Microbial iron acquisition is influenced by spatial and temporal conditions in a glacial influenced river and estuary system.

In Arctic regions, glaciers are major sources of iron to rivers and streams; however, estuaries are considered iron sinks due to the coagulation and flocculation processes that occur at higher salinities. It is unknown how iron dynamics in a glacial influenced river and estuary environment affect microbial mechanisms for iron acquisition. Microbial taxonomic and functional sequencing was performed on samples taken throughout the year from the Kenai River and the estuary, Alaska. Despite distinct iron, sodium, and other nutrient concentrations, the river and estuary did not have statistically different microbial communities nor was time of sampling significant. However, ferrous iron transport (Feo) system genes were more abundant in river environments, while siderophore genes were more abundant and diverse in estuary environments. Siderophore transport and iron storage genes were found in all samples, but gene abundance and distribution were potentially influenced by physical drivers such as discharge rates and nutrient distributions. Differences in iron metabolism between river and estuary ecosystems indicate environmental conditions drive microbial mechanisms to sequester iron. This could have implications for iron transport as the Arctic continues to warm.

Field and laboratory characterization of rotenone attenuation in eight lakes of the Kenai Peninsula, Alaska.

Rotenone is a pesticide commonly used to eradicate Northern Pike (Esox lucius), an invasive species, in Southcentral Alaska. The present work incorporates a field investigation of rotenone attenuation in eight lakes of the Kenai Peninsula, following a CFT Legumine® treatment in October 2018 and a laboratory simulation to determine persistence under light/dark and sterile/nonsterile conditions representative of Southcentral Alaskan winters. In the field, rotenone degraded within <60 days of application in all lakes, while rotenolone, the primary product of rotenone degradation, persisted for up to <280 days post-treatment at two locations. Prolonged rotenolone attenuation was most likely caused by short days and ice cover between October and April. This hypothesis was supported by a laboratory simulation which revealed photolysis as the dominant process driving the overall degradation of rotenone and that microbial degradation will significantly contribute in the absence of sunlight under simulated "winter" conditions of 4 °C. Degradation model fit comparisons (pseudo-first order, multi-parameter linear, and gamma) indicate the most accurate prediction occurred when modeling all eight lakes grouped together in a single dataset, combined and treated with pseudo-first order model kinetics, based on Akaike information criteria (AIC) scores.

The Effect of Spring Water Geochemistry on Copper Proteins in Tengchong Hot Springs, China.

Copper (Cu) is an essential trace metal cofactor for a variety of proteins; however, excess Cu is toxic to most organisms. Cu homeostasis is maintained by a complex machinery of Cu binding proteins that control the uptake, transport, sequestration, and efflux of Cu ions. Despite the importance of Cu binding proteins in electron transfer, substrate oxidation, superoxide dismutation, and denitrification, little information exists about microbial Cu utilization in extreme environments, where the geochemical conditions may affect Cu bioavailability. Using metagenomic data from 9 hot springs in Tengchong, China, which range in temperature from 42°C to 96°C and in pH from 2.3 to 9, the effects of pH, temperature, and spring geochemistry on the distribution of Cu binding domains of proteins and oxidoreductases were studied. Dissolved Cu and Cu binding domains were detected across all temperature and pH gradients. Cu binding domains of cytochrome c oxidase subunits, heavy-metal-associated domains, and nitrous oxide reductase were detected at all sites. DoxB, a quinol oxidase, and other quinol oxidase subunits were the dominant Cu binding oxidoreductase subunits present at low-pH and high-temperature sites, whereas cbb3-type cytochrome c oxidase subunits were dominant at high-pH and high-temperature sites. Additionally, aa3-type cytochrome c oxidase was more prominent than cbb3-type cytochrome c oxidase under circumneutral-pH conditions. This suggests that the type of cytochrome c oxidase pathway and the Cu proteins employed by microbes to carry out important functions such as energy acquisition and efflux of excess Cu are affected by the physicochemical conditions of the springs.IMPORTANCE Copper is present in a variety of proteins and is required to carry out essential functions by all organisms. However, in hot spring environments, copper availability may be limited due to the high temperatures and the wide range in pH. The significance of our research is in relating the physicochemical environment to the distribution of copper proteins across hot spring environments, which provides increased understanding of primary functions and adaptions in these environments.

Bio-production of gaseous alkenes: ethylene, isoprene, isobutene.

