Ecological response of nitrification to oil spills and its impact on the nitrogen cycle.

Marine oil spills are catastrophic events that cause massive damage to ecosystems at all trophic levels. While most of the research has focused on carbon-degrading microorganisms, the potential impacts of hydrocarbons on microbes responsible for nitrification have received far less attention. Nitrifiers are sensitive to hydrocarbon toxicity: ammonia-oxidizing bacteria and archaea being 100 and 1000 times more sensitive than typical heterotrophs respectively. Field studies have demonstrated the response of nitrifiers to hydrocarbons is highly variable and the loss of nitrification activity in coastal ecosystems can be restored within 1-2 years, which is much shorter than the typical recovery time of whole ecosystems (e.g., up to 20 years). Since the denitrification process is mainly driven by heterotrophs, which are more resistant to hydrocarbon toxicity than nitrifiers, the inhibition of nitrification may slow down the nitrogen turnover and increase ammonia availability, which supports the growth of oil-degrading heterotrophs and possibly various phototrophs. A better understanding of the ecological response of nitrification is paramount in predicting impacts of oil spills on the nitrogen cycle under oil spill conditions, and in improving current bioremediation practices.

Vegetation Loss Decreases Salt Marsh Denitrification Capacity: Implications for Marsh Erosion.

Salt marshes play a key role in removing excess anthropogenic nitrogen (N) loads to nearshore marine ecosystems through sediment microbial processes such as denitrification. However, in the Gulf of Mexico, the loss of marsh vegetation because of human-driven disturbances such as sea level rise and oil spills can potentially reduce marsh capacity for N removal. To investigate the effect of vegetation loss on ecosystem N removal, we contrasted denitrification capacity in marsh and subtidal sediments impacted by the Deepwater Horizon oil spill using a combination of 29N2 and 30N2 production (isotope pairing), denitrification potential measurements (acetylene block), and quantitative polymerase chain reaction (qPCR) of functional genes in the denitrification pathway. We found that, on average, denitrification capacity was 4 times higher in vegetated sediments because of a combination of enhanced nitrification and higher organic carbon availability. The abundance of nirS-type denitrifers indicated that marsh vegetation regulates the activity, rather than the abundance, of denitrifier communities. We estimated that marsh sediments remove an average of 3.6 t N km-2 y-1 compared to 0.9 t N km-2 y-1 in unvegetated sediments. Overall, our findings indicate that marsh loss results in a substantial loss of N removal capacity in coastal ecosystems.

Complete genome sequence of Rahnella aquatilis CIP 78.65.

Rahnella aquatilis CIP 78.65 is a gammaproteobacterium isolated from a drinking water source in Lille, France. Here we report the complete genome sequence of Rahnella aquatilis CIP 78.65, the type strain of R. aquatilis.

Complete genome sequence of Rahnella sp. strain Y9602, a gammaproteobacterium isolate from metal- and radionuclide-contaminated soil.

Rahnella sp. strain Y9602 is a gammaproteobacterium isolated from contaminated subsurface soils that is capable of promoting uranium phosphate mineralization as a result of constitutive phosphatase activity. Here we report the first complete genome sequence of an isolate belonging to the genus Rahnella.

Aerobic biotransformation of n-tetradecylbenzyldimethylammonium chloride by an enriched Pseudomonas spp. community.

The biotransformation of n-tetradecylbenzyldimethylammonium chloride (C(14)BDMA-Cl), a quaternary ammonium compound (QAC), under aerobic conditions by an enriched microbial community growing on benzalkonium chlorides (BACs) was investigated. Biotransformation of C(14)BDMA-Cl commenced with cleavage of the C(alkyl)-N bond and formation of benzyldimethylamine (BDMA). BDMA was further degraded, but in contrast to a previously reported BAC biotransformation pathway, neither benzylmethylamine (BMA) nor benzylamine (BA) was detected as a BDMA biotransformation product. Kinetic assays further confirmed that BMA and BA were not intermediates of C(14)BDMA-Cl transformation by the enriched community. Thus, BDMA is thought to be transformed to dimethylamine and benzoic acid via debenzylation. The biomass-normalized rate of C(14)BDMA-Cl biotransformation was 0.09 µmol/[mg of volatile suspended solids (VSS)·h]. The Microtox acute toxicity EC(50) value of BDMA was 500 times higher than that of C(14)BDMA-Cl. Thus, the aerobic biotransformation of C(14)BDMA-Cl to BDMA results in substantial toxicity reduction. Phylogenetic analysis of Bacteria diversity indicated that the majority of the sequenced clones (98% of the clone library) belonged to the genus Pseudomonas.

