Diversity at single nucleotide to pangenome scales among sulfur cycling bacteria in salt marshes.

IMPORTANCE: Salt marshes are known for their significant carbon storage capacity, and sulfur cycling is closely linked with the ecosystem-scale carbon cycling in these ecosystems. Sulfate reducers are key for the decomposition of organic matter, and sulfur oxidizers remove toxic sulfide, supporting the productivity of marsh plants. To date, the complexity of coastal environments, heterogeneity of the rhizosphere, high microbial diversity, and uncultured majority hindered our understanding of the genomic diversity of sulfur-cycling microbes in salt marshes. Here, we use comparative genomics to overcome these challenges and provide an in-depth characterization of sulfur-cycling microbial diversity in salt marshes. We characterize communities across distinct sites and plant species and uncover extensive genomic diversity at the taxon level and specific genomic features present in MAGs affiliated with uncultivated sulfur-cycling lineages. Our work provides insights into the partnerships in salt marshes and a roadmap for multiscale analyses of diversity in complex biological systems.

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.

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.

Denitrification Capacity of a Natural and a Restored Marsh in the Northern Gulf of Mexico.

Anthropogenic pressures, such as diking, construction of dams, and oil spills negatively impact coastal marshes creating growing pressure to preserve and to restore salt marshes due to their critical role in permanently removing nitrate runoff through denitrification as well as other ecosystem services they provide. This study determined denitrification rates across a typical northern Gulf of Mexico salt marsh landscape that included a natural marsh, a tidal creek, and a 21-year-old restored salt marsh. Denitrification capacity, measured with the isotope pairing technique on a membrane inlet mass spectrometer, was comparable across the sites despite significant differences in above and below ground characteristics. Total extractable ammonium concentrations and sediment carbon content were higher at the natural marsh compared to the restored marsh. Hydrogen sulfide concentrations were highest at the creek compared to the vegetated sites and lowest at the restored marsh. This suggests that marsh restoration projects reestablish nitrogen removal capacity at rates similar to those in natural systems and can help to significantly reduce nitrogen loads to the coastal ocean.

Nitrate reduction pathways in the presence of excess nitrogen in a shallow eutrophic estuary.

The eutrophication of estuaries results from increasing anthropogenic nutrient inputs to coastal waters. Ecosystem recovery from eutrophication is partly dependent on the ability of a system to assimilate or remove nutrients, and denitrification and dissimilatory nitrate reduction to ammonium (DNRA) are important pathways for nitrogen (N) removal or retention. We measured rates of denitrification and DNRA over an annual cycle at two stations in Weeks Bay, AL, a shallow microtidal estuary receiving freshwater from two rivers with agricultural watersheds and high N inputs. We hypothesized that rates of DNRA would exceed denitrification in the sulfidogenic sediments in this estuary. Consistent with our hypothesis, we found that DNRA (44.4 ±â€¯5.5 µmol N m-2 hr-1) exceeded in situ denitrification (0.9 ±â€¯2.3 µmol N m-2 hr-1) and that even in the presence of abundant water column nitrate DNRA was favored over denitrification by a factor of two. DNRA is estimated to provide N to the water column at a rate equivalent to 15% of the N input that is retained within the estuary and is a significant component of the N budget in this highly impacted estuary. DNRA by retaining N in the system contributes to the N demand by primary producers and can impact this estuary through enhanced rates of primary production. Weeks Bay, like many coastal estuaries, experiences periods of hypoxia, blooms of harmful algae and fish kills. Future management efforts should focus on reducing nutrient input to this estuary without which the significant retention of N in this system through DRNA will contribute to the undesirable ecosystem attributes associated with eutrophication.

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.

Living oysters and their shells as sites of nitrification and denitrification.

