Rima B. Franklin

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.

Impact of dilution on microbial community structure and functional potential: comparison of numerical simulations and batch culture experiments

ABSTRACT A series of microcosm experiments was performed using serial dilutions of a sewage microbial community to inoculate a set of batch cultures in sterile sewage. After inoculation, the dilution-defined communities were allowed to regrow for several days and a number of community attributes were measured in the regrown assemblages. Based upon a set of numerical simulations, community structure was expected to differ along the dilution gradient; the greatest differences in structure were anticipated between the undiluted–low-dilution communities and the communities regrown from the very dilute (more than 10−4) inocula. Furthermore, some differences were expected among the lower-dilution treatments (e.g., between undiluted and 10−1) depending upon the evenness of the original community. In general, each of the procedures used to examine the experimental community structures separated the communities into at least two, often three, distinct groups. The groupings were consistent with the simulated dilution of a mixture of organisms with a very uneven distribution. Significant differences in community structure were detected with genetic (amplified fragment length polymorphism and terminal restriction fragment length polymorphism), physiological (community level physiological profiling), and culture-based (colony morphology on R2A agar) measurements. Along with differences in community structure, differences in community size (acridine orange direct counting), composition (ratio of sewage medium counts to R2A counts, monitoring of each colony morphology across the treatments), and metabolic redundancy (i.e., generalist versus specialist) were also observed, suggesting that the differences in structure and diversity of communities maintained in the same environment can be manifested as differences in community organization and function.

A global perspective on wetland salinization: ecological consequences of a growing threat to freshwater wetlands

Salinization, a widespread threat to the structure and ecological functioning of inland and coastal wetlands, is currently occurring at an unprecedented rate and geographic scale. The causes of salinization are diverse and include alterations to freshwater flows, land-clearance, irrigation, disposal of wastewater effluent, sea level rise, storm surges, and applications of de-icing salts. Climate change and anthropogenic modifications to the hydrologic cycle are expected to further increase the extent and severity of wetland salinization. Salinization alters the fundamental physicochemical nature of the soil-water environment, increasing ionic concentrations and altering chemical equilibria and mineral solubility. Increased concentrations of solutes, especially sulfate, alter the biogeochemical cycling of major elements including carbon, nitrogen, phosphorus, sulfur, iron, and silica. The effects of salinization on wetland biogeochemistry typically include decreased inorganic nitrogen removal (with implica...

Characterization of microbial communities using randomly amplified polymorphic DNA (RAPD).

Similarity among a number of aquatic microbial communities was examined using randomly amplified polymorphic DNA (RAPD), a common polymerase chain reaction (PCR)-based DNA fingerprinting technique. After amplification of whole-community DNA extracts, the PCR products were resolved by agarose gel electrophoresis and the band patterns compared to determine percent similarity. Twelve different primers were used to amplify approximately 100 fragments (total) from each DNA sample; the bands were scored as present or absent and the similarity between each sample was determined using Jaccards coefficient. From this information. dendrograms were constructed and a bootstrapping procedure was used to assess how well supported the tree topologies were. Principal component analyses were also conducted as a means of visualizing the relationships among samples. Results obtained for two different experimental systems (a pair of tidal creeks and several wells in a shallow groundwater aquifer) correlated well with the temporal and spatial variations in environmental regime at the sites confirming that arbitrarily primed PCR-based DNA fingerprinting techniques such as RAPD are useful means of discriminating among microbial communities and estimating community relatedness. Moreover, this approach has several advantages over other DNA-based procedures for whole-community analysis; it is less laborious and uses smaller quantities of DNA, making it amenable to sample-intensive monitoring, and it does not depend on culturing or the use of selective PCR primers.

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 14C-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.

The spatial distribution of microbes in the environment

Contributing Authors. Preface. Acknowledgements. Introduction Franklin, R.B., Mills, A.L. Statistical Analysis of Spatial Structure in Microbial Communities Franklin, R.B., Mills, A.L. Bacterial Interactions at the Microscale - Linking Habitat to Function In Soil Nunan N., Young, I.M, Crawford, J.W., Ritz, K. Spatial Distribution of Bacteria at the Microscale In Soil Dechesne, A., Pallud, C., Grundmann, G.L. Analysis Of Spatial Patterns Of Rhizoplane Coloniz-Ation Knudsen, G.R., Dandurand, L.-M. Microbial Distributions And Their Potential Control-Ling Factors In Terrestrial Subsurface Environments Lehman, R.M. Spatial Organisation Of Soil Fungi Ritz, K. Spatial Heterogeneity of Planktonic Microorganisms in Aquatic Systems Pinel-Alloul, B., Ghadouani, A. The Interrelationship Between the Spatial Distribution of Microorganisms and Vegetation in Forest Soils Morris S.J., Dress, W.J.

The Distribution of Microbial Communities in Anaerobic and Aerobic Zones of a Shallow Coastal Plain Aquifer

A bstractRandomly amplified polymorphic DNA (RAPD) fingerprinting was used to determine the genetic similarity of whole-community DNA extracts from unattached microorganisms in several groundwater wells. The study site was a shallow coastal plain aquifer on the Eastern Shore of Virginia that contains distinct regions of anaerobic and aerobic groundwater. Several wells in each region were sampled, and principal component and cluster analyses showed a clear separation of the microbial communities from the two chemical zones of the aquifer. Within these zones, there was no relationship between the genetic relatedness of a pair of communities and their spatial separation. Two additional sets of samples were taken at later times, and the same clear separation between communities in the different zones of the aquifer was observed. The specific relationships between wells within each zone changed over time, however, and the magnitude and direction of these changes corresponded to concurrent changes in the groundwater chemistry at each well. Together, these results suggest that local variation in groundwater chemistry can support genetically distinct microbial communities, and that the composition of the microbial communities can follow seasonal fluctuations in groundwater chemistry.

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.

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.

MinION™ nanopore sequencing of environmental metagenomes: a synthetic approach

Abstract Background: 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.