Julie D. Kirshtein

Novel Bacterial and Archaeal Lineages from an In Situ Growth Chamber Deployed at a Mid-Atlantic Ridge Hydrothermal Vent

ABSTRACT The phylogenetic diversity was determined for a microbial community obtained from an in situ growth chamber placed on a deep-sea hydrothermal vent on the Mid-Atlantic Ridge (23°22′ N, 44°57′ W). The chamber was deployed for 5 days, and the temperature within the chamber gradually decreased from 70 to 20°C. Upon retrieval of the chamber, the DNA was extracted and the small-subunit rRNA genes (16S rDNA) were amplified by PCR using primers specific for theArchaea or Bacteria domain and cloned. Unique rDNA sequences were identified by restriction fragment length polymorphisms, and 38 different archaeal and bacterial phylotypes were identified from the 85 clones screened. The majority of the archaeal sequences were affiliated with the Thermococcales (71%) and Archaeoglobales (22%) orders. A sequence belonging to the Thermoplasmales confirms that thermoacidophiles may have escaped enrichment culturing attempts of deep-sea hydrothermal vent samples. Additional sequences that represented deeply rooted lineages in the low-temperature eurarchaeal (marine group II) and crenarchaeal clades were obtained. The majority of the bacterial sequences obtained were restricted to the Aquificales(18%), the ɛ subclass of the Proteobacteria(ɛ-Proteobacteria) (40%), and the genusDesulfurobacterium (25%). Most of the clones (28%) were confined to a monophyletic clade within the ɛ-Proteobacteria with no known close relatives. The prevalence of clones related to thermophilic microbes that use hydrogen as an electron donor and sulfur compounds (S0, SO4, thiosulfate) indicates the importance of hydrogen oxidation and sulfur metabolism at deep-sea hydrothermal vents. The presence of sequences that are related to sequences from hyperthermophiles, moderate thermophiles, and mesophiles suggests that the diversity obtained from this analysis may reflect the microbial succession that occurred in response to the shift in temperature and possible associated changes in the chemistry of the hydrothermal fluid.

A ubiquitous thermoacidophilic archaeon from deep-sea hydrothermal vents

Deep-sea hydrothermal vents are important in global biogeochemical cycles, providing biological oases at the sea floor that are supported by the thermal and chemical flux from the Earths interior. As hot, acidic and reduced hydrothermal fluids mix with cold, alkaline and oxygenated sea water, minerals precipitate to form porous sulphide–sulphate deposits. These structures provide microhabitats for a diversity of prokaryotes that exploit the geochemical and physical gradients in this dynamic ecosystem. It has been proposed that fluid pH in the actively venting sulphide structures is generally low (pH < 4.5), yet no extreme thermoacidophile has been isolated from vent deposits. Culture-independent surveys based on ribosomal RNA genes from deep-sea hydrothermal deposits have identified a widespread euryarchaeotal lineage, DHVE2 (deep-sea hydrothermal vent euryarchaeotic 2). Despite the ubiquity and apparent deep-sea endemism of DHVE2, cultivation of this group has been unsuccessful and thus its metabolism remains a mystery. Here we report the isolation and cultivation of a member of the DHVE2 group, which is an obligate thermoacidophilic sulphur- or iron-reducing heterotroph capable of growing from pH 3.3 to 5.8 and between 55 and 75 °C. In addition, we demonstrate that this isolate constitutes up to 15% of the archaeal population, providing evidence that thermoacidophiles may be key players in the sulphur and iron cycling at deep-sea vents.

