Microbial diversity and gene abundance in denitrifying bioreactors: A comparison of the woodchip surface biofilm versus the interior wood matrix.

Excessive amounts of nitrogen (N) and phosphorus (P) can lead to eutrophication in water sources. Woodchip bioreactors have shown success in removing N from agricultural runoff, but less is known regarding P removal. Woodchip bioreactors are subsurface basins filled with woodchips installed downgradient of agricultural land to collect and treat drainage runoff. Microorganisms use the woodchips as a carbon (C) source to transform N in the runoff, with unresolved biological impacts on P. This study aims to explore microbial communities present in the bioreactor and determine whether milling woodchips to probe the microbial communities within them reveals hidden microbial diversities or potential activities. Metagenomic sequencing and bioinformatic analyses were performed on six woodchip samples (i.e., three unmilled and three milled) collected from a 10-year-old woodchip bioreactor treating agricultural tile drainage. All samples had similar DNA purity, yield, quality, and microbial diversity regardless of milling. However, when sequences were aligned against various protein libraries, our results indicated greater relative abundance of denitrification and P transformation proteins on the outside of the woodchips (unmilled), while the interior of woodchips (milled) exhibited more functional gene abundance for carbohydrate breakdown. Thus, it may be important to characterize microbial communities both within woodchips, and on woodchip surfaces, to gain a more holistic understanding of coupled biogeochemical cycles on N, P, and C in woodchip bioreactors. Based on these findings, we advise that future microbial research on woodchips (and potentially other permeable organic materials) examine both the surface biofilm and the interior organic material during initial studies. Once researchers determine where specific proteins or enzymes of interest are most prevalent, subsequent studies may then focus on either one or both aspects, as needed.

The long-term effect of glutaraldehyde on the bacterial community in anaerobic ammonium oxidation reactor.

A 160-day incubation was performed with two anammox reactors (GA and CK) to investigate the effect of glutaraldehyde. The results indicated that anammox bacteria were very sensitive when glutaraldehyde in GA reactor increased to 40 mg/L, the nitrogen removal efficiency sharply decreased to 11%, only one-quarter of CK. Glutaraldehyde changed spatial distribution of exopolysaccharides, caused anammox bacteria (Brocadia CK_gra75) to disassociate from granules (24.70% of the reads in CK but only 14.09% in GA granules). Metagenome analysis indicated glutaraldehyde led to the denitrifier community succession from strains without nir (nitrite reductase) and nor (nitric oxide reductases) genes to those with them, and the rapid growth of denitrifiers with NodT (an outer membrane factor)-related efflux pumps replacing those with another TolC -related ones. Meanwhile, Brocadia CK_gra75 lacks the NodT proteins. This study provides important insight into community adaptation and potential resistance mechanism in an active anammox community after exposure to disinfectant.

Oxic-anoxic cycling promotes coupling between complex carbon metabolism and denitrification in woodchip bioreactors.

Denitrifying woodchip bioreactors (WBRs) are increasingly used to manage the release of non-point source nitrogen (N) by stimulating microbial denitrification. Woodchips serve as a renewable organic carbon (C) source, yet the recalcitrance of organic C in lignocellulosic biomass causes many WBRs to be C-limited. Prior studies have observed that oxic-anoxic cycling increased the mobilization of organic C, increased nitrate (NO3 - ) removal rates, and attenuated production of nitrous oxide (N2 O). Here, we use multi-omics approaches and amplicon sequencing of fungal 5.8S-ITS2 and prokaryotic 16S rRNA genes to elucidate the microbial drivers for enhanced NO3 - removal and attenuated N2 O production under redox-dynamic conditions. Transient oxic periods stimulated the expression of fungal ligninolytic enzymes, increasing the bioavailability of woodchip-derived C and stimulating the expression of denitrification genes. Nitrous oxide reductase (nosZ) genes were primarily clade II, and the ratio of clade II/clade I nosZ transcripts during the oxic-anoxic transition was strongly correlated with the N2 O yield. Analysis of metagenome-assembled genomes revealed that many of the denitrifying microorganisms also have a genotypic ability to degrade complex polysaccharides like cellulose and hemicellulose, highlighting the adaptation of the WBR microbiome to the ecophysiological niche of the woodchip matrix.

pH-Dependent Hydrogenotrophic Denitratation Based on Self-Alkalization.