To reduce emissions from petrochemical refinement, bio-production has been heralded as a way to create economically valuable compounds with fewer harmful effects. For example, gaseous alkenes are precursor molecules that can be polymerized into a variety of industrially significant compounds and have biological production pathways. Production levels, however, remain low, thus enhancing bio-production of gaseous petrochemicals for chemical precursors is critical. This review covers the metabolic pathways and production levels of the gaseous alkenes ethylene, isoprene, and isobutene. Techniques needed to drive production to higher levels are also discussed.

High Diversity of Myocyanophage in Various Aquatic Environments Revealed by High-Throughput Sequencing of Major Capsid Protein Gene With a New Set of Primers.

Myocyanophages, a group of viruses infecting cyanobacteria, are abundant and play important roles in elemental cycling. Here we investigated the particle-associated viral communities retained on 0.2 µm filters and in sediment samples (representing ancient cyanophage communities) from four ocean and three lake locations, using high-throughput sequencing and a newly designed primer pair targeting a gene fragment (∼145-bp in length) encoding the cyanophage gp23 major capsid protein (MCP). Diverse viral communities were detected in all samples. The fragments of 142-, 145-, and 148-bp in length were most abundant in the amplicons, and most sequences (>92%) belonged to cyanophages. Additionally, different sequencing depths resulted in different diversity estimates of the viral community. Operational taxonomic units obtained from deep sequencing of the MCP gene covered the majority of those obtained from shallow sequencing, suggesting that deep sequencing exhibited a more complete picture of cyanophage community than shallow sequencing. Our results also revealed a wide geographic distribution of marine myocyanophages, i.e., higher dissimilarities of the myocyanophage communities corresponded with the larger distances between the sampling sites. Collectively, this study suggests that the newly designed primer pair can be effectively used to study the community and diversity of myocyanophage from different environments, and the high-throughput sequencing represents a good method to understand viral diversity.

Abundance and taxonomic affiliation of molybdenum transport and utilization genes in Tengchong hot springs, China.

The nitrogen, sulfur and carbon cycles all rely on critical microbial transformations that are carried out by enzymes that require molybdenum (Mo) as a cofactor. Despite Mo importance in these biogeochemical cycles, little information exists about microbial Mo utilization in extreme environments where, due to geochemical conditions, bioavailable Mo may be limited. Using metagenomic data from nine hot springs in Tengchong, Yunnan Province, China, which range in temperature from 42°C to 96°C and pH from 2.3 to 9, the effects of pH, temperature and spring geochemistry on the abundance and taxonomic affiliation of genes related to Mo were studied. Dissolved Mo was only detected at sites with circumneutral pH. However, processes and organisms that require Mo were detected at all sites across all temperature and pH gradients. All sites contained xanthine dehydrogenase, formate dehydrogenase, carbon-monoxide dehydrogenase, nitrate reductase, sulfite oxidase and methionine-sulfoxide reductase despite different community compositions. This suggests that different microbial communities, resulting from different physicochemical conditions, may be performing similar metabolic functions. Furthermore, the abundance and taxonomic diversity of Mo-related annotations increased with higher concentrations of Mo. This study shows that despite geochemical conditions that can limit Mo bioavailability, microbes require Mo for a variety of processes.

Global metagenomic survey reveals a new bacterial candidate phylum in geothermal springs.

Analysis of the increasing wealth of metagenomic data collected from diverse environments can lead to the discovery of novel branches on the tree of life. Here we analyse 5.2 Tb of metagenomic data collected globally to discover a novel bacterial phylum ('Candidatus Kryptonia') found exclusively in high-temperature pH-neutral geothermal springs. This lineage had remained hidden as a taxonomic 'blind spot' because of mismatches in the primers commonly used for ribosomal gene surveys. Genome reconstruction from metagenomic data combined with single-cell genomics results in several high-quality genomes representing four genera from the new phylum. Metabolic reconstruction indicates a heterotrophic lifestyle with conspicuous nutritional deficiencies, suggesting the need for metabolic complementarity with other microbes. Co-occurrence patterns identifies a number of putative partners, including an uncultured Armatimonadetes lineage. The discovery of Kryptonia within previously studied geothermal springs underscores the importance of globally sampled metagenomic data in detection of microbial novelty, and highlights the extraordinary diversity of microbial life still awaiting discovery.

Single-Cell-Genomics-Facilitated Read Binning of Candidate Phylum EM19 Genomes from Geothermal Spring Metagenomes.