Oysters provide a critical habitat, are a food resource for higher trophic levels and support important commercial fisheries throughout the world. Oyster reefs can improve water quality by removing phytoplankton. While sediment denitrification may be enhanced adjacent to oyster reefs, little is known about nitrification and denitrification associated with living oysters and their shells. We measured nitrification and denitrification in living oysters (Crassostrea virginica and Crassostrea gigas) and empty oyster shells. Nitrification was similar between live oysters and empty oyster shells, however, denitrification was enhanced significantly on living oysters compared to shells. This is the first demonstration of nitrification and denitrification associated with living oysters and their shells. Our data suggest that loss of historic oyster reefs has likely affected the resilience of estuaries to eutrophication. The additional benefit of oyster mediated denitrification should be considered in restoration of oyster reefs as a tool for managing eutrophication.

Water uptake of Alaskan tundra evergreens during the winter-spring transition.

PREMISE OF THE STUDY: The cold season in the Arctic extends over 8 to 9 mo, yet little is known about vascular plant physiology during this period. Evergreen species photosynthesize under the snow, implying that they are exchanging water with the atmosphere. However, liquid water available for plant uptake may be limited at this time. The study objective was to determine whether evergreen plants are actively taking up water while under snow and/or immediately following snowmelt during spring thaw. METHODS: In two in situ experiments, one at the plot level and another at the individual species level, (2)H-labeled water was used as a tracer injected beneath the snow, after which plant stems and leaves were tested for the presence of the label. In separate experiments, excised shoots of evergreen species were exposed to (2)H-labeled water for ∼5 s or 60 min and tested for foliar uptake of the label. KEY RESULTS: In both the plot-level and the species-level experiments, some (2)H-labeled water was found in leaves and stems. Additionally, excised individual plant shoots exposed to labeled water for 60 min took up significantly more (2)H-label than shoots exposed ∼5 s. CONCLUSIONS: Evergreen tundra plants take up water under snow cover, some via roots, but also likely by foliar uptake. The ability to take up water in the subnivean environment allows evergreen tundra plants to take advantage of mild spring conditions under the snow and replenish carbon lost by winter respiration.

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.

Impact of crude oil exposure on nitrogen cycling in a previously impacted Juncus roemerianus salt marsh in the northern Gulf of Mexico.

This study investigated potential nitrogen fixation, net nitrification, and denitrification responses to short-term crude oil exposure that simulated oil exposure in Juncus roemerianus salt marsh sediments previously impacted following the Deepwater Horizon accident. Temperature as well as crude oil amount and type affected the nitrogen cycling rates. Total nitrogen fixation rates increased 44 and 194 % at 30 °C in 4,000 mg kg(-1) tar ball and 10,000 mg kg(-1) moderately weathered crude oil treatments, respectively; however, there was no difference from the controls at 10 and 20 °C. Net nitrification rates showed production at 20 °C and consumption at 10 and 30 °C in all oil treatments and controls. Potential denitrification rates were higher than controls in the 10 and 30 ºC treatments but responded differently to the oil type and amount. The highest rates of potential denitrification (12.7 ± 1.0 nmol N g(-1) wet h(-1)) were observed in the highly weathered 4,000 mg kg(-1) oil treatment at 30 °C, suggesting increased rates of denitrification during the warmer summer months. These results indicate that the impacts on nitrogen cycling from a recurring oil spill could depend on the time of the year as well as the amount and type of oil contaminating the marsh. The study provides evidence for impact on nitrogen cycling in coastal marshes that are vulnerable to repeated hydrocarbon exposure.

The degree of urbanization across the globe is not reflected in the δ(15)N of seagrass leaves.

Many studies show that seagrass δ(15)N ratios increase with the amount of urbanization in coastal watersheds. However, there is little information on the relationship between urbanization and seagrass δ(15)N ratios on a global scale. We performed a meta-analysis on seagrass samples from 79 independent locations to test if seagrass δ(15)N ratios correlate with patterns of population density and fertilizer use within a radius of 10-200 km around the sample locations. Our results show that seagrass δ(15)N ratios are more influenced by intergeneric and latitudinal differences than the degree of urbanization or the amount of fertilizer used in nearby watersheds. The positive correlation between seagrass δ(15)N ratios and latitude hints at an underlying pattern in discrimination or a latitudinal gradient in the (15)N isotopic signature of nitrogen assimilated by the plants. The actual mechanisms responsible for the correlation between δ(15)N and latitude remain unknown.