Producing stable nitrite is a necessity for anaerobic ammonium oxidation (anammox) but remains a huge challenge. Here, we describe the design and operation of a hydrogenotrophic denitratation system that stably reduced >90% nitrate to nitrite under self-alkaline conditions of pH up to 10.80. Manually lowering the pH to a range of 9.00-10.00 dramatically decreased the nitrate-to-nitrite transformation ratio to <20%, showing a significant role of high pH in denitratation. Metagenomics combined with metatranscriptomics indicated that six microorganisms, including a Thauera member, dominated the community and encoded the various genes responsible for hydrogen oxidation and the complete denitrification process. During denitratation at high pH, transcription of periplasmic genes napA, nirS, and nirK, whose products perform nitrate and nitrite reduction, decreased sharply compared to that under neutral conditions, while narG, encoding a membrane-associated nitrate reductase, remained transcriptionally active, as were genes involved in intracellular proton homeostasis. Together with no reduction in only nitrite-amended samples, these results disproved the electron competition between reductions of nitrate and nitrite but highlighted a lack of protons outside cells constraining biological nitrite reduction. Overall, our study presents a stably efficient strategy for nitrite production and provides a major advance in the understanding of denitratation.

Linking meta-omics to the kinetics of denitrification intermediates reveals pH-dependent causes of N2O emissions and nitrite accumulation in soil.

Soil pH is a key controller of denitrification. We analysed the metagenomics/transcriptomics and phenomics of two soils from a long-term liming experiment, SoilN (pH 6.8) and un-limed SoilA (pH 3.8). SoilA had severely delayed N2O reduction despite early transcription of nosZ (mainly clade I), encoding N2O reductase, by diverse denitrifiers. This shows that post-transcriptionally hampered maturation of the NosZ apo-protein at low pH is a generic phenomenon. Identification of transcript reads of several accessory genes in the nos cluster indicated that enzymes for NosZ maturation were present across a range of organisms, eliminating their absence as an explanation for the failure to produce a functional enzyme. nir transcript abundances (for NO2- reductase) in SoilA suggest that low NO2- concentrations in acidic soils, often ascribed to abiotic degradation, are primarily due to biological activity. The accumulation of NO2- in neutral soil was ascribed to high nar expression (nitrate reductase). The -omics results revealed dominance of nirK over nirS in both soils while qPCR showed the opposite, demonstrating that standard primer pairs only capture a fraction of the nirK pool. qnor encoding NO reductase was strongly expressed in SoilA, implying an important role in controlling NO. Production of HONO, for which some studies claim higher, others lower, emissions from NO2- accumulating soil, was estimated to be ten times higher from SoilA than from SoilN. The study extends our understanding of denitrification-driven gas emissions and the diversity of bacteria involved and demonstrates that gene and transcript quantifications cannot always reliably predict community phenotypes.

The anammox coupled partial-denitrification process in an integrated granular sludge and fixed-biofilm reactor developed for mainstream wastewater treatment: Performance and community structure.

