Evidence for nontraditional mcr-containing archaea contributing to biological methanogenesis in geothermal springs.

Recent discoveries of methyl-coenzyme M reductase-encoding genes (mcr) in uncultured archaea beyond traditional euryarchaeotal methanogens have reshaped our view of methanogenesis. However, whether any of these nontraditional archaea perform methanogenesis remains elusive. Here, we report field and microcosm experiments based on 13C-tracer labeling and genome-resolved metagenomics and metatranscriptomics, revealing that nontraditional archaea are predominant active methane producers in two geothermal springs. Archaeoglobales performed methanogenesis from methanol and may exhibit adaptability in using methylotrophic and hydrogenotrophic pathways based on temperature/substrate availability. A five-year field survey found Candidatus Nezhaarchaeota to be the predominant mcr-containing archaea inhabiting the springs; genomic inference and mcr expression under methanogenic conditions strongly suggested that this lineage mediated hydrogenotrophic methanogenesis in situ. Methanogenesis was temperature-sensitive , with a preference for methylotrophic over hydrogenotrophic pathways when incubation temperatures increased from 65° to 75°C. This study demonstrates an anoxic ecosystem wherein methanogenesis is primarily driven by archaea beyond known methanogens, highlighting diverse nontraditional mcr-containing archaea as previously unrecognized methane sources.

Controls on the isotopic composition of microbial methane.

Microbial methane production (methanogenesis) is responsible for more than half of the annual emissions of this major greenhouse gas to the atmosphere. Although the stable isotopic composition of methane is often used to characterize its sources and sinks, strictly empirical descriptions of the isotopic signature of methanogenesis currently limit these attempts. We developed a metabolic-isotopic model of methanogenesis by carbon dioxide reduction, which predicts carbon and hydrogen isotopic fractionations, and clumped isotopologue distributions, as functions of the cell's environment. We mechanistically explain multiple isotopic patterns in laboratory and natural settings and show that these patterns constrain the in situ energetics of methanogenesis. Combining our model with data from environments in which methanogenic activity is energy-limited, we provide predictions for the biomass-specific methanogenesis rates and the associated isotopic effects.

Limited Mechanistic Link Between the Monod Equation and Methanogen Growth: a Perspective from Metabolic Modeling.

The Monod equation has been widely applied as the general rate law of microbial growth, but its applications are not always successful. By drawing on the frameworks of kinetic and stoichiometric metabolic models and metabolic control analysis, the modeling reported here simulated the growth kinetics of a methanogenic microorganism and illustrated that different enzymes and metabolites control growth rate to various extents and that their controls peak at either very low, intermediate, or very high substrate concentrations. In comparison, with a single term and two parameters, the Monod equation only approximately accounts for the controls of rate-determining enzymes and metabolites at very high and very low substrate concentrations, but neglects the enzymes and metabolites whose controls are most notable at intermediate concentrations. These findings support a limited link between the Monod equation and methanogen growth, and unify the competing views regarding enzyme roles in shaping growth kinetics. The results also preclude a mechanistic derivation of the Monod equation from methanogen metabolic networks and highlight a fundamental challenge in microbiology: single-term expressions may not be sufficient for accurate prediction of microbial growth. IMPORTANCE The Monod equation has been widely applied to predict the rate of microbial growth, but its application is not always successful. Using a novel metabolic modeling approach, we simulated the growth of a methanogen and uncovered a limited mechanistic link between the Monod equation and the methanogen's metabolic network. Specifically, the equation provides an approximation to the controls by rate-determining metabolites and enzymes at very low and very high substrate concentrations, but it is missing the remaining enzymes and metabolites whose controls are most notable at intermediate concentrations. These results support the Monod equation as a useful approximation of growth rates and highlight a fundamental challenge in microbial kinetics: single-term rate expressions may not be sufficient for accurate prediction of microbial growth.

Combined Effects of Fe(III)-Bearing Nontronite and Organic Ligands on Biogenic U(IV) Oxidation.

