Reaction pathways and Sb(III) minerals formation during the reduction of Sb(V) by Rhodoferax ferrireducens strain YZ-1.

Antimony (Sb), a non-essential metalloid, can be released into the environment through various industrial activities. Sb(III) is considered more toxic than Sb(V), but Sb(III) can be immobilized through the precipitation of insoluble Sb2S3 or Sb2O3. In the subsurface, Sb redox chemistry is largely controlled by microorganisms; however, the exact mechanisms of Sb(V) reduction to Sb(III) are still unclear. In this study, a new strain of Sb(V)-reducing bacterium, designated as strain YZ-1, that can respire Sb(V) as a terminal electron acceptor was isolated from Sb-contaminated soils. 16S-rRNA gene sequencing of YZ-1 revealed high similarity to a known Fe(III)-reducer, Rhodoferax ferrireducens. XRD and XAFS analyses revealed that bioreduction of Sb(V) to Sb(III) proceed through a transition from amorphous valentinite to crystalline senarmontite (allotropes of Sb2O3). Genomic DNA sequencing found that YZ-1 possesses arsenic (As) metabolism genes, including As(V) reductase arsC. The qPCR analysis showed that arsC was highly expressed during Sb(V)-reduction by YZ-1, and thus is proposed as the potential Sb(V) reductase in YZ-1. This study provides new insight into the pathways and products of microbial Sb(V) reduction and demonstrates the potential of a newly isolated bacterium for Sb bioremediation.

Distinct microbial structure and metabolic potential shaped by significant environmental gradient impacted by ferrous slag weathering.

Alkaline ferrous slags pose global environmental issues and long-term risks to ambient environments. To explore the under-investigated microbial structure and biogeochemistry in such unique ecosystems, combined geochemical, microbial, ecological and metagenomic analyses were performed in the areas adjacent to a ferrous slag disposal plant in Sichuan, China. Different levels of exposure to ultrabasic slag leachate had resulted in a significant geochemical gradient of pH (8.0-12.4), electric potential (-126.9 to 437.9 mV), total organic carbon (TOC, 1.5-17.3 mg/L), and total nitrogen (TN, 0.17-1.01 mg/L). Distinct microbial communities were observed depending on their exposure to the strongly alkaline leachate. High pH and Ca2+ concentrations were associated with low microbial diversity and enrichment of bacterial classes Gamma-proteobacteria and Deinococci in the microbial communities exposed to the leachate. Combined metagenomic analyses of 4 leachate-unimpacted and 2-impacted microbial communities led to the assembly of one Serpentinomonas pangenome and 81 phylogenetically diversified metagenome assembled genomes (MAGs). The prevailing taxa in the leachate-impacted habitats (e.g., Serpentinomonas and Meiothermus spp.) were phylogenetically related to those in active serpentinizing ecosystems, suggesting the analogous processes between the man-made and natural systems. More importantly, they accounted for significant abundance of most functional genes associated with environmental adaptation and major element cycling. Their metabolic potential (e.g., cation/H+ antiporters, carbon fixation on lithospheric carbon source, and respiration coupling sulfur oxidization and oxygen or nitrate reduction) may support these taxa to survive and prosper in these unique geochemical niches. This study provides fundamental understandings of the adaptive strategies of microorganisms in response to the strong environmental perturbation by alkali tailings. It also contributes to a better comprehension of how to remediate environments affected by alkaline industrial material.

Tropical lacustrine sediment microbial community response to an extreme El Niño event.

Salinity can influence microbial communities and related functional groups in lacustrine sediments, but few studies have examined temporal variability in salinity and associated changes in lacustrine microbial communities and functional groups. To better understand how microbial communities and functional groups respond to salinity, we examined geochemistry and functional gene amplicon sequence data collected from 13 lakes located in Kiritimati, Republic of Kiribati (2° N, 157° W) in July 2014 and June 2019, dates which bracket the very large El Niño event of 2015-2016 and a period of extremely high precipitation rates. Lake water salinity values in 2019 were significantly reduced and covaried with ecological distances between microbial samples. Specifically, phylum- and family-level results indicate that more halophilic microorganisms occurred in 2014 samples, whereas more mesohaline, marine, or halotolerant microorganisms were detected in 2019 samples. Functional Annotation of Prokaryotic Taxa (FAPROTAX) and functional gene results (nifH, nrfA, aprA) suggest that salinity influences the relative abundance of key functional groups (chemoheterotrophs, phototrophs, nitrogen fixers, denitrifiers, sulfate reducers), as well as the microbial diversity within functional groups. Accordingly, we conclude that microbial community and functional gene groups in the lacustrine sediments of Kiritimati show dynamic changes and adaptations to the fluctuations in salinity driven by the El Niño-Southern Oscillation.

