Microbial Nitrogen cycling in Nevada Geothermal Springs

Hot spring habitats above maximum photosynthetic temperature (73 ºC) are not well understood with respect to nitrogen (N) cycling. Few predictions have been made, and even fewer measurements of in situ activities have been reported. Thermodynamic calculations based on in situ chemical and temperature measurements will be used to predict the occurrence of the specific N-cycling reactions. In addition, these measurements in two springs will aid in an attempt to cultivate ammonia oxidizing species

Exploring diversity of Nitrate reducing thermophiles in Nevada hot springs

High rates of denitrification have been measured in Nevada geothermal hot springs, but little is known about the thermophiles that contribute to this activity. We hypothesize that heterotrophic bacteria in the genus Thermus are the most important denitrifiers in the springs. Alternatively, other microorganisms including chemolithotrophs may also be important. To test these hypotheses, several different strategies will be used to try to enrich and isolate nitrate-reducing microorganisms. Isolates will be identified by 16S rRNA gene PCR and sequencing. Subsequently, representative isolates will be chosen for nitrate reductase gene (narG) sequencing and for studies on the kinetics of nitrate reduction at high temperature. These data will provide information on how these microorganisms may behave in situ and how their activities may affect nitrogen cycling in the hot springs

Researching nitrite oxidation at high temperatures

15N-nitrate (NO3 -) pool dilution experiments show that ammonia (NH3) is oxidized to nitrate in geothermal springs up to at least 85C; however, nitrite (NO2 -)- oxidizing microorganisms are only known to grow up to 66°C. We hypothesize that thermophilic microorganisms oxidize nitrite to nitrate at high temperatures. Alternatively, it is possible that nitrite is oxidized abiotically. We propose to test these hypotheses by setting up microbial enrichments designed to grow thermophilic nitrite oxidizing bacteria by varying incubation temperature (50, 65, 80°C), oxygen concentration (20% and 5%), and cultivation media. A negative control consisting of filtered spring water (0.1 μm) will be used to determine whether nitrite is oxidized abiotically. Enrichments will be monitored for nitrite oxidation activity by using colorimetric assays for nitrite and nitrate. Enrichments showing activity will be used as a source to try to isolate and/or identify responsible microorganisms and to study the kinetics of nitrite oxidation at high temperature

Genomic foundations of carbon fixation in bacteria living in hot springs

Photosynthesis does not occur above 73°C, so organisms living above this temperature must obtain useable carbon by some other mechanism. It is generally assumed that carbon is fixed by thermophiles through the process of chemolithoautotrophy; however, primary production has never been demonstrated to occur in hot springs \u3e73°C. We have shown that two organisms, Thermocrinis and Pyrobaculum, make up more than 90% of the cells in an 80°C Great Basin hot spring, Great Boiling Spring. We hypothesize that these organisms fix carbon in the hot spring via the reverse tricarboxylic acid (rTCA) cycle. To test this hypothesis we will: i) confirm that Thermocrinis and Pyrobaculum dominate in water from the spring; ii) determine whether key genes for the rTCA cycle, citryl co-A lyase (ccl), 2-oxoglutarate:ferredoxin oxidoreductase (korA), pyruvate:ferredoxin oxidoreductase (porA), are present and expressed in the spring; and iii) measure rates of carbon fixation in the spring. Linkage of the genetic data with carbon fixation rate data may help to provide an image of carbon fixation and cycling in Great Basin hot springs

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, andSulfurihydrogenibium, including strains distant enough to likely justify new species ofHydrogenobacter and Hydrogenobaculum. The additional strain may represent a new genus in theHydrogenothermaceae. 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

Novel thermophilic cellulolytic isolates belonging to the phylum Chloroflexi

Current biofuel technologies utilize valuable foodstuffs, such as corn kernels and cane sugar, as sources of easily metabolized sugars. Microbes are used to ferment these sugars into bioethanol, a first-generation biofuel. However, in order to avoid diverting foodstuffs from the food supply, the development of second-generation biofuels technology is necessary. Second-generation biofuels are produced by converting structurally complex lignocellulosic biomass, such as agricultural and municipal wastes, to fermentable sugars or directly to biofuels. The major technological hurdle limiting the mass production of second-generation biofuels is the difficulty in efficiently converting structurally complex lignocellulosic materials to fermentable sugars or directly to biofuels. The discovery of novel thermophilic microorganisms and enzymes that have high activities or broad substrate ranges on plant polymers addresses this challenge

A Spatial and temporal analysis of microbial communities in Great Boiling Spring, Nevada, U.S.A.

