Integration of molecular and computational approaches paints a holistic portrait of obscure metabolisms.

Microorganisms are essential drivers of earth's geochemical cycles. However, the significance of elemental redox cycling mediated by microorganisms is often underestimated beyond the most well-studied nutrient cycles. Phosphite, (per)chlorate, and iodate are each considered esoteric substrates metabolized by microorganisms. However, recent investigations have indicated that these metabolisms are widespread and ubiquitous, affirming a need to continue studying the underlying microbiology to understand their biogeochemical effects and their interface with each other and our biosphere. This review focuses on combining canonical techniques of culturing microorganisms with modern omic approaches to further our understanding of obscure metabolic pathways and elucidate their importance in global biogeochemical cycles. Using these approaches, marker genes of interest have already been identified for phosphite, (per)chlorate, and iodate using traditional microbial physiology and genetics. Subsequently, their presence was queried to reveal the distribution of metabolic pathways in the environment using publicly available databases. In conjunction with each other, computational and experimental techniques provide a more comprehensive understanding of the location of these microorganisms, their underlying biochemistry and genetics, and how they tie into our planet's geochemical cycles.

A description of the genus Denitromonas nom. rev.: Denitromonas iodatirespirans sp. nov., a novel iodate-reducing bacterium, and two novel perchlorate-reducing bacteria, Denitromonas halophila and Denitromonas ohlonensis, isolated from San Francisco Bay intertidal mudflats.

The genus Denitromonas is currently a non-validated taxon that has been identified in several recent publications as members of microbial communities arising from marine environments. Very little is known about the biology of Denitromonas spp., and no pure cultures are presently found in any culture collections. The current epitaph of Denitromonas was given to the organism under the assumption that all members of this genus are denitrifying bacteria. This study performs phenotypic and genomic analyses on three new Denitromonas spp. isolated from tidal mudflats in the San Francisco Bay. We demonstrate that Denitromonas spp. are indeed all facultative denitrifying bacteria that utilize a variety of carbon sources such as acetate, lactate, and succinate. In addition, individual strains also use the esoteric electron acceptors perchlorate, chlorate, and iodate. Both 16S and Rps/Rpl phylogenetic analyses place Denitromonas spp. as a deep branching clade in the family Zoogloeaceae, separate from either Thauera spp., Azoarcus spp., or Aromatoleum spp. Genome sequencing reveals a G + C content ranging from 63.72% to 66.54%, and genome sizes range between 4.39 and 5.18 Mb. Genes for salt tolerance and denitrification are distinguishing features that separate Denitromonas spp. from the closely related Azoarcus and Aromatoleum genera. IMPORTANCE The genus Denitromonas is currently a non-validated taxon that has been identified in several recent publications as members of microbial communities arising from marine environments. Very little is known about the biology of Denitromonas spp., and no pure cultures are presently found in any culture collections. The current epitaph of Denitromonas was given to the organism under the assumption that all members of this genus are denitrifying bacteria. This study performs phenotypic and genomic analyses on three Denitromonas spp., Denitromonas iodatirespirans sp. nov.-a novel iodate-reducing bacterium-and two novel perchlorate-reducing bacteria, Denitromonas halophila and Denitromonas ohlonensis, isolated from San Francisco Bay intertidal mudflats.

Chlorine redox chemistry is widespread in microbiology.

Chlorine is abundant in cells and biomolecules, yet the biology of chlorine oxidation and reduction is poorly understood. Some bacteria encode the enzyme chlorite dismutase (Cld), which detoxifies chlorite (ClO2-) by converting it to chloride (Cl-) and molecular oxygen (O2). Cld is highly specific for chlorite and aside from low hydrogen peroxide activity has no known alternative substrate. Here, we reasoned that because chlorite is an intermediate oxidation state of chlorine, Cld can be used as a biomarker for oxidized chlorine species. Cld was abundant in metagenomes from various terrestrial habitats. About 5% of bacterial and archaeal genera contain a microorganism encoding Cld in its genome, and within some genera Cld is highly conserved. Cld has been subjected to extensive horizontal gene transfer. Genes found to have a genetic association with Cld include known genes for responding to reactive chlorine species and uncharacterized genes for transporters, regulatory elements, and putative oxidoreductases that present targets for future research. Cld was repeatedly co-located in genomes with genes for enzymes that can inadvertently reduce perchlorate (ClO4-) or chlorate (ClO3-), indicating that in situ (per)chlorate reduction does not only occur through specialized anaerobic respiratory metabolisms. The presence of Cld in genomes of obligate aerobes without such enzymes suggested that chlorite, like hypochlorous acid (HOCl), might be formed by oxidative processes within natural habitats. In summary, the comparative genomics of Cld has provided an atlas for a deeper understanding of chlorine oxidation and reduction reactions that are an underrecognized feature of biology.

