Drivers of Antibiotic Resistance Gene Abundance in an Urban River.

In this study, we sought to profile the abundances and drivers of antibiotic resistance genes in an urban river impacted by combined sewage overflow (CSO) events. Water samples were collected weekly during the summer for two years; then, quantitative PCR was applied to determine the abundance of resistance genes associated with tetracycline, quinolones, and ß-lactam antibiotics. In addition to sampling a CSO-impacted site near the city center, we also sampled a less urban site ~12 km upstream with no proximal sewage inputs. The tetracycline genes tetO and tetW were rarely found upstream, but were common at the CSO-impacted site, suggesting that the primary source was untreated sewage. In contrast, ampC was detected in all samples indicating a more consistent and diffuse source. The two other genes, qnrA and blaTEM, were present in only 40-50% of samples and showed more nuanced spatiotemporal patterns consistent with upstream agricultural inputs. The results of this study highlight the complex sources of ARGs in urban riverine ecosystems, and that interdisciplinary collaborations across diverse groups of stakeholders are necessary to combat the emerging threat of antibiotic resistance through anthropogenic pollution.

Online Strategies To Improve Quantitative Skills in Microbiology Laboratory Classes.

Biology is an increasingly quantitative science. Thus, it is important that undergraduate biology curricula include frequent opportunities for students to practice their quantitative skills. This can create a substantial grading burden for faculty teaching online and/or large enrollment courses, but the "formula question" feature present in many learning management systems (LMS) offers a solution. Using this feature, faculty set up a basic scaffold for an algebraic word problem, and the LMS can then automatically generate and grade many different versions of the question. In this paper, we describe the use of "formula questions" in an undergraduate microbiology course and specifically focus on how the strategic use of algebraic word problems at multiple points throughout the semester can help build quantitative literacy. Key to the success of this approach is that faculty provide a review of foundational mathematical skills early in the semester, even in upper-level classes. This should include reacquainting students with formatting conventions (e.g., rounding and scientific notation), familiarizing them with any idiosyncrasies of the technology platforms, and demonstrating how to solve math problems using spreadsheets. This initial effort increases student success when more complex problems are introduced later in the semester. Though the tips summarized in this paper focus on undergraduate microbiology teaching laboratories using Canvas, the approach can easily be modified to help students develop their critical thinking and quantitative reasoning skills at other levels and in other disciplines.

Cooperative microbial interactions mediate community biogeochemical responses to saltwater intrusion in wetland soils.

In freshwater wetlands, competitive and cooperative interactions between respiratory, fermentative and methanogenic microbes mediate the decomposition of organic matter. These interactions may be disrupted by saltwater intrusion disturbances that enhance the activity of sulfate-reducing bacteria (SRB), intensifying their competition with syntrophic bacteria and methanogens for electron donors. We simulated saltwater intrusion into wetland soil microcosms and examined biogeochemical and microbial responses, employing metabolic inhibitors to isolate the activity of various microbial functional groups. Sulfate additions increased total carbon dioxide production but decreased methane production. Butyrate degradation assays showed continued (but lower) levels of syntrophic metabolism despite strong demand by SRB for this key intermediate decomposition product and a shift in the methanogen community toward acetoclastic members. One month after removing SRB competition, total methane production recovered by only ∼50%. Similarly, butyrate assays showed an altered accumulation of products (including less methane), although overall rates of syntrophic butyrate breakdown largely recovered. These effects illustrate that changes in carbon mineralization following saltwater intrusion are driven by more than the oft-cited competition between SRB and methanogens for shared electron donors. Thus, the impacts of disturbances on wetland biogeochemistry are likely to persist until cooperative and competitive microbial metabolic interactions can recover fully.

Persistence of wastewater-associated antibiotic resistant bacteria in river microcosms.

