Hydrologic Connectivity Regulates Riverine N2O Sources and Dynamics.

Indirect nitrous oxide (N2O) emissions from streams and rivers are a poorly constrained term in the global N2O budget. Current models of riverine N2O emissions place a strong focus on denitrification in groundwater and riverine environments as a dominant source of riverine N2O, but do not explicitly consider direct N2O input from terrestrial ecosystems. Here, we combine N2O isotope measurements and spatial stream network modeling to show that terrestrial-aquatic interactions, driven by changing hydrologic connectivity, control the sources and dynamics of riverine N2O in a mesoscale river network within the U.S. Corn Belt. We find that N2O produced from nitrification constituted a substantial fraction (i.e., >30%) of riverine N2O across the entire river network. The delivery of soil-produced N2O to streams was identified as a key mechanism for the high nitrification contribution and potentially accounted for more than 40% of the total riverine emission. This revealed large terrestrial N2O input implies an important climate-N2O feedback mechanism that may enhance riverine N2O emissions under a wetter and warmer climate. Inadequate representation of hydrologic connectivity in observations and modeling of riverine N2O emissions may result in significant underestimations.

Soil microbial community response to ectomycorrhizal dominance in diverse neotropical montane forests.

Ectomycorrhizal (EM) associations can promote the dominance of tree species in otherwise diverse tropical forests. These EM associations between trees and their fungal mutualists have important consequences for soil organic matter cycling, yet the influence of these EM-associated effects on surrounding microbial communities is not well known, particularly in neotropical forests. We examined fungal and prokaryotic community composition in surface soil samples from mixed arbuscular mycorrhizal (AM) and ectomycorrhizal (EM) stands as well as stands dominated by EM-associated Oreomunnea mexicana (Juglandaceae) in four watersheds differing in soil fertility in the Fortuna Forest Reserve, Panama. We hypothesized that EM-dominated stands would support distinct microbial community assemblages relative to the mixed AM-EM stands due to differences in carbon and nitrogen cycling associated with the dominance of EM trees. We expected that this microbiome selection in EM-dominated stands would lead to lower overall microbial community diversity and turnover, with tighter correspondence between general fungal and prokaryotic communities. We measured fungal and prokaryotic community composition via high-throughput Illumina sequencing of the ITS2 (fungi) and 16S rRNA (prokaryotic) gene regions. We analyzed differences in alpha and beta diversity between forest stands associated with different mycorrhizal types, as well as the relative abundance of fungal functional groups and various microbial taxa. We found that fungal and prokaryotic community composition differed based on stand mycorrhizal type. There was lower prokaryotic diversity and lower relative abundance of fungal saprotrophs and pathogens in EM-dominated than AM-EM mixed stands. However, contrary to our prediction, there was lower homogeneity for fungal communities in EM-dominated stands compared to mixed AM-EM stands. Overall, we demonstrate that EM-dominated tropical forest stands have distinct soil microbiomes relative to surrounding diverse forests, suggesting that EM fungi may filter microbial functional groups in ways that could potentially influence plant performance or ecosystem function.

Contemporary evolution rivals the effects of rhizobium presence on community and ecosystem properties in experimental mesocosms.

Because genotypes within a species commonly differ in traits that influence other species, whole communities, or even ecosystem functions, evolutionary change within one key species may affect the community and ecosystem processes. Here we use experimental mesocosms to test how the evolution of reduced cooperation in rhizobium mutualists in response to 20 years of nitrogen fertilization compares to the effects of rhizobium presence on soil nitrogen availability and plant community composition and diversity. The evolution of reduced rhizobium cooperation caused reductions in soil nitrogen, biological nitrogen fixation, and leaf nitrogen concentrations that were as strong as, or even stronger than, experimental rhizobium inoculation (presence/absence) treatments. Effects of both rhizobium evolution and rhizobium inoculation on legume dominance, plant community composition, and plant species diversity were often smaller in magnitude, but suggest that rhizobium evolution can alter the relative abundance of plant functional groups. Our findings indicate that the consequences of rapid microbial evolution for ecosystems and communities can rival the effects resulting from the presence or abundance of keystone mutualists.

Beyond denitrification: The role of microbial diversity in controlling nitrous oxide reduction and soil nitrous oxide emissions.

