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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.

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.

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.

Marine and lacustrine carbonate minerals preserve carbon cycle information, and their stable carbon isotope values (δ¹³C) are frequently used to infer and reconstruct paleoenvironmental changes. However, multiple processes can influence the δ¹³C 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 δ¹³C values of syndepositional carbonate grains of varying morphologies to investigate their origins. Here, we combine stable isotope analyses, metagenomics, and geochemical modeling to interpret δ¹³C values of syndepositional carbonate spherules (>500 μm) and fine-grained micrite (

Nitrogen (N) is often a limiting nutrient in lacustrine systems, and bulk organic matter stable isotope ratios of N (δ15N) are widely used in lake sediment studies to interpret N source inputs and lake trophic status. Although records of lacustrine sedimentary δ15N can provide critical information relating to past environmental change, often productivity interpretations from δ15N and lacustrine fossil records yield conflicting interpretations. Furthermore, components of the internal N cycle have substantial isotopic fractionation factors, and likely wield an enormous influence on bulk lacustrine sedimentary δ15N values. Yet apart from cyanobacteria N-fixation, few studies link specific microbial, N-related activity to δ15N variability in lake sediment records. Here, we assess the relationship between lacustrine sedimentary δ15N and microbiome profiles analyzed from extracted sediment DNA using metagenomics. In a ~ 1600-year-long sediment record from a hypersaline lake located on Kiritimati, Republic of Kiribati (1.9° N, 157.4° W), both δ15N and the taxonomy annotations from five unique metagenomes vary with depth. Despite the relatively high abundance of Cyanobacteria, Bacteroidetes, and N-fixation genes in the uppermost sediment, we find the highest δ15N values of the sediment record there. These high values are likely due to denitrification, supported by a relatively high abundance of denitrification genes and taxa responsible for denitrification, such as those found in family Chromatiaceae within the Gamma-proteobacteria. In the deep sediment, N-related biochemical processes are likely suppressed considering the low energy, low nutrient subsurface environment. Low δ15N values observed in deeper sediments co-occur with genes for assimilatory nitrate reduction and ammonification. Thus, metagenomics provides greater clarity with respect to the specific, microbial processes that alter primary δ15N signatures in the subsurface sediment.

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.

Bacteria affiliated with the phylum Gemmatimonadetes are found in high abundance in many terrestrial and aquatic environments, yet little is known about their metabolic capabilities. Difficulty in their cultivation has prompted interest in identifying better growth conditions for metabolic studies, especially related to their ability to reduce N2O, a potent greenhouse gas. T-27 Gemmatimonas aurantiaca is one of few cultivated strains of Gemmatimonadetes available for physiological studies. Our objective was to test this organism’s ability to use nitrite, nitrate, and N2O, and mineral forms of assimilable NH4 + at concentrations not typically used in tests for compound utilization. Cultures incubated under anaerobic conditions with nitrate, nitrite or N2O failed to grow or show depletion of these substrates. Nitrate and nitrite (1 mM) were not used even when cells were grown aerobically with the O2 allowed to deplete first. N2O reduction only commenced in the presence of O2 and continued to be depleted when refed to the culture under anaerobic, microaerobic and aerobic atmospheres. Carbon mineralization was coupled to the electron-accepting processes, with higher reducing equivalents needed for N2O utilization under aerobic atmospheres. N2O was reduced to N2 in the presence of 20% O2, however the rate of this reaction is reduced in the presence of high O2 concentration. This study demonstrated that G. aurantiaca T-27 possesses unique characteristics for assimilative and dissimilative N processes with new implications for cultivation strategies to better assess the metabolic abilities of Gemmatimonadetes.

The evolutionarily ancient Aquificales bacterium Sulfurihydrogenibium spp. dominates filamentous microbial mat communities in shallow, fast-flowing, and dysoxic hot-spring drainage systems around the world. In the present study, field observations of these fettuccini-like microbial mats at Mammoth Hot Springs in Yellowstone National Park are integrated with geology, geochemistry, hydrology, microscopy, and multi-omic molecular biology analyses. Strategic sampling of living filamentous mats along with the hot-spring CaCO3 (travertine) in which they are actively being entombed and fossilized has permitted the first direct linkage of Sulfurihydrogenibium spp. physiology and metabolism with the formation of distinct travertine streamer microbial biomarkers. Results indicate that, during chemoautotrophy and CO2 carbon fixation, the 87–98% Sulfurihydrogenibium-dominated mats utilize chaperons to facilitate enzyme stability and function. High-abundance transcripts and proteins for type IV pili and extracellular polymeric substances (EPSs) are consistent with their strong mucus-rich filaments tens of centimeters long that withstand hydrodynamic shear as they become encrusted by more than 5 mm of travertine per day. Their primary energy source is the oxidation of reduced sulfur (e.g., sulfide, sulfur, or thiosulfate) and the simultaneous uptake of extremely low concentrations of dissolved O2 facilitated by bd-type cytochromes. The formation of elevated travertine ridges permits the Sulfurihydrogenibium-dominated mats to create a shallow platform from which to access low levels of dissolved oxygen at the virtual exclusion of other microorganisms. These ridged travertine streamer microbial biomarkers are well preserved and create a robust fossil record of microbial physiological and metabolic activities in modern and ancient hot-spring ecosystems.

