No description available.

published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

Deserts are the most abundant terrestrial biome on Earth, and microbial processes assume a major role since environmental stress severely limits higher plant and animal life. A major hurdle to developing an understanding of microbial ecology in deserts has been the lack of knowledge at the fundamental biodiversity level. This is due to lack of research focus and also the inherent bias in ‘traditional’ microbial diversity estimates based upon cultivation. In this thesis an evaluation of culture-independent approaches employing both DNA and RNA from environmental samples was made with comparison to more traditional cultivation techniques. These were then applied to soils from hot and cold deserts, and along stress gradients from semi-arid to hyper-arid. A literature review was first conducted to assess the extent of current knowledge and identify critical knowledge gaps. The scientific study was then carried out as follows. The second chapter presents an evaluation of fungal taxa using cultivation, DNA and RNA based techniques. The findings indicated major taxa are revealed in all approaches, yet differences in less abundant taxa occur. The third chapter describes fungal assemblages in the soils of the McMurdo Dry Valleys, a cold polar desert. In this study, RNA based approaches tracked active fungal assemblages, whilst DNA and cultivation revealed additional taxa. Chapter four analyzed microbial communities in the Thar Desert, a hot monsoon desert in India. This study revealed a diverse community that comprised known desiccation-tolerant taxa but also a phylogenetically broad range of bacteria, archaea and eukarya. Chapter 5 focuses on the delineation of total versus active microorganisms in environmental samples from the hot deserts. As with the initial experiments, this revealed that total and active assemblages track each other broadly in desert soils. A synthesis of the study revealed that certain common microbial phyla are likely well-adapted to xeric stress, although distinct hot and cold desert assemblages also develop. For such low-diversity systems it is likely that DNA-based approaches are reasonable tools for diversity analysis, and will be especially useful in arid systems when long periods of inactivity may confound attempts to estimate active populations. Broader significance of the study includes an increased appreciation of eukaryotic microbial presence in arid soils, and how latent soil microbiota may act as a reservoir for development of future microbial macro-structures (e.g. soil crust) that function in soil stabilization, and should therefore be included in conservation planning for deserts. / published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

The pressing need for clean renewable energy sources has aroused worldwide research interest on the exploration of biofuels produced from lignocellulosic feedstock (e.g. forestry or agricultural residues and municipal wastes). The general absence of cost-effective method to overcome the recalcitrant nature of cellulosic biomass is the major challenge for the industrialization of this so-called second-generation biofuel. With the purpose to enhance our understanding of the fundamental mechanism of thermophilic microbial cellulose conversion process, we used culture-independent metagenomic analysis based on Next Generation Sequencing to explore the physiological ecology of thermophilic cellulolytic microbial community and more importantly to discover metabolic potentials. During the enrichment of thermophilic cellulolytic consortium, noticeable effects of co-substrate and pH was observed and subsequently investigated. Based on the community structure revealed by 16S rRNA gene sequencing at various pH values, we concluded that keeping pH higher than 6.0 was crucial to maintain effective cellulose conversion because the growth of Thermoanaerobacterium over other more efficient cellulolytic populations could be practically avoided. Given in mind that uncharacterized microbial populations may possess critical enzymatic components that are essential for the breakdown of cellulosic feedstock, gene-centric metagenomic pipeline was developed to discover genes that are functionally beneficial for thermophilic cellulose hydrolysis. Aside from that, metagenomic gene mining based on functional prediction using HMM (Hidden Markov Model) showed higher positive ratio in identifying novel carbohydrate-active genes than that of functional screening. Without cultivation, near complete genomes of the major thermophilic cellulose degraders were recovered from the metagenome by a gene binning pipeline combining tetranucleotide frequency based primary k-means clustering and subsequent scaffolding with paired-end relationship between two reads (sequences). Furthermore, by quantifying the transcriptional activities of various carbohydrate-active genes in the metatranscriptome of the enriched thermophilic cellulose-degrading consortium, we disclosed significance of enzymes of GH09 and GH48 which had been underestimated by previous metagenomic studies. Eventually, metagenomic survey of various sludge samples collected at specific operational conditions helped to confirm the metabolism potential of thermophilic sludge in cellulose up taking by possessing more enzymes of GH05 and GH04 families. / published_or_final_version / Civil Engineering / Doctoral / Doctor of Philosophy

