Microorganisms oxidize glucose through distinct pathways in permeable and cohesive sediments.

In marine sediments, microbial degradation of organic matter under anoxic conditions is generally thought to proceed through fermentation to volatile fatty acids, which are then oxidized to CO2 coupled to the reduction of terminal electron acceptors (e.g. nitrate, iron, manganese, and sulfate). It has been suggested that, in environments with a highly variable oxygen regime, fermentation mediated by facultative anaerobic bacteria (uncoupled to external terminal electron acceptors) becomes the dominant process. Here, we present the first direct evidence for this fermentation using a novel differentially labeled glucose isotopologue assay that distinguishes between CO2 produced from respiration and fermentation. Using this approach, we measured the relative contribution of respiration and fermentation of glucose in a range of permeable (sandy) and cohesive (muddy) sediments, as well as four bacterial isolates. Under anoxia, microbial communities adapted to high-energy sandy or bioturbated sites mediate fermentation via the Embden-Meyerhof-Parnas pathway, in a manner uncoupled from anaerobic respiration. Prolonged anoxic incubation suggests that this uncoupling lasts up to 160 h. In contrast, microbial communities in anoxic muddy sediments (smaller median grain size) generally completely oxidized 13C glucose to 13CO2, consistent with the classical redox cascade model. We also unexpectedly observed that fermentation occurred under oxic conditions in permeable sediments. These observations were further confirmed using pure cultures of four bacteria isolated from permeable sediments. Our results suggest that microbial communities adapted to variable oxygen regimes metabolize glucose (and likely other organic molecules) through fermentation uncoupled to respiration during transient anoxic conditions.

Molecular hydrogen in seawater supports growth of diverse marine bacteria.

Molecular hydrogen (H2) is an abundant and readily accessible energy source in marine systems, but it remains unknown whether marine microbial communities consume this gas. Here we use a suite of approaches to show that marine bacteria consume H2 to support growth. Genes for H2-uptake hydrogenases are prevalent in global ocean metagenomes, highly expressed in metatranscriptomes and found across eight bacterial phyla. Capacity for H2 oxidation increases with depth and decreases with oxygen concentration, suggesting that H2 is important in environments with low primary production. Biogeochemical measurements of tropical, temperate and subantarctic waters, and axenic cultures show that marine microbes consume H2 supplied at environmentally relevant concentrations, yielding enough cell-specific power to support growth in bacteria with low energy requirements. Conversely, our results indicate that oxidation of carbon monoxide (CO) primarily supports survival. Altogether, H2 is a notable energy source for marine bacteria and may influence oceanic ecology and biogeochemistry.

Pulses of labile carbon cause transient decoupling of fermentation and respiration in permeable sediments.

Dihydrogen (H2) is an important intermediate in anaerobic microbial processes, and concentrations are tightly controlled by thermodynamic limits of consumption and production. However, recent studies reported unusual H2 accumulation in permeable marine sediments under anoxic conditions, suggesting decoupling of fermentation and sulfate reduction, the dominant respiratory process in anoxic permeable marine sediments. Yet, the extent, prevalence and potential triggers for such H2 accumulation and decoupling remain unknown. We surveyed H2 concentrations in situ at different settings of permeable sand and found that H2 accumulation was only observed during a coral spawning event on the Great Barrier Reef. A flume experiment with organic matter addition to the water column showed a rapid accumulation of hydrogen within the sediment. Laboratory experiments were used to explore the effect of oxygen exposure, physical disturbance and organic matter inputs on H2 accumulation. Oxygen exposure had little effect on H2 accumulation in permeable sediments suggesting both fermenters and sulfate reducers survive and rapidly resume activity after exposure to oxygen. Mild physical disturbance mimicking sediment resuspension had little effect on H2 accumulation; however, vigorous shaking led to a transient accumulation of H2 and release of dissolved organic carbon suggesting mechanical disturbance and cell destruction led to organic matter release and transient decoupling of fermenters and sulfate reducers. In summary, the highly dynamic nature of permeable sediments and its microbial community allows for rapid but transient decoupling of fermentation and respiration after a C pulse, leading to high H2 levels in the sediment.

Hydrodynamic disturbance controls microbial community assembly and biogeochemical processes in coastal sediments.