Many biotic and abiotic processes contribute to nitrous oxide (N2 O) production in the biosphere, but N2 O consumption in the environment has heretofore been attributed primarily to canonical denitrifying microorganisms. The nosZ genes encoding the N2 O reductase enzyme, NosZ, responsible for N2 O reduction to dinitrogen are now known to include two distinct groups: the well-studied Clade I which denitrifiers typically possess, and the novel Clade II possessed by diverse groups of microorganisms, most of which are non-denitrifiers. Clade II N2 O reducers could play an important, previously unrecognized role in controlling N2 O emissions for several reasons, including: (1) the consumption of N2 O produced by processes other than denitrification, (2) hypothesized non-respiratory functions of NosZ as an electron sink or for N2 O detoxification, (3) possible differing enzyme kinetics of Clade II NosZ compared to Clade I NosZ, and (4) greater nosZ gene abundance for Clade II compared to Clade I in soils of many ecosystems. Despite the potential ecological significance of Clade II NosZ, a census of 800 peer-reviewed original research articles discussing nosZ and published from 2013 to 2019 showed that the percentage of articles evaluating or mentioning Clade II nosZ increased from 5% in 2013 to only 22% in 2019. The census revealed that the slowly spreading awareness of Clade II nosZ may result in part from disciplinary silos, with the percentage of nosZ articles mentioning Clade II nosZ ranging from 0% in Agriculture and Agronomy journals to 32% in Multidisciplinary Sciences journals. In addition, inconsistent nomenclature for Clade I nosZ and Clade II nosZ, with 17 different terminologies used in the literature, may have created confusion about the two distinct groups of N2 O reducers. We provide recommendations to accelerate advances in understanding the role of the diversity of N2 O reducers in regulating soil N2 O emissions.

Soils' dirty little secret: Depth-based comparisons can be inadequate for quantifying changes in soil organic carbon and other mineral soil properties.

Quantifying changes in soil organic carbon (SOC) stocks and other soil properties is essential for understanding how soils will respond to land management practices and global change. Although they are widely used, comparisons of SOC stocks at fixed depth (FD) intervals are subject to errors when changes in bulk density or soil organic matter occur. The equivalent soil mass (ESM) method has been recommended in lieu of FD for assessing changes in SOC stocks in mineral soils, but ESM remains underutilized for SOC stocks and has rarely been used for other soil properties. In this paper, we draw attention to the limitations of the FD method and demonstrate the advantages of the ESM approach. We provide illustrations to show that the FD approach is susceptible to errors not only for quantifying SOC stocks but also for soil mass-based properties such as SOC mass percent, C:N mass ratio, and δ13 C. We describe the ESM approach and show how it mitigates the FD method limitations. Using bulk density change simulations applied to an empirical dataset from bioenergy cropping systems, we show that the ESM method provides consistently lower errors than FD when quantifying changes in SOC stocks and other soil properties. To simplify the use of ESM, we detail how the method can be integrated into sampling schemes, and we provide an example R computer script that can perform ESM calculations on large datasets. We encourage future studies, whether temporal or comparative, to utilize sampling methods that are amenable to the ESM approach. Overall, we agree with previous recommendations that ESM should be the standard method for evaluating SOC stock changes in mineral soils, but we further suggest that ESM may also be preferred for comparisons of other soil properties including mass percentages, elemental mass ratios, and stable isotope composition.

Light availability and rhizobium variation interactively mediate the outcomes of legume-rhizobium symbiosis.

PREMISE: Nutrients, light, water, and temperature are key factors limiting the growth of individual plants in nature. Mutualistic interactions between plants and microbes often mediate resource limitation for both partners. In the mutualism between legumes and rhizobia, plants provide rhizobia with carbon in exchange for fixed nitrogen. Because partner quality in mutualisms is genotype-dependent, within-species genetic variation is expected to alter the responses of mutualists to changes in the resource environment. Here we ask whether partner quality variation in rhizobia mediates the response of host plants to changing light availability, and conversely, whether light alters the expression of partner quality variation. METHODS: We inoculated clover hosts with 11 strains of Rhizobium leguminosarum that differed in partner quality, grew plants under either ambient or low light conditions in the greenhouse, and measured plant growth, nodule traits, and foliar nutrient composition. RESULTS: Light availability and rhizobium inoculum interactively determined plant growth, and variation in rhizobium partner quality was more apparent in ambient light. CONCLUSIONS: Our results suggest that variation in the costs and benefits of rhizobium symbionts mediate host responses to light availability and that rhizobium strain variation might more important in higher-light environments. Our work adds to a growing appreciation for the role of microbial intraspecific and interspecific diversity in mediating extended phenotypes in their hosts and suggests an important role for light availability in the ecology and evolution of legume-rhizobium symbiosis.

