D. radiodurans were highly resistant to UV light. Seventy percent of IPA and 7.5% of H2O2 treatments effectively sterilized D. radiodurans and B. diminuta but showed no immediate bactericidal effect against B. atrophaeus spores. IPA immediately sterilized A. niger spores, but H2O2 did not. During VHP treatment under reduced pressure, viable B. atrophaeus spores and A. niger spores were quickly reduced by approximately two log orders. Oxygen plasma sterilized D. radiodurans but did not eliminate B. atrophaeus spores. In contrast, argon plasma sterilized B. atrophaeus but not D. radiodurans. Therefore, dry heat could be used for heat-resistant component bioburden reduction, and VHP or plasma for non-heat-resistant components in bulk bioburden reduction. Furthermore, IPA, H2O2, or UV could be used for additional surface bioburden reduction during assembly and testing. The systemic comparison of sterilization efficiencies under identical experimental conditions in this study provides basic criteria for determining which sterilization techniques should be selected during bioburden reduction for forward planetary protection.

Unique H2-utilizing lithotrophy in serpentinite-hosted systems.

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.

Insights into the physiological and genomic characterization of three bacterial isolates from a highly alkaline, terrestrial serpentinizing system.

The terrestrial serpentinite-hosted ecosystem known as "The Cedars" is home to a diverse microbial community persisting under highly alkaline (pH ~ 12) and reducing (Eh < -550 mV) conditions. This extreme environment presents particular difficulties for microbial life, and efforts to isolate microorganisms from The Cedars over the past decade have remained challenging. Herein, we report the initial physiological assessment and/or full genomic characterization of three isolates: Paenibacillus sp. Cedars ('Paeni-Cedars'), Alishewanella sp. BS5-314 ('Ali-BS5-314'), and Anaerobacillus sp. CMMVII ('Anaero-CMMVII'). Paeni-Cedars is a Gram-positive, rod-shaped, mesophilic facultative anaerobe that grows between pH 7-10 (minimum pH tested was 7), temperatures 20-40°C, and 0-3% NaCl concentration. The addition of 10-20 mM CaCl2 enhanced growth, and iron reduction was observed in the following order, 2-line ferrihydrite > magnetite > serpentinite ~ chromite ~ hematite. Genome analysis identified genes for flavin-mediated iron reduction and synthesis of a bacillibactin-like, catechol-type siderophore. Ali-BS5-314 is a Gram-negative, rod-shaped, mesophilic, facultative anaerobic alkaliphile that grows between pH 10-12 and temperatures 10-40°C, with limited growth observed 1-5% NaCl. Nitrate is used as a terminal electron acceptor under anaerobic conditions, which was corroborated by genome analysis. The Ali-BS5-314 genome also includes genes for benzoate-like compound metabolism. Anaero-CMMVII remained difficult to cultivate for physiological studies; however, growth was observed between pH 9-12, with the addition of 0.01-1% yeast extract. Anaero-CMMVII is a probable oxygen-tolerant anaerobic alkaliphile with hydrogenotrophic respiration coupled with nitrate reduction, as determined by genome analysis. Based on single-copy genes, ANI, AAI and dDDH analyses, Paeni-Cedars and Ali-BS5-314 are related to other species (P. glucanolyticus and A. aestuarii, respectively), and Anaero-CMMVII represents a new species. The characterization of these three isolates demonstrate the range of ecophysiological adaptations and metabolisms present in serpentinite-hosted ecosystems, including mineral reduction, alkaliphily, and siderophore production.

COSPAR Sample Safety Assessment Framework (SSAF).

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.

Involvement of the azorhizobial chromosome partition gene (parA) in the onset of bacteroid differentiation during Sesbania rostrata stem nodule development.

A parA gene in-frame deletion mutant of Azorhizobium caulinodans ORS571 (ORS571-ΔparA) was constructed to evaluate the roles of the chromosome-partitioning gene on various bacterial traits and on the development of stem-positioned nodules. The ΔparA 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 ΔparA 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 ΔparA 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.

Comparison of lung microbiota between antineutrophil cytoplasmic antibody-associated vasculitis and sarcoidosis.

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.

Root-determined hypernodulation mutant of Lotus japonicus shows high-yielding characteristics.

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.

Origin of Short-Chain Organic Acids in Serpentinite Mud Volcanoes of the Mariana Convergent Margin.

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, Fantangisña, and Asùt 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, δ13C-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.

The genome of the versatile nitrogen fixer Azorhizobium caulinodans ORS571.

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.

