Scott D. Wankel

Hydrogen is an energy source for hydrothermal vent symbioses

The discovery of deep-sea hydrothermal vents in 1977 revolutionized our understanding of the energy sources that fuel primary productivity on Earth. Hydrothermal vent ecosystems are dominated by animals that live in symbiosis with chemosynthetic bacteria. So far, only two energy sources have been shown to power chemosynthetic symbioses: reduced sulphur compounds and methane. Using metagenome sequencing, single-gene fluorescence in situ hybridization, immunohistochemistry, shipboard incubations and in situ mass spectrometry, we show here that the symbionts of the hydrothermal vent mussel Bathymodiolus from the Mid-Atlantic Ridge use hydrogen to power primary production. In addition, we show that the symbionts of Bathymodiolus mussels from Pacific vents have hupL, the key gene for hydrogen oxidation. Furthermore, the symbionts of other vent animals such as the tubeworm Riftia pachyptila and the shrimp Rimicaris exoculata also have hupL. We propose that the ability to use hydrogen as an energy source is widespread in hydrothermal vent symbioses, particularly at sites where hydrogen is abundant.

Spatial Variability in Nitrification Rates and Ammonia-Oxidizing Microbial Communities in the Agriculturally Impacted Elkhorn Slough Estuary, California

ABSTRACT Ammonia oxidation—the microbial oxidation of ammonia to nitrite and the first step in nitrification—plays a central role in nitrogen cycling in coastal and estuarine systems. Nevertheless, questions remain regarding the connection between this biogeochemical process and the diversity and abundance of the mediating microbial community. In this study, we measured nutrient fluxes and rates of sediment nitrification in conjunction with the diversity and abundance of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing betaproteobacteria (β-AOB). Sediments were examined from four sites in Elkhorn Slough, a small agriculturally impacted coastal California estuary that opens into Monterey Bay. Using an intact sediment core flowthrough incubation system, we observed significant correlations among NO3 −, NO2 −, NH4 +, and PO4 3+ fluxes, indicating a tight coupling of sediment biogeochemical processes. 15N-based measurements of nitrification rates revealed higher rates at the less impacted, lower-nutrient sites than at the more heavily impacted, nutrient-rich sites. Quantitative PCR analyses revealed that β-AOB amoA (encoding ammonia monooxygenase subunit A) gene copies outnumbered AOA amoA gene copies by factors ranging from 2- to 236-fold across the four sites. Sites with high nitrification rates primarily contained marine/estuarine Nitrosospira-like bacterial amoA sequences and phylogenetically diverse archaeal amoA sequences. Sites with low nitrification rates were dominated by estuarine Nitrosomonas-like amoA sequences and archaeal amoA sequences similar to those previously described in soils. This is the first report measuring AOA and β-AOB amoA abundance in conjunction with 15N-based nitrification rates in estuary sediments.

Dominance of sulfur-fueled iron oxide reduction in low-sulfate freshwater sediments.

A central tenant in microbial biogeochemistry is that microbial metabolisms follow a predictable sequence of terminal electron acceptors based on the energetic yield for the reaction. It is thereby oftentimes assumed that microbial respiration of ferric iron outcompetes sulfate in all but high-sulfate systems, and thus sulfide has little influence on freshwater or terrestrial iron cycling. Observations of sulfate reduction in low-sulfate environments have been attributed to the presumed presence of highly crystalline iron oxides allowing sulfate reduction to be more energetically favored. Here we identified the iron-reducing processes under low-sulfate conditions within columns containing freshwater sediments amended with structurally diverse iron oxides and fermentation products that fuel anaerobic respiration. We show that despite low sulfate concentrations and regardless of iron oxide substrate (ferrihydrite, Al-ferrihydrite, goethite, hematite), sulfidization was a dominant pathway in iron reduction. This process was mediated by (re)cycling of sulfur upon reaction of sulfide and iron oxides to support continued sulfur-based respiration—a cryptic sulfur cycle involving generation and consumption of sulfur intermediates. Although canonical iron respiration was not observed in the sediments amended with the more crystalline iron oxides, iron respiration did become dominant in the presence of ferrihydrite once sulfate was consumed. Thus, despite more favorable energetics, ferrihydrite reduction did not precede sulfate reduction and instead an inverse redox zonation was observed. These findings indicate that sulfur (re)cycling is a dominant force in iron cycling even in low-sulfate systems and in a manner difficult to predict using the classical thermodynamic ladder.

