Estimating denitrification rates in estuarine sediments: A comparison of stoichiometric and acetylene based methods

We compared denitrification rates obtained using an adaptation of the acetylene block technique to rates estimated from benthic flux nutrient stoichiometry in the subtidal sediments of Tomales Bay, California (USA). By amending whole cores with acetylene and saturating nitrate concentrations, we obtained potential denitrification rates, which ranged between 4 and 30 mmol N m^–2 d^–1. We determined the apparent Michaelis constant (K_app) and the maximum potential rate (V_mp) of the denitrifying community and used these constants in a rectangular hyperbola to estimatein situ denitrification rates. Both the K_app and V_mp of the denitrifying community exhibited significant variation over both depth in the sediment column and time of sampling.Estimates ofin situ denitrification obtained using our kinetic-fix adaptation of the acetylene block ranged between 1.8 (March) and 9 (Sept.) mmol N m^–1 d^–1. Denitrification rates obtained using benthic flux stoichiometry ranged between 0.7 and 4.1 mmol N m^–2 d^–1. Average denitrification rates obtained using the kinetic-fix acetylene block approach exceeded those obtained from net benthic flux stoichiometry; however, these differences were not significant. We conclude that our kinetic-fix adaptation of the acetylene block technique provides realistic estimates of denitrification in sediments, even when pore water nitrate concentrations are low and nitrification and denitrification are closely coupled.     

Pelagic denitrification and methane oxidation in oxygen-depleted waters of the Louisiana shelf

Anthropogenic nutrient inputs fuel eutrophication and hypoxia ([O₂]?<?2 mg L^?1), threatening coastal and near shore environments across the globe. The world’s second largest anthropogenic coastal hypoxic zone occurs annually along the Louisiana (LA) shelf. Springtime loading of dissolved inorganic nitrogen (DIN) from the Mississippi River, combined with summertime stratification and increased water residence time on the shelf, promotes establishment of an extensive hypoxic zone that persists throughout the summer. We investigated the patterns of pelagic denitrification and methane (CH₄) oxidation along the LA shelf. Microbial activity rates were determined along with concentrations of dissolved nutrients and greenhouse gases, nitrous oxide (N₂O) and CH₄, during summer in 2013, 2015, and 2016. We documented denitrification rates up to 1900 nmol N L^?1 d^?1 and CH₄ oxidation rates as high as 192 nmol L^?1 d^?1 in hypoxic waters characterized by high concentrations of N₂O (range: 1 to 102 nM) and CH₄ (range: 3 to 641 nM). Ecosystem scaling estimates suggest that pelagic denitrification could remove between 0.1 and 47% of the DIN input from the Mississippi River, whereas CH₄ oxidation does not function as an effective removal process with CH₄ escaping to the atmosphere. Denitrification and CH₄ oxidizing bacteria within the LA shelf hypoxic zone were largely unable to keep up with the DIN and CH₄ inputs to the water column. Rates were variable and physiochemical dynamics appeared to regulate the microbial removal capacity for both nitrate/nitrite and CH₄ in this ecosystem.

     

Methyl-compounds driven benthic carbon cycling in the sulfate-reducing sediments of South China Sea

Methane is a potent greenhouse gas; methane production and consumption within seafloor sediments has generated intense interest. Anaerobic oxidation of methane (AOM) and methanogenesis (MOG) primarily occur at the depth of the sulfate–methane transition zone or underlying sediment respectively. Methanogenesis can also occur in the sulfate-reducing sediments through the utilization of non-competitive methylated compounds; however, the occurrence and importance of this process are not fully understood. Here, we combined a variety of data, including geochemical measurements, rate measurements and molecular analyses to demonstrate the presence of a cryptic methane cycle in sulfate-reducing sediments from the continental shelf of the northern South China Sea. The abundance of methanogenic substrates as well as the high MOG rates from methylated compounds indicated that methylotrophic methanogenesis was the dominant methanogenic pathway; this conclusion was further supported by the presence of the methylotrophic genus Methanococcoides. High potential rates of AOM were observed in the sediments, indicating that methane produced in situ could be oxidized simultaneously by AOM, presumably by ANME-2a/b as indicated by 16S rRNA gene analysis. A significant correlation between the relative abundance of methanogens and methanotrophs was observed over sediment depth, indicating that methylotrophic methanogenesis could potentially fuel AOM in this environment. In addition, higher potential rates of AOM than sulfate reduction rates at in situ methane conditions were observed, making alternative electron acceptors important to support AOM in sulfate-reducing sediment. AOM rates were stimulated by the addition of Fe/Mn oxides, suggesting AOM could be partially coupled to metal oxide reduction. These results suggest that methyl-compounds driven methane production drives a cryptic methane cycling and fuels AOM coupled to the reduction of sulfate and other electron acceptors.     

