Leafy greens like lettuce and spinach are a common vehicle for foodborne illness in United States. It is unknown if native plant epiphytic bacteria may play a role in the establishment of enteric pathogens on leaf surfaces. The objective of this study was to characterize the bacterial communities of fresh and packaged spinach leaves and to explore interactions with E. coli O157:H7. We assessed the bacterial diversity present on the spinach leaf surfaces and how parameters such as spinach cultivar, field conditions, post-harvest operations and the presence of E. coli O157:H7 affected its diversity. Differences in bacterial population size and species richness were associated with differences in plant topography; flat leaves had smaller bacterial populations than savoy leaves, which correlated with larger number of stomata and trichomes in savoy leaves. During spinach growing season shifts in environmental conditions affected richness and population size of the spinach bacterial community. Decreases in the overall soil and ambient temperature and increased rainfall decreased richness and bacterial population size. Fresh spinach richness and composition assessed by parallel pyrosequencing of 16S rRNA elucidated 600 operational taxonomic units, with 11 different bacterial phyla. During postharvest operations diversity indexes and evenness tended to decrease, likely attributed to storage at low temperature and time of storage (4°C and 10°C), that promoted the dominance of g-Proteobacteria. Bacteria isolated from fresh spinach elicited growth inhibition of E. coli O157:H7 in vitro, which was associated with nutrient competition. In contrast growth enhancement produced by epiphytes was associated to low correlations in carbon source utilization and the ability of E. coli O157:H7 to rapidly utilize carbon resources. In packaged spinach, E. coli O157:H7 altered the composition of the bacterial community and its growth was promoted on packaged spinach when a disinfection and temperature abuse occurred, removal of the epiphytic bacteria resulted in significant increases in numbers of E. coli O157:H7 at 10°C and was associated with increased expression of E. coli O157:H7 virulence and stress response genes. The large diversity present on the surface of spinach leaves significantly impacted the ecology of enteric pathogens like E. coli O157:H7 on the phyllosphere. / Ph. D.

Bt-maize is engineered to express insecticidal toxins derived from the bacterium Bacillus thuringiensis and has been shown to be very effective against pests like Busseola fusca. However, resistance of this pest against Bt-maize has developed and spread throughout South Africa. This study was inspired by the lack of knowledge over the microorganisms associated with the gut of these insects as they play a vital role in insect growth and development. Microbial-derived enzymes may have a role during an insect‟s adaption in different environmental conditions and to new diets. Previous studies suggest (1) that gut bacteria are required for B. thuringiensis-induced mortality in most Lepidoptera species and (2) that the toxicity of B. thuringiensis depends on microbial community interactions within the gut. The aim of this study was to determine the microbial diversity present in the midgut of B. fusca larvae occurring in maize. Busseola fusca larvae were collected from 30 sites throughout South Africa and dissected to collect their midgut contents. Serial dilutions were made of the contents and spread plated onto nutrient agar after which morphotypes were identified. One-hundred and five morphotypes were identified; DNA were extracted from the selected morphotypes and subjected to PCR analysis followed by secquencing. Sequencing results revealed the dominance of Enterococcus spp., specifically Enterococcus casseliflavus and Enterococcus gallinarum, Klebsiella spp., espesially Klebsiella pneumoniae and Klebsiella oxytoca and Bacillus spp. such as .B. thuringiensis and B. subtilis. Other organisms isolated, included Achromobacter spp., Brevudimonas spp., Caulobacter spp., Enterobacter spp., Halomonas spp., Ochrobactrum spp., Pantoea spp., Pseudomonas spp., Serratia spp., Stenotrophomonas spp., Arthrobacter spp., Brevibacterium spp., Leucobacter spp., Microbacterium spp., Planomicrobium spp. and Staphylococcus spp. The microbial diversity of larvae collected at the respective sampling sites were determined with the Shannon diversity index. The data were compared to several factors regarding the sampling sites. No significant differences were observed between the microbial diversities isolated at the respective sites. This may imply that the microbial community within B. fusca larvae are relative consistent throughout the maize production area. It is important to understand the distribution and structure of gut microbial communities within insects and whether the gut community is influenced by the geographical distribution of the insects. A better understanding of the distribution of the insects and community structure of their gut microbiota may aid in the development of better insect control strategies. / MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2015