Bioreduction of soluble U(VI) to sparingly soluble U(IV) solids was proposed as a remediation method for uranium contamination. Therefore, the stability and longevity of biogenic U(IV) are critical to the success of uranium remediation. However, co-occurrence of clay minerals and organic ligands could potentially reoxidize U(IV) to U(VI). Herein, we report a combined effect of Fe(III)-rich nontronite (NAu-2) and environmentally prevalent organic ligands on reoxidation of biogenic U(IV) at circumneutral pH. After 30 days of incubation, structural Fe(III) in NAu-2 oxidized 45.50% U(IV) with an initial rate of 2.7 × 10-3 mol m-2 d-1. Addition of citrate and ethylenediaminetetraacetic acid (EDTA) greatly promoted the oxidative dissolution of U(IV) by structural Fe(III) in NAu-2, primarily through the formation of aqueous ligand-U(IV) complexes. In contrast, a model siderophore, desferrioxamine B (DFOB), partially inhibited U(IV) oxidation due to the formation of stable DFOB-Fe3+ complexes. The resulting U(VI) species intercalated into an NAu-2 interlayer or adsorbed onto an NAu-2 surface. Our results highlight the importance of organic ligands in oxidative dissolution of U(IV) minerals by Fe(III)-bearing clay minerals and have important implications for the design of nuclear waste storage and remediation strategies, especially in clay- and organic-rich environments.

Influences of pH and substrate supply on the ratio of iron to sulfate reduction.

Iron reduction and sulfate reduction often occur simultaneously in anoxic systems, and where that is the case, the molar ratio between the reactions (i.e., Fe/SO42- reduced) influences their impact on water quality and carbon storage. Previous research has shown that pH and the supply of electron donors and acceptors affect that ratio, but it is unclear how their influences compare and affect one another. This study examines impacts of pH and the supply of acetate, sulfate, and goethite on the ratio of iron to sulfate reduction in semi-continuous sediment bioreactors. We examined which parameter had the greatest impact on that ratio and whether the parameter influences depended on the state of each other. Results show that pH had a greater influence than acetate supply on the ratio of iron to sulfate reduction, and that the impact of acetate supply on the ratio depended on pH. In acidic reactors (pH 6.0 media), the ratio of iron to sulfate reduction decreased from 3:1 to 2:1 as acetate supply increased (0-1 mM). In alkaline reactors (pH 7.5 media), iron and sulfate were reduced in equal proportions, regardless of acetate supply. Secondly, a comparison of experiments with and without sulfate shows that the extent of iron reduction was greater if sulfate reduction was occurring and that the effect was larger in alkaline reactors than acidic reactors. Thus, the influence of sulfate supply on iron reduction extent also depended on pH and suggests that iron reduction grows more dependent on sulfate reduction as pH increases. Our results compare well to trends in groundwater geochemistry and provide further evidence that pH is a major control on iron and sulfate reduction in systems with crystalline (oxyhydr)oxides. pH not only affects the ratio between the reactions but also the influences of other parameters on that ratio.

Promotion of Microbial Oxidation of Structural Fe(II) in Nontronite by Oxalate and NTA.

Iron redox cycling occurs extensively in soils and sediments. Previous research has focused on microbially mediated redox cycling of aqueous Fe. At circumneutral pH, most Fe occurs in solid phase, where Fe and organic ligands interact closely. However, the role of organic ligands in microbial oxidation of solid-phase Fe(II) is not well understood. Here, we incubated reduced nontronite NAu-2 (rNAu-2) with an iron-oxidizing bacterium and in the presence of oxalate and nitrilotriacetic acid. These ligands significantly enhanced the rate and extent of microbial oxidation of structural Fe(II) in rNAu-2. Aqueous and solid-phase analyses, coupled with biogeochemical modeling, revealed a pathway for ligand-enhanced bio-oxidation of solid-phase Fe(II): (1) dissolution of rNAu-2 to form aqueous Fe(II)-ligand complex; (2) bio-oxidation to Fe(III)-ligand complex; (3) rapid reduction of Fe(III)-ligand complex to Fe(II)-ligand complex by structural Fe(II) in rNAu-2. In this process, the Fe(II)-ligand and Fe(III)-ligand complexes effectively serve as electron shuttle to expand the bioavailable pool of solid-phase Fe(II). These results have important implications for a better understanding of the bioavailability and reactivity of solid-phase Fe pool in the environment.

Influence of pH on the balance between methanogenesis and iron reduction.

