Increases in impervious surfaces caused by urbanization has led to higher volumes and rates of stormwater runoff that transports urban pollutants directly into natural waterways. Bioretention cells (BRCs) are vegetated soil systems designed to intercept stormwater runoff and reduce loads of water and contaminants discharged to surface waters. Nitrogen removal efficiency is highly variable and improvements are constrained by a poor understanding of the physical, biological, and chemical processes that occur within a BRC. The objectives of this study are to characterize and quantify the microbial communities in a range of existing BRCs, and determine which design factors have the greatest impact on denitrification, a microbial process responsible for removing nitrogen from stormwater. We sampled 23 BRCs throughout MD, VA, and NC, and quantified patterns in populations of denitrifying bacteria, denitrification potential, and microbial community structure within the soil medium. We found the greatest denitrifier populations and denitrification potential in the upper layer of the soil medium, which does not coincide with the internal water storage zone that is engineered to harbor anaerobic conditions favorable to denitrifying bacteria at the bottom of recent BRC designs. Results indicate that BRC vegetative cover, soil media nitrogen, and organic carbon concentrations are among the variables that facilitate nitrifying and denitrifying bacteria populations in BRCs. Bacterial community composition was most different between the top and bottom samples of the BRCs while fungal community composition differed most by BRC vegetative cover. Both fungal and bacterial community compositions were influenced by nitrogen and carbon concentrations. / Master of Science

Amphibian populations have undergone unprecedented declines in recent decades. Many of these declines are due to the spread of the cutaneous fungal pathogen Batrachochytrium dendrobatidis (Bd), which causes the disease chytridiomycosis. The Panamanian golden frog (Atelopus zeteki) has not been seen in the wild since Bd spread through western Panama. In response to initial declines, golden frogs were collected from wild populations and placed in captive colonies with the goal of future reintroductions. An understanding of this species' natural defense mechanisms against Bd is needed for reintroduction to be successful. Previous studies indicate that cutaneous bacteria are an important defense mechanism for some amphibians and applying antifungal bacteria to the skin of Bd-susceptible amphibians (probiotic therapy) can prevent chytridiomycosis. Therefore, the goals of my dissertation were to characterize the bacterial community of A. zeteki and determine if probiotic therapy could be used to prevent chytridiomycosis in this species. I initially characterized the bacterial community of wild and captive golden frogs using samples collected prior to the initial declines and after approximately eight years in captivity. I found that the community structure of the microbiota was significantly different between wild and captive frogs; however, the offspring of the original captive frogs still shared 70% of their microbial community with wild frogs. Then, I characterized the Bd-inhibitory properties of 484 bacteria isolated from 11 species of free-living Panamanian amphibians. I found a large proportion of bacteria (75.2%) had the ability to inhibit Bd and this trait was widely distributed among bacterial taxa, although there was also significant variation within bacterial genera in their ability to inhibit Bd growth. I then experimentally tested the ability of four of these isolates to prevent chytridiomycosis in captive golden frogs. None of them successfully prevented infection; however, there were several frogs that cleared infection and this was correlated with composition of the bacteria initially present on their skin. Overall these results demonstrate that the structure of microbial communities of A. zeteki are important to host health and building on this might provide the best hope for reintroducing this iconic species back to its native habitat. / Ph. D.

Thesis (M. Sc.), Memorial University of Newfoundland, 1998. / Bibliography: leaves 138-163.

Thesis (Ph. D.)--University of Canterbury, 2006. / Typescript (photocopy). Includes bibliographical references (leaves 237-268). Also available via the World Wide Web.

Use of an induced blanket reactor (IBR) to break down organic matter into methane is a financially attractive method to reduce the environmental impact of animal or industrial waste. In order to better understand the biological processes involved with the conversion of waste to biogas by an IBR, it is necessary to gain a better understanding of the microorganisms and their roles in the reactor. Molecular techniques based on the isolation of 16S rDNA were used in order to avoid the limitations posed by conventional culture-based techniques. Total DNA was extracted and amplified using universal primers specific to eubacteria and archaea with the purpose of identifying the dominant microorganisms in the IBR. The amplified DNA was separated based on its sequence composition by denaturing gradient gel electrophoresis (DGGE). Several bands were then excised, cloned, and sequenced, in order to characterize the phylogenetic affiliation of many of the microorganisms and create a useful molecular fingerprint. By using this approach, close relatives of several microorganisms that are typical in anaerobic digestion have been identified, including species of Clostridium, Flavobacterium, Bacteroides, Spirochaeta, Methanobrevibacter, and Methanosarcina. Several species were also identified whose role in the reactor is not completely understood, consisting of relatives of Dehalococcoides, Planctomyces, Aequorivita, and Sedimentibacter species. The information obtained in this project may enable refinements that promote desirable reactions and enhance reactor efficiency.

