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. George's 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. George's 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. George's 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.

Isolation of nitrate-reducing bacteria from an offshore reservoir and the associated biosurfactant production.

Biosurfactant producing nitrate-reducing bacteria (NRB) in anaerobic reservoir environments are closely associated with souring (H2S) control in the offshore oil and gas industry. Five NRB strains were screened from offshore produced water samples and all were identified as Pseudomonas stutzeri. Their biosurfactant producing abilities when fed on either glucose or glycerol media were investigated. P. stutzeri CX3 reduced the medium surface tension to 33.5 and 29.6 mN m-1, respectively, while growing on glucose or glycerol media. The CX3 strain was further inoculated to examine its growth performance, resulting in 32.4% and 94.5% of nitrate consumption over 228 hours of monitoring in two media, respectively. The composition analysis of the biosurfactant product generated by P. stutzeri CX3 was conducted through thin-layer chromatography, gas chromatography with a flame ionization detector (FID) and Fourier transform infrared spectroscopy (FT-IR). The biosurfactant product was identified as a mixture of a small part of lipopeptides and a large part of glycolipids while its critical micellar concentration (CMC) was as low as 35 mg L-1. The biosurfactant product demonstrated high stability over a wide range of temperature (4-121 °C), pH (2-10), and salinity (0-20% w/v) concentration. The results provided valuable technical and methodological support for effective offshore reservoir souring control and associated enhanced oil recovery activities.

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.

Methane cycling. Nonequilibrium clumped isotope signals in microbial methane.

Methane is a key component in the global carbon cycle, with a wide range of anthropogenic and natural sources. Although isotopic compositions of methane have traditionally aided source identification, the abundance of its multiply substituted "clumped" isotopologues (for example, (13)CH3D) has recently emerged as a proxy for determining methane-formation temperatures. However, the effect of biological processes on methane's clumped isotopologue signature is poorly constrained. We show that methanogenesis proceeding at relatively high rates in cattle, surface environments, and laboratory cultures exerts kinetic control on (13)CH3D abundances and results in anomalously elevated formation-temperature estimates. We demonstrate quantitatively that H2 availability accounts for this effect. Clumped methane thermometry can therefore provide constraints on the generation of methane in diverse settings, including continental serpentinization sites and ancient, deep groundwaters.

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 (13)C. 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 δ(13)C 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.

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'.

Bacterial communities associated with subsurface geochemical processes in continental serpentinite springs.

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.

Evaluation of isotopic enrichment factors for the biodegradation of chlorinated ethenes using a parameter estimation model: toward an improved quantification of biodegradation.

A model was developed to predict the concentrations of chlorinated ethenes and ethene during sequential reductive dechlorination of tetrachloroethene (PCE) from stable carbon isotope values using Rayleigh model principles and specified isotopic enrichment factors for each step of dechlorination. The model was tested using three separate datasets of concentration and isotope values measured during three experiments involving the degradation of PCE to vinyl chloride (VC), trichloroethene (TCE) to ethene, and cis-1,2-dichloroethene (cDCE) to ethene. The model was then coupled to a parameter estimation method to estimate values for the isotopic enrichment factors of TCE, cDCE, and VC when they are intermediates in the dechlorination to ethene. The enrichment factors estimated for TCE and cDCE when they were intermediates in biodegradation experiments were close to or within the published range of enrichment factors determined from experiments where TCE or cDCE were the initial substrates. In contrast, the enrichment factors determined by parameter estimation for experiments in which VC was an intermediate in biodegradation experiments were consistently more negative (by approximately 10 per thousandth) than the most negative published enrichment factor determined from experiments where VC was the initial substrate. This finding suggests that the range of enrichment factors for VC dechlorination may not be as narrow as previously suggested (-21.5 per thousandth to -26.6 per thousandth) and that fractionation during VC dechlorination when VC is an intermediate compound may be significantly larger than when VC is the initial substrate. These findings have important implications both for the current practice of extrapolating laboratory-derived isotopic enrichment factors to quantify biodegradation of chlorinated ethenes in the field and for understanding the details of enzymatic reductive dechlorination.

Biological enhancement of tetrachloroethene dissolution and associated microbial community changes.

A bench-scale study was performed to evaluate the enhancement of tetrachloroethene (PCE) dissolution from a dense nonaqueous phase liquid (DNAPL) source zone due to reductive dechlorination. The study was conducted in a pair of two-dimensional bench-scale aquifer systems using soil and groundwater from Dover Air Force Base, DE. After establishment of PCE source zones in each aquifer system, one was biostimulated (addition of electron donor) while the other was biostimulated and then bioaugmented with the KB1 dechlorinating culture. Biostimulation resulted in the growth of iron-reducing bacteria (Geobacter) in both systems as a result of the high iron content of the Dover soil. After prolonged electron donor addition methanogenesis dominated, but no dechlorination was observed. Following bioaugmentation of one system, dechlorination to ethene was achieved, coincident with growth of introduced Dehalococcoides and other microbes in the vicinity and downgradient of the PCE DNAPL (detected using DGGE and qPCR). Dechlorination was not detected in the nonbioaugmented system over the course of the study, indicating that the native microbial community, although containing a member of the Dehalococcoides group, was not able to dechlorinate PCE. Over 890 days, 65% of the initial emplaced PCE was removed in the bioaugmented, dechlorinating system, in comparison to 39% removal by dissolution from the nondechlorinating system. The maximum total ethenes concentration (3 mM) in the bioaugmented system occurred approximately 100 days after bioaugmentation, indicating that there was at least a 3-fold enhancement of PCE dissolution atthis time. Removal rates decreased substantially beyond this time, particularly during the last 200 days of the study, when the maximum concentrations of total ethenes were only about 0.5 mM. However, PCE removal rates in the dechlorinating system remained more than twice the removal rates of the nondechlorinating system. The reductions in removal rates over time are attributed to both a shrinking DNAPL source area, and reduced flow through the DNAPL source area due to bioclogging and pore blockage from methane gas generation.