Frontiers in alley cropping: Transformative solutions for temperate agriculture.

Annual row crops dominate agriculture around the world and have considerable negative environmental impacts, including significant greenhouse gas emissions. Transformative land-use solutions are necessary to mitigate climate change and restore critical ecosystem services. Alley cropping (AC)-the integration of trees with crops-is an agroforestry practice that has been studied as a transformative, multifunctional land-use solution. In the temperate zone, AC has strong potential for climate change mitigation through direct emissions reductions and increases in land-use efficiency via overyielding compared to trees and crops grown separately. In addition, AC provides climate change adaptation potential and ecological benefits by buffering alley crops to weather extremes, diversifying income to hedge financial risk, increasing biodiversity, reducing soil erosion, and improving nutrient- and water-use efficiency. The scope of temperate AC research and application has been largely limited to simple systems that combine one timber tree species with an annual grain. We propose two frontiers in temperate AC that expand this scope and could transform its climate-related benefits: (i) diversification via woody polyculture and (ii) expanded use of tree crops for food and fodder. While AC is ready now for implementation on marginal lands, we discuss key considerations that could enhance the scalability of the two proposed frontiers and catalyze widespread adoption.

Gross nitrous oxide production drives net nitrous oxide fluxes across a salt marsh landscape.

Sea level rise will change inundation regimes in salt marshes, altering redox dynamics that control nitrification - a potential source of the potent greenhouse gas, nitrous oxide (N2 O) - and denitrification, a major nitrogen (N) loss pathway in coastal ecosystems and both a source and sink of N2 O. Measurements of net N2 O fluxes alone yield little insight into the different effects of redox conditions on N2 O production and consumption. We used in situ measurements of gross N2 O fluxes across a salt marsh elevation gradient to determine how soil N2 O emissions in coastal ecosystems may respond to future sea level rise. Soil redox declined as marsh elevation decreased, with lower soil nitrate and higher ferrous iron in the low marsh compared to the mid and high marshes (P < 0.001 for both). In addition, soil oxygen concentrations were lower in the low and mid-marshes relative to the high marsh (P < 0.001). Net N2 O fluxes differed significantly among marsh zones (P = 0.009), averaging 9.8 ± 5.4 µg N m(-2)  h(-1) , -2.2 ± 0.9 µg N m(-2)  h(-1) , and 0.67 ± 0.57 µg N m(-2)  h(-1) in the low, mid, and high marshes, respectively. Both net N2 O release and uptake were observed in the low and high marshes, but the mid-marsh was consistently a net N2 O sink. Gross N2 O production was highest in the low marsh and lowest in the mid-marsh (P = 0.02), whereas gross N2 O consumption did not differ among marsh zones. Thus, variability in gross N2 O production rates drove the differences in net N2 O flux among marsh zones. Our results suggest that future studies should focus on elucidating controls on the processes producing, rather than consuming, N2 O in salt marshes to improve our predictions of changes in net N2 O fluxes caused by future sea level rise.

High potential for iron reduction in upland soils.

Changes in the redox state of iron (Fe) can be coupled to the biogeochemical cycling of carbon (C), nitrogen, and phosphorus, and thus regulate soil C, ecosystem nutrient availability, and greenhouse gas production. However, its importance broadly in non-flooded upland terrestrial ecosystems is unknown. We measured Fe reduction in soil samples from an annual grassland, a drained peatland, and a humid tropical forest We incubated soil slurries in an anoxic glovebox for 5.5 days and added sodium acetate daily at rates up to 0.4 mg C x (g soil)(-1) x d(-1). Soil moisture, poorly crystalline Fe oxide concentrations, and Fe(II) concentrations differed among study sites in the following order: annual grassland < drained peatland < tropical forest (P < 0.001 for all characteristics). All of the soil samples demonstrated high Fe reduction potential with maximum rates over the course of the incubation averaging 1706 ± 66, 2016 ± 12, and 2973 ± 115 µg Fe x (g soil)(-1) x d(-1) (mean ± SE) for the tropical forest, annual grassland, and drained peatland, respectively. Our results suggest that upland soils from diverse ecosystems have the potential to exhibit high short-term rates of Fe reduction that may play an important role in driving soil biogeochemical processes during periods of anaerobiosis.

