abstract: Reductive dechlorination by members of the bacterial genus Dehalococcoides is a common and cost-effective avenue for in situ bioremediation of sites contaminated with the chlorinated solvents, trichloroethene (TCE) and perchloroethene (PCE). The overarching goal of my research was to address some of the challenges associated with bioremediation timeframes by improving the rates of reductive dechlorination and the growth of Dehalococcoides in mixed communities. Biostimulation of contaminated sites or microcosms with electron donor fails to consistently promote dechlorination of PCE/TCE beyond cis-dichloroethene (cis-DCE), even when the presence of Dehalococcoides is confirmed. Supported by data from microcosm experiments, I showed that the stalling at cis-DCE is due a H2 competition in which components of the soil or sediment serve as electron acceptors for competing microorganisms. However, once competition was minimized by providing selective enrichment techniques, I illustrated how to obtain both fast rates and high-density Dehalococcoides using three distinct enrichment cultures. Having achieved a heightened awareness of the fierce competition for electron donor, I then identified bicarbonate (HCO3-) as a potential H2 sink for reductive dechlorination. HCO3- is the natural buffer in groundwater but also the electron acceptor for hydrogenotrophic methanogens and homoacetogens, two microbial groups commonly encountered with Dehalococcoides. By testing a range of concentrations in batch experiments, I showed that methanogens are favored at low HCO3 and homoacetogens at high HCO3-. The high HCO3- concentrations increased the H2 demand which negatively affected the rates and extent of dechlorination. By applying the gained knowledge on microbial community management, I ran the first successful continuous stirred-tank reactor (CSTR) at a 3-d hydraulic retention time for cultivation of dechlorinating cultures. I demonstrated that using carefully selected conditions in a CSTR, cultivation of Dehalococcoides at short retention times is feasible, resulting in robust cultures capable of fast dechlorination. Lastly, I provide a systematic insight into the effect of high ammonia on communities involved in dechlorination of chloroethenes. This work documents the potential use of landfill leachate as a substrate for dechlorination and an increased tolerance of Dehalococcoides to high ammonia concentrations (2 g L-1 NH4+-N) without loss of the ability to dechlorinate TCE to ethene. / Dissertation/Thesis / Ph.D. Microbiology 2013