The spread of antibiotic-resistant bacteria (ARB) associated with wastewater is a significant environmental concern, but little is known about the persistence and proliferation of these organisms in receiving water bodies after discharge. To address this knowledge gap, we performed a series of microcosm experiments in which river water was amended with either untreated or treated wastewater, and the abundance of viable ciprofloxacin-, Bactrim-, and erythromycin-resistant bacteria was monitored for 72 h. Both types of wastewater amendments significantly increased the initial abundance of ARB compared to microcosms containing only river water (all p < 0.03). The increase was greatest with untreated wastewater, but that effect decreased steadily over time. In contrast, microcosms amended with treated wastewater saw a smaller initial increase and more complex temporal dynamics. Following a brief lag, ARB abundance bloomed for all three of the antibiotics that we considered. This suggests that ARB that survive wastewater treatment are particularly hardy and may proliferate in riverine conditions after a short recovery period. To determine how interactions with the native river microbial community impacted the persistence of wastewater-associated ARB, an additional set of microcosms was prepared using filter-sterilized river water. Peak abundance in these microcosms was significantly higher by 1-2 orders of magnitude compared to microcosms containing an intact river microbial community (all p < 0.05), which suggests that biotic interactions play a significant role in regulating the persistence and proliferation of ARB. The data presented in this paper are among the first available that specifically consider persistence of viable ARB and represent an important step toward understanding AR-related human health risks downstream from wastewater discharge points and following sewer overflow events. Additional studies that consider longer time scales and the interplay of biotic and abiotic variables are essential for modeling public health risks associated with wastewater inputs of ARB to rivers and other aquatic environments.

Phylogenetic organization in the assimilation of chemically distinct substrates by soil bacteria.

Soils are among the most biodiverse habitats on earth and while the species composition of microbial communities can influence decomposition rates and pathways, the functional significance of many microbial species and phylogenetic groups remains unknown. If bacteria exhibit phylogenetic organization in their function, this could enable ecologically meaningful classification of bacterial clades. Here, we show non-random phylogenetic organization in the rates of relative carbon assimilation for both rapidly mineralized substrates (amino acids and glucose) assimilated by many microbial taxa and slowly mineralized substrates (lipids and cellulose) assimilated by relatively few microbial taxa. When mapped onto bacterial phylogeny using ancestral character estimation this phylogenetic organization enabled the identification of clades involved in the decomposition of specific soil organic matter substrates. Phylogenetic organization in substrate assimilation could provide a basis for predicting the functional attributes of uncharacterized microbial taxa and understanding the significance of microbial community composition for soil organic matter decomposition.

Trace Metal Availability Affects Greenhouse Gas Emissions and Microbial Functional Group Abundance in Freshwater Wetland Sediments.

We investigated the effects of trace metal additions on microbial nitrogen (N) and carbon (C) cycling using freshwater wetland sediment microcosms amended with micromolar concentrations of copper (Cu), molybdenum (Mo), iron (Fe), and all combinations thereof. In addition to monitoring inorganic N transformations (NO3 -, NO2 -, N2O, NH4 +) and carbon mineralization (CO2, CH4), we tracked changes in functional gene abundance associated with denitrification (nirS, nirK, nosZ), dissimilatory nitrate reduction to ammonium (DNRA; nrfA), and methanogenesis (mcrA). With regards to N cycling, greater availability of Cu led to more complete denitrification (i.e., less N2O accumulation) and a higher abundance of the nirK and nosZ genes, which encode for Cu-dependent reductases. In contrast, we found sparse biochemical evidence of DNRA activity and no consistent effect of the trace metal additions on nrfA gene abundance. With regards to C mineralization, CO2 production was unaffected, but the amendments stimulated net CH4 production and Mo additions led to increased mcrA gene abundance. These findings demonstrate that trace metal effects on sediment microbial physiology can impact community-level function. We observed direct and indirect effects on both N and C biogeochemistry that resulted in increased production of greenhouse gasses, which may have been mediated through the documented changes in microbial community composition and shifts in functional group abundance. Overall, this work supports a more nuanced consideration of metal effects on environmental microbial communities that recognizes the key role that metal limitation plays in microbial physiology.

Wood Decay Characteristics and Interspecific Interactions Control Bacterial Community Succession in Populus grandidentata (Bigtooth Aspen).