Application of the N(2)/Ar technique to measuring soil-atmosphere N(2) fluxes.

RATIONALE: The emission of dinitrogen (N(2) ) gas from soil is the most poorly constrained flux in terrestrial nitrogen (N) budgets because the high background atmospheric N(2) concentration makes soil N(2) emissions difficult to measure. In this study, we tested the theoretical and analytical feasibility of using the N(2) /Ar technique to measure soil-atmosphere N(2) fluxes. METHODS: Dual inlet isotope ratio mass spectrometry was used to measure δAr/N(2) values of gas sampled from surface flux chambers. In laboratory experiments using dry sand in a diffusion box, we induced a known steady-state flux of N(2) , and then measured the change in the N(2) /Ar ratio of chamber headspace air samples to test our ability to reconstruct this flux. We m\odeled solubility, thermal, and water vapor flux fractionation effects on the N(2) /Ar ratio to constrain physical effects on the measured N(2) flux. RESULTS: In dry sand, an actual N(2) flux of 108 mg N m(-2) day(-1) was measured as 111 ± 19 mg N m(-2) day(-1) (± standard error (SE)). In wet sand, an actual N(2) flux of 160 mg N m(-2) day(-1) was measured as 146 ± 20 mg N m(-2) day(-1) when solubility and water vapor flux fractionation were taken into account. Corrections for thermal fractionation did not improve estimates of N(2) fluxes. CONCLUSIONS: We conclude that our application of the N(2) /Ar technique to soil surface fluxes is valid only above a detection limit of approximately 108 mg N m(-2) day(-1) . The N(2) /Ar method is currently best used as a validation tool for other methods in ecosystems with high soil N(2) fluxes, but, with future improvements, it holds promise to provide high-resolution measurements in systems with low soil N(2) fluxes.

Nitrogen Cycling Microbial Diversity and Operational Taxonomic Unit Clustering: When to Prioritize Accuracy Over Speed.

Background: Assessments of the soil microbiome provide valuable insight to ecosystem function due to the integral role microorganisms play in biogeochemical cycling of carbon and nutrients. For example, treatment effects on nitrogen cycling functional groups are often presented alongside one another to demonstrate how agricultural management practices affect various nitrogen cycling processes. However, the functional groups commonly evaluated in nitrogen cycling microbiome studies range from phylogenetically narrow (e.g., N-fixation, nitrification) to broad [e.g., denitrification, dissimilatory nitrate reduction to ammonium (DNRA)]. The bioinformatics methods used in such studies were developed for 16S rRNA gene sequence data, and how these tools perform across functional genes of different phylogenetic diversity has not been established. For example, an OTU clustering method that can accurately characterize sequences harboring comparatively little diversity may not accurately resolve the diversity within a gene comprised of a large number of clades. This study uses two nitrogen cycling genes, nifH, a gene which segregates into only three distinct clades, and nrfA, a gene which is comprised of at least eighteen clades, to investigate differences which may arise when using heuristic OTU clustering (abundance-based greedy clustering, AGC) vs. true hierarchical OTU clustering (Matthews Correlation Coefficient optimizing algorithm, Opti-MCC). Detection of treatment differences for each gene were evaluated to demonstrate how conclusions drawn from a given dataset may differ depending on clustering method used. Results: The heuristic and hierarchical methods performed comparably for the more conserved gene, nifH. The hierarchical method outperformed the heuristic method for the more diverse gene, nrfA; this included both the ability to detect treatment differences using PERMANOVA, as well as higher resolution in taxonomic classification. The difference in performance between the two methods may be traced to the AGC method's preferential assignment of sequences to the most abundant OTUs: when analysis was limited to only the largest 100 OTUs, results from the AGC-assembled OTU table more closely resembled those of the Opti-MCC OTU table. Additionally, both AGC and Opti-MCC OTU tables detected comparable treatment differences using the rank-based ANOSIM test. This demonstrates that treatment differences were preserved using both clustering methods but were structured differently within the OTU tables produced using each method. Conclusion: For questions which can be answered using tests agnostic to clustering method (e.g., ANOSIM), or for genes of relatively low phylogenetic diversity (e.g., nifH), most upstream processing methods should lead to similar conclusions from downstream analyses. For studies involving more diverse genes, however, care should be exercised to choose methods that ensure accurate clustering for all genes. This will mitigate the risk of introducing Type II errors by allowing for detection of comparable treatment differences for all genes assessed, rather than disproportionately detecting treatment differences in only low-diversity genes.