The biogeochemical cycling of chlorine.

Chlorine has important roles in the Earth's systems. In different forms, it helps balance the charge and osmotic potential of cells, provides energy for microorganisms, mobilizes metals in geologic fluids, alters the salinity of waters, and degrades atmospheric ozone. Despite this importance, there has not been a comprehensive summary of chlorine's geobiology. Here, we unite different areas of recent research to describe a biogeochemical cycle for chlorine. Chlorine enters the biosphere through volcanism and weathering of rocks and is sequestered by subduction and the formation of evaporite sediments from inland seas. In the biosphere, chlorine is converted between solid, dissolved, and gaseous states and in oxidation states ranging from -1 to +7, with the soluble, reduced chloride ion as its most common form. Living organisms and chemical reactions change chlorine's form through oxidation and reduction and the addition and removal of chlorine from organic molecules. Chlorine can be transported through the atmosphere, and the highest oxidation states of chlorine are produced by reactions between sunlight and trace chlorine gases. Partial oxidation of chlorine occurs across the biosphere and creates reactive chlorine species that contribute to the oxidative stress experienced by living cells. A unified view of this chlorine cycle demonstrates connections between chlorine biology, chemistry, and geology that affect life on the Earth.

Genetic and phylogenetic analysis of dissimilatory iodate-reducing bacteria identifies potential niches across the world's oceans.

Iodine is oxidized and reduced as part of a biogeochemical cycle that is especially pronounced in the oceans, where the element naturally concentrates. The use of oxidized iodine in the form of iodate (IO3-) as an electron acceptor by microorganisms is poorly understood. Here, we outline genetic, physiological, and ecological models for dissimilatory IO3- reduction to iodide (I-) by a novel estuarine bacterium, Denitromonas sp. IR-12. Our results show that dissimilatory iodate reduction (DIR) by strain IR-12 is molybdenum-dependent and requires an IO3- reductase (idrA) and likely other genes in a mobile cluster with a conserved association across known and predicted DIR microorganisms (DIRM). Based on genetic and physiological data, we propose a model where three molecules of IO3- are likely reduced to three molecules of hypoiodous acid (HIO), which rapidly disproportionate into one molecule of IO3- and two molecules of iodide (I-), in a respiratory pathway that provides an energy yield equivalent to that of nitrate or perchlorate respiration. Consistent with the ecological niche expected of such a metabolism, idrA is enriched in the metagenome sequence databases of marine sites with a specific biogeochemical signature (high concentrations of nitrate and phosphate) and diminished oxygen. Taken together, these data suggest that DIRM help explain the disequilibrium of the IO3-:I- concentration ratio above oxygen-minimum zones and support a widespread iodine redox cycle mediated by microbiology.

The diversity and evolution of microbial dissimilatory phosphite oxidation.

Phosphite is the most energetically favorable chemotrophic electron donor known, with a half-cell potential (Eo') of -650 mV for the PO43-/PO33- couple. Since the discovery of microbial dissimilatory phosphite oxidation (DPO) in 2000, the environmental distribution, evolution, and diversity of DPO microorganisms (DPOMs) have remained enigmatic, as only two species have been identified. Here, metagenomic sequencing of phosphite-enriched microbial communities enabled the genome reconstruction and metabolic characterization of 21 additional DPOMs. These DPOMs spanned six classes of bacteria, including the Negativicutes, Desulfotomaculia, Synergistia, Syntrophia, Desulfobacteria, and Desulfomonilia_A Comparing the DPO genes from the genomes of enriched organisms with over 17,000 publicly available metagenomes revealed the global existence of this metabolism in diverse anoxic environments, including wastewaters, sediments, and subsurface aquifers. Despite their newfound environmental and taxonomic diversity, metagenomic analyses suggested that the typical DPOM is a chemolithoautotroph that occupies low-oxygen environments and specializes in phosphite oxidation coupled to CO2 reduction. Phylogenetic analyses indicated that the DPO genes form a highly conserved cluster that likely has ancient origins predating the split of monoderm and diderm bacteria. By coupling microbial cultivation strategies with metagenomics, these studies highlighted the unsampled metabolic versatility latent in microbial communities. We have uncovered the unexpected prevalence, diversity, biochemical specialization, and ancient origins of a unique metabolism central to the redox cycling of phosphorus, a primary nutrient on Earth.