Denitrification in a large river: consideration of geomorphic controls on microbial activity and community structure.

Ecological theory argues that the controls over ecosystem processes are structured hierarchically, with broader-scale drivers acting as constraints over the interactions and dynamics at nested levels of organization. In river ecosystems, these interactions may arise from broadscale variation in channel form that directly shapes benthic habitat structure and indirectly constrains resource supply and biological activity within individual reaches. To evaluate these interactions, we identified sediment characteristics, water chemistry, and denitrifier community structure as factors influencing benthic denitrification rates in a sixth-order river that flows through two physiographic provinces and the transitional zone between them, each with distinct geomorphological properties. We found that denitrification rates tracked spatial changes in sediment characteristics and varied seasonally with expected trends in stream primary production. Highest rates were observed during the spring and summer seasons in the physiographic province dominated by fine-grained sediments, illustrating how large-scale changes in river structure can constrain the location of denitrification hotspots. In addition, nirS and nirK community structure each responded differently to variation in channel form, possibly due to changes in dissolved oxygen and organic matter supply. This shift in denitrifier community structure coincident with higher rates of N removal via denitrification suggests that microbial community structure may influence biogeochemical processes.

Validation and application of cavity-enhanced, near-infrared tunable diode laser absorption spectrometry for measurements of methane carbon isotopes at ambient concentrations.

Methane is an effective greenhouse gas but has a short residence time in the atmosphere, and therefore, reductions in emissions can alleviate its greenhouse gas warming effect within a decadal time frame. Continuous and high temporal resolution measurements of methane concentrations and carbon isotopic ratios (δ(13)CH4) can inform on mechanisms of formation, provide constraints on emissions sources, and guide future mitigation efforts. We describe the development, validation, and deployment of a cavity-enhanced, near-infrared tunable diode laser absorption spectrometry system capable of quantifying δ(13)CH4 at ambient methane concentrations. Laboratory validation and testing show that the instrument is capable of operating over a wide dynamic range of methane concentration and provides a measurement precision for δ(13)CH4 of better than ± 0.5 ‰ (1σ) over 1000 s of data averaging at ambient methane concentrations. The analyzer is accurate to better than ± 0.5 ‰, as demonstrated by measurements of characterized methane/air samples with minimal dependence (<1 ‰) of measured carbon isotope ratio on methane concentration. Deployment of the instrument at a marsh over multiple days demonstrated how methane fluxes varied by an order of magnitude over 2 day deployment periods, and showed a 17 ‰ variability in δ(13)CH4 of the emitted methane during the growing season.

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.

Diurnal patterns of gas-exchange and metabolic pools in tundra plants during three phases of the arctic growing season.

Arctic tundra plant communities are subject to a short growing season that is the primary period in which carbon is sequestered for growth and survival. This period is often characterized by 24-h photoperiods for several months a year. To compensate for the short growing season tundra plants may extend their carbon uptake capacity on a diurnal basis, but whether this is true remains unknown. Here, we examined in situ diurnal patterns of physiological activity and foliar metabolites during the early, mid, and late growing season in seven arctic species under light-saturated conditions. We found clear diurnal patterns in photosynthesis and respiration, with midday peaks and midnight lulls indicative of circadian regulation. Diurnal patterns in foliar metabolite concentrations were less distinct between the species and across seasons, suggesting that metabolic pools are likely governed by proximate external factors. This understanding of diurnal physiology will also enhance the parameterization of process-based models, which will aid in better predicting future carbon dynamics for the tundra. This becomes even more critical considering the rapid changes that are occurring circumpolarly that are altering plant community structure, function, and ultimately regional and global carbon budgets.

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.

Microbial community analysis of a coastal salt marsh affected by the Deepwater Horizon oil spill.