Anaerobic Methane Oxidation in Metalliferous Hydrothermal Sediments: Influence on Carbon Flux and Decoupling from Sulfate Reduction

The anaerobic oxidation of methane (AOM) is a globally significant sink that regulates methane flux from sediments into the oceans and atmosphere. Here we examine mesophilic to thermophilic AOM in hydrothermal sediments recovered from the Middle Valley vent field, on the Juan de Fuca Ridge. Using continuous-flow sediment bioreactors and batch incubations, we characterized (i) the degree to which AOM contributes to net dissolved inorganic carbon flux, (ii) AOM and sulfate reduction (SR) rates as a function of temperature and (iii) the distribution and density of known anaerobic methanotrophs (ANMEs). In sediment bioreactors, inorganic carbon stable isotope mass balances results indicated that AOM accounted for between 16% and 86% of the inorganic carbon produced, underscoring the role of AOM in governing inorganic carbon flux from these sediments. At 90°C, AOM occurred in the absence of SR, demonstrating a striking decoupling of AOM from SR. An abundance of Fe(III)-bearing minerals resembling mixed valent Fe oxides, such as green rust, suggests the potential for a coupling of AOM to Fe(III) reduction in these metalliferous sediments. While SR bacteria were only observed in cooler temperature sediments, ANMEs allied to ANME-1 ribotypes, including a putative ANME-1c group, were found across all temperature regimes and represented a substantial proportion of the archaeal community. In concert, these results extend and reshape our understanding of the nature of high temperature methane biogeochemistry, providing insight into the physiology and ecology of thermophilic anaerobic methanotrophy and suggesting that AOM may play a central role in regulating biological dissolved inorganic carbon fluxes to the deep ocean from the organic-poor, metalliferous sediments of the global mid-ocean ridge hydrothermal vent system.

Enriched iron(III)-reducing bacterial communities are shaped by carbon substrate and iron oxide mineralogy

Iron (Fe) oxides exist in a spectrum of structures in the environment, with ferrihydrite widely considered the most bioavailable phase. Yet, ferrihydrite is unstable and rapidly transforms to more crystalline Fe(III) oxides (e.g., goethite, hematite), which are poorly reduced by model dissimilatory Fe(III)-reducing microorganisms. This begs the question, what processes and microbial groups are responsible for reduction of crystalline Fe(III) oxides within sedimentary environments? Further, how do changes in Fe mineralogy shape oxide-hosted microbial populations? To address these questions, we conducted a large-scale cultivation effort using various Fe(III) oxides (ferrihydrite, goethite, hematite) and carbon substrates (glucose, lactate, acetate) along a dilution gradient to enrich for microbial populations capable of reducing Fe oxides spanning a wide range of crystallinities and reduction potentials. While carbon source was the most important variable shaping community composition within Fe(III)-reducing enrichments, both Fe oxide type and sediment dilution also had a substantial influence. For instance, with acetate as the carbon source, only ferrihydrite enrichments displayed a significant amount of Fe(III) reduction and the well-known dissimilatory metal reducer Geobacter sp. was the dominant organism enriched. In contrast, when glucose and lactate were provided, all three Fe oxides were reduced and reduction coincided with the presence of fermentative (e.g., Enterobacter spp.) and sulfate-reducing bacteria (e.g., Desulfovibrio spp.). Thus, changes in Fe oxide structure and resource availability may shift Fe(III)-reducing communities between dominantly metal-respiring to fermenting and/or sulfate-reducing organisms which are capable of reducing more recalcitrant Fe phases. These findings highlight the need for further targeted investigations into the composition and activity of speciation-directed metal-reducing populations within natural environments.

