Nitrate-reducing Microorganisms Research Articles

Survival of a microbial inoculant in soil after recurrent inoculations

Microbial inoculants are attracting growing interest in agriculture, but their efficacy remains unreliable in relation to their poor survival, partly due to the competition with the soil resident community. We hypothesised that recurrent inoculation could gradually alleviate this competition and improve the survival of the inoculant while increasing its impact on the resident bacterial community. We tested the effectiveness of such strategy with four inoculation sequences of Pseudomonas fluorescens strain B177 in soil microcosms with increasing number and frequency of inoculation, compared to a non-inoculated control. Each sequence was carried out at two inoculation densities (106 and 108 cfu.g soil−1). The four-inoculation sequence induced a higher abundance of P. fluorescens, 2 weeks after the last inoculation. No impact of inoculation sequences was observed on the resident community diversity and composition. Differential abundance analysis identified only 28 out of 576 dominants OTUs affected by the high-density inoculum, whatever the inoculation sequence. Recurrent inoculations induced a strong accumulation of nitrate, not explained by the abundance of nitrifying or nitrate-reducing microorganisms. In summary, inoculant density rather than inoculation pattern matters for inoculation effect on the resident bacterial communities, while recurrent inoculation allowed to slightly enhance the survival of the inoculant and strongly increased soil nitrate content.

Nitrogen removal crash of denitrification in anaerobic biofilm reactor due to dissimilatory nitrate reduction to ammonium (DNRA) for tofu processing wastewater treatment: Based on microbial community and functional genes

Nitrogen removal crash of denitrification in an anaerobic biofilm reactor (ABR) occurred during tofu processing wastewater treatment. This study discovered that the crash occurred due to dissimilatory nitrate reduction to ammonium (DNRA) based on the microbial community and their functional genes. Metagenomic analysis and enzyme activity measurements were carried out to identify variations of microbial communities and their functional genes encoded for denitrification and DNRA. Total nitrogen removal efficiency of the ABR was maintained at approximately 78 ± 2 % during Stage 1 but decreased from 82 to 52 % during Stage 2 (nitrogen removal crash phase). Moreover, NO2−-N and NH4+-N increased during this crash phase with stable NO3−-N removal rate (86 ± 2 %), indicating DNRA in the ABR. Functional gene analysis found a decrease of bacteria with nitrite reductase and nitric oxide reductase, especially NirK and NorC, excluding microorganisms, containing nitrous oxide reductase, variation during the ABR operation. Most DNRA bacteria increased and replaced denitrifies as the dominant nitrate-reducing microorganisms in Stage 2. Moreover, the activities of denitrification functional enzymes, especially nitrite reductase and nitric oxide reductase, decreased from Stage 1 to Stage 2, while the DNRA nitrite reductase showed a noticeable increase. During stages 1 and 2, the tofu processing wastewater contained a high COD:TN ratio of 7.3–9.1, directly inhibiting denitrification and promoting DNRA. The COD:TN ratio reduction in Stage 3 led to denitrification recovery and DNRA inhibition. Therefore, the nitrogen removal crash in the ABR was due to DNRA, which resulted from the high COD:TN ratio.

INFLUENCE OF NITROGEN NUTRITION CONDITIONS AND PHYTHORMONES ON CHANGE OF FATTY ACID COMPOSITION OF VEGETATIVE ORGANS OF SPRING WHEAT TRITICUM AESTIVUM L.

