Microbial decomposition of biodegradable plastics on the deep-sea floor.

Microbes can decompose biodegradable plastics on land, rivers and seashore. However, it is unclear whether deep-sea microbes can degrade biodegradable plastics in the extreme environmental conditions of the seafloor. Here, we report microbial decomposition of representative biodegradable plastics (polyhydroxyalkanoates, biodegradable polyesters, and polysaccharide esters) at diverse deep-sea floor locations ranging in depth from 757 to 5552 m. The degradation of samples was evaluated in terms of weight loss, reduction in material thickness, and surface morphological changes. Poly(L-lactic acid) did not degrade at either shore or deep-sea sites, while other biodegradable polyesters, polyhydroxyalkanoates, and polysaccharide esters were degraded. The rate of degradation slowed with water depth. We analysed the plastic-associated microbial communities by 16S rRNA gene amplicon sequencing and metagenomics. Several dominant microorganisms carried genes potentially encoding plastic-degrading enzymes such as polyhydroxyalkanoate depolymerases and cutinases/polyesterases. Analysis of available metagenomic datasets indicated that these microorganisms are present in other deep-sea locations. Our results confirm that biodegradable plastics can be degraded by the action of microorganisms on the deep-sea floor, although with much less efficiency than in coastal settings.

The etherase system of Novosphingobium sp. MBES04 functions as a sensor of lignin fragments through phenylpropanone production to induce specific transcriptional responses.

The MBES04 strain of Novosphingobium accumulates phenylpropanone monomers as end-products of the etherase system, which specifically and reductively cleaves the ß-O-4 ether bond (a major bond in lignin molecules). However, it does not utilise phenylpropanone monomers as an energy source. Here, we studied the response to the lignin-related perturbation to clarify the physiological significance of its etherase system. Transcriptome analysis revealed two gene clusters, each consisting of four tandemly linked genes, specifically induced by a lignin preparation extracted from hardwood (Eucalyptus globulus) and a ß-O-4-type lignin model biaryl compound, but not by vanillin. The most strongly induced gene was a 2,4'-dihydroxyacetophenone dioxygenase-like protein, which leads to energy production through oxidative degradation. The other cluster was related to multidrug resistance. The former cluster was transcriptionally regulated by a common promoter, where a phenylpropanone monomer acted as one of the effectors responsible for gene induction. These results indicate that the physiological significance of the etherase system of the strain lies in its function as a sensor for lignin fragments. This may be a survival strategy to detect nutrients and gain tolerance to recalcitrant toxic compounds, while the strain preferentially utilises easily degradable aromatic compounds with lower energy demands for catabolism.

Unique evolution of foraminiferal calcification to survive global changes.

Foraminifera, the most ancient known calcium carbonate-producing eukaryotes, are crucial players in global biogeochemical cycles and well-used environmental indicators in biogeosciences. However, little is known about their calcification mechanisms. This impedes understanding the organismal responses to ocean acidification, which alters marine calcium carbonate production, potentially leading to biogeochemical cycle changes. We conducted comparative single-cell transcriptomics and fluorescent microscopy and identified calcium ion (Ca2+) transport/secretion genes and α-carbonic anhydrases that control calcification in a foraminifer. They actively take up Ca2+ to boost mitochondrial adenosine triphosphate synthesis during calcification but need to pump excess intracellular Ca2+ to the calcification site to prevent cell death. Unique α-carbonic anhydrase genes induce the generation of bicarbonate and proton from multiple CO2 sources. These control mechanisms have evolved independently since the Precambrian to enable the development of large cells and calcification despite decreasing Ca2+ concentrations and pH in seawater. The present findings provide previously unknown insights into the calcification mechanisms and their subsequent function in enduring ocean acidification.

Isolation and Polyphasic Characterization of Desulfuromonas versatilis sp. Nov., an Electrogenic Bacteria Capable of Versatile Metabolism Isolated from a Graphene Oxide-Reducing Enrichment Culture.

In this study, a novel electrogenic bacterium denoted as strain NIT-T3 of the genus Desulfuromonas was isolated from a graphene-oxide-reducing enrichment culture that was originally obtained from a mixture of seawater and coastal sand. Strain NIT-T3 utilized hydrogen and various organic acids as electron donors and exhibited respiration using electrodes, ferric iron, nitrate, and elemental sulfur. The strain contained C16:1ω7c, C16:0, and C15:0 as major fatty acids and MK-8, 9, and 7 as the major respiratory quinones. Strain NIT-T3 contained four 16S rRNA genes and showed 95.7% similarity to Desulfuromonasmichiganensis BB1T, the closest relative. The genome was 4.7 Mbp in size and encoded 76 putative c-type cytochromes, which included 6 unique c-type cytochromes (<40% identity) compared to those in the database. Based on the physiological and genetic uniqueness, and wide metabolic capability, strain NIT-T3 is proposed as a type strain of 'Desulfuromonas versatilis' sp. nov.

Prospects for multi-omics in the microbial ecology of water engineering.