Few studies have investigated bacterial community succession and the role of bacterial decomposition over a continuum of wood decay. Here, we identified how (i) the diversity and abundance of bacteria changed along a chronosequence of decay in Populus grandidentata (bigtooth aspen); (ii) bacterial community succession was dependent on the physical and chemical characteristics of the wood; (iii) interspecific bacterial interactions may mediate community structure. Four hundred and fifty-nine taxa were identified through Illumina sequencing of 16S rRNA amplicons from samples taken along a continuum of decay, representing standing dead trees, downed wood, and soil. Community diversity increased as decomposition progressed, peaking in the most decomposed trees. While a small proportion of taxa displayed a significant pattern in regards to decay status of the host log, many bacterial taxa followed a stochastic distribution. Changes in the water availability and chemical composition of standing dead and downed trees and soil were strongly coupled with shifts in bacterial communities. Nitrogen was a major driver of succession and nitrogen-fixing taxa of the order Rhizobiales were abundant early in decomposition. Recently downed logs shared 65% of their bacterial abundance with the microbiomes of standing dead trees while only sharing 16% with soil. As decay proceeds, bacterial communities appear to respond less to shifting resource availability and more to interspecific bacterial interactions - we report an increase in both the proportion (+9.3%) and the intensity (+62.3%) of interspecific interactions in later stages of decomposition, suggesting the emergence of a more complex community structure as wood decay progresses.

Novel microbial community composition and carbon biogeochemistry emerge over time following saltwater intrusion in wetlands.

Sea level rise and changes in precipitation can cause saltwater intrusion into historically freshwater wetlands, leading to shifts in microbial metabolism that alter greenhouse gas emissions and soil carbon sequestration. Saltwater intrusion modifies soil physicochemistry and can immediately affect microbial metabolism, but further alterations to biogeochemical processing can occur over time as microbial communities adapt to the changed environmental conditions. To assess temporal changes in microbial community composition and biogeochemical activity due to saltwater intrusion, soil cores were transplanted from a tidal freshwater marsh to a downstream mesohaline marsh and periodically sampled over 1 year. This experimental saltwater intrusion produced immediate changes in carbon mineralization rates, whereas shifts in the community composition developed more gradually. Salinity affected the composition of the prokaryotic community but did not exert a strong influence on the community composition of fungi. After only 1 week of saltwater exposure, carbon dioxide production doubled and methane production decreased by three orders of magnitude. By 1 month, carbon dioxide production in the transplant was comparable to the saltwater controls. Over time, we observed a partial recovery in methane production which strongly correlated with an increase in the relative abundance of three orders of hydrogenotrophic methanogens. Taken together, our results suggest that ecosystem responses to saltwater intrusion are dynamic over time as complex interactions develop between microbial communities and the soil organic carbon pool. The gradual changes in microbial community structure we observed suggest that previously freshwater wetlands may not experience an equilibration of ecosystem function until long after initial saltwater intrusion. Our results suggest that during this transitional period, likely lasting years to decades, these ecosystems may exhibit enhanced greenhouse gas production through greater soil respiration and continued methanogenesis.

MinION™ nanopore sequencing of environmental metagenomes: a synthetic approach.