Sulfate adenylyl transferase kinetics and mechanisms of metabolic inhibitors of microbial sulfate respiration.

Sulfate analog oxyanions that function as selective metabolic inhibitors of dissimilatory sulfate reducing microorganisms (SRM) are widely used in ecological studies and industrial applications. As such, it is important to understand the mode of action and mechanisms of tolerance or adaptation to these compounds. Different oxyanions vary widely in their inhibitory potency and mechanism of inhibition, but current evidence suggests that the sulfate adenylyl transferase/ATP sulfurylase (Sat) enzyme is an important target. We heterologously expressed and purified the Sat from the model SRM, Desulfovibrio alaskensis G20. With this enzyme we determined the turnover kinetics (kcat, KM) for alternative substrates (molybdate, selenate, arsenate, monofluorophosphate, and chromate) and inhibition constants (KI) for competitive inhibitors (perchlorate, chlorate, and nitrate). These measurements enable the first quantitative comparisons of these compounds as substrates or inhibitors of a purified Sat from a respiratory sulfate reducer. We compare predicted half-maximal inhibitory concentrations (IC50) based on Sat kinetics with measured IC50 values against D. alaskensis G20 growth and discuss our results in light of known mechanisms of sensitivity or resistance to oxyanions. This analysis helps with the interpretation of recent adaptive laboratory evolution studies and illustrates the value of interpreting gene-microbe-environment interactions through the lens of enzyme kinetics.

Isolation of a Dissimilatory Iodate-Reducing Aromatoleum sp. From a Freshwater Creek in the San Francisco Bay Area.

Recent reports of dissimilatory iodate-reducing microorganisms (DIRM) have arisen from studies of bacteria in marine environments. These studies described the physiology and distribution of DIRM while also demonstrating their presence in iodine-rich marine environments. We posited that despite lower iodine concentrations, terrestrial and freshwater ecosystems should also harbor DIRM. We established numerous enrichments from coastal and freshwater environments that actively remove amended iodate. We describe the physiology and genome of a new DIRM isolate, Aromatoleum toluclasticum sp. TC-10, emerging from a freshwater creek microcosm. Like other DIRM, A. toluclasticum sp. TC-10 couples acetate oxidation to iodate reduction with a concomitant increase in the OD600. Our results indicate that A. toluclasticum sp. TC-10 performs dissimilatory iodate reduction (DIR) using the recently described iodate reductase (Idr). We provide further evidence of horizontal gene transfer of the idr genes by demonstrating the lack of Idr in the closely related (99.93% 16S rDNA sequence identity) A. toluclasticum sp. MF63 and describe the heterogeneity of the accessory proteins associated with the iodate reduction island (IRI). These observations provide additional evidence that DIR is a horizontally acquired metabolism with broad environmental distribution beyond exclusively marine environments.

Tungstate Control of Microbial Sulfidogenesis and Souring of the Engineered Environment.