Advances in high-throughput sequencing technologies and bioinformatics approaches over almost the last three decades have substantially increased our ability to explore microorganisms and their functions - including those that have yet to be cultivated in pure isolation. Genome-resolved metagenomic approaches have enabled linking powerful functional predictions to specific taxonomical groups with increasing fidelity. Additionally, related developments in both whole community gene expression surveys and metabolite profiling have permitted for direct surveys of community-scale functions in specific environmental settings. These advances have allowed for a shift in microbiome science away from descriptive studies and towards mechanistic and predictive frameworks for designing and harnessing microbial communities for desired beneficial outcomes. Water engineers, microbiologists, and microbial ecologists studying activated sludge, anaerobic digestion, and drinking water distribution systems have applied various (meta)omics techniques for connecting microbial community dynamics and physiologies to overall process parameters and system performance. However, the rapid pace at which new omics-based approaches are developed can appear daunting to those looking to apply these state-of-the-art practices for the first time. Here, we review how modern genome-resolved metagenomic approaches have been applied to a variety of water engineering applications from lab-scale bioreactors to full-scale systems. We describe integrated omics analysis across engineered water systems and the foundations for pairing these insights with modeling approaches. Lastly, we summarize emerging omics-based technologies that we believe will be powerful tools for water engineering applications. Overall, we provide a framework for microbial ecologists specializing in water engineering to apply cutting-edge omics approaches to their research questions to achieve novel functional insights. Successful adoption of predictive frameworks in engineered water systems could enable more economically and environmentally sustainable bioprocesses as demand for water and energy resources increases.

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.

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.

Complete Genome Sequence of Sphingobium sp. Strain YG1, a Lignin Model Dimer-Metabolizing Bacterium Isolated from Sediment in Kagoshima Bay, Japan.

Sphingobium sp. strain YG1 is a lignin model dimer-metabolizing bacterium newly isolated from sediment in Kagoshima, Japan, at a depth of 102 m. Here, we report the complete genome nucleotide sequence of strain YG1.

Complete Genome Sequence of Altererythrobacter sp. Strain B11, an Aromatic Monomer-Degrading Bacterium, Isolated from Deep-Sea Sediment under the Seabed off Kashima, Japan.

Altererythrobacter sp. strain B11 is an aromatic monomer-degrading bacterium newly isolated from sediment under the seabed off Kashima, Japan, at a depth of 2,100 m. Here, we report the complete nucleotide sequence of the genome of strain B11.

Metagenomic insights into the ecology and physiology of microbes in bioelectrochemical systems.

In bioelectrochemical systems (BESs), electrons are transferred between electrochemically active microbes (EAMs) and conductive materials, such as electrodes, via extracellular electron transfer (EET) pathways, and electrons thus transferred stimulate intracellular catabolic reactions. Catabolic and EET pathways have extensively been studied for several model EAMs, such as Shewanella oneidensis MR-1 and Geobacter sulfurreducens PCA, whereas it is also important to understand the ecophysiology of EAMs in naturally occurring microbiomes, such as those in anode biofilms in microbial fuel cells treating wastewater. Recent studies have exploited metagenomics and metatranscriptomics (meta-omics) approaches to characterize EAMs in BES-associated microbiomes. Here we review recent BES studies that used meta-omics approaches and show that these studies have discovered unexpected features of EAMs and deepened our understanding of functions and behaviors of microbes in BESs. It is desired that more studies will employ meta-omics approaches for advancing our knowledge on microbes in BESs.

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.

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.

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.

Non-autotrophic methanogens dominate in anaerobic digesters.

Anaerobic digesters are man-made habitats for fermentative and methanogenic microbes, and are characterized by extremely high concentrations of organics. However, little is known about how microbes adapt to such habitats. In the present study, we report phylogenetic, metagenomic, and metatranscriptomic analyses of microbiomes in thermophilic packed-bed digesters fed acetate as the major substrate, and we have shown that acetoclastic and hydrogenotrophic methanogens that utilize acetate as a carbon source dominate there. Deep sequencing and precise binning of the metagenomes reconstructed complete genomes for two dominant methanogens affiliated with the genera Methanosarcina and Methanothermobacter, along with 37 draft genomes. The reconstructed Methanosarcina genome was almost identical to that of a thermophilic acetoclastic methanogen Methanosarcina thermophila TM-1, indicating its cosmopolitan distribution in thermophilic digesters. The reconstructed Methanothermobacter (designated as Met2) was closely related to Methanothermobacter tenebrarum, a non-autotrophic hydrogenotrophic methanogen that grows in the presence of acetate. Met2 lacks the Cdh complex required for CO2 fixation, suggesting that it requires organic molecules, such as acetate, as carbon sources. Although the metagenomic analysis also detected autotrophic methanogens, they were less than 1% in abundance of Met2. These results suggested that non-autotrophic methanogens preferentially grow in anaerobic digesters containing high concentrations of organics.

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.

Functional and taxonomic dynamics of an electricity-consuming methane-producing microbial community.

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.

Physiological and genomic features of highly alkaliphilic hydrogen-utilizing Betaproteobacteria from a continental serpentinizing site.

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'.

Microbial population and functional dynamics associated with surface potential and carbon metabolism.

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.

Identifying the microbial communities and operational conditions for optimized wastewater treatment in microbial fuel cells.

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 Ω 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.

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.