Environmental metagenomic analysis is typically accomplished by assigning taxonomy and/or function from whole genome sequencing or 16S amplicon sequences. Both of these approaches are limited, however, by read length, among other technical and biological factors. A nanopore-based sequencing platform, MinION™, produces reads that are ≥1 × 104 bp in length, potentially providing for more precise assignment, thereby alleviating some of the limitations inherent in determining metagenome composition from short reads. We tested the ability of sequence data produced by MinION (R7.3 flow cells) to correctly assign taxonomy in single bacterial species runs and in three types of low-complexity synthetic communities: a mixture of DNA using equal mass from four species, a community with one relatively rare (1%) and three abundant (33% each) components, and a mixture of genomic DNA from 20 bacterial strains of staggered representation. Taxonomic composition of the low-complexity communities was assessed by analyzing the MinION sequence data with three different bioinformatic approaches: Kraken, MG-RAST, and One Codex. Results: Long read sequences generated from libraries prepared from single strains using the version 5 kit and chemistry, run on the original MinION device, yielded as few as 224 to as many as 3497 bidirectional high-quality (2D) reads with an average overall study length of 6000 bp. For the single-strain analyses, assignment of reads to the correct genus by different methods ranged from 53.1% to 99.5%, assignment to the correct species ranged from 23.9% to 99.5%, and the majority of misassigned reads were to closely related organisms. A synthetic metagenome sequenced with the same setup yielded 714 high quality 2D reads of approximately 5500 bp that were up to 98% correctly assigned to the species level. Synthetic metagenome MinION libraries generated using version 6 kit and chemistry yielded from 899 to 3497 2D reads with lengths averaging 5700 bp with up to 98% assignment accuracy at the species level. The observed community proportions for "equal" and "rare" synthetic libraries were close to the known proportions, deviating from 0.1% to 10% across all tests. For a 20-species mock community with staggered contributions, a sequencing run detected all but 3 species (each included at <0.05% of DNA in the total mixture), 91% of reads were assigned to the correct species, 93% of reads were assigned to the correct genus, and >99% of reads were assigned to the correct family. Conclusions: At the current level of output and sequence quality (just under 4 × 103 2D reads for a synthetic metagenome), MinION sequencing followed by Kraken or One Codex analysis has the potential to provide rapid and accurate metagenomic analysis where the consortium is comprised of a limited number of taxa. Important considerations noted in this study included: high sensitivity of the MinION platform to the quality of input DNA, high variability of sequencing results across libraries and flow cells, and relatively small numbers of 2D reads per analysis limit. Together, these limited detection of very rare components of the microbial consortia, and would likely limit the utility of MinION for the sequencing of high-complexity metagenomic communities where thousands of taxa are expected. Furthermore, the limitations of the currently available data analysis tools suggest there is considerable room for improvement in the analytical approaches for the characterization of microbial communities using long reads. Nevertheless, the fact that the accurate taxonomic assignment of high-quality reads generated by MinION is approaching 99.5% and, in most cases, the inferred community structure mirrors the known proportions of a synthetic mixture warrants further exploration of practical application to environmental metagenomics as the platform continues to develop and improve. With further improvement in sequence throughput and error rate reduction, this platform shows great promise for precise real-time analysis of the composition and structure of more complex microbial communities.

Evolutionary history influences the salinity preference of bacterial taxa in wetland soils.

Salinity is a major driver of bacterial community composition across the globe. Despite growing recognition that different bacterial species are present or active at different salinities, the mechanisms by which salinity structures community composition remain unclear. We tested the hypothesis that these patterns reflect ecological coherence in the salinity preferences of phylogenetic groups using a reciprocal transplant experiment of fresh- and saltwater wetland soils. The salinity of both the origin and host environments affected community composition (16S rRNA gene sequences) and activity (CO2 and CH4 production, and extracellular enzyme activity). These changes in community composition and activity rates were strongly correlated, which suggests the effect of environment on function could be mediated, at least in part, by microbial community composition. Based on their distribution across treatments, each phylotype was categorized as having a salinity preference (freshwater, saltwater, or none) and phylogenetic analyses revealed a significant influence of evolutionary history on these groupings. This finding was corroborated by examining the salinity preferences of high-level taxonomic groups. For instance, we found that the majority of α- and γ-proteobacteria in these wetland soils preferred saltwater, while many ß-proteobacteria prefer freshwater. Overall, our results indicate the effect of salinity on bacterial community composition results from phylogenetically-clustered salinity preferences.

Metagenomic analysis of planktonic microbial consortia from a non-tidal urban-impacted segment of James River.

Knowledge of the diversity and ecological function of the microbial consortia of James River in Virginia, USA, is essential to developing a more complete understanding of the ecology of this model river system. Metagenomic analysis of James River's planktonic microbial community was performed for the first time using an unamplified genomic library and a 16S rDNA amplicon library prepared and sequenced by Ion PGM and MiSeq, respectively. From the 0.46-Gb WGS library (GenBank:SRR1146621; MG-RAST:4532156.3), 4 × 10(6) reads revealed >3 × 10(6) genes, 240 families of prokaryotes, and 155 families of eukaryotes. From the 0.68-Gb 16S library (GenBank:SRR2124995; MG-RAST:4631271.3; EMB:2184), 4 × 10(6) reads revealed 259 families of eubacteria. Results of the WGS and 16S analyses were highly consistent and indicated that more than half of the bacterial sequences were Proteobacteria, predominantly Comamonadaceae. The most numerous genera in this group were Acidovorax (including iron oxidizers, nitrotolulene degraders, and plant pathogens), which accounted for 10 % of assigned bacterial reads. Polaromonas were another 6 % of all bacterial reads, with many assignments to groups capable of degrading polycyclic aromatic hydrocarbons. Albidiferax (iron reducers) and Variovorax (biodegraders of a variety of natural biogenic compounds as well as anthropogenic contaminants such as polycyclic aromatic hydrocarbons and endocrine disruptors) each accounted for an additional 3 % of bacterial reads. Comparison of these data to other publically-available aquatic metagenomes revealed that this stretch of James River is highly similar to the upper Mississippi River, and that these river systems are more similar to aquaculture and sludge ecosystems than they are to lakes or to a pristine section of the upper Amazon River. Taken together, these analyses exposed previously unknown aspects of microbial biodiversity, documented the ecological responses of microbes to urban effects, and revealed the noteworthy presence of 22 human-pathogenic bacterial genera (e.g., Enterobacteriaceae, pathogenic Pseudomonadaceae, and 'Vibrionales') and 6 pathogenic eukaryotic genera (e.g., Trypanosomatidae and Vahlkampfiidae). This information about pathogen diversity may be used to promote human epidemiological studies, enhance existing water quality monitoring efforts, and increase awareness of the possible health risks associated with recreational use of James River.