Les données présentées dans ce manuscrit de thèse sont principalement basées sur l'analyse de séquences d'ADN extraites directement de l'environnement (et en particulier du sol) en les comparant aux données cumulées au fil des siècles sur les microorganismes cultivés en laboratoire. Les objectifs, difficultés, résultats et perspectives de cette approche, que l'on nomme « métagénomique », sont décrits. Un métagénome représente la diversité génétique d'un habitat défini (par exemple un sol du champ agricole) dans sa globalité. Ainsi, générer un métagénome a notamment pour but d’accéder à la diversité génétique de la majorité d'espèces récalcitrantes à la culture. Elle permet aussi d'estimer le potentiel fonctionnel d'un jeu de données métagénomiques représentant un écosystème, puis de le comparer à d'autres jeux de données, représentant quant à eux d'autres environnements. Enfin, un autre intérêt de cette approche est la possibilité, dans certains cas, d'assembler partiellement ou entièrement un métagénome, et ainsi de permettre la reconstruction de structures génétiques complexes, qu'elles représentent des plasmides ou des génomes, circulaires ou non. Ce manuscrit de thèse présente ces aspects de la métagénomique (extraction, séquençage, annotation, comparaison, assemblage) sur l'environnement sol, en se basant sur une prairie anglaise (Park Grass, Rothamsted Research) étudiée depuis plus de 150 ans. Les communautés microbiennes prédominantes ont été partiellement caractérisées, leur potentiel fonctionnel comparé à d'autre environnements majeurs de notre planète (océans, sédiments, neige, etc.). Parce que l'assemblage de ces données est très limité dû à une trop grande diversité, des expérimentations ont été faites en microcosmes afin de stimuler une partie de la communauté avant de générer de nouveaux jeux de données, hautement simplifiés. Cette approche a permis l'assemblage et l'étude structurelle de génomes correspondant à des espèces résistant à des conditions extrêmes (par exemple un apport considérable de mercure, ou de métaux lourds).Lorsque l'on regarde la métagénomique dans sa globalité, les perspectives apparaissent clairement : usant d'outils de plus en plus performants (séquençage, annotation, assemblage, etc.), les microbiologistes peuvent d'ores et déjà récolter le fruit de plus de trois milliards d'années d'évolution et d'adaptation microbienne. / Microbial ecology is beginning to interact with metagenomics and many microbiologists are attracted to metagenomics in the hope of discovering novel relationships between microorganisms and/or confirming that work done on isolates applies to the remaining uncultured members of the different ecosystems. With a growing number of available metagenomic datasets, metagenomes can be intensively mined by microbial ecologists in search of previously undetected correlations (both structural and functional). Here, we provide a preliminary exploration of 77 publically available metagenomes corresponding to DNA samples extracted from oceans, atoll corals, deep oceans, Antarctic aquatic environments, Arctic snows, terrestrial environments (sediments, soils, sludges, microbial fuel cell anode biofilms, acid mine drainage biofilms), polluted air, and animal and human microbiomes (human feces, mouse and chicken cecum, and cow rumen). Results show well-defined environmental specificities that emphasize microbial adaptation and evolution capabilities. Unexpected observations were also made for several ecosystems, thus providing new hypotheses about the life style of their microbial communities. Available metagenomes are a gold mine of underexploited information that could be used to explore specific microbial structural and functional relationships. The statistical analysis provided here depends in part on replicates from the different ecosystems. With the continued emphasis on metagenomic sequencing, future analyses should support rigorous statistical treatment. This preliminary metagenomic decryption could represent a pilot-scale test for a future Earth microbiome global comparison

Membrane bioreactors (MBRs) offer promising solution for wastewater treatment and reuse to address the problem of water scarcity. Nevertheless, this technology is still facing challenges associated with membrane biofouling. This phenomenon has been mainly investigated in lab-scale MBRs with little or no insight on biofouling in full-scale MBR plants. Furthermore, the temporal dynamics of biofouling microbial communities and their extracellular polymeric substances (EPS) are less studied. Herein, a multidisciplinary approach was adopted to address the above knowledge gaps in lab- and full-scale MBRs. In the full-scale MBR study, 16S rRNA gene pyrosequencing with multivariate statistical analysis revealed that the early and mature biofilm communities from five full-scale MBRs differed significantly from the source community (i.e. activated sludge), and random immigration of species from the source community was unlikely to shape the community structure of biofilms. Also, a core biofouling community was shared between the five MBR plants sampled despite differences in their operating conditions. In the lab-scale MBR studies, temporal dynamics of microbial communities and their EPS products were monitored on different hydrophobic and hydrophilic membranes during 30 days. At the early stages of filtration (1 d), the same early colonizers belonging to the class Betaproteobacteria were identified on all the membranes. However, their relative abundance decreased on day 20 and 30, and sequence reads belonging to the phylum Firmicutes and Chlorobi became dominant on all the membranes on day 20 and 30. In addition, the intrinsic membrane characteristic did not select any specific EPS fractions at the initial stages of filtration and the same EPS foulants developed with time on the hydrophobic and hydrophilic membranes. Our results indicated that the membrane surface characteristics did not select for specific biofouling communities or EPS foulants, and the same early colonizers were selected from the source community (i.e. activated sludge), and then went through significant changes to form a mature biofilm. Our findings from these studies could support future research aimed at developing enhanced biological-based strategies to control biofouling in MBRs.

No description available.