Arbuscular mycorrhizal (AM) fungi form an obligate symbiosis with many wild and cultivated plants. The plant may benefit from improved nutrient uptake (particularly phosphorus) and resistance to drought, pests and disease. Compared to more natural habitats, arable systems support reduced diversity of AM fungi, with community structure shifted in favour of ruderal types. Physical disturbance is an important driver of these differences and reduced tillage systems might facilitate greater utilisation of the AM symbiosis as part of more sustainable production systems. In this study, the structure of root-associated AM fungal communities in barley grown under a range of tillage regimes was characterised, using high-throughput molecular methodology. AM fungal community structure was significantly influenced by tillage intensity, with soils subject to high tillage burden richest in ruderal types. These findings were consistent in both winter and spring barley cultivars. Significant temporal changes in AM fungal community structure suggested an important role for plant growth stage in determining AM fungal community dynamics. Functional differences can occur between fungal species and different fungus-plant combinations, and in the second part of this study the functional consequences of changes in community structure due to tillage were tested in a model system using intact field-soil cores. There were clear differences in AM fungal community structure due to differential physical disturbance. Although no functional effects were observed, this may have been due to the inherent limitations of recreating field conditions in microcosm experiments. The results of this comprehensive assessment of fine-scale spatial variation in AM fungal community structure in an arable system identify tillage as an important driver of AM fungal community dynamics, and plant growth stage is also a key factor which has rarely been addressed. Elucidating the functional significance of these changes remains essential in order to justify future changes to arable management practices.

Microorganisms play central roles in planet Earth’s geochemical cycles, in food production, and in health and disease of humans and livestock. In spite of this, most microbial life forms remain unknown and unnamed, their ecological importance and potential technological applications beyond the realm of speculation. This is due both to the magnitude of microbial diversity and to technological limitations. Of the many advances that have enabled microbiology to reach new depth and breadth in the past decade, one of the most important is affordable high-throughput DNA sequencing. This technology plays a central role in each paper in this thesis. Papers I and II are focused on developing methods to survey microbial diversity based on marker gene amplification and sequencing. In Paper I we proposed a computational strategy to design primers with the highest coverage among a given set of sequences and applied it to drastically improve one of the most commonly used primer pairs for ecological surveys of prokaryotes. In Paper II this strategy was applied to an eukaryotic marker gene. Despite their importance in the food chain, eukaryotic microbes are much more seldom surveyed than bacteria. Paper II aimed at making this domain of life more amenable to high-throughput surveys. In Paper III, the primers designed in papers I and II were applied to water samples collected up to twice weekly from 2011 to 2013 at an offshore station in the Baltic proper, the Linnaeus Microbial Observatory. In addition to tracking microbial communities over these three years, we created predictive models for hundreds of microbial populations, based on their co-occurrence with other populations and environmental factors. In paper IV we explored the entire metagenomic diversity in the Linnaeus Microbial Observatory. We used computational tools developed in our group to construct draft genomes of abundant bacteria and archaea and described their phylogeny, seasonal dynamics and potential physiology. We were also able to establish that, rather than being a mixture of genomes from fresh and saline water, the Baltic Sea plankton community is composed of brackish specialists which diverged from other aquatic microorganisms thousands of years before the formation of the Baltic itself. / <p>QC 20150505</p>