Stable isotope evidence for biodegradation of chlorinated ethenes at a fractured bedrock site.

Stable carbon isotope analysis of chlorinated ethenes and ethene was performed at a site contaminated with trichloroethene (TCE), a dense non-aqueous phase liquid (DNAPL). The site is located in fractured bedrock and had variable groundwater hydraulic gradients during the study due to a local excavation project. Previous attempts to biostimulate a pilot treatment area at the site resulted in the production of cis-1,2-dichloroethene (cis-DCE), the first product of reductive dechlorination of TCE. Cis-DCE concentrations accumulated however, and there was no appreciable production of the breakdown products from further reductive dechlorination, vinyl chloride (VC) and ethene (ETH). Consequently, the pilot treatment area was bioaugmented with a culture of KB-1, a natural microbial consortium known to completely reduce TCE to nontoxic ETH. Due to ongoing dissolution of TCE from DNAPL in the fractured bedrock, and to variable hydraulic gradients, concentration profiles of dissolved TCE and its degradation products cis-DCE, VC, and ETH could not convincingly confirm biodegradation of the chlorinated ethenes. Isotopic analysis of cis-DCE and VC, however, demonstrated that biodegradation was occurring in the pilot treatment area. The isotope values of cis-DCE and VC became significantly more enriched in 13C over the last two sampling dates (in one well from -17.6%o to -12.8%o and from -22.5%o to -18.2%o for cis-DCE and VC, respectively). Quantification of the extent of biodegradation in the pilot treatment area using the Rayleigh model indicated that, depending on the well, between 21.3% and 40.7% of the decrease in cis-DCE and between 15.2% and 36.7% of the decrease in VC concentrations can be attributed to the effects of biodegradation during this time period. Within each well, the isotope profile of TCE remained relatively constant due to the continuous input of undegraded TCE due to DNAPL dissolution.

Quantifying chlorinated ethene degradation during reductive dechlorination at Kelly AFB using stable carbon isotopes.

Stable isotope analysis of chlorinated ethene contaminants was carried out during a bioaugmentation pilot test at Kelly Air Force Base (AFB) in San Antonio Texas. In this pilot test, cis-1,2-dichloroethene (cDCE) was the primary volatile organic compound. A mixed microbial enrichment culture, KB-1, shown in laboratory experiments to reduce chlorinated ethenes to non-toxic ethene, was added to the pilot test area. Following bioaugmentation with KB-1, perchloroethene (PCE), trichloroethene (TCE) and cDCE concentrations declined, while vinyl chloride (VC) concentrations increased and subsequently decreased as ethene became the dominant transformation product. Shifts in carbon isotopic values up to 2.7 per thousand, 6.4 per thousand, 10.9 per thousand and 10.6 per thousand were observed for PCE, TCE, cDCE and VC, respectively, after bioaugmentation, consistent with the effects of biodegradation. While a rising trend of VC concentrations and the first appearance of ethene were indicative of biodegradation by 72 days post-bioaugmentation, the most compelling evidence of biodegradation was the substantial carbon isotope enrichment (2.0 per thousand to 5.0 per thousand) in ä13C(cDCE). Fractionation factors obtained in previous laboratory studies were used with isotope field measurements to estimate first-order cDCE degradation rate constants of 0.12 h(-1) and 0.17 h(-1) at 115 days post-bioaugmentation. These isotope-derived rate constants were clearly lower than, but within a factor of 2-4 of the previously published rate constant calculated in a parallel study at Kelly AFB using chlorinated ethene concentrations. Stable carbon isotopes can provide not only a sensitive means for early identification of the effects of biodegradation, but an additional means to quantify the rates of biodegradation in the field.

Dynamic headspace: a single-step extraction for isotopic analysis of microg/L concentrations of dissolved chlorinated ethenes.

In this study a dynamic headspace method was developed to measure the carbon isotope values of dissolved chlorinated ethenes at microg/L concentrations. A gas chromatograph/combustion/isotope ratio mass spectrometer (GC/C/IRMS) was modified to include a headspace extraction system followed by a cryogenic trap. Extracting headspace from a 160 mL vial with 80 mL of aqueous solution and 40 g of NaCl for 8-12 min resulted in accurate and reproducible delta13C values for trichloroethene (TCE) and cis-1,2-dichloroethene (cDCE) at concentrations of 50-75 microg/L. Based on these results a conservative lower limit of quantitation of 38 microg/L can be calculated for these compounds. For more volatile compounds such as tetrachloroethene (PCE) and vinyl chloride (VC), field data analyzed using this method indicate a lower limit of quantitation in the tens of microg /L range.