Resource effects on denitrification are mediated by community composition in tidal freshwater wetlands soils.

Accurate prediction of denitrification rates remains difficult, potentially owing to complex uncharacterized interactions between resource conditions and denitrifier communities. To better understand how the availability of organic matter (OM) and nitrate (NO3 (-) ), two of the resources most fundamental to denitrifiers, affect these populations and their activity, we performed an in situ resource manipulation in tidal freshwater wetland soils. Soils were augmented with OM to double ambient concentrations, using either compost or plant litter, and fertilized with KNO3 at two levels (low: ∼ 5 mg l(-1) NO3 (-) -N and high: ∼ 50 mg l(-1) NO3 (-) -N) in a full factorial design. Community composition of nirS-denitrifers (assessed using terminal restriction fragment length polymorphism) was interactively regulated by both NO3 (-) concentration and OM type, and the associated shifts in community composition were relatively consistent across sampling dates (6, 9 and 12 months of incubation). Denitrification potential (pDNF) rates were also strongly affected by NO3 (-) fertilization and increased by ∼ 10-100-fold. Path analysis revealed that the influence of resource availability on pDNF rates was largely mediated through changes in nirS-denitrifier community composition. These results suggest that a greater understanding of denitrifier community ecology may enable more accurate prediction of denitrification rates.

Exposure to the cyanotoxin microcystin arising from interspecific differences in feeding habits among fish and shellfish in the James River Estuary, Virginia.

The cyanotoxin, microcystin (MC), is known to accumulate in the tissues of diverse aquatic biota although factors influencing exposure, such as feeding habits and seasonal patterns in toxin production, are poorly known. We analyzed seasonal variation in the MC content of primary and secondary consumers, and used dietary analysis (gut contents and stable isotopes) to improve understanding of cyanotoxin transport in food webs. Periods of elevated toxin concentration were associated with peaks in the abundance of genes specific to Microcystis and MC toxin production (mcyD). Peak toxin levels in consumer tissues coincided with peak MC concentrations in seston. However, toxins in tissues persisted in overwintering populations suggesting that potential health impacts may not be limited to bloom periods. Interspecific differences in tissue MC concentrations were related to feeding habits and organic matter sources as pelagic fishes ingested a greater proportion of algae in their diet, which resulted in greater MC content in liver and muscle tissues. Sediments contained a greater proportion of allochthonous (terrestrial) organic matter and lower concentrations of MC, resulting in lower toxin concentrations among benthic detritivores. Among shellfish, the benthic suspension feeder Rangia cuneata (wedge clam) showed seasonal avoidance of toxin ingestion due to low feeding rates during periods of elevated MC. Among predators, adult Blue Catfish had low MC concentrations, whereas Blue Crabs exhibited high levels of MC in both muscle and viscera.

Salinity affects microbial activity and soil organic matter content in tidal wetlands.