Inactivation of mismatch repair increases the diversity of Vibrio parahaemolyticus.

Inactivation of mismatch repair (MMR) has been shown to increase the accumulation of spontaneous mutations and frequency of recombination for diverse pathogenic bacteria. Currently, little is known regarding the role of mutator phenotypes for the diversification of natural populations of opportunistic human pathogens in marine environments. In this study, a higher frequency of mutators was detected among V. parahaemolyticus strains obtained from environmental sources compared with clinical sources. Inactivation of the MMR gene mutS caused increased antibiotic resistance and phase variation resulting in translucent colony morphologies. Increased nucleotide diversity in mutS and rpoB alleles from mutator compared with wild-type strains indicated a significant contribution of the mutator phenotype to the evolution of select genes. The results of this study indicate that the inactivation of MMR in V. parahaemolyticus leads to increased genetic and phenotypic diversity. This study is the first to report a higher frequency of natural mutators among Vibrio environmental strains and to provide evidence that inactivation of MMR increases the diversity of V. parahaemolyticus.

Rapid identification of Vibrio parahaemolyticus by whole-cell matrix-assisted laser desorption ionization-time of flight mass spectrometry.

Vibrio parahaemolyticus is a pathogenic marine bacterium that is the main causative agent of bacterial seafood-borne gastroenteritis in the United States. An increase in the frequency of V. parahaemolyticus-related infections during the last decade has been attributed to the emergence of an O3:K6 pandemic clone in 1995. The diversity of the O3:K6 pandemic clone and its serovariants has been examined using multiple molecular techniques including multilocus sequence analysis, pulsed-field gel electrophoresis, and group-specific PCR analysis. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has become a powerful tool for rapidly distinguishing between related bacterial species. In the current study, we demonstrate the development of a whole-cell MALDI-TOF MS method for the distinction of V. parahaemolyticus from other Vibrio spp. We identified 30 peaks that were present only in the spectra of the V. parahaemolyticus strains examined in this study that may be developed as MALDI-TOF MS biomarkers for identification of V. parahaemolyticus. We detected variation in the MALDI-TOF spectra of V. parahaemolyticus strains isolated from different geographical locations and at different times. The MALDI-TOF MS spectra of the V. parahaemolyticus strains examined were distinct from those of the other Vibrio species examined including the closely related V. alginolyticus, V. harveyi, and V. campbellii. The results of this study demonstrate the first use of whole-cell MALDI-TOF MS analysis for the rapid identification of V. parahaemolyticus.

Horizontal gene transfer and mobile genetic elements in marine systems.

The pool of mobile genetic elements (MGE) in microbial communities consists of viruses, plasmids, and associated elements (insertion sequences, transposons, and integrons) that are either self-transmissible or use mobile plasmids and viruses as vehicles for their dissemination. This mobilome facilitates the horizontal transfer of genes that promote the evolution and adaptation of microbial communities. Efforts to characterize MGEs from microbial populations resident in a variety of ecological habitats have revealed a surprisingly novel and seemingly untapped biodiversity. To better understand the impact of horizontal gene transfer (HGT), as well as the agents that promote HGT in marine ecosystems and to determine whether or not environmental parameters can effect the composition and structure of the mobilome in marine microbial communities, information on the distribution, diversity, and ecological traits of the marine mobilome is presented. In this chapter we discuss recent insights gained from different methodological approaches used to characterize the biodiversity and ecology of MGE in marine environments and their contributions to HGT. In addition, we present case studies that highlight specific HGT examples in coastal, open-ocean, and deep-sea marine ecosystems.

Horizontal gene transfer in metal and radionuclide contaminated soils.