The rhizosphere is characterized by the presence of high microbial activities which are influenced by plant root exudates. This study examined bacterial diversity and physiological functions plants rhizosphere using both culture-dependent and culture-independent techniques of seven cultivated. Physico-chemical properties of soil samples revealed that the rhizobacteria adapted well to pH ranging from 7.5 to 9.1. Macronutrients (carbon, nitrogen, calcium, magnesium, phosphorous, potassium, sodium and iron) had a wide range of concentration between 0 to 4380.1 mg/kg. Concentrations of metal elements (cadmium, cobalt, chromium, copper and zinc) from all rhizosphere samples were below the amount of 3.1 mg/kg, indicating that the samples were free from metal contaminations. Sole carbon substrates utilization of bacteria in rhizosphere samples were measured as Average Well Colour Development (A WCD) and Group-wise Average Well Colour Development (AWCDg) patterns. At seventy two hours, there was no significant difference in AWCD patterns between bacteria in all samples and there was a significant difference in AWCDg patterns. Biochemical tests showed majority of isolates had similar physiological properties to members of Bacillus genus. All the bacterial isolates exhibited positive antifungal trait, fifteen solubilized phosphate and three had cyanide production traits during in vitro plant growth promotion assays. In vitro plant growth revealed that bacterial isolate RL1 (Bacillus licheniformis) produced the highest concentration of indole acetic acid (IAA) at 25 mg/ml. Bacterial isolate RG3 (Bacillus pumilus) had the highest amino cyclopropane carboxylase (ACC) deaminase activity indicated by the high production of α-ketobutyrate produced at 4.8 mg/ml. There were significant differences in shoot length at P ≤ 5% level of significance and there was no significant difference in the number of leaves across all three inoculated plants at P ≥ 5% level of significance. Sequence and phylogenetic analysis of identified culture-dependent bacteria revealed a homologous similarity of 94 to 100% between isolates sequences and GenBank sequences. From this, 81% of the sequences were closely related to Firmicutes, 13% to Actinobacteria and 6% to Proteobacteria. From cultureindependent method, only 8 PCR-DGGE bands were detected, the 200 bp sequences in the 16S rRNA fragment showed 91 to 100% homologous similarity to GenBank sequences. Their 16S rRNA sequences was closely related to 50% uncultured bacterium clones, 25% Firmicutes, 13% Proteobacteria and 12% Bacteroidetes sequences. Both culture-dependent and cultureindependent techniques were precise in the identification and description of bacterial community in rhizosphere. / Thesis (M.Sc) North-West University, Mafikeng Campus, 2014

Bt-maize is engineered to express insecticidal toxins derived from the bacterium Bacillus thuringiensis and has been shown to be very effective against pests like Busseola fusca. However, resistance of this pest against Bt-maize has developed and spread throughout South Africa. This study was inspired by the lack of knowledge over the microorganisms associated with the gut of these insects as they play a vital role in insect growth and development. Microbial-derived enzymes may have a role during an insect‟s adaption in different environmental conditions and to new diets. Previous studies suggest (1) that gut bacteria are required for B. thuringiensis-induced mortality in most Lepidoptera species and (2) that the toxicity of B. thuringiensis depends on microbial community interactions within the gut. The aim of this study was to determine the microbial diversity present in the midgut of B. fusca larvae occurring in maize. Busseola fusca larvae were collected from 30 sites throughout South Africa and dissected to collect their midgut contents. Serial dilutions were made of the contents and spread plated onto nutrient agar after which morphotypes were identified. One-hundred and five morphotypes were identified; DNA were extracted from the selected morphotypes and subjected to PCR analysis