The paper shows the effect of nitrogen nutrition, exogenous auxin, and rhizosphere auxin-synthesizing microorganisms on the variability of the composition of fatty acids (FA) in the vegetative organs of spring wheat. The object of the study was seedlings of spring soft wheat Triticum aestivum L. The determination of FAs was carried out by gas chromatography with mass spectrometry (GC-MS). Analysis of FAs showed that in the control variants (without auxin), nitrogen nutrition conditions did not affect the localization of polyunsaturated FAs in vegetative organs; the maximum content of triene FAs was observed in leaves of 48.30% (N-deficient variant) and 44.8% (NO-3-variant ) and the absence of these FAs in the roots. It was found that in the presence of nitrates, the proportion of unsaturated FAs in the leaves and roots of wheat decreases. The use of exogenous auxin (5–50 μg/ml) in the early stages of ontogenesis leads to an increase in the amount of saturated (palmitic and stearic) acids and a decrease in unsaturated acids in vegetative organs, regardless of the conditions of nitrogen nutrition. During the introduction of spring wheat seedlings by auxin-synthesizing microorganisms, it was noted that nitrogen-fixing bacteria affect the leaves of plants most effectively, the content of saturated FAs increases by 72%, and only 16% increases in these FAs in the leaves of nitrate-reducing microorganisms.

Production and compatibility assessment of denitrifying biogranules tailored for self-healing concrete applications

Microbial granules have been mostly used for wastewater treatment. Recently, biogranules consisting nitrate-reducing microorganisms have appeared as a unique healing agent providing simultaneous self-healing of cracks and corrosion inhibition of rebar in concrete. Yet, information about the production process and microbial activity of these biogranules as well as their compatibility with cementitious materials remains unknown. This study presents the biogranule production procedure in detail and evaluates the compatibility of the produced biogranules with the cementitious composites. In the form of biogranules, bacteria doses varying between 0.25% and 3.00% w/w cement were incorporated into mortar and the variations in fresh and hardened properties of mortars were evaluated with respect to abiotic mortars. Biogranules were also tested for their compatibility with concrete at minimum and the defined maximum tolerable doses. Biogranules with a NOx-N reduction activity of 0.10 g NOx-N.g−1 bacteria.d−1 and organic carbon oxidation activity of 1.50 g HCOO⁻.g−1 bacteria.d−1 were produced successfully by using minimal medium. It was found out that biogranules enable bacteria incorporation into mortar up to a dose of 2.50% w/w cement without compromising fresh and hardened properties of cementitious composites. It was revealed that the compatibility of the biogranules was due to the mineral layer surrounding the biogranules which prevented interaction between the cement matrix and the microbial content. The thickness of the protective mineral layer around the granules was varying between 50 and 300 μm depending on the granule size. Net yield for concrete compatible biogranule production was determined as 0.05 g biogranule.g−1 HCOO⁻.

Importance of thermodynamics dependent kinetic parameters in nitrate-based souring mitigation studies

Souring is the unwanted formation of hydrogen sulfide (H2S) by sulfate-reducing microorganisms (SRM) in sewer systems and seawater flooded oil reservoirs. Nitrate treatment (NT) is one of the major methods to alleviate souring: The mechanism of souring remediation by NT is stimulation of nitrate reducing microorganisms (NRM) that depending on the nitrate reduction pathway can outcompete SRM for common electron donors, or oxidize sulfide to sulfate. However, some nitrate reduction pathways may challenge the efficacy of NT. Therefore, a precise understanding of souring rate, nitrate reduction rate and pathways is crucial for efficient souring management. Here, we investigate the necessity of incorporating two thermodynamic dependent kinetic parameters, namely, the growth yield (Y), and FT, a parameter related to the minimum catabolic energy production required by cells to utilize a given catabolic reaction. We first show that depending on physiochemical conditions, Y and FT for SRM change significantly in the range of [0-0.4] mole biomass per mole electron donor and [0.0006-0.5], respectively, suggesting that these parameters should not be considered constant and that it is important to couple souring models with thermodynamic models. Then, we highlight this further by showing an experimental dataset that can be modeled very well by considering variable FT. Next, we show that nitrate based lithotrophic sulfide oxidation to sulfate (lNRM3) is the dominant nitrate reduction pathway. Then, arguing that thermodynamics would suggest that S° consumption should proceed faster than S0 production, we infer that the reason for frequently observed S0 accumulation is its low solubility. Last, we suggest that nitrate based souring treatment will suffer less from S0 accumulation if we (i) act early, (ii) increase temperature and (iii) supplement stoichiometrically sufficient nitrate.