The horizontal transfer of genes encoded on mobile genetic elements (MGEs) such as plasmids and phage and their associated hitchhiking elements (transposons, integrons, integrative and conjugative elements, and insertion sequences) rapidly accelerate genome diversification of microorganisms, thereby affecting their physiology, metabolism, pathogenicity,and ecological character. The analyses of completed prokaryotic genomes reveal that horizontal gene transfer (HGT) continues to be an important factor contributing to the innovation of microbial genomes. Indeed, microbial genomes are remarkably dynamic and a considerable amount of genetic information is inserted or deleted by HGT mechanisms. Thus, HGT and the vast pool of MGEs provide microbial communities with an unparalleled means by which to respond rapidly to changing environmental conditions and exploit new ecological niches. Metals and radionuclide contamination in soils, the subsurface, and aquifers poses a serious challenge to microbial growth and survival because these contaminants cannot be transformed or biodegraded into non-toxic forms as often occurs with organic xenobiotic contaminants. In this chapter we present cases in which HGT has been demonstrated to contribute to the dissemination of genes that provide adaptation to contaminant stress (i.e., toxic heavy metals and radionuclides). In addition, we present directions for future studies that could provide even greater insights into the contributions of HGT to adaptation for survival in mixed waste sites.

Salt marsh denitrification is impacted by oiling intensity six years after the Deepwater Horizon oil spill.

Coastal salt marshes provide the valuable ecosystem service of removing anthropogenic nitrogen (N) via microbially-mediated denitrification. During the 2010 Deepwater Horizon (DWH) spill, oil exposure killed marsh plants in some regions and contributed to rapid compositional shifts in sediment microbial communities, which can impact ecosystem denitrification capacity. Within 3-5 years of the spill, plant biomass and microbial communities in some impacted marshes can recover to a new stable state. The objective of this study was to determine whether marsh recovery 6 years after the DWH oil spill results in subsequent recovery of denitrification capacity. We measured denitrification capacity (isotope pairing technique), microbial 16S rRNA gene composition, and denitrifier abundance (quantitative PCR) at sites subjected to light, moderate, and heavy oiling during the spill that were not targeted by any clean-up efforts. There were no differences in plant belowground biomass, sediment extractable NH4+, inorganic nitrogen flux, 16S rRNA composition, 16S rRNA diversity, or denitrifier functional gene (nirS, norB, and nosZ) abundances associated with oiling status, indicating that certain drivers of ecosystem denitrification capacity have recovered or achieved a new stable state six years after the spill. However, on average, denitrification capacities at the moderately and heavily oiled sites were less than 49% of that of the lightly oiled site (27.7 ±â€¯14.7 and 37.2 ±â€¯24.5 vs 71.8 ±â€¯33.8 µmol N m-2 h-1, respectively). The presence of heavily weathered oiled residue (matched and non-matched for MC252) had no effect on process rates or microbial composition. The loss of function at the moderately and heavily oiled sites compared to the lightly oiled site despite the comparable microbial and environmental factors suggests that oiling intensity plays a role in the long-term recovery of marsh ecosystem services.

Sequence characterization and comparative analysis of three plasmids isolated from environmental Vibrio spp.

The horizontal transfer of genes by mobile genetic elements such as plasmids and phages can accelerate genome diversification of Vibrio spp., affecting their physiology, pathogenicity, and ecological character. In this study, sequence analysis of three plasmids from Vibrio spp. previously isolated from salt marsh sediment revealed the remarkable diversity of these elements. Plasmids p0908 (81.4 kb), p23023 (52.5 kb), and p09022 (31.0 kb) had a predicted 99, 64, and 32 protein-coding sequences and G+C contents of 49.2%, 44.7%, and 42.4%, respectively. A phylogenetic tree based on concatenation of the host 16S rRNA and rpoA nucleotide sequences indicated p23023 and p09022 were isolated from strains most closely related to V. mediterranei and V. campbellii, respectively, while the host of p0908 forms a clade with V. fluvialis and V. furnissii. Many predicted proteins had amino acid identities to proteins of previously characterized phages and plasmids (24 to 94%). Predicted proteins with similarity to chromosomally encoded proteins included RecA, a nucleoid-associated protein (NdpA), a type IV helicase (UvrD), and multiple hypothetical proteins. Plasmid p0908 had striking similarity to enterobacteria phage P1, sharing genetic organization and amino acid identity for 23 predicted proteins. This study provides evidence of genetic exchange between Vibrio plasmids, phages, and chromosomes among diverse Vibrio spp.