Methanogenesis and iron reduction play major roles in determining global fluxes of greenhouse gases. Despite their importance, environmental factors that influence their interactions are poorly known. Here, we present evidence that pH significantly influences the balance between each reaction in anoxic environments that contain ferric (oxyhydr)oxide minerals. In sediment bioreactors that contained goethite as a source of ferric iron, both iron reduction and methanogenesis occurred but the balance between them varied significantly with pH. Compared to bioreactors receiving acidic media (pH 6), electron donor oxidation was 85% lower for iron reduction and 61% higher for methanogenesis in bioreactors receiving alkaline media (pH 7.5). Thus, methanogenesis displaced iron reduction considerably at alkaline pH. Geochemistry data collected from U.S. aquifers demonstrate that a similar pattern also exists on a broad spatial scale in natural settings. In contrast, in bioreactors that were not augmented with goethite, clay minerals served as the source of ferric iron and the balance between each reaction did not vary significantly with pH. We therefore conclude that pH can regulate the relative contributions of microbial iron reduction and methanogenesis to carbon fluxes from terrestrial environments. We further propose that the availability of ferric (oxyhydr)oxide minerals influences the extent to which the balance between each reaction is sensitive to pH. The results of this study advance our understanding of environmental controls on microbial methane generation and provide a basis for using pH and the occurrence of ferric minerals to refine predictions of greenhouse gas fluxes.

Thermodynamic and Kinetic Response of Microbial Reactions to High CO2.

Geological carbon sequestration captures CO2 from industrial sources and stores the CO2 in subsurface reservoirs, a viable strategy for mitigating global climate change. In assessing the environmental impact of the strategy, a key question is how microbial reactions respond to the elevated CO2 concentration. This study uses biogeochemical modeling to explore the influence of CO2 on the thermodynamics and kinetics of common microbial reactions in subsurface environments, including syntrophic oxidation, iron reduction, sulfate reduction, and methanogenesis. The results show that increasing CO2 levels decreases groundwater pH and modulates chemical speciation of weak acids in groundwater, which in turn affect microbial reactions in different ways and to different extents. Specifically, a thermodynamic analysis shows that increasing CO2 partial pressure lowers the energy available from syntrophic oxidation and acetoclastic methanogenesis, but raises the available energy of microbial iron reduction, hydrogenotrophic sulfate reduction and methanogenesis. Kinetic modeling suggests that high CO2 has the potential of inhibiting microbial sulfate reduction while promoting iron reduction. These results are consistent with the observations of previous laboratory and field studies, and highlight the complexity in microbiological responses to elevated CO2 abundance, and the potential power of biogeochemical modeling in evaluating and quantifying these responses.

Broad-Scale Evidence That pH Influences the Balance Between Microbial Iron and Sulfate Reduction.

Understanding basic controls on aquifer microbiology is essential to managing water resources and predicting impacts of future environmental change. Previous theoretical and laboratory studies indicate that pH can influence interactions between microorganisms that reduce ferric iron and sulfate. In this study, we test the environmental relevance of this relationship by examining broad-scale geochemical data from anoxic zones of aquifers. We isolated data from the U.S. Geological Survey National Water Information System for 19 principal aquifer systems. We then removed samples with chemical compositions inconsistent with iron- and sulfate-reducing environments and evaluated the relationships between pH and other geochemical parameters using Spearman's rho rank correlation tests. Overall, iron concentration and the iron-sulfide concentration ratio of groundwater share a statistically significant negative correlation with pH (P < 0.0001). These relationships indicate that the significance of iron reduction relative to sulfate reduction tends to increase with decreasing pH. Moreover, thermodynamic calculations show that, as the pH of groundwater decreases, iron reduction becomes increasingly favorable relative to sulfate reduction. Hence, the relative significance of each microbial reaction may vary in response to thermodynamic controls on microbial activity. Our findings demonstrate that trends in groundwater geochemistry across different regional aquifer systems are consistent with pH as a control on interactions between microbial iron and sulfate reduction. Environmental changes that perturb groundwater pH can affect water quality by altering the balance between these microbial reactions.

Microbial physiology-based model of ethanol metabolism in subsurface sediments.

A biogeochemical reaction model was developed based on microbial physiology to simulate ethanol metabolism and its influence on the chemistry of anoxic subsurface environments. The model accounts for potential microbial metabolisms that degrade ethanol, including those that oxidize ethanol directly or syntrophically by reducing different electron acceptors. Out of the potential metabolisms, those that are active in the environment can be inferred by fitting the model to experimental observations. This approach was applied to a batch sediment slurry experiment that examined ethanol metabolism in uranium-contaminated aquifer sediments from Area 2 at the U.S. Department of Energy Field Research Center in Oak Ridge, TN. According to the simulation results, complete ethanol oxidation by denitrification, incomplete ethanol oxidation by ferric iron reduction, ethanol fermentation to acetate and H(2), hydrogenotrophic sulfate reduction, and acetoclastic methanogenesis: all contributed significantly to the degradation of ethanol in the aquifer sediments. The assemblage of the active metabolisms provides a frame work to explore how ethanol amendment impacts the chemistry of the environment, including the occurrence and levels of uranium. The results can also be applied to explore how diverse microbial metabolisms impact the progress and efficacy of bioremediation strategies.

