Evaluation of nutrient limitation of CO2 and N2 fixation in marine microbial mats

Photosynthetic CO2 fixation and N2 fixation are fundamentally important in mediating production dynamics of intertidal and subtidal marine microbial mat communities. We examined nutrient [N, P, Fe, MO, dissolved organic carbon (DOC)] limitation of CO, and NZ fixation in geographically and physiologically diverse mats. Nitrogen enrichment (as NO3-) infrequently stimulated CO2 fixation. Phosphorus, Fe and MO enrichment generally failed to stimulate CO2 fixation. The frequent absence of N limitation appears linked to the ability of mat microbial communities to fix N2 and effectively recycle fixed N. Nitrogen fixation was enhanced by DOC, while no P, Fe or trace element stimulation was observed. The lack of nutrient stimulation of CO2 fixation appeared related to low net growth rates of some mats. Slow growing mats, including hypersaline, stromatolitic (Storrs Lake, Bahamas) and certain hypersaline, lagoonal mats (Guerrero Negro, Baja California, Mexico) exhibited virtually no nutrient stimulation of either CO2 or N2 fixation. More productive coastal (North Carolina, USA) and estuarine (Tomales Bay, California, USA) mats showed higher frequencies of nutrient limitation of either process. Seasonally, N and DOC stimulation were most profound during periods of maximum growth. Mats are able to minimize C and N limitation by metabolically coupling CO2 and N2 fixation as sources of 'new' C and N inputs respectively. Phototrophic-heterotrophic microbial consortia appear to mediate coupling, which minimizes losses of fixed C and N to overlying waters

Microbial enzymatic activity and secondary production in sediments affected by the sedimentation pulse following the Deepwater Horizon oil spill

A large fraction of the spilled oil from the Deepwater Horizon (DwH) blowout in April 2010 reached the seafloor via sinking oil aggregates (oil snow) in a massive sedimentation that continued until late summer 2010 ("Dirty blizzard"). We measured heterotrophic microbial metabolic rates as well as porewater and sedimentary geochemical parameters at sites proximate to and distant from the wellhead to investigate microbial responses to the "Dirty Blizzard". Lipase activity and rates of bacterial protein production were highest and leucine-aminopeptidase activity was lowest in 0-2 cm sediment layers at the sites proximate to the wellhead. These results suggest that the presence of the oil snow stimulated benthic microbial enzymatic hydrolysis of oil-derived organic matter that was depleted in peptide substrates at the time of our sampling. The strong gradients in porewater DOC, NH4+, and HPO43- concentrations in the upper 6 cm of the sediments near the wellhead likewise indicate elevated heterotrophic responses to recently-sedimented organic matter. In addition to enhanced microbial activities in the 0-2 cm sediment layers, we found peaks of total organic carbon and elevated microbial metabolic rates down to 10 cm at the sites closest to the wellhead. Our results indicate distinct benthic metabolic responses of heterotrophic microbial communities, even three months after the ending of the "Dirty Blizzard". Compared to other deep-sea environments, however, metabolic rates associated with the recently deposited particulate matter around the wellhead were only moderately enhanced. Oil contaminants at the seafloor may therefore have prolonged residence times, enhancing the potential for longer-term ecological consequences in deep-sea environments

The metabolic pathways and environmental controls of hydrocarbon biodegradation in marine ecosystems

Hydrocarbon-degrading microorganisms are ubiquitous in the world’s oceans (Head et al., 2006; Yakimov et al., 2007), and biodegradation mediated by indigenous microbial communities is the ultimate fate of the majority of oil hydrocarbon that enters the marine environment (Leahy and Colwell, 1990; Prince, 2010; Atlas and Hazen, 2011). In response to the natural complexity of hydrocarbon compounds found in petroleum deposits, diverse marine microorganisms have evolved with an equal complexity of metabolic pathways to take advantage of hydrocarbons as a rich carbon and energy source. To minimize the environmental impact of oil spills and to optimize the environmental benefits of biodegradation, it is essential to uncover the metabolic potential of hydrocarbon-degrading bacteria and to address the factors that limit microbially-catalyzed biodegradation in situ