Electrochemical enrichment of haloalkaliphilic nitrate-reducing microbial biofilm at the cathode of bioelectrochemical systems

SummaryElectrotrophic microorganisms have not been well studied in extreme environments. Here, we report on the nitrate-reducing cathodic microbial biofilm from a haloalkaline environment. The biofilm enriched via electrochemical approach under 9.5 pH and 20 g NaCl/L salinity conditions achieved current density and nitrate reduction efficiency via partial and complete denitrification. Voltammetric characterization of the biocathodes revealed a redox center with (vs. Ag/AgCl) formal potential putatively involved in the electron uptake process. The lack of soluble redox mediators and hydrogen-driven nitrate reduction suggests direct-contact cathodic electron uptake by the nitrate-reducing microorganisms in the enriched biofilm. 16S-rRNA amplicon sequencing of the cathodic biofilm revealed the presence of unreported Pseudomonas, Natronococcus, and Pseudoalteromonas spp. at , and relative sequence abundances, respectively. The enriched nitrate-reducing microorganisms also reduced nitrate efficiently using soluble electron donors found in the lake sediments, thereby suggesting their role in N-cycling in such environments.

Enzyme-Specific Coupling of Oxygen and Nitrogen Isotope Fractionation of the Nap and Nar Nitrate Reductases.

Dissimilatory nitrate reduction (DNR) to nitrite is the first step in denitrification, the main process through which bioavailable nitrogen is removed from ecosystems. DNR is catalyzed by both cytosolic (Nar) and periplasmic (Nap) nitrate reductases and fractionates the stable isotopes of nitrogen (14N, 15N) and oxygen (16O, 18O), which is reflected in residual environmental nitrate pools. Data on the relationship between the pattern in oxygen vs nitrogen isotope fractionation (18ε/15ε) suggests that systematic differences exist between marine and terrestrial ecosystems that are not fully understood. We examined the 18ε/15ε of nitrate-reducing microorganisms that encode Nar, Nap, or both enzymes, as well as gene deletion mutants of Nar and Nap to test the hypothesis that enzymatic differences alone could explain the environmental observations. We find that the distribution of 18ε/15ε fractionation ratios of all examined nitrate reductases forms two distinct peaks centered around an 18ε/15ε proportionality of 0.55 (Nap) and 0.91 (Nar), with the notable exception of the Bacillus Nar reductases, which cluster isotopically with the Nap reductases. Our findings may explain differences in 18ε/15ε fractionation between marine and terrestrial systems and challenge current knowledge about Nar 18ε/15ε signatures.

Anaerobic microbial methanol conversion in marine sediments.

SummaryMethanol is an ubiquitous compound that plays a role in microbial processes as a carbon and energy source, intermediate in metabolic processes or as end product in fermentation. In anoxic environments, methanol can act as the sole carbon and energy source for several guilds of microorganisms: sulfate‐reducing microorganisms, nitrate‐reducing microorganisms, acetogens and methanogens. In marine sediments, these guilds compete for methanol as their common substrate, employing different biochemical pathways. In this review, we will give an overview of current knowledge of the various ways in which methanol reaches marine sediments, the ecology of microorganisms capable of utilizing methanol and their metabolism. Furthermore, through a metagenomic analysis, we shed light on the unknown diversity of methanol utilizers in marine sediments which is yet to be explored.

Functional Gene Expression in Shark Bay Hypersaline Microbial Mats: Adaptive Responses.