Sulfide accumulation in oil reservoir fluids (souring) from the activity of sulfate-reducing microorganisms (SRM) is of grave concern because of the associated health and facility failure risks. Here, we present an assessment of tungstate as a selective and potent inhibitor of SRM. Dose-response inhibitor experiments were conducted with a number of SRM isolates and enrichments at 30-80 °C and an increase in the effectiveness of tungstate treatment at higher temperatures was observed. To explore mixed inhibitor treatment modes, we tested synergy or antagonism between several inhibitors with tungstate, and found synergism between WO42- and NO2-, while additive effects were observed with ClO4- and NO3-. We also evaluated SRM inhibition by tungstate in advective upflow oil-sand-packed columns. Although 2 mM tungstate was initially sufficient to inhibit sulfidogenesis, subsequent temporal CaWO4 precipitation resulted in loss of the bioavailable inhibitor from solution and a concurrent increase in effluent sulfide. Mixing 4 mM sodium carbonate with the 2 mM tungstate was enough to promote tungstate solubility to reach inhibitory concentrations, without precipitation, and completely inhibit SRM activity. Overall, we demonstrate the effectiveness of tungstate as a potent SRM inhibitor, particularly at higher temperatures, and propose a novel carbonate-tungstate formulation for application to soured oil reservoirs.

An uncharacterized clade in the DMSO reductase family of molybdenum oxidoreductases is a new type of chlorate reductase.

The dimethylsulfoxide (DMSO) reductase family of enzymes has many subfamilies catalysing unique biogeochemical reactions. It also has many uncharacterized subfamilies. Comparative genomics predicted one such subfamily to participate in a key step of the chlorine cycle because of a conserved genetic association with chlorite dismutase, implying they produce chlorite through chlorate or perchlorate reduction. We determined the activity of the uncharacterized enzyme by comparing strains in the phototrophic genus Rhodoplanes that encode either a typical perchlorate reductase or the uncharacterized enzyme. Rpl. piscinae and Rpl. elegans, which encode perchlorate reductase, grew by using perchlorate as an electron acceptor. In contrast, Rpl. roseus, which encodes the uncharacterized enzyme, grew by chlorate reduction but not by perchlorate reduction. This is the first report of perchlorate and chlorate being used as respiratory electron acceptors by phototrophs. When both chlorate and perchlorate were present, Rpl. roseus consumed only chlorate. Highly concentrated Rpl. roseus cells showed some perchlorate consumption, but chlorate consumption occurred at a 10-fold higher rate. Together, these genomic and physiological data define a new group of chlorate reductases. Some organisms encode both this chlorate reductase and a perchlorate reductase, raising new questions about the physiology and evolution of chlorine oxyanion respiration.

Identification of a parasitic symbiosis between respiratory metabolisms in the biogeochemical chlorine cycle.

A key step in the chlorine cycle is the reduction of perchlorate (ClO4-) and chlorate (ClO3-) to chloride by microbial respiratory pathways. Perchlorate-reducing bacteria and chlorate-reducing bacteria differ in that the latter cannot use perchlorate, the most oxidized chlorine compound. However, a recent study identified a bacterium with the chlorate reduction pathway dominating a community provided only perchlorate. Here we confirm a metabolic interaction between perchlorate- and chlorate-reducing bacteria and define its mechanism. Perchlorate-reducing bacteria supported the growth of chlorate-reducing bacteria to up to 90% of total cells in communities and co-cultures. Chlorate-reducing bacteria required the gene for chlorate reductase to grow in co-culture with perchlorate-reducing bacteria, demonstrating that chlorate is responsible for the interaction, not the subsequent intermediates chlorite and oxygen. Modeling of the interaction suggested that cells specialized for chlorate reduction have a competitive advantage for consuming chlorate produced from perchlorate, especially at high concentrations of perchlorate, because perchlorate and chlorate compete for a single enzyme in perchlorate-reducing cells. We conclude that perchlorate-reducing bacteria inadvertently support large populations of chlorate-reducing bacteria in a parasitic relationship through the release of the intermediate chlorate. An implication of these findings is that undetected chlorate-reducing bacteria have likely negatively impacted efforts to bioremediate perchlorate pollution for decades.

Anion transport as a target of adaption to perchlorate in sulfate-reducing communities.