Carbonate Minerals and Dissimilatory Iron-Reducing Organisms Trigger Synergistic Abiotic and Biotic Chain Reactions under Elevated CO2 Concentration.

Increasing CO2 emission has resulted in pressing climate and environmental issues. While abiotic and biotic processes mediating the fate of CO2 have been studied separately, their interactions and combined effects have been poorly understood. To explore this knowledge gap, an iron-reducing organism, Orenia metallireducens, was cultured under 18 conditions that systematically varied in headspace CO2 concentrations, ferric oxide loading, and dolomite (CaMg(CO3)2) availability. The results showed that abiotic and biotic processes interactively mediate CO2 acidification and sequestration through "chain reactions", with pH being the dominant variable. Specifically, dolomite alleviated CO2 stress on microbial activity, possibly via pH control that transforms the inhibitory CO2 to the more benign bicarbonate species. The microbial iron reduction further impacted pH via the competition between proton (H+) consumption during iron reduction and H+ generation from oxidization of the organic substrate. Under Fe(III)-rich conditions, microbial iron reduction increased pH, driving dissolved CO2 to form bicarbonate. Spectroscopic and microscopic analyses showed enhanced formation of siderite (FeCO3) under elevated CO2, supporting its incorporation into solids. The results of these CO2-microbe-mineral experiments provide insights into the synergistic abiotic and biotic processes that alleviate CO2 acidification and favor its sequestration, which can be instructive for practical applications (e.g., acidification remediation, CO2 sequestration, and modeling of carbon flux).

Stable carbon isotope values of syndepositional carbonate spherules and micrite record spatial and temporal changes in photosynthesis intensity.

Marine and lacustrine carbonate minerals preserve carbon cycle information, and their stable carbon isotope values (δ13 C) are frequently used to infer and reconstruct paleoenvironmental changes. However, multiple processes can influence the δ13 C values of bulk carbonates, confounding the interpretation of these values in terms of conditions at the time of mineral precipitation. Co-existing carbonate forms may represent different environmental conditions, yet few studies have analyzed δ13 C values of syndepositional carbonate grains of varying morphologies to investigate their origins. Here, we combine stable isotope analyses, metagenomics, and geochemical modeling to interpret δ13 C values of syndepositional carbonate spherules (>500 µm) and fine-grained micrite (<63 µm) from a ~1600-year-long sediment record of a hypersaline lake located on the coral atoll of Kiritimati, Republic of Kiribati (1.9°N, 157.4°W). Petrographic, mineralogic, and stable isotope results suggest that both carbonate fractions precipitate in situ with minor diagenetic alterations. The δ13 C values of spherules are high compared to the syndepositional micrite and cannot be explained by mineral differences or external perturbations, suggesting a role for local biological processes. We use geochemical modeling to test the hypothesis that the spherules form in the surface microbial mat during peak diurnal photosynthesis when the δ13 C value of dissolved inorganic carbon is elevated. In contrast, we hypothesize that the micrite may precipitate more continuously in the water as well as in sub-surface, heterotrophic layers of the microbial mat. Both metagenome and geochemical model results support a critical role for photosynthesis in influencing carbonate δ13 C values. The down-core spherule-micrite offset in δ13 C values also aligns with total organic carbon values, suggesting that the difference in the δ13 C values of spherules and micrite may be a more robust, inorganic indicator of variability in productivity and local biological processes through time than the δ13 C values of individual carbonate forms.

Differential structure and functional gene response to geochemistry associated with the suspended and attached shallow aquifer microbiomes from the Illinois Basin, IL.