This study describes an integrated granular sludge and fixed-biofilm (iGB) reactor innovatively designed to carry out the anammox/partial-denitrification (A/PD) process for nitrogen removal with mainstream municipal wastewater. The iGB-A/PD reactor consists of anammox granules inoculated in the lower region of reactor and an acclimated fixed-biofilm positioned in the upper region. Compared to the other reported A/PD systems for mainstream wastewater treatment, this iGB-A/PD reactor is notable due to its higher quality effluent with a total inorganic nitrogen (TIN) of ∼3 mg•L-1 and operation at a high nitrogen removal rate (NRR) of 0.8 ± 0.1 kg-N•m-3•d-1. Reads-based metatranscriptomic analysis found that the expression values of hzsA and hdh, key genes associated with anammox, were much higher than other functional genes on nitrogen conversion, confirming the major roles of the anammox bacteria in nitrogen bio-removal. In both regions of the reactor, the nitrate reduction genes (napA/narG) had expression values of 56-99 RPM, which were similar to that of the nitrite reduction genes (nirS/nirK). The expression reads from genes for dissimilatory nitrate reduction to ammonium (DNRA), nrfA and nirB, were unexpectedly high, and were over the half of the levels of reads from genes required for nitrate reduction. Kinetic assays confirmed that the granules had an anammox activity of 16.2 g-NH4+-N•kg-1-VSS•d-1 and a nitrate reduction activity of 4.1 g-N•kg-1-VSS•d-1. While these values were changed to be 4.9 g- NH4+-N•kg-1-VSS•d-1and 4.3 g-N•kg-1-VSS•d-1 respectively in the fixed-biofilm. Mass flux determination found that PD and DNRA was responsible for ∼50% and ∼25% of nitrate reduction, respectively, in the whole reactor, consistent with high effluent quality and treatment efficiency via a nitrite loop. Metagenomic binning analysis revealed that new and unidentified anammox species, affiliated with Candidatus Brocadia, were the dominant anammox organisms. Myxococcota and Planctomycetota were the principal organisms associated with the PD and DNRA processes, respectively.

Metagenomics and metatranscriptomics uncover the microbial community associated with high S0 production in a denitrifying desulfurization granular sludge reactor.

The denitrification desulfurization process is a promising technology for elemental sulfur (S0) production from sulfide containing wastewater. However, the microbial community associated with high S0 production still is not well studied. This study describes an efficient denitrification S0 production bioreactor based on inoculation with anaerobic granular sludge. At an optimal S/N molar ratio of 7:2, 80 % of the influent sulfide was transformed to high quality elemental sulfur with a purity of 92.5% while the total inorganic nitrogen removal efficiency was stable at ∼80%. Metatranscriptomic analysis found that community expression of the gene encoding the sulfide-quinone reductase (SQR) was 10-fold greater than that of the flavocytochrome-c sulfide dehydrogenase subunit B (fccB). Moreover, the expression level of SQR was also significantly higher than the Dsr gene encoding for dissimilatory sulfate reductase, which encodes a critical S0 oxidation enzyme. Metagenomic binning analysis confirmed that sulfide-oxidizing bacteria (SOB) utilizing SQR were common in the community and most likely accounted for high S0 production. An unexpected enrichment in methanogens and high expression activity of bacteria carrying out Stickland fermentation as well as in other bacteria with reduced genomes indicated a complex community supporting stable sulfide oxidation to S0, likely aiding in performance stability. This study establishes this treatment approach as an alternative biotechnology for sulfide containing wastewater treatment and sheds light on the microbial interactions associated with high S0 production.

Competition for electrons favours N2 O reduction in denitrifying Bradyrhizobium isolates.

Bradyrhizobia are common members of soil microbiomes and known as N2 -fixing symbionts of economically important legumes. Many are also denitrifiers, which can act as sinks or sources for N2 O. Inoculation with compatible rhizobia is often needed for optimal N2 -fixation, but the choice of inoculant may have consequences for N2 O emission. Here, we determined the phylogeny and denitrification capacity of Bradyrhizobium strains, most of them isolated from peanut-nodules. Analyses of genomes and denitrification end-points showed that all were denitrifiers, but only ~1/3 could reduce N2 O. The N2 O-reducing isolates had strong preference for N2 O- over NO3 - -reduction. Such preference was also observed in a study of other bradyrhizobia and tentatively ascribed to competition between the electron pathways to Nap (periplasmic NO3 - reductase) and Nos (N2 O reductase). Another possible explanation is lower abundance of Nap than Nos. Here, proteomics revealed that Nap was instead more abundant than Nos, supporting the hypothesis that the electron pathway to Nos outcompetes that to Nap. In contrast, Paracoccus denitrificans, which has membrane-bond NO3 - reductase (Nar), reduced N2 O and NO3 - simultaneously. We propose that the control at the metabolic level, favouring N2 O reduction over NO3 - reduction, applies also to other denitrifiers carrying Nos and Nap but lacking Nar.