Tracing plant-environment interactions from organismal to planetary scales using stable isotopes: a mini review.

Natural isotope variation forms a mosaic of isotopically distinct pools across the biosphere and flows between pools integrate plant ecology with global biogeochemical cycling. Carbon, nitrogen, and water isotopic ratios (among others) can be measured in plant tissues, at root and foliar interfaces, and in adjacent atmospheric, water, and soil environments. Natural abundance isotopes provide ecological insight to complement and enhance biogeochemical research, such as understanding the physiological conditions during photosynthetic assimilation (e.g. water stress) or the contribution of unusual plant water or nutrient sources (e.g. fog, foliar deposition). While foundational concepts and methods have endured through four decades of research, technological improvements that enable measurement at fine spatiotemporal scales, of multiple isotopes, and of isotopomers, are advancing the field of stable isotope ecology. For example, isotope studies now benefit from the maturation of field-portable infrared spectroscopy, which allows the exploration of plant-environment sensitivity at physiological timescales. Isotope ecology is also benefiting from, and contributing to, new understanding of the plant-soil-atmosphere system, such as improving the representation of soil carbon pools and turnover in land surface models. At larger Earth-system scales, a maturing global coverage of isotope data and new data from site networks offer exciting synthesis opportunities to merge the insights of single-or multi-isotope analysis with ecosystem and remote sensing data in a data-driven modeling framework, to create geospatial isotope products essential for studies of global environmental change.

Complete Genome Sequence of Geobacter sp. Strain FeAm09, a Moderately Acidophilic Soil Bacterium.

A moderately acidophilic Geobacter sp. strain, FeAm09, was isolated from forest soil. The complete genome sequence is 4,099,068 bp with an average GC content of 61.1%. No plasmids were detected. The genome contains a total of 3,843 genes and 3,608 protein-coding genes, including genes supporting iron and nitrogen biogeochemical cycling.

Looking back to look ahead: a vision for soil denitrification research.

Denitrification plays a critical role in regulating ecosystem nutrient availability and anthropogenic reactive nitrogen (N) production. Its importance has inspired an increasing number of studies, yet it remains the most poorly constrained term in terrestrial ecosystem N budgets. We censused the peer-reviewed soil denitrification literature (1975-2015) to identify opportunities for future studies to advance our understanding despite the inherent challenges in studying the process. We found that only one-third of studies reported estimates of both nitrous oxide (N2 O) and dinitrogen (N2 ) production fluxes, often the dominant end products of denitrification, while the majority of studies reported only net N2 O fluxes or denitrification potential. Of the 236 studies that measured complete denitrification to N2 , 49% used the acetylene inhibition method, 84% were conducted in the laboratory, 81% were performed on surface soils (0-20 cm depth), 75% were located in North America and Europe, and 78% performed treatment manipulations, mostly of N, carbon, or water. To improve understanding of soil denitrification, we recommend broadening access to technologies for new methodologies to measure soil N2 production rates, conducting more studies in the tropics and on subsoils, performing standardized experiments on unmanipulated soils, and using more precise terminology to refer to measured process rates (e.g., net N2 O flux or denitrification potential). To overcome the greater challenges in studying soil denitrification, we envision coordinated research efforts based on standard reporting of metadata for all soil denitrification studies, standard protocols for studies contributing to a Global Denitrification Research Network, and a global consortium of denitrification researchers to facilitate sharing ideas, resources, and to provide mentorship for researchers new to the field.

Hierarchical detection of diverse Clade II (atypical) nosZ genes using new primer sets for classical- and multiplex PCR array applications.