The vast majority of microbial life remains uncatalogued due to the inability to cultivate these organisms in the laboratory. This "microbial dark matter" represents a substantial portion of the tree of life and of the populations that contribute to chemical cycling in many ecosystems. In this work, we leveraged an existing single-cell genomic data set representing the candidate bacterial phylum "Calescamantes" (EM19) to calibrate machine learning algorithms and define metagenomic bins directly from pyrosequencing reads derived from Great Boiling Spring in the U.S. Great Basin. Compared to other assembly-based methods, taxonomic binning with a read-based machine learning approach yielded final assemblies with the highest predicted genome completeness of any method tested. Read-first binning subsequently was used to extract Calescamantes bins from all metagenomes with abundant Calescamantes populations, including metagenomes from Octopus Spring and Bison Pool in Yellowstone National Park and Gongxiaoshe Spring in Yunnan Province, China. Metabolic reconstruction suggests that Calescamantes are heterotrophic, facultative anaerobes, which can utilize oxidized nitrogen sources as terminal electron acceptors for respiration in the absence of oxygen and use proteins as their primary carbon source. Despite their phylogenetic divergence, the geographically separate Calescamantes populations were highly similar in their predicted metabolic capabilities and core gene content, respiring O2, or oxidized nitrogen species for energy conservation in distant but chemically similar hot springs.

Reduction of hexavalent chromium by the thermophilic methanogen Methanothermobacter thermautotrophicus.

Despite the significant progress on iron reduction by thermophilic microorganisms, studies on their ability to reduce toxic metals are still limited, despite their common co-existence in high temperature environments (up to 70°C). In this study, Methanothermobacter thermautotrophicus, an obligate thermophilic methanogen, was used to reduce hexavalent chromium. Experiments were conducted in a growth medium with H2/CO2 as substrate with various Cr6+ concentrations (0.2, 0.4, 1, 3, and 5 mM) in the form of potassium dichromate (K2Cr2O7). Time-course measurements of aqueous Cr6+ concentrations with the 1, 5-diphenylcarbazide colorimetric method showed complete reduction of the 0.2 and 0.4 mM Cr6+ solutions by this methanogen. However, much lower reduction extents of 43.6%, 13.0%, and 3.7% were observed at higher Cr6+ concentrations of 1, 3 and 5 mM, respectively. These lower extents of bioreduction suggest a toxic effect of aqueous Cr6+ to cells at this concentration range. At these higher Cr6+ concentrations, methanogenesis was inhibited and cell growth was impaired as evidenced by decreased total cellular protein production and live/dead cell ratio. Likewise, Cr6+ bioreduction rates decreased with increased initial concentrations of Cr6+ from 13.3 to1.9 µM h-1. X-ray absorption near-edge structure (XANES) spectroscopy revealed a progressive reduction of soluble Cr6+ to insoluble Cr3+ precipitates, which was confirmed as amorphous chromium hydroxide by X-ray diffraction and selected area electron diffraction pattern. However, a small fraction of reduced Cr occurred as aqueous Cr3+. Scanning and transmission electron microscope observations of M. thermautotrophicus cells after Cr6+ exposure suggest both extra- and intracellular chromium reduction mechanisms. Results of this study demonstrate the ability of M. thermautotrophicus cells to reduce toxic Cr6+ to less toxic Cr3+ and its potential application in metal bioremediation, especially at high temperature subsurface radioactive waste disposal sites, where the temperature may reach ∼70°C.

Isolation of diverse members of the Aquificales from geothermal springs in Tengchong, China.

The order Aquificales (phylum Aquificae) consists of thermophilic and hyperthermophilic bacteria that are prominent in many geothermal systems, including those in Tengchong, Yunnan Province, China. However, Aquificales have not previously been isolated from Tengchong. We isolated five strains of Aquificales from diverse springs (temperature 45.2-83.3°C and pH 2.6-9.1) in the Rehai Geothermal Field from sites in which Aquificales were abundant. Phylogenetic analysis showed that four of the strains belong to the genera Hydrogenobacter, Hydrogenobaculum, and Sulfurihydrogenibium, including strains distant enough to likely justify new species of Hydrogenobacter and Hydrogenobaculum. The additional strain may represent a new genus in the Hydrogenothermaceae. All strains were capable of aerobic respiration under microaerophilic conditions; however, they had variable capacity for chemolithotrophic oxidation of hydrogen and sulfur compounds and nitrate reduction.

Greater temporal changes of sediment microbial community than its waterborne counterpart in Tengchong hot springs, Yunnan Province, China.

