Penny L. Morrill

Bacterial Communities Associated with Subsurface Geochemical Processes in Continental Serpentinite Springs

ABSTRACT Reactions associated with the geochemical process of serpentinization can generate copious quantities of hydrogen and low-molecular-weight organic carbon compounds, which may provide energy and nutrients to sustain subsurface microbial communities independently of the photosynthetically supported surface biosphere. Previous microbial ecology studies have tested this hypothesis in deep sea hydrothermal vents, such as the Lost City hydrothermal field. This study applied similar methods, including molecular fingerprinting and tag sequencing of the 16S rRNA gene, to ultrabasic continental springs emanating from serpentinizing ultramafic rocks. These molecular surveys were linked with geochemical measurements of the fluids in an interdisciplinary approach designed to distinguish potential subsurface organisms from those derived from surface habitats. The betaproteobacterial genus Hydrogenophaga was identified as a likely inhabitant of transition zones where hydrogen-enriched subsurface fluids mix with oxygenated surface water. The Firmicutes genus Erysipelothrix was most strongly correlated with geochemical factors indicative of subsurface fluids and was identified as the most likely inhabitant of a serpentinization-powered subsurface biosphere. Both of these taxa have been identified in multiple hydrogen-enriched subsurface habitats worldwide, and the results of this study contribute to an emerging biogeographic pattern in which Betaproteobacteria occur in near-surface mixing zones and Firmicutes are present in deeper, anoxic subsurface habitats.

Investigations of potential microbial methanogenic and carbon monoxide utilization pathways in ultra-basic reducing springs associated with present-day continental serpentinization: the Tablelands, NL, CAN.

Ultra-basic reducing springs at continental sites of serpentinization act as portals into the biogeochemistry of a subsurface environment with H2 and CH4 present. Very little, however, is known about the carbon substrate utilization, energy sources, and metabolic pathways of the microorganisms that live in this ultra-basic environment. The potential for microbial methanogenesis with bicarbonate, formate, acetate, and propionate precursors and carbon monoxide (CO) utilization pathways were tested in laboratory experiments by adding substrates to water and sediment from the Tablelands, NL, CAD, a site of present-day continental serpentinization. Microbial methanogenesis was not observed after bicarbonate, formate, acetate, or propionate addition. CO was consumed in the live experiments but not in the killed controls and the residual CO in the live experiments became enriched in 13C. The average isotopic enrichment factor resulting from this microbial utilization of CO was estimated to be 11.2 ± 0.2‰. Phospholipid fatty acid concentrations and δ13C values suggest limited incorporation of carbon from CO into microbial lipids. This indicates that in our experiments, CO was used primarily as an energy source, but not for biomass growth. Environmental DNA sequencing of spring fluids collected at the same time as the addition experiments yielded a large proportion of Hydrogenophaga-related sequences, which is consistent with previous metagenomic data indicating the potential for these taxa to utilize CO.

Exploring the metabolic potential of microbial communities in ultra‐basic, reducing springs at The Cedars, CA, USA: Experimental evidence of microbial methanogenesis and heterotrophic acetogenesis

Present-day serpentinization generates groundwaters with conditions (pH > 11, Eh < −550 mV) favorable for the microbial and abiotic production of organic compounds from inorganic precursors. Elevated concentrations of methane, C2-C6 alkanes, acetate, and formate have been detected at these sites, but the microbial or abiotic origin of these compounds remains unclear. While geochemical data indicate that methane at most sites of present-day serpentinization is abiogenic, the stable carbon, hydrogen, and clumped isotope data as well as the hydrocarbon gas composition from The Cedars, CA, USA, are consistent with a microbial origin for methane. However, there is no direct evidence of methanogenesis at this site of serpentinization. We report on laboratory experiments in which the microbial communities in fluids and sediments from The Cedars were incubated with 13C labeled substrates. Increasing methane concentrations and the incorporation of 13C into methane in live experiments, but not in killed controls, demonstrated that methanogens converted methanol, formate, acetate (methyl group), and bicarbonate to methane. The apparent fractionation between methane and potential substrates (α13CCH4-CO2(g) = 1.059 to 1.105, α13CCH4-acetate = 1.042 to 1.119) indicated that methanogenesis was dominated by the carbonate reduction pathway. Increasing concentrations of volatile organic acid anions indicated microbial acetogenesis. α13CCO2(g)-acetate values (0.999 to 1.000), however, were inconsistent with autotrophic acetogenesis, thus suggesting that acetate was produced through fermentation. This is the first study to show direct evidence of microbial methanogenesis and acetogenesis by the native microbial community at a site of present-day serpentinization.

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.

Phospholipid fatty acid (PLFA) analysis for profiling microbial communities in offshore produced water

A method based on phospholipid fatty acid (PLFA) analysis for profiling microbial communities in offshore produced water was optimized. The operation parameters affecting final PLFA profiling performance from the solid phase extraction (SPE) purification and fatty acid methyl esters (FAMEs) yielding process were investigated. Under the selected conditions, 92.9%, 96.3% and 92.8% of the spiked phospholipid standards C16:1 (cis-9) PC, C18:1 (cis-9) PC, and C19:0 PC were recovered, respectively, using 10mL methanol as elution solvent on a non-commercial SPE column. Over 90% of spiked C19:0 PC was recovered before sample transesterification. Four parameters including alkaline reagent, volume of acid for neutralization, time and temperature for FAMEs derivatization were examined. Gas Chromatography-Mass Spectrometry (GC-MS) was used to analyze FAMEs and the method linearities, recoveries of 29 FAMEs during transesterification, detection limits, relative standard deviations were presented. The results provided valuable information for biological reservoir souring control.

The Potential Impact of Hydrocarbons on Mussels in Port au Port Bay, Newfoundland

Since 2012, the scallop fishery in Port au Port Bay, Newfoundland, Canada has experienced a drastic decline, while no decline was observed in adjacent St. Georges Bay. Local fishermen have raised concerns about an abandoned oil exploration well in the Port au Port Bay. This study investigated the potential impact of petroleum hydrocarbons on sediments and blue mussels [Mytilus edulis] (a proxy organism for scallops) in the area. Sediments from both bays were characterized for their hydrocarbons and compared to potential petroleum hydrocarbon sources. Mussels were analysed for health indices and their 14C content. The results showed that the concentration of hydrocarbons found in the sediments of the fishing ground was within the range of unpolluted marine sediments and that the hydrocarbons present were likely from a mixture of sources. The health indices of the mussels in Port au Port Bay were similar to the health indices of mussels in St. Georges Bay and the 14C content of the mussels from both bays was modern. These data suggest that the Port au Port fishing ground was not solely contaminated from crude oil leaking from an oil exploration well, that the mussels were not contaminated with petroleum hydrocarbons, and that Port au Port mussels were just as healthy as the mussels of St. Georges Bay. Therefore, whatever caused the scallop decline was most likely scallop- and bay-specific. During this study a fast and efficient method for extracting petroleum hydrocarbons from sediment using accelerated solvent extraction with integrated silica gel was developed.