Background: Symbioses throughout the animal kingdom are known to extend physiological and ecological capabilities to hosts. Insect-microbe associations are extremely common and are often related to novel niche exploitation, fitness advantages, and even speciation events. These phenomena include expansions in host diet, detoxification of insecticides and toxins, and increased defense against pathogens. However, dissecting the contributions of individual groups of symbionts at the molecular level is often underexplored due to methodological and analytical limitations. Termites are one of the best studied systems for physiological collaborations between host and symbiota; however, most work in lower termites (those with bacterial and protist symbionts) focuses on the eukaryotic members of this symbiotic consortium. Here we present a metatranscriptomic analysis which provides novel insights into bacterial contributions to the holobiont of the eastern subterranean termite, Reticulitermes flavipes, in the presence and absence of a fungal pathogen. Results: Using a customized ribodepletion strategy, a metatranscriptome assembly was obtained representing the host termite as well as bacterial and protist symbiota. Sequence data provide new insights into biosynthesis, catabolism, and transport of major organic molecules and ions by the gut consortium, and corroborate previous findings suggesting that bacteria play direct roles in nitrogen fixation, amino acid biosynthesis, and lignocellulose digestion. With regard to fungal pathogen challenge, a total of 563 differentially expressed candidate host and symbiont contigs were identified (162 up-and 401 downregulated; a/FDR = 0.05) including an upregulated bacterial amidohydrolase. Conclusions: This study presents the most complete bacterial metatranscriptome from a lower termite and provides a framework on which to build a more complete model of termite-symbiont interactions including, but not limited to, digestion and pathogen defense.

With respect to microbiological food safety, beer is thought to be very safe. This is due to the inability of pathogenic organisms to survive in the harsh environment that beer presents, due to low pH, alcohol content and hop acids. However, there are some organisms which have adapted to brewery conditions and can cause off-flavours, hazes or low ethanol yield. The effects of spoilage and subsequent product recall can result in massive economic losses for brewing companies affected. Silver nano particle coatings for pipes and vessels have been suggested as a means of eliminating or reducing contaminants in the brewery. In this study the sensitivity of several brewery contaminants to silver has been investigated. Pichia membranaefaciens, Brettanomyces anomalous, Candida krusei, Hansenula saturnus, Kloeckera apiculata, Rhodotorula mucilaginosa, Saccharomyces ellipsoids var. diastaticus, Lactobacillus brevis, Pediococcus damnosus, were all tested against a range of silver nitrate concentrations (0-1 mM) in YPD, wort and beer. It was found that sensitivity to silver varied between organisms, but no tolerance exceeded 0.55 mM. It was also found that for the majority of organisms, tolerance to silver decreased under simulated brewery conditions i.e. wort, beer and microaerophillic conditions. In the investigation of potential silver tolerance mechanisms, gene microarrays of Saccharomyces ellipsoids var. diastaticus in wort and beer in the presence and absence of silver found that genes most up-regulated during silver stress were those with transmembrane transporter functions. Silver tolerance testing with gene deletion strains of selected potential silver tolerance genes demonstrated reduced silver tolerance for the deletion strains of the HIS1, COX17 and CUP1 genes. All three of these have known functions in copper tolerance. The data collected in this study would suggest that silver (particularily in nanoparticle form) is an effective means of microbial brewery contamination control especially under brewery conditions. However, further study is needed into the effect of silver antimicrobial surfaces on brewery microbial contaminants, silver concentrations needed in antimicrobial surfaces and silver leaching etc.

Thesis (PhD (Microbiology))--University of Pretoria, 1996. / This study analysed the bacteria numbers of Hake products manufactured at 6 different levels of processing complexity, analyses were conducted at 20°C on Salt Water Agar (SWA) for a 48h incubation period. This enumeration method was suitable for indicating time and temperature related abuse in the processing system. This method was sensitive in indicating the level of processing complexity. The aerobic bacteria enumerations at 20°C (SWA) were compared to the current EU/FDA (European Union/Federal Drug Administration USA) standard procedure testing samples on Plate Count Agar, (PCA) incubated at 30 - 35°C. The method using SWA incubated at 20°C provided better and more accurate bacterial counts under "good" and "poor" manufacturing practice. The hurdle concept was applied, using gamma irradiation, temperature (Time), chemical preservatives and Vacuum packaging in an attempt to prolong the shelf life of fresh, uncooked Cape hake and Kingklip products. Radurization was effective only when the treated product was kept at refrigerated temperatures. Temperature was the most important control measure in extending the shelf life of the products. No shelf life extension was achieved with the use of chemical preservatives. Vacuum packaging was not effective in prolonging shelf life of the products and did not show any benefit when compared to oxygen permeable vacuum (microaerophilic) packaging. Determination of the total aerobic viable bacteria numbers at 20°C SWA, were suitable for estimating the shelflife of deepsea whitefish products.