Temporal variation in geochemistry can cause changes in microbial community structure and diversity. Here we studied temporal changes of microbial communities in Tengchong hot springs of Yunnan Province, China in response to geochemical variations by using microbial and geochemical data collected in January, June and August of 2011. Greater temporal variations were observed in individual taxa than at the whole community structure level. Water and sediment communities exhibited different temporal variation patterns. Water communities were largely stable across three sampling times and dominated by similar microbial lineages: Hydrogenobaculum in moderate-temperature acidic springs, Sulfolobus in high-temperature acidic springs, and Hydrogenobacter in high-temperature circumneutral to alkaline springs. Sediment communities were more diverse and responsive to changing physicochemical conditions. Most of the sediment communities in January and June were similar to those in waters. However, the August sediment community was more diverse and contained more anaerobic heterotrophs than the January and June: Desulfurella and Acidicaldus in moderate-temperature acidic springs, Ignisphaera and Desulfurococcus in high-temperature acidic springs, the candidate division OP1 and Fervidobacterium in alkaline springs, and Thermus and GAL35 in neutral springs. Temporal variations in physicochemical parameters including temperature, pH, and dissolved organic carbon may have triggered the observed microbial community shifts.

Taxonomic and functional diversity provides insight into microbial pathways and stress responses in the saline Qinghai Lake, China.

Microbe-mediated biogeochemical cycles contribute to the global climate system and have sensitive responses and feedbacks to environmental stress caused by climate change. Yet, little is known about the effects of microbial biodiversity (i.e., taxonmic and functional diversity) on biogeochemical cycles in ecosytems that are highly sensitive to climate change. One such sensitive ecosystem is Qinghai Lake, a high-elevation (3196 m) saline (1.4%) lake located on the Tibetan Plateau, China. This study provides baseline information on the microbial taxonomic and functional diversity as well as the associated stress response genes. Illumina metagenomic and metatranscriptomic datasets were generated from lake water samples collected at two sites (B and E). Autotrophic Cyanobacteria dominated the DNA samples, while heterotrophic Proteobacteria dominated the RNA samples at both sites. Photoheterotrophic Loktanella was also present at both sites. Photosystem II was the most active pathway at site B; while, oxidative phosphorylation was most active at site E. Organisms that expressed photosystem II or oxidative phosphorylation also expressed genes involved in photoprotection and oxidative stress, respectively. Assimilatory pathways associated with the nitrogen cycle were dominant at both sites. Results also indicate a positive relationship between functional diversity and the number of stress response genes. This study provides insight into the stress resilience of microbial metabolic pathways supported by greater taxonomic diversity, which may affect the microbial community response to climate change.

Seasonal patterns in microbial communities inhabiting the hot springs of Tengchong, Yunnan Province, China.

Studies focusing on seasonal dynamics of microbial communities in terrestrial and marine environments are common; however, little is known about seasonal dynamics in high-temperature environments. Thus, our objective was to document the seasonal dynamics of both the physicochemical conditions and the microbial communities inhabiting hot springs in Tengchong County, Yunnan Province, China. The PhyloChip microarray detected 4882 operational taxonomic units (OTUs) within 79 bacterial phylum-level groups and 113 OTUs within 20 archaeal phylum-level groups, which are additional 54 bacterial phyla and 11 archaeal phyla to those that were previously described using pyrosequencing. Monsoon samples (June 2011) showed increased concentrations of potassium, total organic carbon, ammonium, calcium, sodium and total nitrogen, and decreased ferrous iron relative to the dry season (January 2011). At the same time, the highly ordered microbial communities present in January gave way to poorly ordered communities in June, characterized by higher richness of Bacteria, including microbes related to mesophiles. These seasonal changes in geochemistry and community structure are likely due to high rainfall influx during the monsoon season and indicate that seasonal dynamics occurs in high-temperature environments experiencing significant changes in seasonal recharge. Thus, geothermal environments are not isolated from the surrounding environment and seasonality affects microbial ecology.

Archaeal and bacterial diversity in acidic to circumneutral hot springs in the Philippines.

The microbial diversity was investigated in sediments of six acidic to circumneutral hot springs (Temperature: 60-92 °C, pH 3.72-6.58) in the Philippines using an integrated approach that included geochemistry and 16S rRNA gene pyrosequencing. Both bacterial and archaeal abundances were lower in high-temperature springs than in moderate-temperature ones. Overall, the archaeal community consisted of sequence reads that exhibited a high similarity (nucleotide identity > 92%) to phyla Crenarchaeota, Euryarchaeota, and unclassified Archaea. The bacterial community was composed of sequence reads moderately related (nucleotide identity > 90%) to 17 phyla, with Aquificae and Firmicutes being dominant. These phylogenetic groups were correlated with environmental conditions such as temperature, dissolved sulfate and calcium concentrations in spring water, and sediment properties including total nitrogen, pyrite, and elemental sulfur. Based on the phylogenetic inference, sulfur metabolisms appear to be key physiological functions in these hot springs. Sulfobacillus (within phylum Firmicutes) along with members within Sulfolobales were abundant in two high-temperature springs (> 76 °C), and they were hypothesized to play an important role in regulating the sulfur cycling under high-temperature conditions. The results of this study improve our understanding of microbial diversity and community composition in acidic to circumneutral terrestrial hot springs and their relationships with geochemical conditions.