Despite the clear ecological significance of the microbiomes inhabiting groundwater and connected ecosystems, our current understanding of their habitats, functionality, and the ecological processes controlling their assembly have been limited. In this study, an efficient pipeline combining geochemistry, high-throughput FluidigmTM functional gene amplification and sequencing was developed to analyze the suspended and attached microbial communities inhabiting five groundwater monitoring wells in the Illinois Basin, USA. The dominant taxa in the suspended and the attached microbial communities exhibited significantly different spatial and temporal changes in both alpha- and beta-diversity. Further analyses of representative functional genes affiliated with N2 fixation (nifH), methane oxidation (pmoA), and sulfate reduction (dsrB, and aprA), suggested functional redundancy within the shallow aquifer microbiomes. While more diversified functional gene taxa were observed for the suspended microbial communities than the attached ones except for pmoA, different levels of changes over time and space were observed between these functional genes. Notably, deterministic and stochastic ecological processes shaped the assembly of microbial communities and functional gene reservoirs differently. While homogenous selection was the prevailing process controlling assembly of microbial communities, the neutral processes (e.g., dispersal limitation, drift and others) were more important for the functional genes. The results suggest complex and changing shallow aquifer microbiomes, whose functionality and assembly vary even between the spatially proximate habitats and fractions. This research underscored the importance to include all the interface components for a more holistic understanding of the biogeochemical processes in aquifer ecosystems, which is also instructive for practical applications.

Impact of antibiotic concentration gradients on nitrate reduction and antibiotic resistance in a microfluidic gradient chamber.

In order to explore the impact of antibiotics on the bacterial metabolic cycling of nitrate within contaminated soil and groundwater environments, we compared the effects of polymyxin B (PMB) and ciprofloxacin (CIP) concentration gradients on the distribution and activity of a wild type (WT) and a flagella deficient mutant (Δflag) of Shewanella oneidensis MR-1 in a microfluidic gradient chamber (MGC). Complementary batch experiments were performed to measure bacteriostatic versus bactericidal concentrations of the two antibiotics, as well as their effect on nitrate reduction. Prior work demonstrated that PMB disrupts cell membranes while CIP inhibits DNA synthesis. Consistent with these modes of action, batch results from this work show that PMB is bactericidal at lower concentrations than CIP relative to their respective minimum inhibitory concentrations (MICs) (≥5× MICPMB vs. ≥20× MICCIP). Concentration gradients from 0 to 50× the MIC of both antibiotics were established in the MGC across a 2-cm interconnected pore network, with nutrients injected at both concentration boundaries. The WT cells could only access and reduce nitrate in regions of the MGC with PMB at <18× MICPMB, whereas this occurred with CIP up to 50× MICCIP; and cells extracted from these MGCs showed no antibiotic resistance. The distribution of Δflag cells was further limited to lower antibiotic concentrations (≤1× MICPMB, ≤43× MICCIP) due to inability of movement. These results indicate that S. oneidensis access and reduce nitrate in bactericidal regions via chemotactic migration without development of antibiotic resistance, and that this migration is inhibited by acutely lethal bactericidal levels of antibiotics.

The role of chemotaxis and efflux pumps on nitrate reduction in the toxic regions of a ciprofloxacin concentration gradient.

Spatial concentration gradients of antibiotics are prevalent in the natural environment. Yet, the microbial response in these heterogeneous systems remains poorly understood. We used a microfluidic reactor to create an artificial microscopic ecosystem that generates diffusive gradients of solutes across interconnected microenvironments. With this reactor, we showed that chemotaxis toward a soluble electron acceptor (nitrate) allowed Shewanella oneidensis MR-1 to inhabit and sustain metabolic activity in highly toxic regions of the antibiotic ciprofloxacin (>80× minimum inhibitory concentration, MIC). Acquired antibiotic resistance was not observed for cells extracted from the reactor, so we explored the role of transient adaptive resistance by probing multidrug resistance (MDR) efflux pumps, ancient elements that are important for bacterial physiology and virulence. Accordingly, we constructed an efflux pump deficient mutant (∆mexF) and used resistance-nodulation-division (RND) efflux pump inhibitors (EPIs). While batch results showed the importance of RND efflux pumps for microbial survival, microfluidic studies indicated that these pumps were not necessary for survival in antibiotic gradients. Our work contributes to an emerging body of knowledge deciphering the effects of antibiotic spatial heterogeneity on microorganisms and highlights differences of microbial response in these systems versus well-mixed batch conditions.

Microbial Taxonomy Run Amok.

DNA sequencing has led to an explosion in discovery of microbial phylogenetic novelty, especially that represented by uncultivated taxa, to which the traditional system of prokaryotic taxonomy has not adapted. A lack of expansion of the International Code of Nomenclature of Prokaryotes (ICNP, 'the Code') to effectively capture this information has created a 'wild west' situation where names are published or appear in popular reference databases without further verification or validation. The rapid propagation of variant and questionable naming methods has led to widespread confusion and undermines prior accomplishments. We exemplify inconsistencies that have arisen from this practice and endanger the interoperability of scientific findings. The immediate solution to this problem is to develop and adopt universal best practices that are accepted by expert researchers, major publishers, the International Committee on Systematics of Prokaryotes (ICSP), and international microbiological societies.