The Arctic is undergoing a rapid environmental shift with increasing temperatures and precipitations expected to continue over the next century. Yet, little is known about how microbial communities and their underlying metabolic processes will respond to ongoing climatic changes. To address this question, we focused on Lake Hazen, NU, Canada. As the largest High Arctic lake by volume, it is a unique site to investigate microbial responses to environmental changes. Over the past decade, glacial coverage of the lake has declined. Increasing glacial runoff and sedimentation rates in the lake has resulted in differential influx of nutrients through spatial gradients. I used these spatial gradients to study how environmental changes might affect microbial community structure and functional capacity in Arctic lakes. I performed a metagenomic analysis of microbial communities from hydrological regimes representing high, low, and negligible influence of glacial runoff and compared the observed structure and function to the natural geochemical gradients. Genes and reconstructed genomes found in different abundances across these sites suggest that high-runoff regimes alter geochemical gradients, homogenise the microbial structure, and reduce genetic diversity. This work shows how a genome-centric metagenomics approach can be used to predict future microbial responses to a changing climate.

This dissertation aims to expand our knowledge of glycerol-driven engineered biological nitrogen removal processes by elucidating the link between operational controls and the structure and function of the microbial ecology grown under stoichiometrically-limited and excess glycerol conditions. Specific objectives were to: 1. Develop and experimentally evaluate an improved metric for denitratation performance that can be objectively compared across studies; 2. characterize the process kinetics, nitrogen conversion efficiencies, and microbial ecology of a glycerol-driven, stoichiometrically-limited denitratation process; 3. elucidate the impact of kinetic limitation on microbial community structure and function in a glycerol-driven, stoichiometrically-limited denitratation process; 4. explore the biological mechanisms contributing to nitrite (NO2-) accumulation in a glycerol-driven denitratating microbial community; and, 5. characterize the nitrogen conversion efficiencies and microbial ecology that favor dissimilatory nitrate reduction to ammonium (DNRA) in a glycerol-driven denitrification process at stoichiometric excess. Accordingly, a nitrate (NO3-) conversion ratio (NaCR) was first proposed as an improved metric of denitratation performance metric. Previous metrics used throughout literature were deemed insufficient as they provided an incomplete and subjective representation of denitratation performance by not accounting for residual NO3- remaining in the system following the selective reduction of NO3- to NO2-. The NaCR represented a singular metric that better signifies true denitratation performance and can be compared across studies regardless of carbon source or system configuration. Second, a glycerol-driven denitratation process was optimized according to different operational controls. Steady-state reactor operation and in situ and ex situ batch assays indicated that the influent chemical oxygen demand to NO3- (COD:NO3--N) ratio was determined to influence process kinetics and nitrogen conversion efficiencies leading to significant NO2- accumulation. A singular microbial community structure correlated to system performance was identified. Third, the application of kinetic limitation (by imposing different solids retention times [SRTs]) at a given influent COD:NO3--N ratio was demonstrated as an effective mechanism in the selection for a denitratating microbial ecology capable of significant NO2- accumulation. Steady-state reactor operation was used to characterize process kinetics and nitrogen conversion ratios supporting the determination of the optimal SRT for reactor operation. Analysis of the microbial community structure elucidated the impacts of kinetic limitation on the microbial ecology which were correlated to system performance. Functional denitrification gene transcripts were found to be significantly different under kinetic limitation, indicating that NO2- accumulation was driven more by differences in microbial community structure as opposed to differential expression at different operating SRTs. Fourth, ex situ batch assays were used to elucidate the microbial transcriptional response to the presence of varied sequences of electron acceptors. The microbial community was found to be enriched with NO3--respirers, or microorganisms incapable of NO2- reduction, and progressive onset denitrifiers, which express functional denitrification genes in sequence. The presence or re-introduction of NO3- in a NO2--reducing community was found to elicit an immediate transcriptional change and shift of electron flow to NO3- reductase. Electron competition as the primary contribution to NO2- accumulation was confirmed through the artificial inactivation of NO3- reductase. Lastly, an influent COD:NO3--N ratio was applied in stoichiometric excess to create the conditions necessary to support DNRA over denitrification. System performance at steady-state was found to vary under different kinetic regimes. The induction of DNRA was found to be far more complex than simply providing glycerol in stoichiometric excess. Additionally, glycerol does not appear to be an optimal COD source for DNRA under these conditions. In sum, the optimization of engineered biological nitrogen removal processes through the manipulation of process kinetics and the resulting impacts on nitrogen conversion efficiencies and microbial community structure and function was investigated in detail. From an engineering perspective, this knowledge can help guide the design and operation of biological nitrogen removal processes to systematically maximize the accumulation of targeted nitrogenous products or mitigate unintentional and undesired products.