Evidence for iron‐mediated anaerobic methane oxidation in a crude oil‐contaminated aquifer

In a methanogenic crude oil contaminated aquifer near Bemidji, Minnesota, the decrease in dissolved CH(4) concentrations along the groundwater flow path, along with the positive shift in δ(13) C(CH) (4) and negative shift in δ(13) C(DIC) , is indicative of microbially mediated CH(4) oxidation. Calculations of electron acceptor transport across the water table, through diffusion, recharge, and the entrapment and release of gas bubbles, suggest that these processes can account for at most 15% of the observed total reduced carbon oxidation, including CH(4) . In the anaerobic plume, the characteristic Fe(III)-reducing genus Geobacter was the most abundant of the microbial groups tested, and depletion of labile sediment iron is observed over time, confirming that reduced carbon oxidation coupled to iron reduction is an important process. Electron mass balance calculations suggest that organic carbon sources in the aquifer, BTEX and non-volatile dissolved organic carbon, are insufficient to account for the loss in sediment Fe(III), implying that CH(4) oxidation may also be related to Fe(III) reduction. The results support a hypothesis of Fe(III)-mediated CH(4) oxidation in the contaminated aquifer.

Heterogeneous Occupancy and Density Estimates of the Pathogenic Fungus Batrachochytrium dendrobatidis in Waters of North America

Biodiversity losses are occurring worldwide due to a combination of stressors. For example, by one estimate, 40% of amphibian species are vulnerable to extinction, and disease is one threat to amphibian populations. The emerging infectious disease chytridiomycosis, caused by the aquatic fungus Batrachochytrium dendrobatidis (Bd), is a contributor to amphibian declines worldwide. Bd research has focused on the dynamics of the pathogen in its amphibian hosts, with little emphasis on investigating the dynamics of free-living Bd. Therefore, we investigated patterns of Bd occupancy and density in amphibian habitats using occupancy models, powerful tools for estimating site occupancy and detection probability. Occupancy models have been used to investigate diseases where the focus was on pathogen occurrence in the host. We applied occupancy models to investigate free-living Bd in North American surface waters to determine Bd seasonality, relationships between Bd site occupancy and habitat attributes, and probability of detection from water samples as a function of the number of samples, sample volume, and water quality. We also report on the temporal patterns of Bd density from a 4-year case study of a Bd-positive wetland. We provide evidence that Bd occurs in the environment year-round. Bd exhibited temporal and spatial heterogeneity in density, but did not exhibit seasonality in occupancy. Bd was detected in all months, typically at less than 100 zoospores L−1. The highest density observed was ∼3 million zoospores L−1. We detected Bd in 47% of sites sampled, but estimated that Bd occupied 61% of sites, highlighting the importance of accounting for imperfect detection. When Bd was present, there was a 95% chance of detecting it with four samples of 600 ml of water or five samples of 60 mL. Our findings provide important baseline information to advance the study of Bd disease ecology, and advance our understanding of amphibian exposure to free-living Bd in aquatic habitats over time.

Characterization of a microbial consortium capable of rapid and simultaneous dechlorination of 1,1,2,2-tetrachloroethane and chlorinated ethane and ethene intermediates

ABSTRACT Mixed cultures capable of dechlorinating chlorinated ethanes and ethenes were enriched from contaminated wetland sediment at Aberdeen Proving Ground (APG) Maryland. The “West Branch Consortium” (WBC-2) was capable of degrading 1,1,2,2-tetrachloroethane (TeCA), trichloroethene (TCE), cis and trans 1,2-dichloroethene (DCE), 1,1,2-trichloroethane (TCA), 1,2-dichloroethane, and vinyl chloride to nonchlorinated end products ethene and ethane. WBC-2 dechlorinated TeCA, TCA, and cisDCE rapidly and simultaneously. A Clostridium sp. phylogenetically closely related to an uncultured member of a TCE-degrading consortium was numerically dominant in the WBC-2 clone library after 11 months of enrichment in culture. Clostridiales, including Acetobacteria, comprised 65% of the bacterial clones in WBC-2, with Bacteroides (14%), and epsilon Proteobacteria (14%) also numerically important. Methanogens identified in the consortium were members of the class Methanomicrobia, which includes acetoclastic methanogens. Dehalococcoides did not become dominant in the culture, although it was present at about 1% in the microbial population. The WBC-2 consortium provides opportunities for the in situ bioremediation of sites contaminated with mixtures of chlorinated ethenes and ethanes.

