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.

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.

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.

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.

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.

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.

Modularity of nitrogen-oxide reducing soil bacteria: linking phenotype to genotype.

Model denitrifiers convert NO3- to N2 , but it appears that a significant fraction of natural populations are truncated, conducting only one or two steps of the pathway. To better understand the diversity of partial denitrifiers in soil and whether discrepancies arise between the presence of known N-oxide reductase genes and phenotypic features, bacteria able to reduce NO3- to NO2- were isolated from soil, N-oxide gas products were measured for eight isolates, and six were genome sequenced. Gas phase analyses revealed that two were complete denitrifiers, which genome sequencing corroborated. The remaining six accumulated NO and N2 O to varying degrees and genome sequencing of four indicated that two isolates held genes encoding nitrate reductase as the only dissimilatory N-oxide reductase, one contained genes for both nitrate and nitric oxide reductase, and one had nitrate and nitrite reductase. The results demonstrated that N-oxide production was not always predicted by the genetic potential and suggested that partial denitrifiers could be readily isolated among soil bacteria. This supported the hypothesis that each N-oxide reductase could provide a selectable benefit on its own, and therefore, reduction of nitrate to dinitrogen may not be obligatorily linked to complete denitrifiers but instead a consequence of a functionally diverse community.

The Role of Denitrification in Stormwater Detention Basin Treatment of Nitrogen.

The nitrogen (N) cycling dynamics of four stormwater basins, two often saturated sites ("Wet Basins") and two quick draining sites ("Dry Basins"), were monitored over a ∼ 1-year period. This study paired stormwater and greenhouse gas monitoring with microbial analyses to elucidate the mechanisms controlling N treatment. Annual dissolved inorganic N (DIN) mass reductions (inflow minus outflow) were greater in the Dry Basin than in the Wet Basin, 2.16 vs 0.75 g N m-2 yr-1, respectively. The Dry Basin infiltrated a much larger volume of water and thus had greater DIN mass reductions, even though incoming and outgoing DIN concentrations were statistically the same for both sites. Wet Basins had higher proportions of denitrification genes and potential denitrification rates. The Wet Basin was capable of denitrifying 58% of incoming DIN, whereas the Dry Basin only denitrified 1%. These results emphasize the need for more mechanistic attention to basin design because the reductions calculated by comparing inflow and outflow loads may not be relevant at watershed scales. Denitrification is the only way to fully remove DIN from the terrestrial environment and receiving waterbodies. Consequently, at the watershed scale the Wet Basin may have better overall DIN treatment.

Role of norEF in denitrification, elucidated by physiological experiments with Rhodobacter sphaeroides.

Many denitrifying organisms contain the norEF gene cluster, which codes for two proteins that are thought to be involved in denitrification because they are expressed during the reduction of nitrite and nitric oxide. The products of both genes are predicted to be membrane associated, and the norE product is a member of the cytochrome c oxidase subunit III family. However, the specific role of norEF is unknown. The denitrification phenotypes of Rhodobacter sphaeroides strains with and without norEF genes were studied, and it was found that loss of norEF lowered the rate of denitrification from nitrate and resulted in accumulation of micromolar concentrations of nitric oxide during denitrification from nitrite. norEF appears to have no direct role in the reduction of nitric oxide; however, since deletion of norEF in the wild-type 2.4.3 strain had essentially no influence on the kinetics of potential nitric oxide reduction (Vmax and Ks), as measured by monitoring the depletion of a bolus of nitric oxide injected into anoxic cultures without any other electron acceptors. However, norEF-deficient cells that had undergone a more chronic exposure to micromolar concentrations of nitric oxide showed an ∼50% reduction in Vmax but no change in apparent Ks. These results can explain the occurrence of norEF in the 2.4.3 strain of R. sphaeroides, which can reduce nitrate to nitrous oxide, and their absence from strains such as 2.4.1, which likely use nitric oxide reductase to mitigate stress due to episodic exposure to nitric oxide from exogenous sources.

Metatranscriptomic analyses of plankton communities inhabiting surface and subpycnocline waters of the Chesapeake Bay during oxic-anoxic-oxic transitions.