The communities of microorganisms associated with humans and other animals are characterized by a large degree of diversity and unexplained variation across individual hosts. While efforts to explain this variation in host-associated systems have focused heavily on the effects of host selection, community assembly theory emphasizes the role of dispersal and stochastic demographic processes, otherwise known as ecological drift. In this dissertation, I characterize the communities of microorganisms associated with the zebrafish, Danio rerio, intestine, and assess the importance of microbial dispersal and drift to their assembly. First, I describe changes in the composition and diversity of the zebrafish intestinal microbiome over zebrafish development and show that while host development is a major driver of community composition over time, there remains a large amount of unexplained variation among similar hosts of the same age. I go on to show that random dispersal and ecological drift alone in the absence of host selection are sufficient to explain a substantial amount of this variation, but the ability of these processes to predict the distribution of microorganisms across hosts decreases over host development. Finally, I present an experimental test of dispersal in host-associated systems, and show that not only does dispersal among individual zebrafish hosts have a large impact on the composition and diversity of associated microbial communities, but it can also overwhelm the effects of important host factors, such as the innate immune system. As a whole, this work demonstrates that the composition and diversity of microbial communities associated with animal hosts are not solely the result of selection by the host environment, but rather dispersal and stochastic processes have important and often overwhelming effects on their assembly. To fully understand the assembly of host-microbe systems, we must broaden our focus to include scales beyond that of an individual host and their associated microorganisms. / 10000-01-01

The skin microbiome is a critical component of human health, however, little is understood about the daily dynamics of skin microbiome community assembly and the skin’s potential to acquire microorganisms from the external environment. I performed a series of microbial transfers using three skin habitat types (dry, moist, sebaceous) on human subject volunteers. Microbial communities were transferred to recipient skin using a sterile swab 1) from other skin sites on the same individual, 2) from other skin sites on a different individual, 3) and from two environmental donor sources (plant leaf surfaces and farm soil). With these experiments I was able to test for the presence of initial transfer effects and for the persistence of those effects over the time period sampled (2-, 4-, 8-, and 24-hours post-transfer). The sebaceous skin community was associated with the strongest initial effect of transfer and persistence on the moist recipient skin site, and to a lesser extent the dry skin site. The soil donor community when transferred to dry skin resulted in the strongest initial transfer effect and was persistent over 8- and even 24-hours post-transfer. These experiments are the first in scope and scale to directly demonstrate that dispersal from other human or environmental microbial communities are plausible drivers of community dynamics in the skin microbiome.

The colonial, N2 fixing cyanobacterium Trichodesmium is a keystone species in oligotrophic ocean ecosystems. Trichodesmium is responsible for approximately 50% of the total biologically fixed N2 in the ocean, and this “new” nitrogen fuels primary productivity and the amount of carbon sequestered by the ocean. Trichodesmium does not exist in isolation. Colonies occur ubiquitously with an assemblage of epibiotic microorganisms that are distinct from planktonic microbes and modulated across environments, yet the implications of this relationship have not been explored. In this thesis, the ecology, physiology, and potential geochemical impact of interactions within the Trichodesmium host-microbiome system were examined across three different oligotrophic ocean environments. First, to establish the metabolic diversity contributed by the microbiome to Trichodesmium consortia, a whole community metagenomic sequencing approach was used across a transect the western North Atlantic. This study demonstrated that the microbiome contributes a large amount of unique functional potential and is modulated across a geochemical gradient. In the following study, metatranscriptomics was used to show that such metabolic potential in Trichodesmium and the microbiome was expressed and modulated across the environment. Colonies were sampled in the western tropical South Pacific and gene expression dynamics indicated co-limitation by iron and phosphorus, and revealed a mechanism for phosphate reduction by Trichodesmium and subsequent utilization by the microbiome. These activities were verified with phosphate reduction rate measurements and indicated cryptic phosphorus cycling within colonies. Next, the suite of potential physiological interactions between host and microbiome was assessed with metatranscriptome sequencing on high frequency samples of Trichodesmium colonies from the North Pacific subtropical gyre. Synchronized day-night gene expression periodicity between consortia members indicated tightly linked metabolisms. The functional annotations of these synchronous genes indicated intra-consortia cycling of nitrogen, phosphorus and iron, as well as a microbiome dependence on Trichodesmium-derived cobalamin—interactions that could alter the transfer of these resources to the surrounding water column. In the final study, the effect of the microbiome on Trichodesmium N2 fixation was assessed. Using colonies obtained from the North Atlantic, activity in the microbiome was selectively modified using quorum sensing acyl homoserine lactone cell-cell signaling, a mechanism that Trichodesmium itself does not possess. These experiments indicated that the microbiome has the potential to increase or decrease Trichodesmium N2 fixation to a degree that rivals the effects of alterations in nutrient concentration, but at a more rapid rate. In all, the research presented in this thesis demonstrates the integral importance of the microbiome to Trichodesmium physiology and ecology, highlighting the importance of an unexplored facet of marine microbial systems that likely influences the biogeochemistry of the planet.

No description available.

Graduation date: 2005