Chemically mediated competition between microbes and animals: microbes as consumers in food webs.

Microbes are known to affect ecosystems and communities as decomposers, pathogens, and mutualists. However, they also may function as classic consumers and competitors with animals if they chemically deter larger consumers from using rich food-falls such as carrion, fruits, and seeds that can represent critical windfalls to both microbes and animals. Microbes often use chemicals (i.e., antibiotics) to compete against other microbes. Thus using chemicals against larger competitors might be expected and could redirect significant energy subsidies from upper trophic levels to the detrital pathway. When we baited traps in a coastal marine ecosystem with fresh vs. microbe-laden fish carrion, fresh carrion attracted 2.6 times as many animals per trap as microbe-laden carrion. This resulted from fresh carrion being found more frequently and from attracting more animals when found. Microbe-laden carrion was four times more likely to be uncolonized by large consumers than was fresh carrion. In the lab, the most common animal found in our traps (the stone crab Menippe mercenaria) ate fresh carrion 2.4 times more frequently than microbe-laden carrion. Bacteria-removal experiments and feeding bioassays using organic extracts of microbe-laden carrion showed that bacteria produced noxious chemicals that deterred animal consumers. Thus bacteria compete with large animal scavengers by rendering carcasses chemically repugnant. Because food-fall resources such as carrion are major food subsidies in many ecosystems, chemically mediated competition between microbes and animals could be an important, common, but underappreciated interaction within many communities.

Chromium(VI) transport and fate in unsaturated zone and aquifer: 3D Sandbox results.

The simulation of Cr(VI) behavior in an unsaturated zone and aquifer, using a 3D experimental set-up were performed to illustrate the distribution, transport and transformation of Cr(VI), and further to reveal the potential harm of Cr(VI) after entering the groundwater. The result indicated that chromium(VI) was transported in the vertical direction, meanwhile, was transported in the horizontal direction under the influence of groundwater flow. The direction and distance away from the pollution source zone had great effect on the chromium(VI) concentration. At the sampling sites near the pollution source zone, there was a sudden increase of chromium(VI) concentration. The concentration of chromium(III) concentration in some random effluent samples was not detected. Chromium had not only transported but also had fraction and specie transformation in the unsaturated zone and aquifer. The relative concentration of residue fraction chromium was decreased with time. The content of Fe-Mn oxide fraction chromium was increased with time. The relative content of exchangeable and carbonate-bound fraction chromium was lower and the content variations were not obvious. Chromium(VI) (91-98%) was first reduced to chromium(III) rapidly. The oxidation reaction occurred later and the relative content of chromium(VI) was increased again. The presence of manganese oxides under favorable soil conditions can promote the reoxidation of Cr(III) to Cr(VI).

Input of organic matter enhances degradation of weathered diesel fuel in sub-tropical sediments.

We investigated different types of biostimulation practices to enhance degradation of weathered conventional diesel fuel in sandy beach sediments from coastal Alabama. Biodegradation rates were measured following the addition of either inorganic nutrients, or organic matter derived from either plant material (Spartina alterniflora) or fish tissue (Chloroscombrus chrysurus) both common to the region. The greatest hydrocarbon degradation rates were observed in the C. chrysurus amended treatments (k=0.0119 d(-1)). Treatment with fish-derived organic matter increased the degradation rates by 104% as compared to control treatments, while inorganic nutrient addition increased the degradation rates by 57%. The addition of plant derived organic matter, however, only marginally enhanced the degradation rates (~7%) during the course of the study. Bacterial 16S rRNA analyses revealed that most sediment microorganisms belonged to the classes; Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, and Bacteroidetes. The most frequently abundant hydrocarbon degraders were mostly similar to Achromobater sp., Microbulbifer sp., Ruegeria sp., and Pseudomonas sp.

Insights into the environmental reservoir of pathogenic Vibrio parahaemolyticus using comparative genomics.