Thesis (PhD)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: The grape and wine ecosystem contains fungi, bacteria and yeasts whose interactions contribute to the final wine product. While the non-Saccharomyces yeasts are dominant in the early stage of alcoholic fermentation, the later stage is always dominated by Saccharomyces cerevisiae. Although their presence in wine fermentation is often short-lived, the non-Saccharomyces yeasts are known to produce an array of extracellular hydrolytic enzymes which facilitate the extraction and release of aroma compounds, but might also play a role in microbial interactions. The present study aimed to investigate the microbial diversity of grape juice and to evaluate the potential of non-Saccharomyces yeasts to produce hydrolytic enzymes and display anti-fungal properties. To capture the microbial diversity, culture-dependent (plating) and –independent (Automated Ribosomal Intergenic Spacer Analysis (ARISA)) techniques were used in parallel. The fungal and bacterial ARISA displayed a wider range of operational taxonomic units (OTUs) in comparison to cultivation-based technique, demonstrating that ARISA is a powerful culture-independent technique applicable to ecological studies in wine. Some of the uncommon yeast isolates derived from our cultivation-based study were subjected to an enzymatic screening process. Hydrolases, such as chitinases, β-1,4-cellulases, β-1,3-1,6-glucanases, β-glucosidases, pectinases and acid proteases were specifically sought. Most of the yeast isolates exhibited chitinase, β-1,4-cellulase as well as β-1,3-1,6-glucanase activities. Only Metschnikowia chrysoperlae exhibited β-glucosidase activity. We also retrieved the partial chitinase gene sequences from M. chrysoperlae, Pichia burtonii, Hyphopichia pseudoburtonii that exhibited chitinase activity. Among the isolates, Pseudozyma fusiformata exhibited a strong antagonistic activity against the wine spoilage yeasts B. bruxellensis AWRI 1499 and B. anomalus IWBT Y105. Furthermore, we showed that the killer phenotype of P. fusiformata cannot be attributed to a viral encoded dsRNA. Finally, two metagenomic approaches were employed in an attempt to explore the indigenous microbiome in a more holistic manner, where we adopted whole metagenome Roche GS-FLX 454-pyrosequencing and construction of a fosmid library. The whole metagenome sequencing revealed a wide range of hydrolytic enzymes that showed homology to enzymes from different fungal and non-Saccharomyces yeast species. Moreover, the metagenomic library screening resulted in the retrieval of 22 chitinase and 11 β-glucosidase positive fosmid clones originating from yeasts. Two clones of interest, BgluFos-G10 and ChiFos-C21, were subjected to next generation sequencing. BgluFos-G10 revealed 2 ORFs exhibiting homology to glycosyl hydrolase family 16 proteins whereas no ORFs encoding chitinase enzymes could be identified in the ChiFos-C21 clone. However, all the potential ORFs identified exhibited homology to a gene cluster from Clavispora lusitaniae ATCC 42720, suggesting that the cloned DNA fragments belonged to a yeast species closely related to C. lusitaniae or members of the family Metschnikowiaceae. Overall, our study identified a variety of novel hydrolytic enzymes. However, retrieving the full gene sequences of these identified enzymes would be the immediate follow-up of our study. Moreover, the hydrolytic and antifungal activities exhibited by the yeast isolate could be of major interest in evaluating their potential as biocontrol agents against grapevine fungal pathogens and subsequently the wine spoilage yeasts. It would be interesting to evaluate as well the potential impact of these enzymes under wine making condition and could be our next step of investigation. / AFRIKAANSE OPSOMMING: Die druif en wyn ekosisteme bevat swamme, bakterië en giste en die interaksies van hierdie organismes dra by tot die finale wyn produk. Die nie-Saccharomyces giste is dominant in die vroeë stadium van die alkoholiese fermentasie, maar die latere fase word altyd gedomineer deur Saccharomyces cerevisiae. Alhoewel hulle teenwoordigheid in wyngistings gewoonlik kortstondig is, is die nie-Saccharomyces giste bekend vir die produksie van ‘n verskeidenheid ekstrasellulêre hidrolitiese ensieme wat die ekstraksie en vrylating van aroma komponente fasiliteer, en ook moontlik ‘n rol kan speel in mikrobiese interaksie. Hierdie studie beoog om die mikrobiese diversiteit van druiwesap te bestudeer en die potensiaal van nie-Saccharomyces giste te evalueer ten opsigte van die produksie van hidrolitiese ensieme, asook die demonstrasie van anti-swam eienskappe. Kweking-afhanklike (uitplating), asook –onafhanklike (Automatiese Ribosomale Intergeniese Spasieerder Analise (ARISA)) tegnieke is in parallel gebruik om die mikrobiese diversiteit te bepaal. Die swam en bakteriële ARISA het ‘n groter verskeidenheid van operasionele taksinomiese eenhede (OTUe) vertoon in vergelyking met die kweking-gebasseerde tegniek en dit demonstreer dat ARISA ‘n kragtige kweking-onafhanklike tegniek is, wat toepasbaar is in ekologiese studies van wyn . Sommige van die skaarser gisisolate, uit ons kweking -gebasseerde studie was vir ensiemaktiwiteite geskandeer. Daar is spesifiek gesoek vir hidrolases soos chitinases,β-1,4-sellulases, β-1,3-1,6-glukunases, β-glukosidases, pektinases en suur proteases. Die meeste gisisolate het chitinase,β-1,4-sellulase asook β-1,3-1,6-glukunase aktiwiteit vertoon. Slegs Metschinikowia chrysoperlae het β-glukosidase aktiwiteit vertoon. Ons het verder die gedeeltelike chitinase geensekwensies van M. chrysoperlae, Pichia burtonii en Hyphopichia pseudoburtonii wat chitinase aktiwiteit vertoon het, bepaal. Een isolaat, Pseudozyma fusiformata, het ‘n sterk antagonistiese aktiwiteit teenoor die wyn bederfgiste, Bretanomyces bruxellensis AWRI 1499 en B. anomalus IWBT Y105 vertoon. Verder het ons gewys dat die killer fenotipe van P. fusiformata nie gekoppel kan word aan’n viraal gekodeerde dsRNA nie. Ten laaste is twee metagenomiese benaderings, naamlik die volledige metagenoom Roche GS-FLX 454-pirovolgordebepaling en konstruksie van ‘n fosmied biblioteek, gebruik om die inheemse mikrobioom op ‘n meer holistiese wyse te bestudeer. Die volgordebepaling van die volledige metagenoom het ‘n wye verskeidenheid hidrolitiese ensieme aan die lig gebring wat homologie met ensieme van verskillende swamme en nie-Saccharomyces gisspesies getoon het. Verder het die skandering van die metagenomiese biblioteek die isolasie van fosmiedklone van gisoorsprong wat positief is vir chitinase aktiwiteit (22 klone) en β-glukosidase aktiwiteit (11 klone) tot gevolg gehad. Twee van hierdie klone, BgluFos-G10 en ChiFos-C21, is met volgende generasie volgordebepaling ontleed. BgluFos-G10 het twee oopleesrame (OLRe) wat homologie met glikosiel hidrolase familie 16 proteïene het, vertoon maar geen OLRe wat chitinase ensieme enkodeer kon in die ChiFos-C21 kloon geïdentifiseer word nie. Al die potensiële OLRe wat geïdentifiseer is, het homologie aan ‘n genepoel van Clavispora lusitaniae ATCC 42720 vertoon, wat daarop dui dat die gekloneerde DNS fragmente aan ‘n gisspesie behoort wat naverwant aan C. lusitaniae of lede van die Metschinikowiaceae familie is. In geheel gesien het ons studie ‘n verskeidenheid van nuwe hidrolitiese ensieme geïdentifiseer. Die bepaling van die volledige geenvolgordes van hierdie geïdentifiseerde ensieme sal die onmiddelike opvolg aksie van hierdie studie wees. Verder is die hidrolitiese en anti-swam aktiwiteite wat deur die gisisolate gedemonstreer is, van hoof belang, asook die evaluering van hulle potensiaal as biokontrole agente teen wingerd swampatogene en wyn bederfgiste. Dit sal ook interessant wees om die potensiële impak van hierdie ensieme onder wynmaakkondisies te bepaal, en dit kan dus ons volgende ondersoek stap wees.