An outer membrane autotransporter, AoaA, of Azorhizobium caulinodans is required for sustaining high N2-fixing activity of stem nodules.

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.

Genomic and in-situ Transcriptomic Characterization of the Candidate Phylum NPL-UPL2 From Highly Alkaline Highly Reducing Serpentinized Groundwater.

Serpentinization is a process whereby water interacts with reduced mantle rock called peridotite to produce a new suite of minerals (e.g., serpentine), a highly alkaline fluid, and hydrogen. In previous reports, we identified abundance of microbes of the candidate phylum NPL-UPA2 in a serpentinization site called The Cedars. Here, we report the first metagenome assembled genome (MAG) of the candidate phylum as well as the in-situ gene expression. The MAG of the phylum NPL-UPA2, named Unc8, is only about 1 Mbp and its biosynthetic properties suggest it should be capable of independent growth. In keeping with the highly reducing niche of Unc8, its genome encodes none of the known oxidative stress response genes including superoxide dismutases. With regard to energy metabolism, the MAG of Unc8 encodes all enzymes for Wood-Ljungdahl acetogenesis pathway, a ferredoxin:NAD+ oxidoreductase (Rnf) and electron carriers for flavin-based electron bifurcation (Etf, Hdr). Furthermore, the transcriptome of Unc8 in the waters of The Cedars showed enhanced levels of gene expression in the key enzymes of the Wood-Ljungdahl pathway [e.g., Carbon monoxide dehydrogenase /Acetyl-CoA synthase complex (CODH/ACS), Rnf, Acetyl-CoA synthetase (Acd)], which indicated that the Unc8 is an acetogen. However, the MAG of Unc8 encoded no well-known hydrogenase genes, suggesting that the energy metabolism of Unc8 might be focused on CO as the carbon and energy sources for the acetate formation. Given that CO could be supplied via abiotic reaction associated with deep subsurface serpentinization, while available CO2 would be at extremely low concentrations in this high pH environment, CO-associated metabolism could provide advantageous approach. The CODH/ACS in Unc8 is a Bacteria/Archaea hybrid type of six-subunit complex and the electron carriers, Etf and Hdr, showed the highest similarity to those in Archaea, suggesting that archaeal methanogenic energy metabolism was incorporated into the bacterial acetogenesis in NPL-UPA2. Given that serpentinization systems are viewed as potential habitats for early life, and that acetogenesis via the Wood-Ljungdahl pathway is proposed as an energy metabolism of Last Universal Common Ancestor, a phylogenetically distinct acetogen from an early earth analog site may provide important insights in primordial lithotrophs and their habitat.

Comparative metatranscriptomics reveals extracellular electron transfer pathways conferring microbial adaptivity to surface redox potential changes.

Some microbes can capture energy through redox reactions with electron flow to solid-phase electron acceptors, such as metal-oxides or poised electrodes, via extracellular electron transfer (EET). While diverse oxide minerals, exhibiting different surface redox potentials, are widely distributed on Earth, little is known about how microbes sense and use the minerals. Here we show electrochemical, metabolic, and transcriptional responses of EET-active microbial communities established on poised electrodes to changes in the surface redox potentials (as electron acceptors) and surrounding substrates (as electron donors). Combination of genome-centric stimulus-induced metatranscriptomics and metabolic pathway investigation revealed that nine Geobacter/Pelobacter microbes performed EET activity differently according to their preferable surface potentials and substrates. While the Geobacter/Pelobacter microbes coded numerous numbers of multi-heme c-type cytochromes and conductive e-pili, wide variations in gene expression were seen in response to altering surrounding substrates and surface potentials, accelerating EET via poised electrode or limiting EET via an open circuit system. These flexible responses suggest that a wide variety of EET-active microbes utilizing diverse EET mechanisms may work together to provide such EET-active communities with an impressive ability to handle major changes in surface potential and carbon source availability.

Population dynamics of electrogenic microbial communities in microbial fuel cells started with three different inoculum sources.

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 MFCs 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 δ-Proteobacteria, γ-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 MFCs. 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.

Unusual metabolic diversity of hyperalkaliphilic microbial communities associated with subterranean serpentinization at The Cedars.

Water from The Cedars springs that discharge from serpentinized ultramafic rocks feature highly basic (pH=~12), highly reducing (Eh<-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.

Alkaliphilic Bacteria with Impact on Industrial Applications, Concepts of Early Life Forms, and Bioenergetics of ATP Synthesis.

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 metabolic networks in a complex electrogenic biofilm recovered from a stimulus-induced metatranscriptomics approach.

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.