Nitrogen is an essential element for life, with the availability of fixed nitrogen limiting productivity in many ecosystems. The return of oxidized nitrogen species to the atmospheric N2 pool is predominately catalyzed by microbial denitrification (NO3- → NO2- → NO → N2O → N2)1. Incomplete denitrification can produce N2O as a terminal product, leading to an increase in atmospheric N2O, a potent greenhouse and ozone-depleting gas2. The production of N2O is catalyzed by nitric oxide reductase (NOR) members of the heme-copper oxidoreductase (HCO) superfamily3. Here we use phylogenomics to identify a number of previously uncharacterized HCO families and propose that many of them (eNOR, sNOR, gNOR, and nNOR) perform nitric oxide reduction. These families have novel active-site structures and several have conserved proton channels, suggesting that they might be able to couple nitric oxide reduction to energy conservation. We isolated and biochemically characterized a member of the eNOR family from Rhodothermus marinus, verifying that it performs nitric oxide reduction both in vitro and in vivo. These newly identified NORs exhibit broad phylogenetic and environmental distributions, expanding the diversity of microbes that can perform denitrification. Phylogenetic analyses of the HCO superfamily demonstrate that nitric oxide reductases evolved multiple times independently from oxygen reductases, suggesting that complete denitrification evolved after aerobic respiration.

Nonnative conifers are widespread in the southern hemisphere, where their use as plantation species has led to adverse ecosystem impacts sometimes intensified by invasion. Mechanical removal is a common strategy used to reduce or eliminate the negative impacts of nonnative conifers, and encourage native regeneration. However, a variety of factors may preclude active ecological restoration following removal. As a result, passive restoration – unassisted natural vegetation regeneration – is common following conifer removal. We asked, ‘what is the response of understorey cover to removal of nonnative conifer stands followed by passive restoration?' We sampled understorey cover in three site types: two‐ to ten‐year‐old clearcuts, native forest and current plantations. We then grouped understorey species by origin (native/nonnative) and growth form, and compared proportion and per cent cover of these groups as well as of bare ground and litter between the three site types. For clearcuts, we also analysed the effect of time since clearcut on the studied variables. We found that clearcuts had a significantly higher average proportion of nonnative understorey vegetation cover than native forest sites, where nonnative vegetation was nearly absent. The understorey of clearcut sites also averaged more overall vegetation cover and more nonnative vegetation cover (in particular nonnative shrubs and herbaceous species) than either plantation or native forest sites. Notably, 99% of nonnative shrub cover in clearcuts was the invasive nonnative species Scotch broom (Cytisus scoparius). After ten years of passive recovery since clearcutting, the proportion of understorey vegetation cover that is native has not increased and remains far below the proportion observed in native forest sites. Reduced natural regeneration capacity of the native ecosystem, presence of invasive species in the surrounding landscape and land‐use legacies from plantation forestry may inhibit native vegetation recovery and benefit opportunistic invasives, limiting the effectiveness of passive restoration in this context. Abstract in Spanish is available with online material.

Microbial sulfate reduction is associated with characteristic S isotope partitioning, which can aid in determining the rate of this ubiquitous reactive pathway in a wide variety of reducing environments. Here we introduce a new simple yet predictive model that expands the range of environmental conditions over which this functional relationship can be applied through the use of a modified Monod rate expression constrained by a novel set of experiments. A series of continuously-fed batch reactors containing an equivilant biomass of Desulfovibrio vulgaris, a sulfate reducing bacteria, were subjected to differing continuous mass addition rates of formate to precisely control the rate of sulfate reduction via electron donor limitation. Based on growth yield calculations, additional biomass accumulation was negligible. The isotopic composition (δ34S) of the residual sulfate pool was measured through time for each experiment. This approach resulted in five steady state reduction rates of 2.06, 1.22, 0.83, 0.52 and 0.28 μmol * h−1 that enriched the unreacted sulfate in 34S by unique characteristic enrichment factors (αobs) of 0.9976, 0.9962, 0.9938, 0.9924 and 0.9903, respectively. This relationship was used to calibrate a new coupled set of isotope-specific Monod rate laws that have been modified to incorporate (1) a minimum (α1) and maximum (α2) fractionation factor and (2) a rate-controlling electron donor factor (DF). Application of these parameters in the updated model reproduce most of our data and simulate realistic shifts in the observed fractionation factor (αobs) as a function of reduction rate in an electron donor limited system. The model was applied to our current dataset as well as three previous studies, which collectively provide a broad range in both rates and αobs, and support validation across substantially different conditions. We show that this approach offers a reasonable approximation to more detailed microbial reactive network models, while still maintaining sufficient simplicity and versatility to allow incorporation into multi-component reactive transport simulations. Thus, the current study provides a foundation for accurate simulation of the relationship between αobs and sulfate reduction rates in open, transient, and through-flowing systems.