Microbial mat communities possess extensive taxonomic and functional diversity, which drive high metabolic rates and rapid cycling of major elements. Modern microbial mats occurring in hypersaline environments are considered as analogs to extinct geobiological formations dating back to ∼ 3.5 Gyr ago. Despite efforts to understand the diversity and metabolic potential of hypersaline microbial mats in Shark Bay, Western Australia, there has yet to be molecular analyses at the transcriptional level in these microbial communities. In this study, we generated metatranscriptomes for the first time from actively growing mats comparing the type of mat, as well as the influence of diel and seasonal cycles. We observed that the overall gene transcription is strongly influenced by microbial community structure and seasonality. The most transcribed genes were associated with tackling the low nutrient conditions by the uptake of fatty acids, phosphorus, iron, and nickel from the environment as well as with protective mechanisms against elevated salinity conditions and to prevent build-up of ammonium produced by nitrate reducing microorganisms. A range of pathways involved in carbon, nitrogen, and sulfur cycles were identified in mat metatranscriptomes, with anoxygenic photosynthesis and chemoautotrophy using the Arnon–Buchanan cycle inferred as major pathways involved in the carbon cycle. Furthermore, enrichment of active anaerobic pathways (e.g., sulfate reduction, methanogenesis, Wood–Ljungdahl) in smooth mats corroborates previous metagenomic studies and further advocates the potential of these communities as modern analogs of ancient microbialites.

Genome Analysis of a Marine Bacterium Halomonas sp. and Its Role in Nitrate Reduction under the Influence of Photoelectrons

The solar light response and photoelectrons produced by widespread semiconducting mineral play important roles in biogeochemical cycles on Earth’s surface. To explore the potential influence of photoelectrons generated by semiconducting mineral particles on nitrate-reducing microorganisms in the photic zone, a marine heterotrophic denitrifier Halomonas sp. strain 3727 was isolated from seawater in the photic zone of the Yellow Sea, China. This strain was classified as a Halomonadaceae. Whole-genome analysis indicated that this strain possessed genes encoding the nitrogen metabolism, i.e., narG, nasA, nirBD, norZ, nosB, and nxr, which sustained dissimilatory nitrate reduction, assimilatory nitrate reduction, and nitrite oxidation. This strain also possessed genes related to carbon, sulfur, and other metabolisms, hinting at its substantial metabolic flexibility. A series of microcosm experiments in a simulative photoelectron system was conducted, and the results suggested that this bacterial strain could use simulated photoelectrons with different energy for nitrate reduction. Nitrite, as an intermediate product, was accumulated during the nitrate reduction with limited ammonia residue. The nitrite and ammonia productions differed with or without different energy electron supplies. Nitrite was the main product accounting for 30.03% to 68.40% of the total nitrogen in photoelectron supplement systems, and ammonia accounted for 3.77% to 8.52%. However, in open-circuit systems, nitrite and ammonia proportions were 26.77% and 11.17%, respectively, and nitrogen loss in the liquid was not observed. This study reveals that photoelectrons can serve as electron donors for nitrogen transformation mediated by Halomonas sp. strain 3727, which reveals an underlying impact on the nitrogen biogeochemical cycle in the marine photic zone.

Anaerobic Dissolution Rates of U(IV)-Oxide by Abiotic and Nitrate-Dependent Bacterial Pathways.

The long-term stability of U(IV) solid phases in anaerobic aquifers depends upon their reactivity in the presence of oxidizing chemical species and microbial catalysts. We performed flow-through column experiments under anaerobic conditions to investigate the mechanisms and dissolution rates of biogenic, noncrystalline UO2(s) by chemical oxidants (nitrate and/or nitrite) or by Thiobacillus denitrificans, a widespread, denitrifying, chemolithoautotrophic model bacterium. Dissolution rates of UO2(s) with dissolved nitrite were approximately 5 to 10 times greater than with nitrate alone. In the presence of wild-type T. denitrificans and nitrate, UO2(s) dissolution rates were similar to those of abiotic experiments with nitrite (from 1.15 × 10-14 to 4.94 × 10-13 mol m-2 s-1). Experiments with a T. dentrificans mutant strain defective in U(IV) oxidation supported microbially mediated U(IV) oxidation. X-ray absorption spectroscopy (XAS) analysis of post-reaction solids showed the presence of mononuclear U(VI) species rather than a solid U(VI) phase. At steady-state U release, kinetic and spectroscopic results suggest detachment of oxidized U(VI) from the UO2(s) surface as the rate-determining step rather than electron transfer or ion diffusion. Under anaerobic conditions, production of nitrite by nitrate-reducing microorganisms and enzymatically catalyzed, nitrate-dependent U(IV) oxidation are likely dual processes by which reduced U solids may be oxidized and mobilized in the aqueous phase.