The microbial community composition and biogeochemical dynamics of coastal permeable (sand) sediments differs from cohesive (mud) sediments. Tide- and wave-driven hydrodynamic disturbance causes spatiotemporal variations in oxygen levels, which select for microbial generalists and disrupt redox cascades. In this work, we profiled microbial communities and biogeochemical dynamics in sediment profiles from three sites varying in their exposure to hydrodynamic disturbance. Strong variations in sediment geochemistry, biogeochemical activities, and microbial abundance, composition, and capabilities were observed between the sites. Most of these variations, except for microbial abundance and diversity, significantly correlated with the relative disturbance level of each sample. In line with previous findings, metabolically flexible habitat generalists (e.g., Flavobacteriaceae, Woeseaiceae, Rhodobacteraceae) dominated in all samples. However, we present evidence that aerobic specialists such as ammonia-oxidizing archaea (Nitrosopumilaceae) were more abundant and active in more disturbed samples, whereas bacteria capable of sulfate reduction (e.g., uncultured Desulfobacterales), dissimilatory nitrate reduction to ammonium (DNRA; e.g., Ignavibacteriaceae), and sulfide-dependent chemolithoautotrophy (e.g., Sulfurovaceae) were enriched and active in less disturbed samples. These findings are supported by insights from nine deeply sequenced metagenomes and 169 derived metagenome-assembled genomes. Altogether, these findings suggest that hydrodynamic disturbance is a critical factor controlling microbial community assembly and biogeochemical processes in coastal sediments. Moreover, they strengthen our understanding of the relationships between microbial composition and biogeochemical processes in these unique environments.

Metabolic flexibility allows bacterial habitat generalists to become dominant in a frequently disturbed ecosystem.

Ecological theory suggests that habitat disturbance differentially influences distributions of habitat generalist and specialist species. While well-established for macroorganisms, this theory has rarely been explored for microorganisms. Here we tested these principles in permeable (sandy) sediments, ecosystems with much spatiotemporal variation in resource availability and physicochemical conditions. Microbial community composition and function were profiled in intertidal and subtidal sediments using 16S rRNA gene amplicon sequencing and metagenomics, yielding 135 metagenome-assembled genomes. Community composition and metabolic traits modestly varied with sediment depth and sampling date. Several taxa were highly abundant and prevalent in all samples, including within the orders Woeseiales and Flavobacteriales, and classified as habitat generalists; genome reconstructions indicate these taxa are highly metabolically flexible facultative anaerobes and adapt to resource variability by using different electron donors and acceptors. In contrast, obligately anaerobic taxa such as sulfate reducers and candidate lineage MBNT15 were less abundant overall and only thrived in more stable deeper sediments. We substantiated these findings by measuring three metabolic processes in these sediments; whereas the habitat generalist-associated processes of sulfide oxidation and fermentation occurred rapidly at all depths, the specialist-associated process of sulfate reduction was restricted to deeper sediments. A manipulative experiment also confirmed habitat generalists outcompete specialist taxa during simulated habitat disturbance. Together, these findings show metabolically flexible habitat generalists become dominant in highly dynamic environments, whereas metabolically constrained specialists are restricted to narrower niches. Thus, an ecological theory describing distribution patterns for macroorganisms likely extends to microorganisms. Such findings have broad ecological and biogeochemical ramifications.

Can We Use Functional Genetics to Predict the Fate of Nitrogen in Estuaries?

Increasing nitrogen (N) loads present a threat to estuaries, which are among the most heavily populated and perturbed parts of the world. N removal is largely mediated by the sediment microbial process of denitrification, in direct competition to dissimilatory nitrate reduction to ammonium (DNRA), which recycles nitrate to ammonium. Molecular proxies for N pathways are increasingly measured and analyzed, a major question in microbial ecology, however, is whether these proxies can add predictive power around the fate of N. We analyzed the diversity and community composition of sediment nirS and nrfA genes in 11 temperate estuaries, covering four types of land use in Australia, and analyzed how these might be used to predict N removal. Our data suggest that sediment microbiomes play a central role in controlling the magnitude of the individual N removal rates in the 11 estuaries. Inclusion, however, of relative gene abundances of 16S, nirS, nrfA, including their ratios did not improve physicochemical measurement-based regression models to predict rates of denitrification or DNRA. Co-occurrence network analyses of nirS showed a greater modularity and a lower number of keystone OTUs in pristine sites compared to urban estuaries, suggesting a higher degree of niche partitioning in pristine estuaries. The distinctive differences between the urban and pristine network structures suggest that the nirS gene could be a likely gene candidate to understand the mechanisms by which these denitrifying communities form and respond to anthropogenic pressures.

Bacterial fermentation and respiration processes are uncoupled in anoxic permeable sediments.

Permeable (sandy) sediments cover half of the continental margin and are major regulators of oceanic carbon cycling. The microbial communities within these highly dynamic sediments frequently shift between oxic and anoxic states, and hence are less stratified than those in cohesive (muddy) sediments. A major question is, therefore, how these communities maintain metabolism during oxic-anoxic transitions. Here, we show that molecular hydrogen (H2) accumulates in silicate sand sediments due to decoupling of bacterial fermentation and respiration processes following anoxia. In situ measurements show that H2 is 250-fold supersaturated in the water column overlying these sediments and has an isotopic composition consistent with fermentative production. Genome-resolved shotgun metagenomic profiling suggests that the sands harbour diverse and specialized microbial communities with a high abundance of [NiFe]-hydrogenase genes. Hydrogenase profiles predict that H2 is primarily produced by facultatively fermentative bacteria, including the dominant gammaproteobacterial family Woeseiaceae, and can be consumed by aerobic respiratory bacteria. Flow-through reactor and slurry experiments consistently demonstrate that H2 is rapidly produced by fermentation following anoxia, immediately consumed by aerobic respiration following reaeration and consumed by sulfate reduction only during prolonged anoxia. Hydrogenotrophic sulfur, nitrate and nitrite reducers were also detected, although contrary to previous hypotheses there was limited capacity for microalgal fermentation. In combination, these experiments confirm that fermentation dominates anoxic carbon mineralization in these permeable sediments and, in contrast to the case in cohesive sediments, is largely uncoupled from anaerobic respiration. Frequent changes in oxygen availability in these sediments may have selected for metabolically flexible bacteria while excluding strict anaerobes.