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.

Profiling of Microbial Communities in the Sediments of Jinsha River Watershed Exposed to Different Levels of Impacts by the Vanadium Industry, Panzhihua, China.

The mining, smelting, manufacturing, and disposal of vanadium (V) and associated products have caused serious environmental problems. Although the microbial ecology in V-contaminated soils has been intensively studied, the impacted watershed ecosystems have not been systematically investigated. In this study, geochemistry and microbial structure were analyzed along ~30 km of the Jinsha River and its two tributaries across the industrial areas in Panzhihua, one of the primary V mining and production cities in China. Geochemical analyses showed different levels of contamination by metals and metalloids in the sediments, with high degrees of contamination observed in one of the tributaries close to the industrial park. Analyses of the V4 hypervariable region of 16S rRNA genes of the microbial communities in the sediments showed significant decrease in microbial diversity and microbial structure in response to the environmental gradient (e.g., heavy metals, total sulfur, and total nitrogen). Strong association of the taxa (e.g., Thauera, Algoriphagus, Denitromonas, and Fontibacter species) with the metals suggested selection for these potential metal-resistant and/or metabolizing populations. Further co-occurrence network analysis showed that many identified potential metal-mediating species were among the keystone taxa that were closely associated in the same module, suggesting their strong inter-species interactions but relative independence from other microorganisms in the hydrodynamic ecosystems. This study provided new insight into the microbe-environment interactions in watershed ecosystems differently impacted by the V industries. Some of the phylotypes identified in the highly contaminated samples exhibited potential for bioremediation of toxic metals (e.g., V and Cr).

In Vivo Entombment of Bacteria and Fungi during Calcium Oxalate, Brushite, and Struvite Urolithiasis.

Background: Human kidney stones form via repeated events of mineral precipitation, partial dissolution, and reprecipitation, which are directly analogous to similar processes in other natural and manmade environments, where resident microbiomes strongly influence biomineralization. High-resolution microscopy and high-fidelity metagenomic (microscopy-to-omics) analyses, applicable to all forms of biomineralization, have been applied to assemble definitive evidence of in vivo microbiome entombment during urolithiasis. Methods: Stone fragments were collected from a randomly chosen cohort of 20 patients using standard percutaneous nephrolithotomy (PCNL). Fourier transform infrared (FTIR) spectroscopy indicated that 18 of these patients were calcium oxalate (CaOx) stone formers, whereas one patient formed each formed brushite and struvite stones. This apportionment is consistent with global stone mineralogy distributions. Stone fragments from seven of these 20 patients (five CaOx, one brushite, and one struvite) were thin sectioned and analyzed using brightfield (BF), polarization (POL), confocal, super-resolution autofluorescence (SRAF), and Raman techniques. DNA from remaining fragments, grouped according to each of the 20 patients, were analyzed with amplicon sequencing of 16S rRNA gene sequences (V1-V3, V3-V5) and internal transcribed spacer (ITS1, ITS2) regions. Results: Bulk-entombed DNA was sequenced from stone fragments in 11 of the 18 patients who formed CaOx stones, and the patients who formed brushite and struvite stones. These analyses confirmed the presence of an entombed low-diversity community of bacteria and fungi, including Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria, and Aspergillus niger. Bacterial cells approximately 1 µm in diameter were also optically observed to be entombed and well preserved in amorphous hydroxyapatite spherules and fans of needle-like crystals of brushite and struvite. Conclusions: These results indicate a microbiome is entombed during in vivo CaOx stone formation. Similar processes are implied for brushite and struvite stones. This evidence lays the groundwork for future in vitro and in vivo experimentation to determine how the microbiome may actively and/or passively influence kidney stone biomineralization.

Controls on Iron Reduction and Biomineralization over Broad Environmental Conditions as Suggested by the Firmicutes Orenia metallireducens Strain Z6.