Oxidized humic acids from the soil of heat power plant

Humic acids isolated from the soil of heat power plant (HA-E) contaminated with oil were analyzed by the Fourier Transform Infrared spectra (FTIR). In comparison with humic acids standard (HA-S) luck of intense broad band of stretching vibrations of hydrogen-bonded hydroxyl groups (3600-3200 cm-1) is evident. HA-E have a pick at 1649 cm-1 which could belong to carbonyl groups. HA-E are heavily oxidized and among isolated microorganisms Achromobacter denitrificans may be responsible for such intensive oxidation of HA-E. To phylogenetically diverse nitrate-reducing microorganisms that have the capacity to utilize reduced HA as electron donors in soils, we can ad A. denitrificans.

Nitrate-Utilizing Microorganisms Resistant to Multiple Metals from the Heavily Contaminated Oak Ridge Reservation.

Contamination of environments with nitrate generated by industrial processes and the use of nitrogen-containing fertilizers is a growing problem worldwide. While nitrate can be removed from contaminated areas by microbial denitrification, nitrate frequently occurs with other contaminants, such as heavy metals, that have the potential to impede the process. Here, nitrate-reducing microorganisms were enriched and isolated from both groundwater and sediments at the Oak Ridge Reservation (ORR) using concentrations of nitrate and metals (Al, Mn, Fe, Co, Ni, Cu, Cd, and U) similar to those observed in a contaminated environment at ORR. Seven new metal-resistant, nitrate-reducing strains were characterized, and their distribution across both noncontaminated and contaminated areas at ORR was examined. While the seven strains have various pH ranges for growth, carbon source preferences, and degrees of resistance to individual and combinations of metals, all were able to reduce nitrate at similar rates both in the presence and absence of the mixture of metals found in the contaminated ORR environment. Four strains were identified in groundwater samples at different ORR locations by exact 16S RNA sequence variant analysis, and all four were found in both noncontaminated and contaminated areas. By using environmentally relevant metal concentrations, we successfully isolated multiple organisms from both ORR noncontaminated and contaminated environments that are capable of reducing nitrate in the presence of extreme mixed-metal contamination.IMPORTANCE Nitrate contamination is a global issue that affects groundwater quality. In some cases, cocontamination of groundwater with nitrate and mixtures of heavy metals could decrease microbially mediated nitrate removal, thereby increasing the duration of nitrate contamination. Here, we used metal and nitrate concentrations that are present in a contaminated site at the Oak Ridge Reservation to isolate seven metal-resistant strains. All were able to reduce nitrate in the presence of high concentrations of a mixture of heavy metals. Four of seven strains were located in pristine as well as contaminated sites at the Oak Ridge Reservation. Further study of these nitrate-reducing strains will uncover mechanisms of resistance to multiple metals that will increase our understanding of the effect of nitrate and metal contamination on groundwater microbial communities.