Climate change-associated sea level rise is expected to cause saltwater intrusion into many historically freshwater ecosystems. Of particular concern are tidal freshwater wetlands, which perform several important ecological functions including carbon sequestration. To predict the impact of saltwater intrusion in these environments, we must first gain a better understanding of how salinity regulates decomposition in natural systems. This study sampled eight tidal wetlands ranging from freshwater to oligohaline (0-2 ppt) in four rivers near the Chesapeake Bay (Virginia). To help isolate salinity effects, sites were selected to be highly similar in terms of plant community composition and tidal influence. Overall, salinity was found to be strongly negatively correlated with soil organic matter content (OM%) and C : N, but unrelated to the other studied environmental parameters (pH, redox, and above- and below-ground plant biomass). Partial correlation analysis, controlling for these environmental covariates, supported direct effects of salinity on the activity of carbon-degrading extracellular enzymes (ß-1, 4-glucosidase, 1, 4-ß-cellobiosidase, ß-D-xylosidase, and phenol oxidase) as well as alkaline phosphatase, using a per unit OM basis. As enzyme activity is the putative rate-limiting step in decomposition, enhanced activity due to salinity increases could dramatically affect soil OM accumulation. Salinity was also found to be positively related to bacterial abundance (qPCR of the 16S rRNA gene) and tightly linked with community composition (T-RFLP). Furthermore, strong relationships were found between bacterial abundance and/or composition with the activity of specific enzymes (1, 4-ß-cellobiosidase, arylsulfatase, alkaline phosphatase, and phenol oxidase) suggesting salinity's impact on decomposition could be due, at least in part, to its effect on the bacterial community. Together, these results indicate that salinity increases microbial decomposition rates in low salinity wetlands, and suggests that these ecosystems may experience decreased soil OM accumulation, accretion, and carbon sequestration rates even with modest levels of saltwater intrusion.

Enumerating bacterial cells on bioadhesive coated slides.

Quantifying bacterial abundance and biomass is fundamental to many microbiological studies. Directly counting via epifluorescence microscopy has become the method of choice, especially for environmental samples, and conventional techniques require filtration of cells onto black polycarbonate membrane filters. We investigated the utility of instead capturing stained bacterial suspensions on bioadhesive slides, performing tests using pure cultures of bacteria, mixtures of cultured bacteria, and environmental samples from five habitat types. When compared to the standard filtration and flow cytometric approaches, bioadhesive slides were found to be an accurate and precise platform for rapid enumeration of bacteria. Total bacterial counts made using the three methods were positively correlated for acridine orange and Live/Dead® (L/D) staining (0.81≤r≤0.95, all p≤0.002). All platforms had similar precision, though counts obtained using bioadhesive slides were significantly higher than those made with polycarbonate filters and flow cytometry. The specific bioadhesive slides we used resulted in substantial cell mortality for certain pure cultures and river water samples, limiting their use for L/D determination. Cell enumeration using bioadhesive slides is particularly effective because it is highly precise at a wide range of cell concentrations, allows observation of cells that are not readily discernible on filters, reduces the number of steps and processing materials associated with sample analysis, and increases throughput.

Effects of age and composition of field-produced biofilms on oyster larval setting.

Lack of success in restoring the native Eastern oyster, Crassostrea virginica, to Chesapeake Bay has been linked to the low occurrence of oyster larval setting in tributaries to the Bay. Among the many potential factors that could affect efforts to produce oysters through aquaculture or supplementation of shell beds is substratum condition. The present study examined larval setting on field-produced biofilms from Little Wicomico River (Virginia, USA) to assess whether bacterial community structure (examined by terminal restriction fragment length polymorphism, T-RFLP) or other characteristics of contemporary biofilms in this tributary (biofilm age and mass, algal abundance, and percentage organic matter) inhibited setting of larval oysters. The structure of the natural and heterogenous bacterial community in the biofilms and the success of oyster set were correlated, suggesting that specific microbial species may play a role in oyster setting. Larval set increased with biofilm age and mass, suggesting that established field-produced biofilms have no inhibitory effect. In contrast, the percentage of organic matter was negatively correlated with oyster set, whereas chlorophyll a concentration had no observed effect. The study expands prior knowledge by providing a more realistic timeframe for biofilm development (weeks as opposed to days), recounting effects of biofilms that are more representative of the natural dynamic and disturbance processes that would be expected to occur on submerged structures, and by incorporating seasonal and spatial variation. An important negative effect observed during the study period was heavy predation by Stylochus ellipticus on newly set oysters. Overall, the results of this study, which is the first assessment of the effects of biofilms produced naturally within a Chesapeake Bay tributary, suggest that the absence of large numbers of oysters in Little Wicomico River is not related to microbes or other specific characteristics of biofilms that develop on suitable setting substrata, but rather to heavy predation of newly set larvae.