Microbial iron reduction is a ubiquitous biogeochemical process driven by diverse microorganisms in a variety of environments. However, it is often difficult to separate the biological from the geochemical controls on bioreduction of Fe(III) oxides. Here, we investigated the primary driving factor(s) that mediate secondary iron mineral formation over a broad range of environmental conditions using a single dissimilatory iron reducer, Orenia metallireducens strain Z6. A total of 17 distinct geochemical conditions were tested with differing pH (6.5-8.5), temperature (22-50 °C), salinity (2-20% NaCl), anions (phosphate and sulfate), electron shuttle (anthraquinone-2,6-disulfonate), and Fe(III) oxide mineralogy (ferrihydrite, lepidocrocite, goethite, hematite, and magnetite). The observed rates and extent of iron reduction differed significantly with kint between 0.186 and 1.702 mmol L-1 day-1 and Fe(II) production ranging from 6.3% to 83.7% of the initial Fe(III). Using X-ray absorption and scattering techniques (EXAFS and XRD), we identified and assessed the relationship between secondary minerals and the specific environmental conditions. It was inferred that the observed bifurcation of the mineralization pathways may be mediated by differing extents of Fe(II) sorption on the remaining Fe(III) minerals. These results expand our understanding of the controls on biomineralization during microbial iron reduction and aid the development of practical applications.

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.

Microbial U Isotope Fractionation Depends on the U(VI) Reduction Rate.

U isotope fractionation may serve as an accurate proxy for U(VI) reduction in both modern and ancient environments, if the systematic controls on the magnitude of fractionation (ε) are known. We model the effect of U(VI) reduction kinetics on U isotopic fractionation during U(VI) reduction by a novel Shewanella isolate, Shewanella sp. (NR), in batch incubations. The measured ε values range from 0.96 ± 0.16 to 0.36 ± 0.07‰ and are strongly dependent on the U(VI) reduction rate. The ε decreases with increasing reduction rate constants normalized by cell density and initial U(VI). Reactive transport simulations suggest that the rate dependence of ε is due to a two-step process, where diffusive transport of U(VI) from the bulk solution across a boundary layer is followed by enzymatic reduction. Our results imply that the spatial decoupling of bulk U(VI) solution and enzymatic reduction should be taken into account for interpreting U isotope data from the environment.

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.

Diversity and geochemical community assembly processes of the living rare biosphere in a sand-and-gravel aquifer ecosystem in the Midwestern United States.

Natural microbial communities consist of a limited number of abundant species and an extraordinarily diverse population of rare species referred to as the rare biosphere. Recent studies have revealed that the rare biosphere is not merely an inactive dormant population but may play substantial functional roles in the ecosystem. However, structure, activity and community assembly processes of the rare biosphere are poorly understood. In this study, we evaluated the present and living microbial community structures including rare populations in an aquifer ecosystem, the Mahomet Aquifer, USA, by both 16S rDNA and rRNA amplicon deep sequencing. The 13 groundwater samples formed three distinct groups based on the "entire" community structure, and the same grouping was obtained when focusing on the "rare" subcommunities (<0.1% of total abundance), while the "abundant" subcommunities (>1.0%) gave a different grouping. In the correlation analyses, the observed grouping pattern is associated with several geochemical factors, and structures of not only the entire community but also the rare subcommunity are correlated with geochemical profiles in the aquifer ecosystem. Our findings first indicate that the living rare biosphere in the aquifer system has the metabolic potential to adapt to local geochemical factors which dictate the community assembly processes.

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.

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).

Adaptive Evolution of Escherichia coli to Ciprofloxacin in Controlled Stress Environments: Contrasting Patterns of Resistance in Spatially Varying versus Uniformly Mixed Concentration Conditions.

A microfluidic gradient chamber (MGC) and a homogeneous batch culturing system were used to evaluate whether spatial concentration gradients of the antibiotic ciprofloxacin allow development of greater antibiotic resistance in Escherichia coli strain 307 (E. coli 307) compared to exclusively temporal concentration gradients, as indicated in an earlier study. A linear spatial gradient of ciprofloxacin and Luria-Bertani broth (LB) medium was established and maintained by diffusion over 5 days across a well array in the MGC, with relative concentrations along the gradient of 1.7-7.7× the original minimum inhibitory concentration (MICoriginal). The E. coli biomass increased in wells with lower ciprofloxacin concentrations, and only a low level of resistance to ciprofloxacin was detected in the recovered cells (∼2× MICoriginal). Homogeneous batch culture experiments were performed with the same temporal exposure history to ciprofloxacin concentration, the same and higher initial cell densities, and the same and higher nutrient (i.e., LB) concentrations as in the MGC. In all batch experiments, E. coli 307 developed higher ciprofloxacin resistance after exposure, ranging from 4 to 24× MICoriginal in all replicates. Hence, these results suggest that the presence of spatial gradients appears to reduce the driving force for E. coli 307 adaptation to ciprofloxacin, which suggests that results from batch experiments may over predict the development of antibiotic resistance in natural environments.