Inhibitors can be used to control the functionality of microbial communities by targeting specific metabolisms. The targeted inhibition of dissimilatory sulfate reduction limits the generation of toxic and corrosive hydrogen sulfide across several industrial systems. Sulfate-reducing microorganisms (SRM) are specifically inhibited by sulfate analogs, such as perchlorate. Previously, we showed pure culture SRM adaptation to perchlorate stress through mutation of the sulfate adenylyltransferase, a central enzyme in the sulfate reduction pathway. Here, we explored adaptation to perchlorate across unconstrained SRM on a community scale. We followed natural and bio-augmented sulfidogenic communities through serial transfers in increasing concentrations of perchlorate. Our results demonstrated that perchlorate stress altered community structure by initially selecting for innately more resistant strains. Isolation, whole-genome sequencing, and molecular biology techniques allowed us to define subsequent genetic mechanisms of adaptation that arose across the dominant adapting SRM. Changes in the regulation of divalent anion:sodium symporter family transporters led to increased intracellular sulfate to perchlorate ratios, allowing SRM to escape the effects of competitive inhibition. Thus, in contrast to pure-culture results, SRM in communities cope with perchlorate stress via changes in anion transport and its regulation. This highlights the value of probing evolutionary questions in an ecological framework, bridging the gap between ecology, evolution, genomics, and physiology.

Resistance and Resilience of Sulfidogenic Communities in the Face of the Specific Inhibitor Perchlorate.

Hydrogen sulfide is a toxic and corrosive gas, produced by the activity of sulfate-reducing microorganisms (SRM). Owing to the environmental, economic and human-health consequences of sulfide, there is interest in developing specific inhibitors of SRM. Recent studies have identified perchlorate as a promising emerging inhibitor. The aim of this work is to quantitatively dissect the inhibitory dynamics of perchlorate. Sulfidogenic mixed continuous-flow systems were treated with perchlorate. SRM number, sulfide production and community structure were monitored pre-, during and post-treatment. The data generated was compared to a simple mathematical model, where SRM growth slows as a result of inhibition. The experimental data supports the interpretation that perchlorate largely acts to suppress SRM growth rates, rendering planktonic SRM increasingly susceptible to wash-out. Surface-attachment was identified as an important parameter preventing SRM wash-out and thus governing inhibitory dynamics. Our study confirmed the lesser depletion of surface-attached SRM as compared to planktonic SRM during perchlorate treatment. Indirect effects of perchlorate (bio-competitive exclusion of SRM by dissimilatory perchlorate-reducing bacteria, DPRB) were also assayed by amending reactors with DPRB. Indeed, low concentrations of perchlorate coupled with DRPB amendment can drive sulfide concentrations to zero. Further, inhibition in a complex community was compared to that in a pure culture, highlighting similarities and differences between the two scenarios. Finally, we quantified susceptibility to perchlorate across SRM in various culture conditions, showing that prediction of complex behavior in continuous systems from batch results is possible. This study thus provides an overview of the sensitivity of sulfidogenic communities to perchlorate, as well as mechanisms underlying these patterns.

Adaptation of Desulfovibrio alaskensis G20 to perchlorate, a specific inhibitor of sulfate reduction.

Hydrogen sulfide produced by sulfate-reducing microorganisms (SRM) poses significant health and economic risks, particularly during oil recovery. Previous studies identified perchlorate as a specific inhibitor of SRM. However, constant inhibitor addition to natural systems results in new selective pressures. Consequently, we investigated the ability of Desulfovibrio alaskensis G20 to evolve perchlorate resistance. Serial transfers in increasing concentrations of perchlorate led to robust growth in the presence of 100 mM inhibitor. Isolated adapted strains demonstrated a threefold increase in perchlorate resistance compared to the wild-type ancestor. Whole genome sequencing revealed a single base substitution in Dde_2265, the sulfate adenylyltransferase (sat). We purified and biochemically characterized the Sat from both wild-type and adapted strains, and showed that the adapted Sat was approximately threefold more resistant to perchlorate inhibition, mirroring whole cell results. The ability of this mutation to confer resistance across other inhibitors of sulfidogenesis was also assayed. The generalizability of this mutation was confirmed in multiple evolving G20 cultures and in another SRM, D. vulgaris Hildenborough. This work demonstrates that a single nucleotide polymorphism in Sat can have a significant impact on developing perchlorate resistance and emphasizes the value of adaptive laboratory evolution for understanding microbial responses to environmental perturbations.

Specific inhibitors of respiratory sulfate reduction: towards a mechanistic understanding.