Microbial activity in the marine deep biosphere: progress and prospects

The vast marine deep biosphere consists of microbial habitats within sediment, pore waters, upper basaltic crust and the fluids that circulate throughout it. A wide range of temperature, pressure, pH, and electron donor and acceptor conditions exists—all of which can combine to affect carbon and nutrient cycling and result in gradients on spatial scales ranging from millimeters to kilometers. Diverse and mostly uncharacterized microorganisms live in these habitats, and potentially play a role in mediating global scale biogeochemical processes. Quantifying the rates at which microbial activity in the subsurface occurs is a challenging endeavor, yet developing an understanding of these rates is essential to determine the impact of subsurface life on Earths global biogeochemical cycles, and for understanding how microorganisms in these “extreme” environments survive (or even thrive). Here, we synthesize recent advances and discoveries pertaining to microbial activity in the marine deep subsurface, and we highlight topics about which there is still little understanding and suggest potential paths forward to address them. This publication is the result of a workshop held in August 2012 by the NSF-funded Center for Dark Energy Biosphere Investigations (C-DEBI) “theme team” on microbial activity (www.darkenergybiosphere.org).

Levels of mercury in alligators (Alligator mississippiensis) collected along a transect through the Florida Everglades

As part of a multi-agency study of alligator health, 28 American alligators (Alligator mississippiensis) were captured along a transect through the Florida Everglades in 1999. Liver and tail muscle tissues were sampled and analyzed on a wet weight basis for total mercury (THg) using cold-vapor atomic absorption spectrophotometry. All tissues had detectable concentrations of THg that ranged from 0.6 to 17 mg/kg in liver and from 0.1 to 1.8 mg/kg in tail muscle. THg was more concentrated in liver tissue than tail muscle, but levels were highly correlated between tissues. THg concentrations in tissue differed significantly among locations, with animals from Everglades National Park (ENP) having mean concentrations of THg in liver (10.4 mg/kg) and tail muscle (1.2 mg/kg) that were two-fold higher than basin-wide averages (4.9 and 0.64 mg/kg, respectively). The reasons for higher contamination of ENP alligators were unclear and could not be explained by differences in sex, length, weight or animal age. While delta15N values were positively correlated with THg concentrations in tail muscle, spatial patterns in isotopic composition did not explain the elevated THg levels in ENP alligators. Therefore, it appears that ENP alligators were more highly exposed to mercury in their environment than individuals in other areas. Comparisons to a previous survey by Yanochko et al. [Arch Environ Contam Toxicol 32 (1997) 323] suggest that mercury levels have declined in some Everglades alligators since 1994.

Using sulfur isotopes to elucidate the origin of barite associated with high organic matter accumulation events in marine sediments

Events of widespread deposition of organic-carbon–rich marine sediments, identifi ed as ocean anoxic events, occurred in the middle of the Cretaceous. Similar deposits termed sapropels occurred during the Pliocene and Pleistocene in the Mediterranean Basin. High biological productivity and/or anoxia have been invoked as possible causes for these widespread high organic carbon deposition events. We use the S isotopic composition of barite associated with these events to confi rm that high barite accumulation rates are a result of elevated marine biological productivity and not a diagenetic artifact. The accumulation and good preservation of biogenic barite, which dissolves when pore-water sulfate concentrations are low, in association with high organic matter and authigenic pyrite, indicates that the rate of bacterial sulfate reduction was low enough for downward diffusion of seawater sulfate to replenish the pore water and prevent depletion of sulfate. The organic C to S burial ratios in samples with high barite accumulation is typically high (>5 wt ratio), supporting burial in high-productivity open-ocean regions, where pyrite formation is restricted.