Phenolic acid-degrading Paraburkholderia prime decomposition in forest soil.

Plant-derived phenolic acids are catabolized by soil microorganisms whose activity may enhance the decomposition of soil organic carbon (SOC). We characterized whether phenolic acid-degrading bacteria enhance SOC mineralization in forest soils when primed with 13C-labeled p-hydroxybenzoic acid (pHB). We further tested whether pHB-induced priming could explain differences in SOC content among mono-specific tree plantations in a 70-year-old common garden experiment. pHB addition primed significant losses of SOC (3-13 µmols C g-1 dry wt soil over 7 days) compared to glucose, which reduced mineralization (-3 to -8 µmols C g-1 dry wt soil over 7 days). The principal degraders of pHB were Paraburkholderia and Caballeronia in all plantations regardless of tree species or soil type, with one predominant phylotype (RP11ASV) enriched 23-fold following peak pHB respiration. We isolated and confirmed the phenolic degrading activity of a strain matching this phylotype (RP11T), which encoded numerous oxidative enzymes, including secretion signal-bearing laccase, Dyp-type peroxidase and aryl-alcohol oxidase. Increased relative abundance of RP11ASV corresponded with higher pHB respiration and expression of pHB monooxygenase (pobA), which was inversely proportional to SOC content among plantations. pobA expression proved a responsive measure of priming activity. We found that stimulating phenolic-acid degrading bacteria can prime decomposition and that this activity, corresponding with differences in tree species, is a potential mechanism in SOC cycling in forests. Overall, this study highlights the ecology and function of Paraburkholderia whose associations with plant roots and capacity to degrade phenolics suggest a role for specialized bacteria in the priming effect.

Community Organization and Metagenomics of Bacterial Assemblages Across Local Scale pH Gradients in Northern Forest Soils.

Soil pH has shown to predict bacterial diversity, but mechanisms are still poorly understood. To investigate how bacteria distribute themselves as a function of soil pH, we assessed community composition, diversity, assembly, and gene abundance across local (ca. 1 km) scale gradients in soil pH from ~ 3.8 to 6.5 created by differences in soil parent material in three northern forests. Plant species were the same on all sites, with no evidence of agriculture in the past. Concentrations of extractable calcium, iron, and phosphorus also varied significantly across the pH gradients. Among taxa, Alphaproteobacteria and Acidobacteria were more common in soils with acidic pH values. Overall richness and diversity of OTUs peaked at intermediate pH values. Variations in OTU richness and diversity also had a quadratic fit with concentrations of extractable calcium and phosphorus. Community assembly was via homogeneous deterministic processes in soils with acidic pH values, whereas stochastic processes dominated in soils with near-neutral pH values. Although we expected selection via genes for acid tolerance response in acidic soils, genes for genetic information processing were more selective. Taxa in higher pH soils had differential abundance of transporter genes, suggesting adaptation to acquire metabolic substrates from soils. Soil bacterial communities in northern forest soils are incredibly diverse, and we still have much to learn about how soil pH and co-varying soil parameters directly drive gene selection in this critical component of ecosystem structure.

Application of acidic conditions and inert-gas sparging to achieve high-efficiency nitrous oxide recovery during nitrite denitrification.