Vibrio parahaemolyticus is an aquatic halophilic bacterium that occupies estuarine and coastal marine environments, and is a leading cause of seafood-borne food poisoning cases. To investigate the environmental reservoir and potential gene flow that occurs among V. parahaemolyticus isolates, the virulence-associated gene content and genome diversity of a collection of 133 V. parahaemolyticus isolates were analyzed. Phylogenetic analysis of housekeeping genes, and pulsed-field gel electrophoresis, demonstrated that there is genetic similarity among V. parahaemolyticus clinical and environmental isolates. Whole-genome sequencing and comparative analysis of six representative V. parahaemolyticus isolates was used to identify genes that are unique to the clinical and environmental isolates examined. Comparative genomics demonstrated an O3:K6 environmental isolate, AF91, which was cultured from sediment collected in Florida in 2006, has significant genomic similarity to the post-1995 O3:K6 isolates. However, AF91 lacks the majority of the virulence-associated genes and genomic islands associated with these highly virulent post-1995 O3:K6 genomes. These findings demonstrate that although they do not contain most of the known virulence-associated regions, some V. parahaemolyticus environmental isolates exhibit significant genetic similarity to clinical isolates. This highlights the dynamic nature of the V. parahaemolyticus genome allowing them to transition between aquatic and host-pathogen states.

Microbial community responses to organophosphate substrate additions in contaminated subsurface sediments.

BACKGROUND: Radionuclide- and heavy metal-contaminated subsurface sediments remain a legacy of Cold War nuclear weapons research and recent nuclear power plant failures. Within such contaminated sediments, remediation activities are necessary to mitigate groundwater contamination. A promising approach makes use of extant microbial communities capable of hydrolyzing organophosphate substrates to promote mineralization of soluble contaminants within deep subsurface environments. METHODOLOGY/PRINCIPAL FINDINGS: Uranium-contaminated sediments from the U.S. Department of Energy Oak Ridge Field Research Center (ORFRC) Area 2 site were used in slurry experiments to identify microbial communities involved in hydrolysis of 10 mM organophosphate amendments [i.e., glycerol-2-phosphate (G2P) or glycerol-3-phosphate (G3P)] in synthetic groundwater at pH 5.5 and pH 6.8. Following 36 day (G2P) and 20 day (G3P) amended treatments, maximum phosphate (PO4(3-)) concentrations of 4.8 mM and 8.9 mM were measured, respectively. Use of the PhyloChip 16S rRNA microarray identified 2,120 archaeal and bacterial taxa representing 46 phyla, 66 classes, 110 orders, and 186 families among all treatments. Measures of archaeal and bacterial richness were lowest under G2P (pH 5.5) treatments and greatest with G3P (pH 6.8) treatments. Members of the phyla Crenarchaeota, Euryarchaeota, Bacteroidetes, and Proteobacteria demonstrated the greatest enrichment in response to organophosphate amendments and the OTUs that increased in relative abundance by 2-fold or greater accounted for 9%-50% and 3%-17% of total detected Archaea and Bacteria, respectively. CONCLUSIONS/SIGNIFICANCE: This work provided a characterization of the distinct ORFRC subsurface microbial communities that contributed to increased concentrations of extracellular phosphate via hydrolysis of organophosphate substrate amendments. Within subsurface environments that are not ideal for reductive precipitation of uranium, strategies that harness microbial phosphate metabolism to promote uranium phosphate precipitation could offer an alternative approach for in situ sequestration.

Intrinsic rates of petroleum hydrocarbon biodegradation in Gulf of Mexico intertidal sandy sediments and its enhancement by organic substrates.

The rates of crude oil degradation by the extant microorganisms in intertidal sediments from a northern Gulf of Mexico beach were determined. The enhancement in crude oil degradation by amending the microbial communities with marine organic matter was also examined. Replicate mesocosm treatments consisted of: (i) controls (intertidal sand), (ii) sand contaminated with crude oil, (iii) sand plus organic matter, and (iv) sand plus crude oil and organic matter. Carbon dioxide (CO(2)) production was measured daily for 42 days and the carbon isotopic ratio of CO(2) (δ(13)CO(2)) was used to determine the fraction of CO(2) derived from microbial respiration of crude oil. Bacterial 16S rRNA clone library analyses indicated members of Actinobacteria, Bacteroidetes, and Chloroflexi occurred exclusively in control sediments whereas Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, and Firmicutes occurred in both control and oil contaminated sediments. Members of the hydrocarbon-degrading genera Hydrocarboniphaga, Pseudomonas, and Pseudoxanthomonas were found primarily in oil contaminated treatments. Hydrocarbon mineralization was 76% higher in the crude oil amended with organic matter treatment compared to the rate in the crude oil only treatment indicating that biodegradation of crude oil in the intertidal zone by an extant microbial community is enhanced by input of organic matter.