Biodegradability of dissolved organic matter in Boom Clay pore water under nitrate-reducing conditions: Effect of additional C and P sources

In Belgium, Boom Clay is considered a potential host rock for the deep geological disposal of nuclear waste such as Eurobitum, an intermediate-level nitrate-containing radioactive waste form. The presence of dissolved organic matter (DOM) in the Boom Clay may play an important role in the mobility of radionuclides due to complexation and/or reduction of redox-sensitive radionuclides. The biodegradation of this DOM could therefore affect the barrier function of the Boom Clay. Due to the presence of nitrate leaching from Eurobitum into the clay formation, the biodegradation of DOM by a nitrate-reducing population cannot be ruled out in the Boom Clay surrounding a Eurobitum repository. In this paper, the biodegradability of DOM in Boom Clay pore water (BCPW) under nitrate-reducing conditions was investigated in the presence or absence of additional nutrients (i.e., acetate and phosphate) expected to be present in the clay surrounding a Eurobitum repository. The results of these experiments indicate that microbial nitrate reduction and the related degradation of DOM are performed by a syntrophic microbial community but are characterised by very slow kinetics. Nitrate-reducing microorganisms seem to be (at least partially) dependent on fermentation products such as acetate provided by fermenting microorganisms. The observed slow biodegradation of DOM appears to be linked to the characteristics of Boom Clay DOM, rather than to a limitation of nutrients necessary for microbial growth. The addition of phosphate only boosts the microbial growth of certain microbial species but not the biodegradation of DOM. On the other hand, acetate significantly enhances the microbial degradation of DOM. This result can be attributed to the use of acetate as an efficient C source for certain DOM-degrading microbial species and/or as a co-substrate to stimulate DOM degradation.

Responses of Aromatic-Degrading Microbial Communities to Elevated Nitrate in Sediments.

A high number of aromatic compounds that have been released into aquatic ecosystems have accumulated in sediment because of their low solubility and high hydrophobicity, causing significant hazards to the environment and human health. Since nitrate is an essential nitrogen component and a more thermodynamically favorable electron acceptor for anaerobic respiration, nitrate-based bioremediation has been applied to aromatic-contaminated sediments. However, few studies have focused on the response of aromatic-degrading microbial communities to nitrate addition in anaerobic sediments. Here we hypothesized that high nitrate inputs would stimulate aromatic-degrading microbial communities and their associated degrading processes, thus increasing the bioremediation efficiency in aromatic compound-contaminated sediments. We analyzed the changes of key aromatic-degrading genes in the sediment samples from a field-scale site for in situ bioremediation of an aromatic-contaminated creek in the Pearl River Delta before and after nitrate injection using a functional gene array. Our results showed that the genes involved in the degradation of several kinds of aromatic compounds were significantly enriched after nitrate injection, especially those encoding enzymes for central catabolic pathways of aromatic compound degradation, and most of the enriched genes were derived from nitrate-reducing microorganisms, possibly accelerating bioremediation of aromatic-contaminated sediments. The sediment nitrate concentration was found to be the predominant factor shaping the aromatic-degrading microbial communities. This study provides new insights into our understanding of the influences of nitrate addition on aromatic-degrading microbial communities in sediments.

Plant traits related to nitrogen uptake influence plant-microbe competition.

Plant species are important drivers of soil microbial communities. However, how plant functional traits are shaping these communities has received less attention though linking plant and microbial traits is crucial for better understanding plant-microbe interactions. Our objective was to determine how plant-microbe interactions were affected by plant traits. Specifically we analyzed how interactions between plant species and microbes involved in nitrogen cycling were affected by plant traits related to 'nitrogen nutrition in interaction with soil nitrogen availability. Eleven plant species, selected along an oligotrophic-nitrophilic gradient, were grown individually in a nitrogen-poor soil with two levels of nitrate availability. Plant traits for both carbon and nitrogen nutrition were measured and the genetic structure and abundance of rhizosphere. microbial communities, in particular the ammonia oxidizer and nitrate reducer guilds, were analyzed. The structure of the bacterial community in the rhizosphere differed significantly between plant species and these differences depended on nitrogen availability. The results suggest that the rate of nitrogen uptake per unit of root biomass and per day is a key plant trait, explaining why the effect of nitrogen availability on the structure of the bacterial community depends on the plant species. We also showed that the abundance of nitrate reducing bacteria always decreased with increasing nitrogen uptake per unit of root biomass per day, indicating that there was competition for nitrate between plants and nitrate reducing bacteria. This study demonstrates that nitrate-reducing microorganisms may be adversely affected by plants with a high nitrogen uptake rate. Our work puts forward the role of traits related to nitrogen in plant-microbe interactions, whereas carbon is commonly considered as the main driver. It also suggests that plant traits related to ecophysiological processes, such as nitrogen uptake rates, are more relevant for understanding plant-microbe interactions than composite traits, such as nitrophily, which are related to a number of ecophysiological processes.