Diverse,rare microbial taxa responded to the Deepwater Horizon deep-sea hydrocarbon plume

The Deepwater Horizon (DWH) oil well blowout generated an enormous plume of dispersed hydrocarbons that substantially altered the Gulf of Mexico''s deep-sea microbial community. A significant enrichment of distinct microbial populations was observed, yet, little is known about the abundance and richness of specific microbial ecotypes involved in gas, oil and dispersant biodegradation in the wake of oil spills. Here, we document a previously unrecognized diversity of closely related taxa affiliating with Cycloclasticus, Colwellia and Oceanospirillaceae and describe their spatio-temporal distribution in the Gulf''s deepwater, in close proximity to the discharge site and at increasing distance from it, before, during and after the discharge. A highly sensitive, computational method (oligotyping) applied to a data set generated from 454-tag pyrosequencing of bacterial 16S ribosomal RNA gene V4–V6 regions, enabled the detection of population dynamics at the sub-operational taxonomic unit level (0.2% sequence similarity). The biogeochemical signature of the deep-sea samples was assessed via total cell counts, concentrations of short-chain alkanes (C₁–C₅), nutrients, (colored) dissolved organic and inorganic carbon, as well as methane oxidation rates. Statistical analysis elucidated environmental factors that shaped ecologically relevant dynamics of oligotypes, which likely represent distinct ecotypes. Major hydrocarbon degraders, adapted to the slow-diffusive natural hydrocarbon seepage in the Gulf of Mexico, appeared unable to cope with the conditions encountered during the DWH spill or were outcompeted. In contrast, diverse, rare taxa increased rapidly in abundance, underscoring the importance of specialized sub-populations and potential ecotypes during massive deep-sea oil discharges and perhaps other large-scale perturbations.     

A plasmid-encoded nicotinamidase (PncA) is essential for infectivity of Borrelia burgdorferi in a mammalian host

Borrelia burgdorferi, a spirochaete that causes Lyme borreliosis, contains 21 linear and circular plasmids thought to be important for survival in mammals or ticks. Our results demonstrate that the gene BBE22 encoding a nicotinamidase is capable of replacing the requirement for the 25 kb linear plasmid lp25 during mammalian infection. Transformation of B. burgdorferi lacking lp25 with a shuttle vector containing the lp25 gene BBE22 (pBBE22) restored infectivity in mice to a level comparable to that of wild-type Borrelia. This complementation also restored the growth and host adaptation of lp25-B. burgdorferi in dialysis membrane chambers (DMCs) implanted in rats. A single Cys to Ala conversion at the putative active site of BBE22 abrogated the ability of pBBE22 to re-establish infectivity or growth in DMCs. Additional Salmonella typhimurium complementation studies and enzymatic analysis demonstrated that the BBE22 gene product has nicotinamidase activity and is most probably required for the biosynthesis of NAD. These results indicate that some plasmid-encoded products fulfil physiological functions required in the enzootic cycle of pathogenic Borrelia.     

Barite encrustation of benthic sulfur‐oxidizing bacteria at a marine cold seep

Crusts and chimneys composed of authigenic barite are found at methane seeps and hydrothermal vents that expel fluids rich in barium. Microbial processes have not previously been associated with barite precipitation in marine cold seep settings. Here, we report on the precipitation of barite on filaments of sulfide‐oxidizing bacteria at a brine seep in the Gulf of Mexico. Barite‐mineralized bacterial filaments in the interiors of authigenic barite crusts resemble filamentous sulfide‐oxidizing bacteria of the genus Beggiatoa. Clone library and iTag amplicon sequencing of the 16S rRNA gene show that the barite crusts that host these filaments also preserve DNA of Candidatus Maribeggiatoa, as well as sulfate‐reducing bacteria. Isotopic analyses show that the sulfur and oxygen isotope compositions of barite have lower δ³⁴S and δ¹⁸O values than many other marine barite crusts, which is consistent with barite precipitation in an environment in which sulfide oxidation was occurring. Laboratory experiments employing isolates of sulfide‐oxidizing bacteria from Gulf of Mexico seep sediments showed that under low sulfate conditions, such as those encountered in brine fluids, sulfate generated by sulfide‐oxidizing bacteria fosters rapid barite precipitation localized on cell biomass, leading to the encrustation of bacteria in a manner reminiscent of our observations of barite‐mineralized Beggiatoa in the Gulf of Mexico. The precipitation of barite directly on filaments of sulfide‐oxidizing bacteria, and not on other benthic substrates, suggests that sulfide oxidation plays a role in barite formation at certain marine brine seeps where sulfide is oxidized to sulfate in contact with barium‐rich fluids, either prior to, or during, the mixing of those fluids with sulfate‐containing seawater in the vicinity of the sediment/water interface. As with many other geochemical interfaces that foster mineral precipitation, both biological and abiological processes likely contribute to the precipitation of barite at marine brine seeps such as the one studied here.     