Role of organic carbon, nitrate and ferrous iron on the partitioning between denitrification and DNRA in constructed stormwater urban wetlands.

Denitrification (DNF) and dissimilatory nitrate reduction to ammonium (DNRA) are two competing nitrate reduction pathways that remove or recycle nitrogen, respectively. However, factors controlling the partitioning between these two pathways are manifold and our understanding of these factors is critical for the management of N loads in constructed wetlands. An important factor that controls DNRA in an aquatic ecosystem is the electron donor, commonly organic carbon (OC) or alternatively ferrous iron and sulfide. In this study, we investigated the role of natural organic carbon (NOC) and acetate at different OC/NO3- ratios and ferrous iron on the partitioning between DNF and DNRA using the 15N-tracer method in slurries from four constructed stormwater urban wetlands in Melbourne, Australia. The carbon and nitrate experiments revealed that DNF dominated at all OC/NO3- ratios. The higher DNF and DNRA rates observed after the addition of NOC indicates that nitrate reduction was enhanced more by NOC than acetate. Moreover, addition of NOC in slurries stimulated DNRA more than DNF. Interestingly, slurries amended with Fe2+ showed that Fe2+ had significant control on the balance between DNF and DNRA. From two out of four wetlands, a significant increase in DNRA rates (p < .05) at the cost of DNF in the presence of available Fe2+ suggests DNRA is coupled to Fe2+ oxidation. Rates of DNRA increased 1.5-3.5 times in the Fe2+ treatment compared to the control. Overall, our study provides direct evidence that DNRA is linked to Fe2+ oxidation in some wetland sediments and highlights the role of Fe2+ in controlling the partitioning between removal (DNF) and recycling (DNRA) of bioavailable N in stormwater urban constructed wetlands. In our study we also measured anammox and found that it was always <0.05% of total nitrate reduction in these sediments.

Bedforms as Biocatalytic Filters: A Pumping and Streamline Segregation Model for Nitrate Removal in Permeable Sediments.

Bedforms are a focal point of carbon and nitrogen cycling in streams and coastal marine ecosystems. In this paper, we develop and test a mechanistic model, the "pumping and streamline segregation" or PASS model, for nitrate removal in bedforms. The PASS model dramatically reduces computational overhead associated with modeling nitrogen transformations in bedforms and reproduces (within a factor of 2 or better) previously published measurements and models of biogeochemical reaction rates, benthic fluxes, and in-sediment nutrient and oxygen concentrations. Application of the PASS model to a diverse set of marine and freshwater environments indicates that (1) physical controls on nitrate removal in a bedform include the pore water flushing rate, residence time distribution, and relative rates of respiration and transport (as represented by the Damkohler number); (2) the biogeochemical pathway for nitrate removal is an environment-specific combination of direct denitrification of stream nitrate and coupled nitrification-denitrification of stream and/or sediment ammonium; and (3) permeable sediments are almost always a net source of dissolved inorganic nitrogen. The PASS model also provides a mechanistic explanation for previously published empirical correlations showing denitrification velocity (N2 flux divided by nitrate concentration) declines as a power law of nitrate concentration in a stream (Mulholland et al. Nature, 2008, 452, 202-205).

Transport zonation limits coupled nitrification-denitrification in permeable sediments.

Measurement of biogeochemical processes in permeable sediments (including the hyporheic zone) is difficult because of complex multidimensional advective transport. This is especially the case for nitrogen cycling, which involves several coupled redox-sensitive reactions. To provide detailed insight into the coupling between ammonification, nitrification and denitrification in stationary sand ripples, we combined the diffusion equilibrium thin layer (DET) gel technique with a computational reactive transport biogeochemical model. The former approach provided high-resolution two-dimensional distributions of NO3(-) and (15)N-N2 gas. The measured two-dimensional profiles correlate with computational model simulations, showing a deep pool of N2 gas forming, and being advected to the surface below ripple peaks. Further isotope pairing calculations on these data indicate that coupled nitrification-denitrification is severely limited in permeable sediments because the flow and transport field limits interaction between oxic and anoxic pore water. The approach allowed for new detailed insight into subsurface denitrification zones in complex permeable sediments.