Coastal salt marshes are highly sensitive wetland ecosystems that can sustain long-term impacts from anthropogenic events such as oil spills. In this study, we examined the microbial communities of a Gulf of Mexico coastal salt marsh during and after the influx of petroleum hydrocarbons following the Deepwater Horizon oil spill. Total hydrocarbon concentrations in salt marsh sediments were highest in June and July 2010 and decreased in September 2010. Coupled PhyloChip and GeoChip microarray analyses demonstrated that the microbial community structure and function of the extant salt marsh hydrocarbon-degrading microbial populations changed significantly during the study. The relative richness and abundance of phyla containing previously described hydrocarbon-degrading bacteria (Proteobacteria, Bacteroidetes, and Actinobacteria) increased in hydrocarbon-contaminated sediments and then decreased once hydrocarbons were below detection. Firmicutes, however, continued to increase in relative richness and abundance after hydrocarbon concentrations were below detection. Functional genes involved in hydrocarbon degradation were enriched in hydrocarbon-contaminated sediments then declined significantly (p<0.05) once hydrocarbon concentrations decreased. A greater decrease in hydrocarbon concentrations among marsh grass sediments compared to inlet sediments (lacking marsh grass) suggests that the marsh rhizosphere microbial communities could also be contributing to hydrocarbon degradation. The results of this study provide a comprehensive view of microbial community structural and functional dynamics within perturbed salt marsh ecosystems.

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.

The impact of soil microorganisms on the global budget of delta18O in atmospheric CO2.

Improved global estimates of terrestrial photosynthesis and respiration are critical for predicting the rate of change in atmospheric CO(2). The oxygen isotopic composition of atmospheric CO(2) can be used to estimate these fluxes because oxygen isotopic exchange between CO(2) and water creates distinct isotopic flux signatures. The enzyme carbonic anhydrase (CA) is known to accelerate this exchange in leaves, but the possibility of CA activity in soils is commonly neglected. Here, we report widespread accelerated soil CO(2) hydration. Exchange was 10-300 times faster than the uncatalyzed rate, consistent with typical population sizes for CA-containing soil microorganisms. Including accelerated soil hydration in global model simulations modifies contributions from soil and foliage to the global CO(18)O budget and eliminates persistent discrepancies existing between model and atmospheric observations. This enhanced soil hydration also increases the differences between the isotopic signatures of photosynthesis and respiration, particularly in the tropics, increasing the precision of CO(2) gross fluxes obtained by using the delta(18)O of atmospheric CO(2) by 50%.

Does the (13)C of foliage-respired CO(2) and biochemical pools reflect the (13)C of recently assimilated carbon?

Isotopic labelling experiments were conducted to assess relationships among (13)C of recently assimilated carbon (deltaC(A)), foliage respiration (deltaC(F)), soluble carbohydrate (deltaC(SC)), leaf waxes (deltaC(LW)) and bulk organic matter (deltaC(OM)). Slash pine, sweetgum and maize were grown under (13)C depleted CO(2) to label biomass and then placed under ambient conditions to monitor the loss of label. In pine and sweetgum, deltaC(F) of labelled plants (approximately -44 and -35 per thousand, respectively) rapidly approached control values but remained depleted by approximately 4-6 per thousand after 3-4 months. For these tree species, no or minimal label was lost from deltaC(SC), deltaC(LW) and deltaC(OM) during the observation periods. deltaC(F) and deltaC(SC) of labelled maize plants rapidly changed and were indistinguishable from controls after 1 month, while deltaC(LW) and deltaC(OM) more slowly approached control values and remained depleted by 2-6 per thousand. Changes in deltaC(F) in slash pine and sweetgum fit a two-pool exponential model, with the fast turnover metabolic pool (approximately 3-4 d half-life) constituting only 1-2% of the total. In maize, change in deltaC(F) fits a single pool model with a half-life of 6.4 d. The (13)C of foliage respiration and biochemical pools reflect temporally integrated values of deltaC(A), with change in isotopic composition dampened by the size of metabolic carbon reserves and turnover rates.