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.

Microbiological activity can exert a substantial influence on carbonate mineral precipitation, but linking specific microbiological processes to carbonate minerals in an environmental setting is complex, as both abiotic and biotic factors ultimately influence carbonate mineral precipitation. The coral atoll of Kiritimati, Republic of Kiribati (1.9°N, 157.4°W), in the central tropical Pacific Ocean, contains hundreds of shallow water brackish to hypersaline lakes that contain a range of carbonate and evaporite minerals. Previous studies of Kiritimati lakes have investigated the microbial communities of finely laminated microbial mats and associated microbialites found in several of the more hypersaline lakes on the island. However, the microbial communities of the more brackish lakes are unknown. These brackish lakes precipitate metres of fine‐grained carbonate muds, which are useful for palaeoenvironmental reconstruction. Here, the relationships between carbonate abundance, mineralogy, water chemistry, and bacterial and archaeal communities are investigated in a suite of brackish to hypersaline lakes (8.7‐190 ppt) on Kiritimati. Next generation 16S rRNA gene sequencing of bacteria and archaea indicate that brackish lake sediments contain distinct microbial communities. In relation to carbonate precipitation, the relative abundance of Cyanobacteria, Choloroflexi and Deltaproteobacteria is greater in the brackish lake sediments, suggesting photosynthesis and sulphate reduction associated with these taxa may strongly influence alkalinity and carbonate precipitation in brackish lakes. The presence of dolomite in certain hypersaline lakes also coincided with the presence of a methanogenic family, indicating that methogenesis may contribute to dolomite precipitation in these lakes.

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.

Precipitation events are increasing in intensity in the Midwestern US due to climate change. This is resulting in flooding of poorly-drained upland soils, which can feed back on climate change by altering greenhouse gas (GHG) emissions, including nitrous oxide (N2O) and carbon dioxide (CO2). The objective of this study was to determine if soil drainage history affects the response of soil GHG emissions to rain events. To do this, we measured N2O and CO2 fluxes from poorly-drained (PD) and well-drained (WD) soils in an agricultural field in Urbana, Illinois before and after large rain events. We also performed a lab experiment to separate effects of soil drainage history from contemporary effects of ponding. Finally, we utilized stable isotope techniques to measure gross N2O dynamics and to determine the contributions of nitrifiers and denitrifiers to net N2O fluxes. We found that ponding of WD soils led to pulses of net N2O efflux caused by stimulation of gross N2O production by denitrifiers. In contrast, PD soils had high net N2O effluxes only between large rain events, and gross N2O production was inhibited following ponding. Soil CO2 efflux was greater from PD soils under lab conditions, but autotrophic respiration obscured this trend in the field. Soil GHG emissions were a result of different contemporary ponding status as well as historical soil drainage, suggesting that historical soil redox regimes regulate soil GHG dynamics in response to precipitation. These soil drainage legacy effects are likely important in predicting soil GHG feedback effects on climate change.

Subsurface environments often contain mixtures of contaminants in which the microbial degradation of one pollutant may be inhibited by the toxicity of another. Agricultural settings exemplify these complex environments, where antimicrobial leachates may inhibit nitrate bioreduction, and are the motivation to address this fundamental ecological response. In this study, a microfluidic reactor was fabricated to create diffusion-controlled concentration gradients of nitrate and ciprofloxacin under anoxic conditions in order to evaluate the ability of Shewanella oneidenisis MR-1 to reduce the former in the presence of the latter. Results show a surprising ecological response, where swimming motility allow S. oneidensis MR-1 to accumulate and maintain metabolic activity for nitrate reduction in regions with toxic ciprofloxacin concentrations (i.e., 50× minimum inhibitory concentration, MIC), despite the lack of observed antibiotic resistance. Controls with limited nutrient flux and a nonmotile mutant (Δ flag) show that cells cannot colonize antibiotic rich microenvironments, and this results in minimal metabolic activity for nitrate reduction. These results demonstrate that under anoxic, nitrate-reducing conditions, motility can control microbial habitability and metabolic activity in spatially heterogeneous toxic environments.

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.

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.