Arsenic speciation in food chains from mid-Atlantic hydrothermal vents

Arsenic concentration and speciation were determined in benthic fauna collected from the Mid-Atlantic Ridge hydrothermal vents. The shrimp species, Rimicaris exoculata, the vent chimney-dwelling mussel, Bathymodiolus azoricus, Branchipolynoe seepensis, a commensal worm of B. azoricus, and the gastropod Peltospira smaragdina showed variations in As concentration and in stable isotope (δ13C and δ15N) signature between species, suggesting different sources of As uptake. Arsenic speciation showed arsenobetaine to be the dominant species in R. exoculata, whereas in B. azoricus and B. seepensis arsenosugars were most abundant, although arsenobetaine, dimethylarsinate, and inorganic arsenic were also observed, along with several unidentified species. Scrape samples from outside the vent chimneys, covered with microbial mat, which is a presumed food source for many vent organisms, contained high levels of total As, but organic species were not detectable. The formation of arsenosugars in pelagic environments is typically attributed to marine algae, and the pathway to arsenobetaine is still unknown. The occurrence of arsenosugars and arsenobetaine in these deep sea organisms, where primary production is chemolithoautotrophic and stable isotope analyses indicate food sources are of vent origin, suggests that organic arsenicals can occur in a food web without algae or other photosynthetic life.

Methane oxidation in a crude oil contaminated aquifer: Delineation of aerobic reactions at the plume fringes

High resolution direct-push profiling over short vertical distances was used to investigate CH(4) attenuation in a petroleum contaminated aquifer near Bemidji, Minnesota. The contaminant plume was delineated using dissolved gases, redox sensitive components, major ions, carbon isotope ratios in CH(4) and CO(2), and the presence of methanotrophic bacteria. Sharp redox gradients were observed near the water table. Shifts in δ(13)C(CH4) from an average of -57.6‰ (±1.7‰) in the methanogenic zone to -39.6‰ (±8.7‰) at 105m downgradient, strongly suggest CH(4) attenuation through microbially mediated degradation. In the downgradient zone the aerobic/anaerobic transition is up to 0.5m below the water table suggesting that transport of O(2) across the water table is leading to aerobic degradation of CH(4) at this interface. Dissolved N(2) concentrations that exceeded those expected for water in equilibrium with the atmosphere indicated bubble entrapment followed by preferential stripping of O(2) through aerobic degradation of CH(4) or other hydrocarbons. Multivariate and cluster analysis were used to distinguish between areas of significant bubble entrapment and areas where other processes such as the infiltration of O(2) rich recharge water were important O(2) transport mechanisms.

A Biogeochemical and Genetic Survey of Acetylene Fermentation by Environmental Samples and Bacterial Isolates

Anoxic samples (sediment and groundwater) from 13 chemically diverse field sites were assayed for their ability to consume acetylene (C2H2). Over incubation periods ranging from ∼ 10 to 80 days, selected samples from 7 of the 13 tested sites displayed significant C2H2 removal. No significant formation of ethylene was noted in these incubations; therefore, C2H2 consumption could be attributed to acetylene hydratase (AH) rather than nitrogenase activity. This putative AH (PAH) activity was observed in only 21% of the total of assayed samples, while amplification of AH genes from extracted DNA using degenerate primers derived from Pelobacter acetylenicus occurred in even fewer (9.8%) samples. Acetylene-fermenting bacteria were isolated as a pure culture from the sediments of a tidal mudflat in San Francisco Bay (SFB93) and as an enrichment culture from freshwater Searsville Lake (SV7). Comparison of 16S rDNA clone libraries revealed that SFB93 was closely related to P. carbolinicus, while SV7 consisted of several unrelated bacteria. AH gene was amplified from SFB93 but not SV7. The inability of the primers to generate amplicons in the SV7 enrichment, as well as from several of the environmental samples that displayed PAH activity, implied that either the primers were too highly constrained in their specificity or that there was a different type of AH gene in these environmental samples than occurs in P. acetylenicus. The significance of this work with regard to the search for life in the outer Solar System, where C2H2 is abundant, is discussed.