We used metatranscriptomics to study the gene transcription patterns of microbial plankton (0.2 to 64 µm) at a mesohaline station in the Chesapeake Bay under transitions from oxic to anoxic waters in spring and from anoxic to oxic waters in autumn. Samples were collected from surface (i.e., above pycnocline) waters (3 m) and from waters beneath the pycnocline (16 to 22 m) in both 2010 and 2011. Metatranscriptome profiles based on function and potential phylogeny were different between 2010 and 2011 and strongly variable in 2011. This difference in variability corresponded with a highly variable ratio of eukaryotic to bacterial sequences (0.3 to 5.5), reflecting transient algal blooms in 2011 that were absent in 2010. The similarity between metatranscriptomes changed at a lower rate during the transition from oxic to anoxic waters than after the return to oxic conditions. Transcripts related to photosynthesis and low-affinity cytochrome oxidases were significantly higher in shallow than in deep waters, while in deep water genes involved in anaerobic metabolism, particularly sulfate reduction, succinyl coenzyme A (succinyl-CoA)-to-propionyl-CoA conversion, and menaquinone synthesis, were enriched relative to in shallow waters. Expected transitions in metabolism between oxic and anoxic deep waters were reflected in elevated levels of anaerobic respiratory reductases and utilization of propenediol and acetoin. The percentage of archaeal transcripts increased in both years in late summer (from 0.1 to 4.4% of all transcripts in 2010 and from 0.1 to 6.2% in 2011). Denitrification-related genes were expressed in a predicted pattern during the oxic-anoxic transition. Overall, our data suggest that Chesapeake Bay microbial assemblages express gene suites differently in shallow and deep waters and that differences in deep waters reflect variable redox states.

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.

A novel protein protects bacterial iron-dependent metabolism from nitric oxide.

Reactive nitrogen species (RNS), in particular nitric oxide (NO), are toxic to bacteria, and bacteria have mechanisms to allow growth despite this stress. Understanding how bacteria interact with NO is essential to understanding bacterial physiology in many habitats, including pathogenesis; however, many targets of NO and enzymes involved in NO resistance remain uncharacterized. We performed for the first time a metabolomic screen on NO-treated and -untreated bacteria to define broadly the effects of NO on bacterial physiology, as well as to identify the function of NnrS, a previously uncharacterized enzyme involved in defense against NO. We found many known and novel targets of NO. We also found that iron-sulfur cluster enzymes were preferentially inhibited in a strain lacking NnrS due to the formation of iron-NO complexes. We then demonstrated that NnrS is particularly important for resistance to nitrosative stress under anaerobic conditions. Our data thus reveal the breadth of the toxic effects of NO on metabolism and identify the function of an important enzyme in alleviating this stress.

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.

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.

Physiological roles for two periplasmic nitrate reductases in Rhodobacter sphaeroides 2.4.3 (ATCC 17025).

The metabolically versatile purple bacterium Rhodobacter sphaeroides 2.4.3 is a denitrifier whose genome contains two periplasmic nitrate reductase-encoding gene clusters. This work demonstrates nonredundant physiological roles for these two enzymes. One cluster is expressed aerobically and repressed under low oxygen while the second is maximally expressed under low oxygen. Insertional inactivation of the aerobically expressed nitrate reductase eliminated aerobic nitrate reduction, but cells of this strain could still respire nitrate anaerobically. In contrast, when the anaerobic nitrate reductase was absent, aerobic nitrate reduction was detectable, but anaerobic nitrate reduction was impaired. The aerobic nitrate reductase was expressed but not utilized in liquid culture but was utilized during growth on solid medium. Growth on a variety of carbon sources, with the exception of malate, the most oxidized substrate used, resulted in nitrite production on solid medium. This is consistent with a role for the aerobic nitrate reductase in redox homeostasis. These results show that one of the nitrate reductases is specific for respiration and denitrification while the other likely plays a role in redox homeostasis during aerobic growth.

Oxygen control of nitrogen oxide respiration, focusing on α-proteobacteria.

Denitrification is generally considered to occur under micro-oxic or anoxic conditions. With this in mind, the physiological function and regulation of several steps in the denitrification of model α-proteobacteria are compared in the present review. Expression of the periplasmic nitrate reductase is quite variable, with this enzyme being maximally expressed under oxic conditions in some bacteria, but under micro-oxic conditions in others. Expression of nitrite and NO reductases in most denitrifiers is more tightly controlled, with expression only occurring under micro-oxic conditions. A possible exception to this may be Roseobacter denitrificans, but the physiological role of these enzymes under oxic conditions is uncertain.

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.

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.