followed by secquencing. Sequencing results revealed the dominance of Enterococcus spp., specifically Enterococcus casseliflavus and Enterococcus gallinarum, Klebsiella spp., espesially Klebsiella pneumoniae and Klebsiella oxytoca and Bacillus spp. such as .B. thuringiensis and B. subtilis. Other organisms isolated, included Achromobacter spp., Brevudimonas spp., Caulobacter spp., Enterobacter spp., Halomonas spp., Ochrobactrum spp., Pantoea spp., Pseudomonas spp., Serratia spp., Stenotrophomonas spp., Arthrobacter spp., Brevibacterium spp., Leucobacter spp., Microbacterium spp., Planomicrobium spp. and Staphylococcus spp. The microbial diversity of larvae collected at the respective sampling sites were determined with the Shannon diversity index. The data were compared to several factors regarding the sampling sites. No significant differences were observed between the microbial diversities isolated at the respective sites. This may imply that the microbial community within B. fusca larvae are relative consistent throughout the maize production area. It is important to understand the distribution and structure of gut microbial communities within insects and whether the gut community is influenced by the geographical distribution of the insects. A better understanding of the distribution of the insects and community structure of their gut microbiota may aid in the development of better insect control strategies. / MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2015

The structure and function of microbial communities are critical to the maintenance and sustainability of terrestrial ecosystem processes. Consequently, there is substantial interest in assessing how microbial communities respond to various land management practices, and if alterations to the characteristics of microbial communities has the potential to disrupt ecosystem processes. This thesis was conducted to identify the long term effects of fertilisation and different levels of post-harvest organic matter removal on the characteristics of the FH litter and soil microbial communities in six, second rotation Pinus radiata plantation forests located around New Zealand. The six sites, established between 1986 and 1994, were sampled in 2002 and 2003. Various physical and chemical properties of the sites were measured, and litterfall production was determined. The microbial biomass in the FH litter layer and soil was determined by chloroform fumigation-extraction, and Biolog plates were used to assess the relative differences in microbial community diversity, based on patterns of substrate utilisation. Fertilisation substantially altered the physical and chemical properties of the forest floor, including FH litter moisture content, mass, carbon content, nitrogen content and carbon: nitrogen ratio and soil pH, nitrogen content and carbon: nitrogen ratio. The same range of FH litter and soil properties were also significantly changed by different levels of organic matter removal. The biomass and diversity of the FH litter and soil microbial communities were significantly altered by fertilisation and organic matter removal, and the differences in the microbial community characteristics were significantly correlated to the effects of the fertilisation and organic matter removal treatments on the physical and chemical environment in the majority of cases. The physical and chemical properties of the sites were significantly correlated to estimates of wood production, and it was also found that the characteristics of the microbial community were strongly related to productivity at several sites. The results demonstrated that fertilisation and organic matter removal regimes have had long term effects on the microbial communities at the sites. The persistence of the effects of the organic matter removal treatments were particularly noteworthy, as these treatments were applied at site establishment, and despite no subsequent reinforcement over the life of the trials, were still substantially influencing the physical, chemical and microbiological properties of the FH litter and soil up to 17 years later. The results of this thesis also emphasised the value of long-term experiments in assessing the effects of disturbance on the physical, chemical and microbiological characteristics of forest ecosystems. Further research into the specific nature of the relationship between site productivity and microbial community characteristics was suggested as an important focus for future studies.