How clonal is clonal? Genome plasticity across multicellular segments of a "candidatus marithrix sp." filament from sulfidic, briny seafloor sediments in the Gulf of Mexico

"Candidatus Marithrix" is a recently described lineage within the group of large sulfur bacteria (Beggiatoaceae, Gammaproteobacteria). This genus of bacteria comprises vacuolated, attached-living filaments that inhabit the sediment surface around vent and seep sites in the marine environment. A single filament is ca. 100 μm in diameter, several millimeters long, and consists of hundreds of clonal cells, which are considered highly polyploid. Based on these characteristics, "Candidatus Marithrix" was used as a model organism for the assessment of genomic plasticity along segments of a single filament using next generation sequencing to possibly identify hotspots of microevolution. Using six consecutive segments of a single filament sampled from a mud volcano in the Gulf of Mexico, we recovered ca. 90% of the "Candidatus Marithrix" genome in each segment. There was a high level of genome conservation along the filament with average nucleotide identities between 99.98 and 100%. Different approaches to assemble all reads into a complete consensus genome could not fill the gaps. Each of the six segment datasets encoded merely a few hundred unique nucleotides and 5 or less unique genes-the residual content was redundant in all datasets. Besides the overall high genomic identity, we identified a similar number of single nucleotide polymorphisms (SNPs) between the clonal segments, which are comparable to numbers reported for other clonal organisms. An increase of SNPs with greater distance of filament segments was not observed. The polyploidy of the cells was apparent when analyzing the heterogeneity of reads within a segment. Here, a strong increase in single nucleotide variants, or "intrasegmental sequence heterogeneity" (ISH) events, was observed. These sites may represent hotspots for genome plasticity, and possibly microevolution, since two thirds of these variants were not co-localized across the genome copies of the multicellular filament

Microbial ecology and biogeochemistry of hypersaline sediments in Orca Basin

In deep ocean hypersaline basins, the combination of high salinity, unusual ionic composition and anoxic conditions represents significant challenges for microbial life. We used geochemical porewater characterization and DNA sequencing based taxonomic surveys to enable environmental and microbial characterization of anoxic hypersaline sediments and brines in the Orca Basin, the largest brine basin in the Gulf of Mexico. Full-length bacterial 16S rRNA gene clone libraries from hypersaline sediments and the overlying brine were dominated by the uncultured halophilic KB1 lineage, Deltaproteobacteria related to cultured sulfate-reducing halophilic genera, and specific lineages of heterotrophic Bacteroidetes. Archaeal clones were dominated by members of the halophilic methanogen genus Methanohalophilus, and the ammonia-oxidizing Marine Group I (MG-I) within the Thaumarchaeota. Illumina sequencing revealed higher phylum- and subphylum-level complexity, especially in lower-salinity sediments from the Orca Basin slope. Illumina and clone library surveys consistently detected MG-I Thaumarchaeota and halotolerant Deltaproteobacteria in the hypersaline anoxic sediments, but relative abundances of the KB1 lineage differed between the two sequencing methods. The stable isotopic composition of dissolved inorganic carbon and methane in porewater, and sulfate concentrations decreasing downcore indicated methanogenesis and sulfate reduction in the anoxic sediments. While anaerobic microbial processes likely occur at low rates near their maximal salinity thresholds in Orca Basin, long-term accumulation of reaction products leads to high methane concentrations and reducing conditions within the Orca Basin brine and sediments

Polysaccharide hydrolysis in the presence of oil and dispersants: Insights into potential degradation pathways of exopolymeric substances (EPS) from oil-degrading bacteria