Great Boiling Spring (GBS) is a large, circumneutral, long residence time geothermal spring in the US Great Basin. Twelve samples were taken from four different sediment sites and the planktonic community in the bulk water of GBS on up to four different dates. Microbial community composition and diversity was assessed by using a barcoded, improved universal primer set targeting the V8 portion of the 16S rRNA gene and PCR. Over 200,000 products were sequenced using the Roche 454 GS FLX Titanium System. Sediment and planktonic microbial communities were distinct with very little overlap, regardless of the sampling location or temperature. Planktonic communities were extremely uneven and were dominated by a single phylotype related to Thermocrinis in the Aquificales. Benthic microbial communities grouped according to temperature and sampling location. Two locations, Site A (80-87°C) and Site B (79°C), were predominantly composed of the crenarchaeal class Thermoprotei, the novel archaeal lineage pSL4, and the novel bacterial lineage GAL35. Populations of the ammonia oxidizing archaeon “Candidatus Nitrosocaldus yellowstonii” comprised 5-15% of all samples when Site A was cooler than normal (80°C) and at cooler sites throughout the spring (76-62°C). At cooler temperature sites (76-62°C), the phylum-level diversity and evenness were significantly higher, and bacteria made up a significantly higher percentage of the population. To our knowledge, this is the most detailed study of the spatial and temporal variation in any geothermal spring. The study underscores the distinctness of planktonic and benthic communities and the importance of temperature in driving the spatial variation of microbial phylotypes throughout the mineralogically homogenous source pool. 8

Isolation, characterization, and genome sequence of the first representative of a novel class within the Chloroflexi that is abundant in some U.S. Great Basin hot springs and may play important roles in N and C cycling

A thermophilic, facultatively microaerophilic, heterotrophic bacterium, designated strain JAD2, was isolated from sediments of Great Boiling Spring (GBS), an ~80oC, circumneutral hot spring in the Great Basin GB). The strain grew anaerobically on yeast extract or peptone with an optimal growth temperature of 70-75oC. Growth was stimulated by addition of 0.01 atm O2 to the culture vessel headspace, but was inhibited by higher concentrations (0.2 atm). Cells of JAD2 formed non-motile filaments ranging from 10 to \u3e300 μm in length, which typically decreased in length during stationary phase. 16S rRNA gene-targeted pyrotag sequencing and clone library data suggest that close relatives of this isolate are prominent members of the sediment communities in GBS. Shotgun sequencing of the JAD2 genome produced an assembly consisting of ~3.2 Mbp with an average G+C content of 67.3%. Phylogenies inferred from the 16S rRNA gene and predicted amino acid sequences of various conserved proteins indicate that JAD2 is the first cultivated representative of the GAL35 group, a new class within the Chloroflexi. Predicted genes in the draft genome encoding a putative carbon monoxide dehydrogenase (coxMSL), nitrite reductase (nrfHA) and nitrous oxide reductase (nosZ) suggest that this isolate may play important roles in N and C cycling in GBS sediments

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.This work was supported by NASA exobiology grant EXO-NNX11AR78G, U.S. National Science Foundation grant OISE 0968421, and U.S. Department of Energy grant DE-EE-0000716. B.P.H. acknowledges generous support from Greg Fullmer through the UNLV Foundation, and W.S. acknowledges Northern Illinois University for funding. B.P.H and S.K.M. acknowledge support from an Amazon Web Services Education Research Grant award. The work conducted by the U.S. Department of Energy Joint Genome Institute, a DOE Office of Science User Facility, is supported by the Office of Science of the U.S. Department of Energy under contract no. DE-AC02-05CH11231. This article is made openly accessible in part by an award from the Northern Illinois University Libraries’ Open Access Publishing Fund