Nitrogen removal with energy recovery through denitrification dependent N2O production is garnering recent attention due to its cost advantages. The most effective current method requires alternating COD and nitrite to achieve high N2O production making it incompatible with typical wastewaters and consequently difficult to use in most settings. The work described here introduces a robust and highly efficient N2O recovery approach which has the potential to work with wastewaters containing COD and nitrite simultaneously. This method relies on low pH incubation and inert gas sparging (IGS) to shift a community of mainly N2 producing nitrite denitrifiers to a community that accumulates N2O when incubated in the absence of IGS. Before experiencing IGS, samples from activated sludge incubated at a pH of 4.5 and 6.0 only achieved a maximum N2O production efficiency (PE_N2O) of ∼26%. After IGS the PE_N2O values increased to ∼97.5% and ∼80.2% for samples from these same pH 4.5 and pH 6.0 reactors, respectively. IGS did not lead to N2O production in a pH 7.5 bioreactor. Meta-omics analysis revealed that IGS resulted in an increase in bacteria utilizing the clade I nitrous oxide reductase (nosZI) relative to bacteria utilizing the clade II nitrous oxide reductase (nosZII). This likely results from IGS flushing out N2O leaving nitrite as the principal nitrogen oxide available for respiration, favoring nosZI utilizing bacteria which are more likely to be complete denitrifiers. Metatranscriptomic analysis suggested that the high PE_N2O values that occurred after stopping IGS result from the NO generated by chemodenitrification accumulating to levels that inactivate [4Fe:4S] clusters in the NosR protein essential for N2O reduction in the nosZI denitrifiers. This study provides an efficient and straightforward method for N2O recovery, widening the options for energy recovery from nitrogen-based wastes.

Bacteriophage-mediated extracellular DNA release is important for the structural stability of aerobic granular sludge.

The aim of this study was to investigate the microbial characteristics and the structural role of exDNA in different size AGSs. Metagenomic results showed that exDNA has a significantly lower GC content, ~46.0%, than the ~65.0% GC of intracellular DNA (inDNA). Taxonomic predictions showed most of the reads from the exDNA that could be taxonomically assigned were from members of the phyla Bacteroidetes (55.0-64.2% of the total exDNA reads). Assigned inDNA reads were mainly from Proteobacteria (50.9-57.8%) or Actinobacteria (18.0-28.0%). Reads mapping showed that exDNA read depths were similar across all predicted open reading frames from assembled genomes that were assigned as Bacteroidetes which is consistent with cell lysis as a source of exDNA. Enrichment of CRISPR-CAS proteins in exDNA reads and CRISPR spacers in Bacteroidetes associated draft genomes suggested that bacteriophage infection may be an important cause of lysis of these cells. A critical role for this exDNA was found using DNase I digestion experiments which showed that the exDNA was vital for the structural stability of relatively small sized AGS but not for the larger sized AGS. The characteristics of exDNA in AGSs revealed in this work provide a new perspective on AGS components and structural stability.

Metagenomics revealed the phase-related characteristics during rapid development of halotolerant aerobic granular sludge.

Efforts to produce aerobic granular sludge (AGS) for high-efficient and stable nutrient removal in high saline wastewaters have gained much attention recently. This study was undertaken to describe the phase-related characteristics of the rapid formation of glucose-fed salt-tolerant AGS (SAGS) generated from common municipal activated sludge using metagenomic approaches. The time needed for SAGS formation is about 11 days in a multi-ion matrix salinity of 3%. There were three distinct developmental phases during sludge maturation which were designated: I) the salinity adaptation phase (days 1-2), II) the particle-size transition phase (days 3-5) and III) the maturation and steady-state phase (days 6-11), respectively. Genome-based analysis revealed that during the phase I, members of the genus Mangrovibacter, which has the potential to secrete extracellular polymeric substances (EPS), dominated during the formation of initial SAGS aggregates. During phase II, fungi of the class Saccharomycetes, in particular the genus Geotrichum, became dominant and provided a matrix for bacterial attachment. This mutualistic interaction supported the rapid development and maintenance of mature SAGS. This work characterizes a robust approach for the rapid development of SAGS for efficient saline sewage treatment and provides unique insight into the granulation mechanism occurring during the development process.

Metagenomics reveals microbial community differences lead to differential nitrate production in anammox reactors with differing nitrogen loading rates.