Enhancing the biodegradation of oil in sandy sediments with choline: a naturally methylated nitrogen compound.

We investigated how additions of choline, a naturally occurring methylated nitrogen-containing compound, accelerated hydrocarbon degradation in sandy sediments contaminated with moderately weathered crude oil (4000 mg kg(-1) sediment). Addition of lauroylcholine chloride (LCC) and tricholine citrate (TCC) to oil contaminated sediments resulted in 1.6 times higher hydrocarbon degradation rates compared to treatments without added choline derivatives. However, the degradation rate constant for the oil contaminated sediments amended with LCC was similar to that in contaminated sediments amended with inorganic nitrogen, phosphorus, and glucose. Additions of LLC and TCC to sediments containing extensively weathered oil also resulted in enhanced mineralization rates. Cultivation-free 16S rRNA analysis revealed the presence of an extant microbial community with clones closely related to known hydrocarbon degraders from the Gammaproteobacteria, Alphaproteobacteria, and Firmicutes phyla. The results demonstrate that the addition of minimal amounts of organic compounds to oil contaminated sediments enhances the degradation of hydrocarbons.

Responses of microbial community from northern Gulf of Mexico sandy sediments following exposure to Deepwater Horizon crude oil.

In the present study, microbial community responses to exposure to unweathered Macondo Well crude oil and conventional diesel in a sandy beach environment were determined. Biodegradation was assessed in mesocosm experiments with differing fuel amounts (2,000 and 4,000 mg/kg) and with or without inorganic nutrient amendment. Carbon dioxide production was measured daily for 42 d. Aerobic alkane, total hydrocarbon, and polycyclic aromatic hydrocarbon (PAH) degraders were enumerated in treated and control mesocosms and changes in their abundances were measured weekly. Hydrocarbon mineralization occurred in all treatments. In the inorganic nutrient-amended treatments, the degradation rates were 2.31 and 2.00 times greater in the 2,000 mg/kg diesel and crude oil treatments, respectively, and 3.52 (diesel) and 4.14 (crude) times higher for the fuel types at the 4,000 mg/kg fuel concentrations compared to unamended treatments. Microbial lag phases were short (<3 d) and alkane and total hydrocarbon degrader numbers increased by five orders of magnitude compared to the uncontaminated treatments within 7 d in most treatments. Hydrocarbon degrader numbers in diesel and in crude oil treatments were similar; however, the PAH degraders were more abundant in the crude oil relative to diesel treatment. These findings indicate that hydrocarbon degradation by extant microbial populations in the northern Gulf of Mexico sandy beach environments can be stimulated and enhanced by inorganic nutrient addition.

Evaluation of positron emission tomography as a method to visualize subsurface microbial processes.

Positron emission tomography (PET) provides spatiotemporal monitoring in a nondestructive manner and has higher sensitivity and resolution relative to other tomographic methods. Therefore, this technology was evaluated for its application to monitor in situ subsurface bacterial activity. To date, however, it has not been used to monitor or image soil microbial processes. In this study, PET imaging was applied as a "proof-of-principle" method to assess the feasibility of visualizing a radiotracer labeled subsurface bacterial strain (Rahnella sp. Y9602), previously isolated from uranium contaminated soils and shown to promote uranium phosphate precipitation. Soil columns packed with acid-purified simulated mineral soils were seeded with 2-deoxy-2-[(18)F]fluoro-D-glucose ((18)FDG) labeled Rahnella sp. Y9602. The applicability of [(18)F]fluoride ion as a tracer for measuring hydraulic conductivity and (18)FDG as a tracer to identify subsurface metabolically active bacteria was successful in our soil column studies. Our findings indicate that positron-emitting isotopes can be utilized for studies aimed at elucidating subsurface microbiology and geochemical processes important in contaminant remediation.