Thermodynamics of microbial growth coupled to metabolism of glucose, ethanol, short-chain organic acids, and hydrogen.

A literature compilation demonstrated a linear relationship between microbial growth yield and the free energy of aerobic and anaerobic (respiratory and/or fermentative) metabolism of glucose, ethanol, formate, acetate, lactate, propionate, butyrate, and H(2). This relationship provides a means to estimate growth yields for modeling microbial redox metabolism in soil and sedimentary environments.

Extremely alkaline (pH > 12) ground water hosts diverse microbial community.

Chemically unusual ground water can provide an environment for novel communities of bacteria to develop. Here, we describe a diverse microbial community that inhabits extremely alkaline (pH > 12) ground water from the Lake Calumet area of Chicago, Illinois, where historic dumping of steel slag has filled in a wetland. Using microbial 16S ribosomal ribonucleic acid gene sequencing and microcosm experiments, we confirmed the presence and growth of a variety of alkaliphilic beta-Proteobacteria, Bacillus, and Clostridium species at pH up to 13.2. Many of the bacterial sequences most closely matched those of other alkaliphiles found in more moderately alkaline water around the world. Oxidation of dihydrogen produced by reaction of water with steel slag is likely a primary energy source to the community. The widespread occurrence of iron-oxidizing bacteria suggests that reduced iron serves as an additional energy source. These results extend upward the known range of pH tolerance for a microbial community by as much as 2 pH units. The community may provide a source of novel microbes and enzymes that can be exploited under alkaline conditions.

A new rate law describing microbial respiration.

The rate of microbial respiration can be described by a rate law that gives the respiration rate as the product of a rate constant, biomass concentration, and three terms: one describing the kinetics of the electron-donating reaction, one for the kinetics of the electron-accepting reaction, and a thermodynamic term accounting for the energy available in the microbe's environment. The rate law, derived on the basis of chemiosmotic theory and nonlinear thermodynamics, is unique in that it accounts for both forward and reverse fluxes through the electron transport chain. Our analysis demonstrates how a microbe's respiration rate depends on the thermodynamic driving force, i.e., the net difference between the energy available from the environment and energy conserved as ATP. The rate laws commonly applied in microbiology, such as the Monod equation, are specific simplifications of the general law presented. The new rate law is significant because it affords the possibility of extrapolating in a rigorous manner from laboratory experiment to a broad range of natural conditions, including microbial growth where only limited energy is available. The rate law also provides a new explanation of threshold phenomena, which may reflect a thermodynamic equilibrium where the energy released by electron transfer balances that conserved by ADP phosphorylation.

Cyanobacteria associated with coral black band disease in Caribbean and Indo-Pacific Reefs.

For 30 years it has been assumed that a single species of cyanobacteria, Phormidium corallyticum, is the volumetrically dominant component of all cases of black band disease (BBD) in coral. Cyanobacterium-specific 16S rRNA gene primers and terminal restriction fragment length polymorphism analyses were used to determine the phylogenetic diversity of these BBD cyanobacteria on coral reefs in the Caribbean and Indo-Pacific Seas. These analyses indicate that the cyanobacteria that inhabit BBD bacterial mats collected from the Caribbean and Indo-Pacific Seas belong to at least three different taxa, despite the fact that the corals in each case exhibit similar signs and patterns of BBD mat development.

Kinetics of electron transfer through the respiratory chain.

We show that the rate at which electrons pass through the respiratory chain in mitochondria and respiring prokaryotic cells is described by the product of three terms, one describing electron donation, one acceptance, and a third, the thermodynamic drive. We apply the theory of nonequilibrium thermodynamics in the context of the chemiosmotic model of proton translocation and energy conservation. This approach leads to a closed-form expression that predicts steady-state electron flux as a function of chemical conditions and the proton motive force across the mitochondrial inner membrane or prokaryotic cytoplasmic membrane. The rate expression, derived considering reverse and forward electron flow, is the first to account for both thermodynamic and kinetic controls on the respiration rate. The expression can be simplified under specific conditions to give rate laws of various forms familiar in cellular physiology and microbial ecology. The expression explains the nonlinear dependence of flux on electrical potential gradient, its hyperbolic dependence on substrate concentration, and the inhibiting effects of reaction products. It provides a theoretical basis for investigating life under unusual conditions, such as microbial respiration in alkaline waters.