Microbial sulfate reduction (SR) by sulfate-reducing micro-organisms (SRM) is a primary environmental mechanism of anaerobic organic matter mineralization, and as such influences carbon and sulfur cycling in many natural and engineered environments. In industrial systems, SR results in the generation of hydrogen sulfide, a toxic, corrosive gas with adverse human health effects and significant economic and environmental consequences. Therefore, there has been considerable interest in developing strategies for mitigating hydrogen sulfide production, and several specific inhibitors of SRM have been identified and characterized. Specific inhibitors are compounds that disrupt the metabolism of one group of organisms, with little or no effect on the rest of the community. Putative specific inhibitors of SRM have been used to control sulfidogenesis in industrial and engineered systems. Despite the value of these inhibitors, mechanistic and quantitative studies into the molecular mechanisms of their inhibition have been sparse and unsystematic. The insight garnered by such studies is essential if we are to have a more complete understanding of SR, including the past and current selective pressures acting upon it. Furthermore, the ability to reliably control sulfidogenesis - and potentially assimilatory sulfate pathways - relies on a thorough molecular understanding of inhibition. The scope of this review is to summarize the current state of the field: how we measure and understand inhibition, the targets of specific SR inhibitors and how SRM acclimatize and/or adapt to these stressors.

Mitigating Sulfidogenesis With Simultaneous Perchlorate and Nitrate Treatments.

Sulfide biogenesis (souring) in oil reservoirs is an extensive and costly problem. Nitrate is currently used as a souring inhibitor but often requires high concentrations and yields inconsistent results. Recently, perchlorate has displayed promise as a more potent inhibitor in lab scale studies. However, combining the two treatments to determine synergy and effectiveness in a dynamic system has never been tested. Nitrate inhibits perchlorate consumption by perchlorate reducing bacteria, suggesting that the combined treatment may allow deeper penetration of the perchlorate into the reservoir matrix. Furthermore, the metabolic intermediates of perchlorate and nitrate reduction (nitrite and chlorite, respectively) are synergistic with the primary electron acceptors for inhibition of sulfate reduction. To assess the possible synergies between nitrate and perchlorate treatments, triplicate glass columns packed with pre-soured marine sediment were flushed with media containing sulfate and an inhibitor treatment [(i) perchlorate; (ii) nitrate; (iii) perchlorate and nitrate; or (iv) none]. Internal geochemistry and microbial community changes were monitored along the length of the columns during six phases of increasing treatment concentrations. In a final phase all treatments were removed. Sulfide production decreased in all treated columns in conjunction with increased inhibitor concentrations relative to the untreated control. Interestingly, the potency of the "mixed" treatment was additive relative to the individual treatments suggesting no interaction. Microbial community analyses indicated community shifts and clustering by treatment. The mixed treatment column community's trajectory closely resembled that of the community found in the perchlorate only treatment, suggesting that perchlorate was the dominant control on the "mixed" community structure. In contrast, the nitrate and untreated column communities had unique trajectories. This study indicates that concurrent nitrate and perchlorate treatment is not more effective than perchlorate treatment alone but is more effective than nitrate treatment. As such, treatment decisions may be based on economic factors.

Attenuating Sulfidogenesis in a Soured Continuous Flow Column System With Perchlorate Treatment.

Hydrogen sulfide production by sulfate reducing bacteria (SRB) is the primary cause of oil reservoir souring. Amending environments with chlorate or perchlorate [collectively denoted (per)chlorate] represents an emerging technology to prevent the onset of souring. Recent studies with perchlorate reducing bacteria (PRB) monocultures demonstrated that they have the innate capability to enzymatically oxidize sulfide, thus PRB may offer an effective means of reversing souring. (Per)chlorate may be effective by (i) direct toxicity to SRB; (ii) competitive exclusion of SRB by PRB; or (iii) reversal of souring through re-oxidation of sulfide by PRB. To determine if (per)chlorate could sweeten a soured column system and assign a quantitative value to each of the mechanisms we treated columns flooded with San Francisco bay water with temporally decreasing amounts (50, 25, and 12.5 mM) of (per)chlorate. Geochemistry and the microbial community structure were monitored and a reactive transport model was developed, Results were compared to columns treated with nitrate or untreated. Souring was reversed by all treatments at 50 mM but nitrate-treated columns began to re-sour when treatment concentrations decreased (25 mM). Re-souring was only observed in (per)chlorate-treated columns when concentrations were decreased to 12.5 mM and the extent of re-souring was less than the control columns. Microbial community analyses indicated treatment-specific community shifts. Nitrate treatment resulted in a distinct community enriched in genera known to perform sulfur cycling metabolisms and genera capable of nitrate reduction. (Per)chlorate treatment enriched for (per)chlorate reducing bacteria. (Per)chlorate treatments only enriched for sulfate reducing organisms when treatment levels were decreased. A reactive transport model of perchlorate treatment was developed and a baseline case simulation demonstrated that the model provided a good fit to the effluent geochemical data. Subsequent simulations teased out the relative role that each of the three perchlorate inhibition mechanisms played during different phases of the experiment. These results indicate that perchlorate addition is an effective strategy for both souring prevention and souring reversal. It provides insight into which organisms are involved, and illuminates the interactive effects of the inhibition mechanisms, further highlighting the versatility of perchlorate as a sweetening agent.