Nitrate production during anammox can decrease total nitrogen removal efficiency, which will negatively impact its usefulness for the removal of nitrogen from waste streams. However, neither the performance characteristics nor physiological shifts associated with nitrate accumulation in anammox reactors under different nitrogen loading rates (NLRs) is well understood. Consequently, these parameters were studied in a lower NLR anammox reactor, termed R1, producing higher than expected levels of nitrate and compared with a higher NLR reactor, termed R2, showing no excess nitrate production. While both reactors showed high NH4+-N removal efficiencies (>90%), the total nitrogen removal efficiency (<60%) was much lower in R1 due to higher nitrate production. Metagenomic analysis found that the number of reads derived from anammox bacteria were significantly higher in R2. Another notable trend in reads occurrence was the relatively higher levels of reads from genes predicted to be nitrite oxidoreductases (nxr) in R1. Binning yielded 27 high quality draft genomes from the two reactors. Analysis of bin occurrence found that R1 showing both a decrease in anammox bacteria and an unexpected increase in nxr. In-situ assays confirmed that R1 had higher rates of nitrite oxidation to nitrate and suggested that it was not solely due to obligate NOB, but other nxr-containing bacteria are important contributors as well. Our results demonstrate that nitrate accumulation can be a serious operational concern for the application of anammox technology to low-strength wastewater treatment and provide insight into the community changes leading to this outcome.

Multi-omics reveal various potential antimonate reductases from phylogenetically diverse microorganisms.

While previous work has demonstrated that antimonate (Sb(V)) can be bio-reduced with methane as the sole electron donor, the microorganisms responsible for Sb(V) reduction remain largely uncharacterized. Inspired by the recently reported Sb(V) reductase belonging to the dimethyl sulfoxide reductase (DMSOR) family, this study was undertaken to use metagenomics and metatranscriptomics to unravel whether any DMSOR family genes in the bioreactor had the potential for Sb(V) reduction. A search through metagenomic-assembled genomes recovered from the microbial community found that some DMSOR family genes, designated sbrA (Sb(V) reductase gene), were highly transcribed in four phylogenetically disparate assemblies. The putative catalytic subunits were found to be representatives of two distinct phylogenetic clades of reductases that were most closely related to periplasmic nitrate reductases and respiratory arsenate reductases, respectively. Putative operons containing sbrA possessed many other components, including genes encoding c-type cytochromes, response regulators, and ferredoxins, which together implement Sb(V) reduction. This predicted ability was confirmed by incubating the enrichment culture with 13C-labeled CH4 and Sb(V) in serum bottles, where Sb(V) was reduced coincident with the production of 13C-labeled CO2. Overall, these results increase our understanding of how Sb(V) can be bio-reduced in environments.

Metagenomic analysis reveals distinct patterns of denitrification gene abundance across soil moisture, nitrate gradients.

This study coupled a landscape-scale metagenomic survey of denitrification gene abundance in soils with in situ denitrification measurements to show how environmental factors shape distinct denitrification communities that exhibit varying denitrification activity. Across a hydrologic gradient, the distribution of total denitrification genes (nap/nar + nirK/nirS + cNor/qNor + nosZ) inferred from metagenomic read abundance exhibited no consistent patterns. However, when genes were considered independently, nirS, cNor and nosZ read abundance was positively associated with areas of higher soil moisture, higher nitrate and higher annual denitrification rates, whereas nirK and qNor read abundance was negatively associated with these factors. These results suggest that environmental conditions, in particular soil moisture and nitrate, select for distinct denitrification communities that are characterized by differential abundance of genes encoding apparently functionally redundant proteins. In contrast, taxonomic analysis did not identify notable variability in denitrifying community composition across sites. While the capacity to denitrify was ubiquitous across sites, denitrification genes with higher energetic costs, such as nirS and cNor, appear to confer a selective advantage in microbial communities experiencing more frequent soil saturation and greater nitrate inputs. This study suggests metagenomics can help identify denitrification hotspots that could be protected or enhanced to treat non-point source nitrogen pollution.

Salinity-Aided Selection of Progressive Onset Denitrifiers as a Means of Providing Nitrite for Anammox.