Novel vacuolate sulfur bacteria from the Gulf of Mexico reproduce by reductive division in three dimensions

Large spherical sulfur bacteria, 180-375 microm in diameter, were found regularly and in abundance in surface sediments collected from hydrocarbon seeps (water depth 525-640 m) in the Gulf of Mexico. These bacteria were characterized by a thin 'shell' of sulfur globule-filled cytoplasm that surrounded a central vacuole (roughly 80% of biovolume) containing high concentrations of nitrate (average 460 mM). Approximately 800 base pairs of 16S rRNA gene sequence data, linked to this bacterium by fluorescent in situ hybridization, showed 99% identity with Thiomargarita namibiensis, previously described only from sediments collected off the coast of Namibia (Western Africa). Unlike T. namibiensis, where cells form a linear chain within a common sheath, the Gulf of Mexico strain occurred as single cells and clusters of two, four and eight cells, which were clearly the product of division in one to three planes. In sediment cores maintained at 4 degrees C, which undoubtedly experienced a diminishing flux of hydrogen sulfide over time, the Thiomargarita-like bacterium remained viable for up to 2 years. During that long period, each cell appeared to undergo (as judged by change in biovolume) one to three reductive divisions, perhaps as a dispersal strategy in the face of diminished availability of its putative electron donor.     

Variation in Prokaryotic Community Composition as a Function of Resource Availability in Tidal Creek Sediments

In anaerobic coastal sediments, hydrolytic and/or fermentative bacteria degrade polymeric material and produce labile intermediates, which are used by terminal metabolizers to complete the conversion of organic material to CO₂. We used molecular approaches to evaluate the response of two bacterial terminal metabolizer groups from a coastal tidal creek sediments, sulfate reducers and methanogens, to controlled changes in carbon resource supply. Tidal creek sediment bioreactors were established in April and August 2004. For each date, intact sediment sections were continuously supplied with flowthrough seawater that was either unamended or amended with the high-molecular-weight polysaccharide dextran. Biogeochemical data indicate that the activity of fermenting bacteria and the terminal metabolizers was limited by organic carbon supply during both experiments, with a significant increase in net volatile fatty acid (VFA) production and rates of sulfate reduction and methanogenesis following dextran addition. Community composition (measured by using terminal restriction fragment length polymorphism analysis, and functional gene [dsrA, mcrA] clone libraries) changed from April to August. However, community composition was not different between amended and unamended cores within each month, despite the change in resource level. Moreover, there was no relationship between community richness and evenness with resource level. This lack of variation in community composition with C addition could be attributed to the dynamic environment these sediment communities experience in situ. Fluctuations in VFA concentrations are most likely very high, so that the dominant bacterial species must be able to outcompete other species at both high and low resource levels.     

Field measurements and modeling of groundwater flow and biogeochemistry at Moses Hammock, a backbarrier island on the Georgia coast

A combination of field measurements, laboratory experiments and model simulations were used to characterize the groundwater biogeochemical dynamics along a shallow monitoring well transect on a coastal hammock. A switch in the redox status of the dissolved inorganic nitrogen (DIN) pool in the well at the upland/saltmarsh interface occurred over the spring-neap tidal transition: the DIN pool was dominated by nitrate during spring tide and by ammonium during neap tide. A density-dependent reaction-transport model was used to investigate the relative importance of individual processes to the observed N redox-switch. The observed N redox-switch was evaluated with regard to the roles of nitrification, denitrification, dissimilatory nitrate reduction to ammonium (DNRA), ammonium adsorption, and variations in inflowing water geochemistry between spring and neap tides. Transport was driven by measured pressure heads and process parameterizations were derived from field observations, targeted laboratory experiments, and the literature. Modeling results suggest that the variation in inflow water chemistry was the dominant driver of DIN dynamics and highlight the importance of spring-neap tide variations in the high marsh, which influences groundwater biogeochemistry at the marsh-upland transition.     

On the relationship between methane production and oxidation by anaerobic methanotrophic communities from cold seeps of the Gulf of Mexico

The anaerobic oxidation of methane (AOM) in the marine subsurface is a significant sink for methane in the environment, yet our understanding of its regulation and dynamics is still incomplete. Relatively few groups of microorganisms consume methane in subsurface environments – namely the anaerobic methanotrophic archaea (ANME clades 1, 2 and 3), which are phylogenetically related to methanogenic archaea. Anaerobic oxidation of methane presumably proceeds via a 'reversed' methanogenic pathway. The ANME are generally associated with sulfate-reducing bacteria (SRB) and sulfate is the only documented final electron acceptor for AOM in marine sediments. Our comparative study explored the coupling of AOM with sulfate reduction (SR) and methane generation (MOG) in microbial communities from Gulf of Mexico cold seep sediments that were naturally enriched with methane and other hydrocarbons. These sediments harbour a variety of ANME clades and SRB. Following enrichment under an atmosphere of methane, AOM fuelled 50–100% of SR, even in sediment slurries containing petroleum-associated hydrocarbons and organic matter. In the presence of methane and sulfate, the investigated microbial communities produce methane at a small fraction (∼10%) of the AOM rate. Anaerobic oxidation of methane, MOG and SR rates decreased significantly with decreasing concentration of methane, and in the presence of the SR inhibitor molybdate, but reacted differently to the MOG inhibitor 2-bromoethanesulfonate (BES). The addition of acetate, a possible breakdown product of petroleum in situ and a potential intermediate in AOM/SR syntrophy, did not suppress AOM activity; rather acetate stimulated microbial activity in oily sediment slurries.