Microbial communities are key drivers of biogeochemical cycles and several important industrial processes rely on complex, undefined microbial ecosystems for production or conversion of substrates for example in wastewater treatment or anaerobic digestion plants. Despite their significance, such communities are often poorly defined, if at all. This project concerned previously undefined secondary microbial communities (SMCs) from photobioreactors culturing cyanobacterium Arthrospira platensis, known for producing high-value protein-pigment complex C-phycocyanin (C-PC). C-PC has a range of applications in the biochemical/pharmaceutical and food industries. Next-generation sequencing methods were applied to characterize the SMCs sampled over the course of various batch runs. The bioreactor exerted a strong selective pressure on the SMC, initially diverse and dynamic, succeeded by a stable and predictable SMC dominated by a few species. SMC stability and diversity correlated with reactor performance, especially proliferation and instability of the rare-abundance sub-population; dominant species ratios were likely less important. The substantially larger (compared to other species present) A. platensis filaments may represent a dynamic microenvironment in itself, and if so, constitutes a significant parameter when optimizing culture conditions. Denser and carefully pre-acclimated inocula reduce the ecological space available to undesirable taxa (e.g. pathogens) otherwise below detectable/significant limits. This has implications for other processes that rely on mixed cultures and may be a control strategy in manufacturing active pharmaceutical ingredients to cGMP standards. Molecular data was used to obtain several pure isolates which were characterized further. Strategies to optimize performance with respect to SMCs were explored and evaluated. A significant aspect of this CASE project was an industrial placement with Scottish Bioenergy. The placement involved set-up of a production facility and incremental scale-up of cultivation from 2 L to 1000 L reactors; development of a downstream processing protocol covering harvesting, pigment extraction and protein purification, and some formulation/stability testing. A very low-cost method is described for obtaining relatively high-purities of C-PC, broadly considered the most costly part of the entire production process.

No description available.

Doctor of Philosophy / Biology / Walter K. Dodds / Ari M. Jumpponen / Due to agricultural practices and urbanization, tallgrass prairie ecosystems have become threatened as < 5% of its historical coverage exists today. The small remainder of praire that does exist is further threatened by the encroachment of woody plant species. Woody plant encroachment may not only alter prairie ecosystem function, but also prairie microbial communities responsible for these functional processes. Further, prairies are high disturbance ecosystems, especially prairie streams which are hydrologically harsh. They support communities that frequently undergo succession due to recurring flood and drought conditions, yet little is known about the response of microbial communities to these disturbances. In my dissertation, I first address the degree of woody vegetation expansion in riparian corridors (parallel to streams) in watersheds with variable fire frequency and grazing. I found that the rate of riparian woody expansion declines with higher fire intervals and is not affected by grazing, but even annual burns may not prevent woody plant expansion in riparian zones from occurring. Second, I quantified the effect of using restorations of riparian corridors, through removal of woody plants, on physical, chemical, and microbial community (bacteria and fungi) dynamics across stream to upslope soils. Removal restoration causes a decrease in NH₄⁺ and soil water content, and causes streams and upslope soils to become similar in fungal community richness unlike forested landscapes. Bacterial communities were minimally impacted by removals, but were highly structured among stream to upslope soils due to multiple environmental gradients (i.e., pH, NO₃⁻, soil moisture). Lastly, I examined the successional development of biofilm-associated microbial communities in a prairie stream from both a functional and structural perspective. I found that biofilm microbes exhibited strong successional trajectories, with communities developing towards net autotrophy and therefore becoming reliant upon in-stream derived carbon. Further, bacterial communities displayed spatial differences, but much stronger temporal patterns in community composition were detected. These studies highlight how woody plant encroachment may influence stream ecosystems in addition to spatiotemporal trends in microbial community assembly.