Nitrate ammonification in mangrove soils: a hidden source of nitrite?

Nitrate reduction is considered to be a minor microbial pathway in the oxidation of mangrove-derived organic matter due to a limited supply of nitrate in mangrove soils. At a limited availability of this electron acceptor compared to the supply of degradable carbon, nitrate ammonification is thought to be the preferential pathway of nitrate reduction. Mangrove forest mutually differ in their productivity, which may lead to different available carbon to nitrate ratios in their soil. Hence, nitrate ammonification is expected to be of more importance in high- compared to low-productive forests. The hypothesis was tested in flow-through reactors that contain undisturbed mangrove soils from high-productive Avicennia germinans and Rhizophora mangle forests in Florida and low-productive Avicennia marina forests in Saudi Arabia. Nitrate was undetectable in the soils from both regions. It was assumed that a legacy of nitrate ammonification would be reflected by a higher ammonium production from these soils upon the addition of nitrate. Unexpectedly, the soils from the low-productive forests in Saudi Arabia produced considerably more ammonium than the soils from the high-productive forests in Florida. Hence, other environmental factors than productivity must govern the selection of nitrate ammonification or denitrification. A rather intriguing observation was the 1:1 production of nitrite and ammonium during the consumption of nitrate, more or less independent from sampling region, location, sampling depth, mangrove species and from the absence or presence of additional degradable carbon. This 1:1 ratio points to a coupled production of ammonium and nitrite by one group of nitrate-reducing microorganisms. Such a production of nitrite will be hidden by the presence of active nitrite-reducing microorganisms under the nitrate-limited conditions of most mangrove forest soils.

Microbial redox processes in deep subsurface environments and the potential application of (per)chlorate in oil reservoirs.

The ability of microorganisms to thrive under oxygen-free conditions in subsurface environments relies on the enzymatic reduction of oxidized elements, such as sulfate, ferric iron, or CO2, coupled to the oxidation of inorganic or organic compounds. A broad phylogenetic and functional diversity of microorganisms from subsurface environments has been described using isolation-based and advanced molecular ecological techniques. The physiological groups reviewed here comprise iron-, manganese-, and nitrate-reducing microorganisms. In the context of recent findings also the potential of chlorate and perchlorate [jointly termed (per)chlorate] reduction in oil reservoirs will be discussed. Special attention is given to elevated temperatures that are predominant in the deep subsurface. Microbial reduction of (per)chlorate is a thermodynamically favorable redox process, also at high temperature. However, knowledge about (per)chlorate reduction at elevated temperatures is still scarce and restricted to members of the Firmicutes and the archaeon Archaeoglobus fulgidus. By analyzing the diversity and phylogenetic distribution of functional genes in (meta)genome databases and combining this knowledge with extrapolations to earlier-made physiological observations we speculate on the potential of (per)chlorate reduction in the subsurface and more precisely oil fields. In addition, the application of (per)chlorate for bioremediation, souring control, and microbial enhanced oil recovery are addressed.