Integration of stable carbon isotope, microbial community, dissolved hydrogen gas, and 2HH2O tracer data to assess bioaugmentation for chlorinated ethene degradation in fractured rocks

An in situ bioaugmentation (BA) experiment was conducted to understand processes controlling microbial dechlorination of trichloroethene (TCE) in groundwater at the Naval Air Warfare Center (NAWC), West Trenton, NJ. In the BA experiment, an electron donor (emulsified vegetable oil and sodium lactate) and a chloro-respiring microbial consortium were injected into a well in fractured mudstone of Triassic age. Water enriched in ²H was also injected as a tracer of the BA solution, to monitor advective transport processes. The changes in concentration and the δ¹³C of TCE, cis-dichloroethene (cis-DCE), and vinyl chloride (VC); the δ²H of water; changes in the abundance of the microbial communities; and the concentration of dissolved H₂ gas compared to pre- test conditions, provided multiple lines of evidence that enhanced biodegradation occurred in the injection well and in two downgradient wells. For those wells where the biodegradation was stimulated intensively, the sum of the molar chlorinated ethene (CE) concentrations in post-BA water was higher than that of the sum of the pre-BA background molar CE concentrations. The concentration ratios of TCE/(cis-DCE+VC) indicated that the increase in molar CE concentration may result from additional TCE mobilized from the rock matrix in response to the oil injection or due to desorption/diffusion. The stable carbon isotope mass-balance calculations show that the weighted average ¹³C isotope of the CEs was enriched for around a year compared to the background value in a two year monitoring period, an effective indication that dechlorination of VC was occurring. Insights gained from this study can be applied to efforts to use BA in other fractured rock systems. The study demonstrates that a BA approach can substantially enhance in situ bioremediation not only in fractures connected to the injection well, but also in the rock matrix around the well due to processes such as diffusion and desorption. Because the effect of the BA was intensive only in wells where an amendment was distributed during injection, it is necessary to adequately distribute the amendments throughout the fractured rock to achieve substantial bioremediation. The slowdown in BA effect after a year is due to some extend to the decrease abundant of appropriate microbes, but more likely the decreased concentration of electron donor.

Impact Disruption and Recovery of the Deep Subsurface Biosphere

Although a large fraction of the worlds biomass resides in the subsurface, there has been no study of the effects of catastrophic disturbance on the deep biosphere and the rate of its subsequent recovery. We carried out an investigation of the microbiology of a 1.76 km drill core obtained from the ∼35 million-year-old Chesapeake Bay impact structure, USA, with robust contamination control. Microbial enumerations displayed a logarithmic downward decline, but the different gradient, when compared to previously studied sites, and the scatter of the data are consistent with a microbiota influenced by the geological disturbances caused by the impact. Microbial abundance is low in buried crater-fill, ocean-resurge, and avalanche deposits despite the presence of redox couples for growth. Coupled with the low hydraulic conductivity, the data suggest the microbial community has not yet recovered from the impact ∼35 million years ago. Microbial enumerations, molecular analysis of microbial enrichment cultures, and geochemical analysis showed recolonization of a deep region of impact-fractured rock that was heated to above the upper temperature limit for life at the time of impact. These results show how, by fracturing subsurface rocks, impacts can extend the depth of the biosphere. This phenomenon would have provided deep refugia for life on the more heavily bombarded early Earth, and it shows that the deeply fractured regions of impact craters are promising targets to study the past and present habitability of Mars.