Dissimilatory Sulfate Reduction Under High Pressure by Desulfovibrio alaskensis G20.

Biosouring results from production of H2S by sulfate-reducing microorganisms (SRMs) in oil reservoirs. H2S is toxic, corrosive, and explosive, and as such, represents a significant threat to personnel, production facilities, and transportation pipelines. Since typical oil reservoir pressures can range from 10 to 50 MPa, understanding the role that pressure plays in SRM metabolism is important to improving souring containment strategies. To explore the impact of pressure, we grew an oil-field SRM isolate, Desulfovibrio alaskensis G20, under a range of pressures (0.1-14 MPa) at 30°C. The observed microbial growth rate was an inverse function of pressure with an associated slight reduction in sulfate and lactate consumption rate. Competitive fitness experiments with randomly bar-coded transposon mutant library sequencing (RB-TnSeq) identified several genes associated with flagellar biosynthesis and assembly that were important at high pressure. The fitness impact of specific genes was confirmed using individual transposon mutants. Confocal microscopy revealed that enhanced cell aggregation occurs at later stages of growth under pressure. We also assessed the effect of pressure on SRM inhibitor potency. Dose-response experiments showed a twofold decrease in the sensitivity of D. alaskensis to the antibiotic chloramphenicol at 14 MPa. Fortuitously, pressure had no significant influence on the inhibitory potency of the common souring controlling agent nitrate, or the emerging SRM inhibitors perchlorate, monofluorophosphate, or zinc pyrithione. Our findings improve the conceptual model of microbial sulfate reduction in high-pressure environments and the influence of pressure on souring inhibitor efficacy.

Functional Redundancy in Perchlorate and Nitrate Electron Transport Chains and Rewiring Respiratory Pathways to Alter Terminal Electron Acceptor Preference.

Most dissimilatory perchlorate reducing bacteria (DPRB) are also capable of respiratory nitrate reduction, and preferentially utilize nitrate over perchlorate as a terminal electron acceptor. The similar domain architectures and phylogenetic relatedness of the nitrate and perchlorate respiratory complexes suggests a common evolutionary history and a potential for functionally redundant electron carriers. In this study, we identify key genetic redundancies in the electron transfer pathways from the quinone pool(s) to the terminal nitrate and perchlorate reductases in Azospira suillum PS (hereafter referred to as PS). We show that the putative quinol dehydrogenases, (PcrQ and NapC) and the soluble cytochrome electron carriers (PcrO and NapO) are functionally redundant under anaerobic growth conditions. We demonstrate that, when grown diauxically with both nitrate and perchlorate, the endogenous expression of NapC and NapO during the nitrate reduction phase was sufficient to completely erase any growth defect in the perchlorate reduction phase caused by deletion of pcrQ and/or pcrO. We leveraged our understanding of these genetic redundancies to make PS mutants with altered electron acceptor preferences. Deletion of the periplasmic nitrate reductase catalytic subunit, napA, led to preferential utilization of perchlorate even in the presence of equimolar nitrate, and deletion of the electron carrier proteins napQ and napO, resulted in concurrent reduction of nitrate and perchlorate. Our results demonstrate that nitrate and perchlorate respiratory pathways in PS share key functionally redundant electron transfer proteins and that mutagenesis of these proteins can be utilized as a strategy to alter the preferential usage of nitrate over perchlorate.