The removal of yeast biomass (cropping) at the end of fermentation to inoculate a subsequent fermentation (serial-repitching) is common practice in the brewing industry. Between successive fermentations cropped yeast is stored as a slurry in cooled storage vessels under anaerobic conditions until required for subsequent use. Maintenance of yeast quality during storage is critical for subsequent fermentation performance. An assumption is made in brewing that all strains benefit from storage at 3-4°C. To test this assumption a model working system was initially established to assess cooling times of lager yeast in different suspension media. Preliminary investigations focussing on freshly propagated yeast slurry demonstrated that whilst the deleterious effects of extremely high storage temperatures on lager brewing yeast physiology was in line with expectation, utilization of traditionally recommended storage temperatures does not necessarily benefit yeast physiology when compared to slurry maintenance at slightly higher temperatures. Genome-scale transcriptional analysis in slurries cropped following an initial fermentation suggested that lager yeast might experience cold stress during slurry maintenance at typically recommended storage temperatures. In contrast, maintenance of lager yeast at a slightly higher storage temperature, in this case 10°C, yielded no adverse impact on key indicators of brewing yeast physiological state or on subsequent fermentation profiles following repitching into fermentations. Whilst these observations were not made using full production scale, they do indicate that optimal storage may not be currently being deployed for brewing yeast at full scale.

In this dissertation I explore the ecology of bacteriophage T4, a virus of Escherichia coli. In particular, I argue that the life history of bacteriophage T4 can be divided into the growth and survival of T4 phages in three distinct environments. I argue that these environments are distinguished by at least two T4 phage sensory systems. These include (i) the sensing of secondary adsorption by infecting phages and (ii) the determination of the concentration of monovalent cations and free tryptophan in solution about free T4 phage particles. The first environment consists of high concentrations of uninfected, logarithmic phase E. coli cells. These concentrations are approximately 10⁶ E. coli cells/ml and greater. This environment occurs in the prefecal colonic lumen of animals. Here T4 phages exhibit unimpeded logarithmic growth. The second environment contains high concentrations of infected E. coli cells, low concentrations of uninfected E. coli cells, and high concentrations of free T4 phage particles. This second environment also occurs in the prefecal colonic lumen of animals and represents the maturation of environments supporting logarithmic T4 phage population growth. Such phage phenotypes as secondary exclusion and lysis inhibition characterize T4 phage growth in this environment. The third environment consists of extra-colonic waters. Here T4 phages avoid infecting E. coli cells and exhibit strategies that maximize their stability. These strategies in extra-colonic waters increase the potential of T4 phages to disseminate successfully from colon to colon. I employ this enhanced understanding of T4 phage ecology, outlined above, in an exploration of the ecology of the repair of DNA damage by T4 phages.