The reduction of nitrous oxide (N2O) to N2 represents the key terminal step in canonical denitrification. Nitrous oxide reductase (NosZ), the enzyme associated with this biological step, however, is not always affiliated with denitrifying microorganisms. Such organisms were shown recently to possess a Clade II (atypical) nosZ gene, in contrast to Clade I (typical) nosZ harbored in more commonly studied denitrifiers. Subsequent phylogenetic analyses have shown that Clade II NosZ are affiliated with a much broader diversity of microorganisms than those with Clade I NosZ, the former including both non-denitrifiers and denitrifiers. Most studies attempting to characterize the nosZ gene diversity using DNA-based PCR approaches have only focused on Clade I nosZ, despite recent metagenomic sequencing studies that have demonstrated the dominance of Clade II nosZ genes in many ecosystems, particularly soil. As a result, these studies have greatly underestimated the genetic potential for N2O reduction present in ecosystems. Because the high diversity of Clade II NosZ makes it impossible to design a universal primer set that would effectively amplify all nosZ genes in this clade, we developed a suite of primer sets to specifically target seven of ten designated subclades of Clade II nosZ genes. The new primer sets yield suitable product sizes for paired end amplicon sequencing and qPCR, demonstrated here in their use for both conventional single-reaction and multiplex array platforms. In addition, we show the utility of these primers for detecting nosZ gene transcripts from mRNA extracted from soil.

Invasive perennial forb effects on gross soil nitrogen cycling and nitrous oxide fluxes depend on phenology.

Invasive plants can increase soil nitrogen (N) pools and accelerate soil N cycling rates, but their effect on gross N cycling and nitrous oxide (N2 O) emissions has rarely been studied. We hypothesized that perennial pepperweed (Lepidium latifolium) invasion would increase rates of N cycling and gaseous N loss, thereby depleting ecosystem N and causing a negative feedback on invasion. We measured a suite of gross N cycling rates and net N2 O fluxes in invaded and uninvaded areas of an annual grassland in the Sacramento-San Joaquin River Delta region of northern California. During the growing season, pepperweed-invaded soils had lower microbial biomass N, gross N mineralization, dissimilatory nitrate reduction to ammonium (DNRA), and denitrification-derived net N2 O fluxes (P < 0.02 for all). During pepperweed dormancy, gross N mineralization, DNRA, and denitrification-derived net N2 O fluxes were stimulated in pepperweed-invaded plots, presumably by N-rich litter inputs and decreased competition between microbes and plants for N (P < 0.04 for all). Soil organic carbon and total N concentrations, which reflect pepperweed effects integrated over longer time scales, were lower in pepperweed-invaded soils (P < 0.001 and P = 0.04, respectively). Overall, pepperweed invasion had a net negative effect on ecosystem N status, depleting soil total N to potentially cause a negative feedback to invasion in the long term.

Sequence alignments and validation of PCR primers used to detect phylogenetically diverse nrfA genes associated with dissimilatory nitrate reduction to ammonium (DNRA).

PCR primer sets were designed to target nrfA, the gene encoding the pentaheme nitrite reductase NrfA that catalyzes the nitrite ammonification step in the process of dissimilatory nitrate reduction to ammonium (DNRA). Details of the nucleotide alignments of the primer target regions of 271 nrfA sequences from reference genomes representing 18 distinct clades of NrfA are shown here along with validation of application to PCR-based methodology including the use of amplified fragment length polymorphism (AFLP) profiling and Illumina platform amplicon-based sequencing of environmental samples and selected reference strains. Summary data tables illustrate the specificity of forward primers nrfAF2awMOD and nrfAF2awMODgeo when paired with the new reverse primer nrfAR1MOD in relation to consensus target reference sequences associated with members of 18 NrfA clades. Specificity of the new primers to nrfA sequences in environmental samples is shown in AFLP analysis and amino acid-translated amplicon sequences obtained with the new primer sets. We also provide sequence alignment files of the full length nrfA genes, PCR reference amplicon alignment, NrfA amino-acid alignment and NrfA translated PCR amplicon-amino acid alignment. The full nucleotide and protein alignments contain 271 reference genomes that represent the 18 identified NrfA clades as a tool to further aid practitioners in examining new sequences corresponding to the primer target regions and allow further primer design modifications if deemed pertinent to specific studies. A more comprehensive analysis of this data may be obtained from ("Optimization of PCR primers to detect phylogenetically diverse nrfA genes associated with nitrite ammonification" Cannon et al., 2019).

Optimization of PCR primers to detect phylogenetically diverse nrfA genes associated with nitrite ammonification.