Isotopic overprinting of nitrification on denitrification as a ubiquitous and unifying feature of environmental nitrogen cycling

Significance Stable isotopes of nitrate have long provided a tool for tracking environmental sources and biological transformations. However, divergent interpretations of fundamental nitrate isotope systematics exist among disciplinary divisions. In an effort to transcend disciplinary boundaries of terrestrial and marine biogeochemistry, we use a quantitative model for coupled nitrogen and oxygen isotopes of nitrate founded on benchmarks established from microbial cultures, to reconcile decades of nitrate isotopic measurements in freshwater and seawater and move toward a unified understanding of cycling processes and isotope systematics. Our findings indicate that denitrification operates within the pervasive context of nitrite reoxidation mechanisms, specifically highlighting the relative importance of nitrification in marine denitrifying systems and anammox in groundwater aquifers. Natural abundance nitrogen and oxygen isotopes of nitrate (δ15NNO3 and δ18ONO3) provide an important tool for evaluating sources and transformations of natural and contaminant nitrate (NO3−) in the environment. Nevertheless, conventional interpretations of NO3− isotope distributions appear at odds with patterns emerging from studies of nitrifying and denitrifying bacterial cultures. To resolve this conundrum, we present results from a numerical model of NO3− isotope dynamics, demonstrating that deviations in δ18ONO3 vs. δ15NNO3 from a trajectory of 1 expected for denitrification are explained by isotopic over-printing from coincident NO3− production by nitrification and/or anammox. The analysis highlights two driving parameters: (i) the δ18O of ambient water and (ii) the relative flux of NO3− production under net denitrifying conditions, whether catalyzed aerobically or anaerobically. In agreement with existing analyses, dual isotopic trajectories >1, characteristic of marine denitrifying systems, arise predominantly under elevated rates of NO2− reoxidation relative to NO3− reduction (>50%) and in association with the elevated δ18O of seawater. This result specifically implicates aerobic nitrification as the dominant NO3− producing term in marine denitrifying systems, as stoichiometric constraints indicate anammox-based NO3− production cannot account for trajectories >1. In contrast, trajectories <1 comprise the majority of model solutions, with those representative of aquifer conditions requiring lower NO2− reoxidation fluxes (<15%) and the influence of the lower δ18O of freshwater. Accordingly, we suggest that widely observed δ18ONO3 vs. δ15NNO3 trends in freshwater systems (<1) must result from concurrent NO3− production by anammox in anoxic aquifers, a process that has been largely overlooked.

Nitrate dynamics within the Pajaro River, a nutrient-rich, losing stream

Abstract The major ion chemistry of water from an 11.42-km reach of the Pajaro River, a losing stream in central coastal California, shows a consistent pattern of higher concentrations during the 2nd (dry) half of the water year. Most solutes are conserved during flow along the reach, but [NO3−] decreases by ~30% and is accompanied by net loss of channel discharge and extensive surface–subsurface exchange. The corresponding net NO3− uptake length is 37 ± 13 km (42 ± 12 km when normalized to the conservative solute Cl−), and the areal NO3− uptake rate is 0.5 μmol m−2 s−1. The observed reduction in [NO3−] along the reach results from one or more internal sinks, not dilution by ground water, hill-slope water, or other water inputs. Observed reductions in [NO3−] and channel discharge along the experimental reach result in a net loss of 200–400 kg/d of NO3−-N, ~50% of the input load. High-resolution (temporal and spatial) sampling indicates that most of the NO3− loss occurs along the lower part of the reach, where there is the greatest seepage loss and surface–subsurface exchange of water. Stable isotopes of NO3−, total dissolved P concentrations, and streambed chemical profiles suggest that denitrification is the most significant NO3− sink along the reach. Denitrification efficiency, as expressed through downstream enrichment in 15N-NO3−, varies considerably during the water year. When discharge is greater (typically earlier in the water year), denitrification is least efficient and downstream enrichment in 15N-NO3− is greatest. When discharge is lower, denitrification in the streambed appears to occur with greater efficiency, resulting in lower downstream enrichment in 15N-NO3−.