An anion-exchange method to concentrate dissolved DNA from aquifer water.

A rapid DNA isolation method was developed to concentrate dissolved DNA (dDNA) in aquifer water for molecular analysis. The aquifer dDNA from the Eastern Snake River Plain Aquifer (ESRPA) was extracted and concentrated using a new method with an anion-exchange Mustang® Q membrane. The concentration of aquifer dDNA in this study ranged from 60 to 264.5 ng l−1 in ESRPA aquifer wells. DNA stability in ESRPA aquifer water was also tested in this study. The dDNA extracted from aquifer water samples was used for PCR amplification of bacterial 16S rRNA genes for terminal restriction fragment length polymorphism (T-RFLP) analysis and construction of 16S rRNA gene clone libraries. The ureC gene, IncP, IncQ and IncW plasmid genes were also PCR amplified from dDNA samples. Based on the results, dDNA is relatively stable in aquifer water and can be concentrated by Q membrane method for molecular analysis. The quality of isolated dDNA was suitable as a PCR template.

A comprehensive census of microbial diversity in hot springs of Tengchong, Yunnan Province China using 16S rRNA gene pyrosequencing.

The Rehai and Ruidian geothermal fields, located in Tengchong County, Yunnan Province, China, host a variety of geochemically distinct hot springs. In this study, we report a comprehensive, cultivation-independent census of microbial communities in 37 samples collected from these geothermal fields, encompassing sites ranging in temperature from 55.1 to 93.6°C, in pH from 2.5 to 9.4, and in mineralogy from silicates in Rehai to carbonates in Ruidian. Richness was low in all samples, with 21-123 species-level OTUs detected. The bacterial phylum Aquificae or archaeal phylum Crenarchaeota were dominant in Rehai samples, yet the dominant taxa within those phyla depended on temperature, pH, and geochemistry. Rehai springs with low pH (2.5-2.6), high temperature (85.1-89.1°C), and high sulfur contents favored the crenarchaeal order Sulfolobales, whereas those with low pH (2.6-4.8) and cooler temperature (55.1-64.5°C) favored the Aquificae genus Hydrogenobaculum. Rehai springs with neutral-alkaline pH (7.2-9.4) and high temperature (>80°C) with high concentrations of silica and salt ions (Na, K, and Cl) favored the Aquificae genus Hydrogenobacter and crenarchaeal orders Desulfurococcales and Thermoproteales. Desulfurococcales and Thermoproteales became predominant in springs with pH much higher than the optimum and even the maximum pH known for these orders. Ruidian water samples harbored a single Aquificae genus Hydrogenobacter, whereas microbial communities in Ruidian sediment samples were more diverse at the phylum level and distinctly different from those in Rehai and Ruidian water samples, with a higher abundance of uncultivated lineages, close relatives of the ammonia-oxidizing archaeon "Candidatus Nitrosocaldus yellowstonii", and candidate division O1aA90 and OP1. These differences between Ruidian sediments and Rehai samples were likely caused by temperature, pH, and sediment mineralogy. The results of this study significantly expand the current understanding of the microbiology in Tengchong hot springs and provide a basis for comparison with other geothermal systems around the world.

Bacterial dominance in subseafloor sediments characterized by methane hydrates.

The degradation of organic carbon in subseafloor sediments on continental margins contributes to the largest reservoir of methane on Earth. Sediments in the Andaman Sea are composed of ~ 1% marine-derived organic carbon and biogenic methane is present. Our objective was to determine microbial abundance and diversity in sediments that transition the gas hydrate occurrence zone (GHOZ) in the Andaman Sea. Microscopic cell enumeration revealed that most sediment layers harbored relatively low microbial abundance (10(3)-10(5) cells cm(-3)). Archaea were never detected despite the use of both DNA- and lipid-based methods. Statistical analysis of terminal restriction fragment length polymorphisms revealed distinct microbial communities from above, within, and below the GHOZ, and GHOZ samples were correlated with a decrease in organic carbon. Primer-tagged pyrosequences of bacterial 16S rRNA genes showed that members of the phylum Firmicutes are predominant in all zones. Compared with other seafloor settings that contain biogenic methane, this deep subseafloor habitat has a unique microbial community and the low cell abundance detected can help to refine global subseafloor microbial abundance.