Dissimilatory nitrate reduction to ammonium (DNRA) is now known to be a more prevalent process in terrestrial ecosystems than previously thought. The key enzyme, a pentaheme cytochrome c nitrite reductase NrfA associated with respiratory nitrite ammonification, is encoded by the nrfA gene in a broad phylogeny of bacteria. The lack of reliable and comprehensive molecular tools to detect diverse nrfA from environmental samples has hampered efforts to meaningfully characterize the genetic potential for DNRA in environmental systems. In this study, modifications were made to optimize the amplification efficiency of previously-designed PCR primers, targeting the diagnostic region of NrfA between the conserved third- and fourth heme binding domains, and to increase coverage to include detection of environmentally relevant Geobacteraceae-like nrfA. Using an alignment of the primers to >270 bacterial nrfA genes affiliated with 18 distinct clades, modifications to the primer sequences improved coverage, minimized amplification artifacts, and yielded the predicted product sizes from reference-, soil-, and groundwater DNA. Illumina sequencing of amplicons showed the successful recovery of nrfA gene fragments from environmental DNA based on alignments of the translated sequences. The new primers developed in this study are more efficient in PCR reactions, although gene targets with high GC content affect efficiency. Furthermore, the primers have a broader spectrum of detection and were validated rigorously for use in detecting nrfA from natural environments. These are suitable for conventional PCR, qPCR, and use in PCR access array technologies that allow multiplex gene amplification for downstream high throughput sequencing platforms.

Effects of triclosan and triclocarban on denitrification and N2O emissions in paddy soil.

Triclosan (TCS) and triclocarban (TCC) are two common antimicrobial compounds, which are widely used as ingredients in pharmaceuticals and personal care products. They occur ubiquitously in soil due to biosolid application as agricultural fertilizers, but their influence on microbially mediated soil biogeochemical processes is poorly understood. We tested the effects of varying concentrations of TCS and TCC applied both individually and together on denitrification and N2O emissions in paddy soil. We also quantified denitrification functional gene abundances by q-PCR to elucidate the microbial mechanisms of TCS and TCC's effects. Our results showed that TCS and TCC exposure both individually and together significantly (p < 0.05) inhibited denitrification (7.0-36.7%) and N2O emissions (15.4-86.4%) except for the 0.01 mg kg-1 TCC treatment in which denitrification was slightly but significantly (p < 0.05) stimulated. The inhibitory effects of TCS and TCC exposure were mainly attributed to their negative net effects on denitrifying bacteria as suggested by the decrease in abundances of 16S rRNA, narG, nirK and clade I nosZ genes in the TCS and TCC treatments. Overall, we found that TCS and TCC exposure in paddy soil could substantially alter nitrogen cycling in rice paddy ecosystems by inhibiting denitrification and N2O emissions. These effects should be taken into consideration when evaluating the environmental impacts of TCS and TCC.

Comparing DNA, RNA and protein levels for measuring microbial dynamics in soil microcosms amended with nitrogen fertilizer.

To what extent multi-omic techniques could reflect in situ microbial process rates remains unclear, especially for highly diverse habitats like soils. Here, we performed microcosm incubations using sandy soil from an agricultural site in Midwest USA. Microcosms amended with isotopically labeled ammonium and urea to simulate a fertilization event showed nitrification (up to 4.1 ± 0.87 µg N-NO3- g-1 dry soil d-1) and accumulation of N2O after 192 hours of incubation. Nitrification activity (NH4+ → NH2OH → NO → NO2- → NO3-) was accompanied by a 6-fold increase in relative expression of the 16S rRNA gene (RNA/DNA) between 10 and 192 hours of incubation for ammonia-oxidizing bacteria Nitrosomonas and Nitrosospira, unlike archaea and comammox bacteria, which showed stable gene expression. A strong relationship between nitrification activity and betaproteobacterial ammonia monooxygenase and nitrite oxidoreductase transcript abundances revealed that mRNA quantitatively reflected measured activity and was generally more sensitive than DNA under these conditions. Although peptides related to housekeeping proteins from nitrite-oxidizing microorganisms were detected, their abundance was not significantly correlated with activity, revealing that meta-proteomics provided only a qualitative assessment of activity. Altogether, these findings underscore the strengths and limitations of multi-omic approaches for assessing diverse microbial communities in soils and provide new insights into nitrification.