Anaerobic ammonium oxidation (anammox) combined with partial-denitrification (NO3- → NO2-) is an innovative process for the simultaneous removal of ammonia and nitrate from wastewaters. An efficient method for the selection of partial denitrifying community, which relies on increasing influent salinity, is described. Using this method, a denitratating community was enriched, which showed a nitrite accumulation efficiency higher than 75% as well as a high nitrate conversion efficiency. Community analysis using 16S rDNA indicated that Halomonas became the dominant genus as salinity increased. Metagenomic analysis revealed that there was not a significant difference in reads mapping to downstream denitrification genes in a comparison of samples from cultures with 5% salinity to those without salinity. The majority of the reads mapping to the genes encoding dissimilatory nitrate and nitrite reductases nar and nirS came from Halomonas under high salinity conditions. Two metagenome-assembled genomes taxonomically assigned to Halomonas were obtained, one of which accounted for ∼35% of the reads under high salinity conditions. Both genomes harbored the genes for the complete denitrification pathway. These results indicate progressive onset denitrifiers, a phenotype where nitrite reduction only occurs after nitrate exhaustion, could be successfully enriched with increasing salinity. Progressive onset denitrifiers may be more widespread in natural and artificial habitats than anticipated and are shown here to be valuable for nitrogen mitigating processes.

Plant-Microbe Interactions Drive Denitrification Rates, Dissolved Nitrogen Removal, and the Abundance of Denitrification Genes in Stormwater Control Measures.

The microbial community and function along with nitrate/nitrite (NOx) removal rates, and nitrogen (N) partitioning into "uptake", "denitrification", and "remaining" via isotope tracers, were studied in soil bioretention mesocolumns (8 unique plant species). Total denitrification gene reads per million (rpm) were positively correlated with % denitrified ( r = 0.69) but negatively correlated with total NOx removal following simulated rain events ( r = -0.79). This is likely due to plant-microbe interactions. Plant species with greater root volume, plant and microbial assimilation %, and NOx removal % had lower denitrification genes and rates. This implies that although microorganisms have access to N, advantageous functions, like denitrification, may not increase. At the conclusion of the 1.5-year experiment, the microbial community was strongly influenced by plant species within the Top zone dominated by plant roots, and the presence or absence of a saturated zone influenced the microbial community within the Bottom zone. Leptospermum continentale was an outlier from the other plants and had much lower denitrification gene rpm (average 228) compared to the other species (range: 277 to 413). The antimicrobial properties and large root volume of Leptospermum continentale likely caused this denitrification gene depression.

Metagenomic Evidence for a Methylocystis Species Capable of Bioremediation of Diverse Heavy Metals.

Heavy metal pollution has become an increasingly serious problem worldwide. Co-contamination with toxic mercury (Hg) and arsenic (As) presents a particularly difficult bioremediation trouble. By oxidizing the greenhouse gas methane, methanotrophs have been demonstrated to have high denitrification activity in eutrophic waters, indicating their possible potential for use in bioremediation of Hg(II) and As(V) in polluted water. Using metagenomics, a novel Methylocystis species (HL18), which was one of the most prevalent bacteria (9.9% of the relative abundance) in a CH4-based bio-reactor, is described here. The metagenomic-assembled genome (MAG) HL18 had gene products whose average amino acid identity against other known Methylocystis species varied from 69 to 85%, higher than the genus threshold but lower than the species boundary. Genomic analysis indicated that HL18 possessed all the genes necessary for the reduction of Hg(II) and As(V). Phylogenetic investigation of mercuric reductase (MerA) found that the HL18 protein was most closely affiliated with proteins from two Hg(II)-reducing bacteria, Bradyrhizobium sp. strain CCH5-F6 and Paracoccus halophilus. The genomic organization and phylogeny of the genes in the As(V)-reducing operon (arsRCCB) had significant identity with those from a As(V)-reducing bacterium belonging to the Rhodopseudomonas genus, indicating their reduction capability of As(V). Further analysis found that at least eight genera of methanotrophs possess both Hg(II) and As(V) reductases, illustrating the generally overlooked metabolic potential of methanotrophs. These results suggest that methanotrophs have greater bioremediation potential in heavy metal contaminated water than has been appreciated and could play an important role in the mitigation of heavy metal toxicity of contaminated wastewater.