Metam sodium is a fumigant often used as a crop pretreatment in agriculture to control a wide array of pests that may inhibit plant yields. Previously, there have only been limited studies conducted on the effects of metam sodium on native soil microbial communities and plant pathogens, and results have been inconsistent. This present study utilized control and metam sodium-treated field plots to examine the effects of metam sodium on soil microbes in terms of numbers, activity, and diversity. Metam sodium did not cause significant changes in culturable heterotrophic numbers, as shown by heterotrophic plate counts, but may have adversely affected non-culturable microbes since metam sodium did affect microbial activity. Specifically, the LuminUltra® and dehydrogenase activity assays both showed a significant decrease in total activity in treated plots one day after soil treatment, with a return to pre-application conditions within seven days. Illumina Next-Generation Sequencing of the 16S rRNA gene showed slight changes in richness and community composition throughout the 28-day study, but initial and final communities were similar in both control and treated soils. Overall, some soil microbes were adversely affected by metam sodium, but the resilience of the soil microbial community allowed for an apparent rapid recovery in terms of numbers, activity, and diversity.

Animal hosts provide associated microorganisms with suitable ecological niches in their intestines. Microbes help their hosts to digest food, protect against pathogens, and influence the host’s metabolisms. Compositional variation of gut microbial communities is common among hosts, and may affect the health status of hosts. Diet and genetic factors are well known to influence the assembly of gut microbial communities. This thesis focuses on disentangling the contributions of factors including host genetics (sex), diet, environment, and other ecological processes to the assembly of gut microbial communities in freshwater fish. The association between gut microbial communities and fish condition is also evaluated in this thesis. Applying metacommunity theory, we found environmental factors including fish habitat, fish species, their diet, dispersal factors including microbes from fish diet, and ecological drift contributed to the assembly of fish gut microbial communities. The proportion of their contribution varied between fish species, where ecological drift explained more in perch than in roach. Under natural conditions fish populations face the risk of predation, which can induce competition and impose predation stress within prey individuals. This can therefore lead to changes in their diet qualities and quantities. In this thesis, it was shown that fish diet in terms of qualities and quantities significantly influenced the overall gut microbial composition, and this influence was dependent on fish sex, a host genetic factor. Predation stress was also suggested to significantly decrease the species richness. Furthermore, when fish were experiencing a diet shift, we showed that different bacterial phyla from novel food had different colonization success in the intestine, and this colonization success was positively influenced by predation stress. Fish condition was suggested in this thesis to be affected by gut microbial composition, especially by the contributions of the bacterial phyla Tenericutes and Actinobacteria.

Soils, and the microbial communities contained within them, are vital for most chemical, physical, and biological processes. This study investigated how microbial community structure responded to environmental changes, such as hydrology, across vertical space (depth) and time in an emergent fresh water wetland. Research was conducted in a non-tidal freshwater wetland along the James River (Charles City County, Virginia) by establishing plots in two areas that experienced different hydrologic regimes and plant communities. Soil cores (30 cm) were collected monthly from January 2008 to February 2009, and then every two to three months thereafter until October 2009, for a total of 17 sampling events. The soil cores were divided by depth (Top: 0 – 10 cm, Bottom: 20 – 30 cm) and analyzed for a variety of soil properties including: pH, organic matter (OM), water content (WC), C:N, redox, and root biomass. Additionally, above-ground plant communities were monitored during the growing seasons. Based on preliminary analysis, one date from each season (Winter, Spring, Summer, and Fall) from both sampling years were selected for in depth analysis of the microbial community structure via Terminal Restriction Fragment Length Polymorphism (T-RFLP) of 16S-rRNA. Analysis of variance (ANOVA) found significant differences were found between the environmental parameters in regards to site, depth, and season. Three physical-chemical variables (WC, OM, and redox) were different between sites, but the majority of environmental parameters were significantly different between depths and seasons. The dominant environmental effect on microbial communities was soil depth and, overall, no seasonal patterns were observed in the microbial communities. Further, archaeal communities were most strongly correlated to changes in water content, while redox was strongly correlated to changes across depth in the bacterial communities. Collectively, these results demonstrate that wetland microbial communities are not a product of one separate variable or spatial scale, but result from various factors interlinked to shape microbial communities. More long-term studies are needed to investigate interactions between microbial community structure and environmental variables in these dynamic ecosystems.