Oceanic oil-degrading bacteria produce copious amounts of exopolymeric substances (EPS) that facilitate their access to oil. The fate of EPS in the water column is in part determined by activities of heterotrophic microbes capable of utilizing EPS compounds as carbon and energy sources. To evaluate the potential of natural microbial communities to degrade EPS produced during oil degradation, we measured potential hydrolysis rates of six structurally distinct polysaccharides in two roller bottle experiments, using water from a natural oil seep in the northern Gulf of Mexico. The suite of polysaccharides used to measure the initial step in carbon degradation is indicative of polymers within microbial EPS. The treatments included (i) unamended surface or deep waters (whole water), and water amended with (ii) a water-accommodated fraction of oil (WAF), (iii) oil dispersant Corexit 9500, and (iv) WAF chemically-enhanced with Corexit (CEWAF). The oil and Corexit treatments were employed to simulate conditions during the Deepwater Horizon oil spill. Polysaccharide hydrolysis rates in the surface-water treatments were lowest in the WAF treatment, despite elevated levels of EPS in the form of transparent exopolymer particles (TEP). In contrast, the three deep-water treatments (WAF, Corexit, CEWAF) showed enhanced hydrolysis rates and TEP levels (WAF) compared to the whole water. We also observed variations in the spectrum of polysaccharide-hydrolyzing enzyme activities among the treatments. These substrate specificities were likely driven by activities of oil-degrading bacteria, shaping the pool of EPS and TEP as well as degradation products of hydrocarbons and Corexit compounds. A model calculation of potential turnover rates of organic carbon within the TEP pool suggests extended residence times of TEP in oil-contaminated waters, making them prone to serve as the sticky matrix for oily aggregates known as marine oil snow

Remarkable Capacity for Anaerobic Oxidation of Methane at High Methane Concentration

Anaerobic oxidation of methane (AOM), a central process in the carbon cycle of anoxic environments, moderates the release of methane from soils and sediments to water bodies and, ultimately, the atmosphere. The regulation of AOM in the environment remains poorly constrained. Here we quantified AOM and sulfate reduction (SR) rates in diverse deep seafloor samples at in situ pressure and methane concentration and discovered that, in some cases, AOM exceeded SR rates by more than four times when methane concentrations were above 5 mM. Methane concentration also affected other carbon-cycling processes (e.g., carbon assimilation) in addition to SR. These results illustrate that substantial amounts of methane may be oxidized independent of SR under in situ conditions, reshaping our view of the capacity and mechanism of AOM in methane-rich environments, including the deep biosphere, where sulfate availability is considered to limit AOM

Pulsed blooms and persistent oil-degrading bacterial populations in the water column during and after the Deepwater Horizon blowout

One of the defining features of the Deepwater Horizon oil spill was the rapid formation and persistence of a hydrocarbon plume in deep water. Here we use 16S rRNA gene clone libraries and pyrosequencing of 16S rRNA gene fragments to outline the temporal dynamics of the bacterial community in the water column near the Macondo wellhead. Our timeline starts with the pre-spill (March 2010) status of the water column bacterial community, continues through the bacterial enrichments dominating the hydrocarbon plume after the blowout (DWH Oceanospirillales, Cycloclasticus, Colwellia in late May 2010), and leads towards post-spill bacterial communities with molecular signatures related to degradation of phytoplankton pulses (September and October 2010; July 2011) in the water column near the Macondo wellhead. We document a dramatic transition as the complex bacterial community before the oil spill was temporarily overwhelmed by a few specialized bacterial groups responding to the massive influx of hydrocarbons in May 2010. In September and October 2010, this bacterial bloom had been replaced by a diversified bacterial community which resembled its predecessor prior to the spill. Notably, the post-plume 16S rRNA gene clone libraries and pyrosequencing datasets illustrated the continued presence of oil-degrading bacteria in the water column near the Macondo wellhead which we posit to represent an inherent signature of hydrocarbon catabolic potential to the Gulf of Mexico. The pyroseqencing results detected and tracked minority bacterial populations that were not visible in the conventional 16S rRNA gene clone libraries and allowed us to identify natural reservoirs of the Deepwater Horizon Oceanospirillales within and outside of the Gulf of Mexico