Structural and functional responses of a sewage microbial community to dilution-induced reductions in diversity.

The relationship between functional redundancy and microbial community structure-diversity was examined using laboratory incubations to ensure constant environmental conditions. Serial dilutions of a sewage microbial community were prepared, used to inoculate sterile sewage, and maintained in batch culture. Probability suggests that dilution of the initial community should remove rare organism types, creating mixtures of cells differing in diversity. Regrowth of the diluted mixtures generated communities similar in abundance but differing in community structure and relative diversity (as determined using two DNA fingerprinting techniques and dilution-to-extinction analysis of community-level physiological profiles). The in situ function of each regrown community was examined by monitoring the short-term uptake of five different (14)C-labeled compounds (glucose, acetate, citrate, palmitic acid, and an amino acid mixture). No significant differences were detected between treatments in either the rate of uptake of a substrate or the efficiency with which each community assimilated each compound. The fact that the activity of the original community was the same as that of a community regrown from an inoculum containing fewer that 100 cells (10(-6) dilution) indicates that functional redundancy was quite high in this system. For each organism type eliminated during the dilution process, at least one of the remaining types was able to provide the same function at the same level as the lost one. Further research is necessary to determine what impact this functional redundancy may have on overall ecosystem function and stability.

Multi-scale variation in spatial heterogeneity for microbial community structure in an eastern Virginia agricultural field.

To better understand the distribution of soil microbial communities at multiple spatial scales, a survey was conducted to examine the spatial organization of community structure in a wheat field in eastern Virginia (USA). Nearly 200 soil samples were collected at a variety of separation distances ranging from 2.5 cm to 11 m. Whole-community DNA was extracted from each sample, and community structure was compared using amplified fragment length polymorphism (AFLP) DNA fingerprinting. Relative similarity was calculated between each pair of samples and compared using geostatistical variogram analysis to study autocorrelation as a function of separation distance. Spatial autocorrelation was found at scales ranging from 30 cm to more than 6 m, depending on the sampling extent considered. In some locations, up to four different correlation length scales were detected. The presence of nested scales of variability suggests that the environmental factors regulating the development of the communities in this soil may operate at different scales. Kriging was used to generate maps of the spatial organization of communities across the plot, and the results demonstrated that bacterial distributions can be highly structured, even within a habitat that appears relatively homogeneous at the plot and field scale. Different subsets of the microbial community were distributed differently across the plot, and this is thought to be due to the variable response of individual populations to spatial heterogeneity associated with soil properties.

A geostatistical analysis of small-scale spatial variability in bacterial abundance and community structure in salt marsh creek bank sediments.

Small-scale variations in bacterial abundance and community structure were examined in salt marsh sediments from Virginia's eastern shore. Samples were collected at 5 cm intervals (horizontally) along a 50 cm elevation gradient, over a 215 cm horizontal transect. For each sample, bacterial abundance was determined using acridine orange direct counts and community structure was analyzed using randomly amplified polymorphic DNA fingerprinting of whole-community DNA extracts. A geostatistical analysis was used to determine the degree of spatial autocorrelation among the samples, for each variable and each direction (horizontal and vertical). The proportion of variance in bacterial abundance that could be accounted for by the spatial model was quite high (vertical: 60%, horizontal: 73%); significant autocorrelation was found among samples separated by 25 cm in the vertical direction and up to 115 cm horizontally. In contrast, most of the variability in community structure was not accounted for by simply considering the spatial separation of samples (vertical: 11%, horizontal: 22%), and must reflect variability from other parameters (e.g., variation at other spatial scales, experimental error, or environmental heterogeneity). Microbial community patch size based upon overall similarity in community structure varied between 17 cm (vertical) and 35 cm (horizontal). Overall, variability due to horizontal position (distance from the creek bank) was much smaller than that due to vertical position (elevation) for both community properties assayed. This suggests that processes more correlated with elevation (e.g., drainage and redox potential) vary at a smaller scale (therefore producing smaller patch sizes) than processes controlled by distance from the creek bank.