The development of biofilms on mild and stainless steel surfaces in pure and mixed batch cultures of the bacterial species Pseudomonas fluorescens and Desulfovibrio desulfuricans and the role of these biofilms in corrosion of steel has been investigated. Early events leading to the formation of biofilms have been elucidated by studying the attachment of bacterial cells to steel using epifluorescence microscopy. To identify the nature of the bacterial surface components involved in the initial adhesion to mild steel, lectins, their sugar inhibitors and saccharolytic and proteolytic enzymes have been employed. Polyclonal antibodies have been raised against bacteriallipopolysaccharides (LPS) and their influence on bacterial adhesion assessed. LPS have been analysed chemically by gas-chromatography (GC-FID) and gas chromatography-mass spectrometry (GC-MS) to determine their ~arbohydrate composition and fatty acid content. On the basis of the results obtained the ~nvolv~~~nt of glucose and N-acetylglucosamine, present in O-antigenic fractions of LPS, 10 the lOlnal attachment of the two bacterial species to mild steel is suggested. Both types of carbohydrates are likely to be involved in early attachment of Pseudomonas to mild steel, whereas only a polymeric fonn of N-acetylglucosamine seems to participate in adhesion of Desulfovibrio. The subsequent biofilm development on steel surfaces and their accompanying corrosion h~s been monitored by scanning electron microscopy (SEM). SEM studies reveal very different patterns of bacterial biofilms on mild and stainless steel and show varied degrees of corrosion occurring on these surfaces. Thin and patchy Pseudomonas biofilms are accompanied by little corrosion whilst thick. more continuous, Desul/ovibrio biofilms are associated with higher levels of corrosion. Energy dispersive X-ray analysis (BOAX) of corrosion products present on steel surfaces indicates ferrous sulphides as the major components in Desul/ovibrio biofilms. The corrosion of steel in bacterial cultures has also been investigated by kinetic polarisation measurements. The results obtained from cathodic and anodic polarisation curves, combined with SEM and EDAX analyses confmn the SEM observation. Stainless steel is not subjected to any great degree of fouling or corrosion under the chosen experimental conditions. The EPS associated with biofilms and released into the liquid phase of the culture media (free EPS) has been characterised. Proteins and carbohydrates in these polymers are detected colorimenically and by SDS-gel electrophoresis. Uronic acids, found in biofilmbound BPS. are not detected in free EPS. The GC-MS and GC-FIO analyses have aided in establishing types and quantities of neutral carbohydrates present in bacterial exopolymers and show that the neutral sugar composition of free and surface-associated BPS is not identical for a given bacterial culture. The biofilm-bound BPS are believed not to play a major role in corrosion of mild steel but to provide additional mechanisms in its facilitation. No correlation between levels of free BPS and corrosion of steel is found.

No description available.

Integrases (Int) are enzymes that mediate the integration and excision of viral DNA into or out of their hosts‟ chromosomes and can therefore be exploited to integrate or delete genes in a precise way. In order to establish lysogeny, integrase mediates recombination between the bacterial and phage attachment sites, attB and attP respectively to generate an integrated prophage flanked by attL and attR. This reaction occurs in vitro without any need for accessory proteins prompting the question, how does the prophage excise? Phages use accessory proteins, Recombination Directionality Factors, RDFs to control the directionality of integrase. For the serine integrase family, RDFs have been identified for three phages, TP901-1, φRv1 and Bxb1, and there is no detectable sequence conservation between them. This work has identified the φC31 early protein gp3 as the RDF. Gp3 acts stoichiometrically to activate excision and binds to Int in solution and in complex with DNA. Insight into the mechanism of gp3 action has revealed that it is at the synapse level that gp3 switches the directionality of Int. The properties of the gp3+Int driven reaction was found to be similar to that mediated by a previously characterised mutated Int, IntE449K that triggers gp3 independent excision (Rowley et al., 2008). Despite φC31 and φBT1 Ints being only 21% identical in sequence, the gp3 homologues from these phages cross-react. Both the gp3s bind to the last 200 amino acids of C-terminal domain of φC31 Int to activate excision and inhibit integration. Evidence is presented that gp3, on binding to Int, overcomes an innate mechanism that normally prevents synapsis of the excision substrates. These observations could lead to further exploitation of φC31 system as a tool for genome engineering.