Multiple evidence for methylotrophic methanogenesis as the dominant methanogenic pathway in hypersaline sediments from the Orca Basin, Gulf of Mexico

Among the most extreme habitats on Earth, dark, deep, anoxic brines host unique microbial ecosystems that remain largely unexplored. As the terminal step of anaerobic degradation of organic matter, methanogenesis is a potentially significant but poorly constrained process in deep-sea hypersaline environments. We combined biogeochemical and phylogenetic analyses with incubation experiments to unravel the origin of methane in the hypersaline sediments of Orca Basin in the northern Gulf of Mexico. Substantial concentrations of methane, up to 3.4 mM, coexisted with high concentrations of sulfate from 16 to 43 mM in two sediment cores retrieved from the northern and southern parts of Orca Basin. The strong depletion of 13C in methane (-77‰ to -89‰) points towards a biological source. While low concentrations of competitive substrates limited the significance of hydrogenotrophic and acetoclastic methanogenesis, the presence of non-competitive methylated substrates (methanol, trimethylamine, dimethyl sulfide, dimethylsulfoniopropionate) supported the potential for methane generation through methylotrophic methanogenesis. Thermodynamic calculations demonstrated that hydrogenotrophic and acetoclastic methanogenesis were unlikely to occur under in situ conditions, while methylotrophic methanogenesis from a variety of substrates was highly favorable. Likewise, carbon isotope relationships between methylated substrates and methane suggested methylotrophic methanogenesis was the major source of methane. Stable and radio-isotope tracer experiments with 13C-labeled bicarbonate, acetate and methanol and 14C-labeled methylamine indicated that methylotrophic methanogenesis was the predominant methanogenic pathway. Based on 16S rRNA gene sequences, halophilic methylotrophic methanogens related to the genus Methanohalophilus dominated the benthic archaeal community in the northern basin and also occurred in the southern basin. High abundances of methanogen lipid biomarkers such as intact polar and polyunsaturated hydroxyarchaeols were detected in sediments from the northern basin, with lower abundances in the southern basin. Strong 13C-depletion of saturated and monounsaturated hydroxyarchaeol were consistent with methylotrophic methanogenesis as the major methanogenic pathway. Collectively, the availability of methylated substrates, thermodynamic calculations, experimentally determined methanogenic activity as well as lipid and gene biomarkers support the hypothesis that methylotrophic methanogenesis is the predominant pathway of methane formation in the presence of sulfate in Orca Basin sediments

Generation and Utilization of Volatile Fatty Acids and Alcohols in Hydrothermally Altered Sediments in the Guaymas Basin, Gulf of California

Volatile fatty acids (VFAs) and alcohols are key intermediates of anaerobic carbon metabolism, yet their biogeochemical cycling remains poorly constrained in hydrothermal systems. We investigated the abundance, stable carbon isotopic composition, and metabolic cycling of VFAs and alcohols to elucidate their generation and utilization pathways in hydrothermally influenced sediments (4 °C to 90 °C) from the Guaymas Basin. Acetate (up to 229 μM) and methanol (up to 37 μM) were abundant in porewaters. The δ13C values of acetate varied between −35.6‰ and −18.1‰. Carbon isotopic signatures, thermodynamic predictions, and experimental incubations suggested biological sources such as fermentation and acetogenesis for acetate. Acetate and methanol were predominantly consumed by nonmethanogenic processes (e.g., sulfate reduction), as reflected in high oxidation rates versus low methanogenesis rates, and further evidenced through inhibition experiments with molybdate. These results reveal an important role for VFAs and alcohols as energy sources for diverse chemoheterotrophs in organic-rich hydrothermally influenced sediments