A mixture of nitrite-oxidizing and denitrifying microorganisms affects the δ18O of dissolved nitrate during anaerobic microbial denitrification depending on the δ18O of ambient water

The stable isotopes 15N/14N and 18O/16O of nitrate are frequently used to determine sources of nitrate and to assess denitrification processes in the environment. Nitrate isotope ratios are thought to be conservative unless involved in (bio-) chemical conversion processes. Thus, stable isotopes are considered to be a reliable tool to determine sources of nitrate in aquatic habitats even after transport and dilution has occurred. Denitrification is known to shift both isotope ratios towards higher δ-values. A fixed ratio of 0.5 for Δδ18O/Δδ15N has been proposed and has been widely used to detect denitrification in terrestrial environments, predominantly in aquifers. However, it is observed in environmental and laboratory studies that this ratio actually varies between less than 0.5 and 1 for uncertain reasons with laboratory studies usually describing a ratio close to 1.Here we report results of anoxic incubation experiments with natural populations of nitrate-reducing microorganisms using sediments from three different environments. In our experiments we used water with a δ18O in excess of 500‰ and found a microbially mediated influence of the oxygen isotopic composition of ambient water on the isotopic composition of the residual dissolved nitrate. We found up to 5.7±2.3% of the oxygen-atoms in the residual dissolved nitrate was exchanged by oxygen-atoms from ambient water within the limited timeframe of the experiments. The fastest incorporation of oxygen-atoms from water into dissolved nitrate correlated with the highest intermittent nitrite concentrations observed in our experiments. In a second series of batch experiments we also found that pure cultures of the nitrite-oxidizing bacterium Nitrobacter vulgaris promoted the incorporation of oxygen atoms from ambient water into dissolved nitrate under anoxic conditions. Presumably this happens via a reoxidation of intermediary formed nitrite by the enzyme “nitrite oxidoreductase” (NXR) in concurrence with respiratory nitrate reduction. In this context, our hypothesis is a reversibility of the reactions at the NXR enzyme even in the absence of external electron acceptors for nitrite oxidation. We suggest that the presence of nitrite-oxidizing microorganisms in aquatic environments may catalyse such an incorporation of oxygen-atoms stemming from ambient water into nitrate. This process may thus mask the original δ18O value of nitrate sources during denitrification and also distort the observed enrichment of 18O that is ascribed to denitrification. Our results are highly likely an explanation of the deviation of the described variable Δδ18O/Δδ15N ratios for denitrification in terrestrial field studies from the values observed in the laboratory on pure cultures.

Humic Acid-Oxidizing, Nitrate-Reducing Bacteria in Agricultural Soils

ABSTRACTThis study demonstrates the prevalence, phylogenetic diversity, and physiology of nitrate-reducing microorganisms capable of utilizing reduced humic acids (HA) as electron donors in agricultural soils. Most probable number (MPN) enumeration of agricultural soils revealed large populations (104 to 106 cells g−1 soil) of microorganisms capable of reducing nitrate while oxidizing the reduced HA analog 2,6-anthrahydroquinone disulfonate (AH2DS) to its corresponding quinone. Nitrate-dependent HA-oxidizing organisms isolated from agricultural soils were phylogenetically diverse and included members of the Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria. Advective up-flow columns inoculated with corn plot soil and amended with reduced HA and nitrate supported both HA oxidation and enhanced nitrate reduction relative to no-donor or oxidized HA controls. The additional electron donating capacity of reduced HA could reasonably be attributed to the oxidation of reduced functional groups. Subsequent 16S rRNA gene-based high-density oligonucleotide microarray (PhyloChip) indicated that reduced HA columns supported the development of a bacterial community enriched with members of the Acidobacteria, Firmicutes, and Betaproteobacteria relative to the no-donor control and initial inoculum. This study identifies a previously unrecognized role for HA in stimulating denitrification processes in saturated soil systems. Furthermore, this study indicates that reduced humic acids impact soil geochemistry and the indigenous bacterial community composition.