Habitat heterogeneity is a driving factor for speciation and ecosystem functioning and is well studied in macro-ecology. Yet our understanding of microbial adaptations, and governing processes is incomplete. The here presented thesis aims at giving us a better understanding of patterns in micro-heterogeneity, and microbial adaptations to such heterogeneity with particular focus on surface-dominated, aquatic habitats. The most prominent microbial adaptation to surface associated mode of life is biofilm formation. Biofilms rely heavily on type IV pili. These pili systems are well studied in Bacteria, but largely unknown in Archaea. Therefore, the first part of this thesis focuses on resolving genetic and structural feature of the type IV like aap-pilus of the thermo-acidophilic Sulfolobus acidocaldarius. We found the aap-pilus to be indispensible for biofilm formation, and to be unparalleled in variability of its quaternary structure and cross regulation with other filaments. The second part of this thesis investigates particle colonization in the water column, focusing on diatoms as a model system, allowing an in situ assessment of different stages of particle colonization, and potential particle-specificity of the associated bacterial community. Opposing reports from marine systems, we did not observe diatom-specificity in the associated bacterial community. Instead we found bacterial community subsets, one likely originating from sediment resuspension, and the other being controlled by biofilm-forming populations (e.g. Flexibacter), able to attach to newly formed particle surfaces and subsequently facilitate secondary colonization by other bacteria. Finally, the habitat heterogeneity in top-layers of lake sediments were investigated in experimental microcosms. Cell-specific oxygen consumption rates were determined, to assess microbial activity across different scales. Individual activity rates differed strongly across all investigated scales, likely due to spatially heterogeneous distribution of nutrients with differing quality. Vice versa, the influence of microbial activity on micro-habitat-heterogeneity was investigated. We correlated sediment redox-state with bacterial community composition and populations. Our results indicate that habitat heterogeneity is generally beneficial for microorganism, and greater heterogeneity results in greater bacterial diversity. However, this heterogeneity-diversity relationship is limited and microorganisms actively stabilize their immediate redox environment to a preferred, community-specific, stable state, if cell abundances exceed a minimum threshold.

The two main objectives in this study were investigating the microbial quality and microbial communities of 'use-by date' Mung bean sprouts by using conventional culture and 16S/18S rDNA PCR-DGGE methods, and evaluating the efficacy of natural antimicrobial substances, chemical disinfectants, and thermal treatments in reducing and inhibiting the growth of the pathogens on mung bean seeds. Retail samples of pre-packed mung bean sprouts were obtained from three retailers in the local area. The microbial quality and communities were evaluated on the 'use-by date'. The highest counts of total aerobic counts (7.86 log10 CFU/g), yeasts and moulds (7.0 log10 CFU/g), total lactic acid bacteria (6.24 log10 CFU/g) and total coliforms (6.63 log10 CFU/g) were found in samples from one shop and the DGGE band sequences also identified major populations of LAB from the same samples, These indicated poor quality and spoilage of the samples from this location and could be related to improper storage at temperatures above 5°C. The combination of conventional culture methods with the PCR-DGGE technique revealed a larger diversity of bacterial communities than eukaryotic ones based on the relative number of amplimers present on most of the OGGE gels. Identification based on band analysis revealed that the Enterobacteriaceae (29.6%), soil bacteria (20.4%), lactic acid bacteria (18.5%). yeast (14.8%). Pseudomonas spp. (13%), and Flavobacterium (3.7%) constituted the major populations in bean sprout samples. Cluster analysis of the OGGE patterns of both 16S and 18S rDNA amplimers found no strong relationship between sample sources and batches indicating the variability of natural populations. The use of natural antimicrobial products, such as a mixture of lime juice and vinegar (1: 1, pH 2.83) and bacteriocin-like substances produced by Pediococcus acidilactici, failed to reduce and inhibit the growth of Listeria monocytogenes on mung bean seeds. The former solution had higher antimicrobial efficiency in reducing the pathogen on seeds (1.93 log10 CFU/g) compared to the Pediococcus broth culture (1.22 log10 CFU/g), but both solutions failed to inhibit the re-growth of the pathogen during the sprouting process and also reduced seed germination percentage by 13-18%. The evaluation of efficacy of sequential washing using a combination of chemical treatments (two-step dipping) against the pathogens on seeds showed that a two-step dipping treatment in a solution containing 2% sodium hypochlorite for to min followed by 5% lactic acid solution for 5 min was the most effective treatment. This treatment achieved the highest reductions of L. monocytogenes (2.91 log10 CFU/g) and Salmonella Typhimurium (3.20 l0g10 CFU/g) after treatment and continued to reduce the pathogen during the sprouting process. This may be due to the chemical residues on treated seeds which lowered both pathogens on sprouted seeds to below the limit of detection <50 CFU/g) by direct plating without significantly affecting seed viability. The use of thermal treatments based on a hot and cold water dipping was found to be more effective in reducing the normal flora on seeds and less affecting of seed germination compared to microwave heating. The use of a hot and cold water dipping treatment at 92°c for 1 min followed by ice-cold water at 5°C for 30 sec achieved the highest reduction of L. monocytogenes on seeds (>5 log10 CFU/g) but had the lowest germination percentage (89%) compared to other hot and cold water dipping treatments. Microwave heating at 1-4 kW showed a poorer efficiency in reducing nonnal flora on seeds and severely affected seed viability. Overall, a two-step washing with 2% sodium hypochlorite followed by 5% lactic acid seems to be the most successful treatment in reducing and inhibiting the recovery of the pathogen during the sprouting process. However, the chemical residues on treated seed may become a negative image to apply this treatment in the sprout industry.

published_or_final_version / Biochemistry / Master / Master of Philosophy