The aim of this study was to optimize methane production by investigating hydrothermal pretreatment of sugarcane bagasse impregnated with acid (H2SO4) and alkaline H2O2 using substrate (g kg-1) -inoculum (g kg-1) ratio of 1:2. Batch reactors were realized under mesophilic conditions (37 °C). A central composite design (CCD) involving three factors; temperature (°C), time (min), and chemical compound concentration (H2O2 (%v/v) and H2SO4 (%w/v)) was utilized to optimize hydrothermal pretreatment. Thirty-two hydrothermal pretreatments were conducted according to CCD. H2O2 assisted hydrothermal pretreatment resulted in higher solid recovery (93.13%), higher percent glucan increase (139.52%), and lower lignin recovery (76.48%) in pretreated solid fraction in comparison to H2SO4 impregnated hydrothermal pretreatment. In the latter case, lower solid yield (12.27%), glucan recovery (187.01%) and higher lignin recovery (358.85%) was recorded. Higher COD solubilization (25.20 g L-1), lower total phenolic (content 658.13 ppm), higher sulfate (7240 mg L-1), furfural (925.77-2216.47 mg L-1) and 5-hydroxymehtylfurfural (70.95-970.08 mg L-1) were observed in liquid hydrolysate of H2SO4 assisted hydrothermal pretreatment. While lower COD solubilization (17.75 mg L-1), higher total phenolic content (3005.63 ppm), lower concentration of furfural (0-56.91 mg L-1) and 5-hydroxymethylfurfural (2.56-56.60 mg L-1) was recorded with H2O2 assisted hydrothermal pretreatment. Concerning methane production for H2O2 assisted conditions, 5.59 Nmmol g-1 TVS (2% H2O2) to 13.49 Nmmol g-1 TVS (6% H2O2) was recorded. However, pretreatment with 7.36% H2O2 resulted in 14.43 Nmmol g-1 TVS, which was 118.16% higher comparing to untreated sugarcane bagasse (6.60 Nmmol g-1 TVS). Methanogenic inhibition was recorded for most of the H2SO4.pretreated reactor (1 – 3 %w/v H2SO4). Minimum CH4 production observed was 0.58 Nmmol g-1 TVS in pretreatment O-HSO (2% w/v H2SO4). Acetic acid was the predominant volatile fatty acid observed in digestion process of H2O2 treated batch reactor however was not recorded in H2SO4 treated batch reactors. Microbial community analysis indicated the prevalence of unclassified AUTHM297, Clostridium, and Treponema related genera in H2O2 treated reactors. Genera related aromatic compound degradation were identified and abundant in H2SO4 treated reactors. Methanolinea, Methanobacterium, and Methanosaeta were abundant methanogens in both pretreatments. Hydrogen peroxide assisted hydrothermal pretreatment was verified as a better choice for methane production comparing to sulfuric acid assisted hydrothermal pretreatment primarily on account of higher lignin solubilization, higher glucan recovery, and lower furanic compounds production. / O objetivo deste estudo foi otimizar a produção de metano investigando as condições do pré-tratamento hidrotérmico assistido do bagaço da cana de açúcar sob impregnação de ácido (H2SO4) e álcali (H2O2) utilizando-se a razão substrato (g kg-1) inóculo (g kg-1) de 1:2. Os reatores em batelada foram mantidos em condições mesofílicas (37 ºC). Para otimizar as condições de pré-tratamento hidrotérmico, o design de composto central rotacional (DCCR) foi realizado utilizando três fatores: temperatura (ºC), tempo (min) e concentração do composto químico (H2O2 (%v/v) e H2SO4 (% p/v)). Trinta e dois pré-tratamentos hidrotérmicos foram realizados de acordo com a concepção do DCCR. O pré-tratamento hidrotérmico assistido do bagaço, com H2O2 resultou em maior recuperação de sólidos (93,13%), elevado percentual de glicana (139,52%) e menor recuperação de lignina (76,48%) da fração sólida pré-tratada, se comparada aquele com H2SO4. Nesse último caso, observou-se menor rendimento de sólidos (12,27%) e glucanas (187,01%) e maior recuperação de lignina (358,85%). No líquido hidrolisado do pré-tratamento hidrotérmico assistido do bagaço com H2SO4 foi observada elevada solubilização de DQO (25,20 g L-1), menor teor de fenóis totais (658,13 mg L-1), elevado sulfato (7240 mg L-1), furfural (925,77 - 2216,47 mg L-1 e 5-hidroximetilfurfural (70,95 - 970,08 mg L-1). Enquanto, foi registrado menor solubilização de DQO (17,27 g L-1), maior teor de fenóis totais (3005,63 ppm), e menor concentração de furfural (0 - 56.91 mg L-1), 5-hidroximetilfurfural (2,56 - 50,60 mg L--1 com H2O2. Em relação ao a produção de metano nas condições com H2O2, observou-se 5.59 Nmmol g-1 STV (2%v/v H2O2) a 13.49 Nmmol g-1 STV (6%v/v H2O2). No tratamento com 7.36% de H2O2 observou-se 14,43 Nmmol g-1 STV que foi 118.16% maior se comparado com o bagaço não-tratado (6,60 Nmmol g-1 STV). Inibição metanogênica foi observada na maioria dos reatores pré-tratados com H2SO4 (1 – 3% p/v), e a produção mínima observada foi de 0.58 Nmmol g-1 TVS no pré-tratamento com 2% p/v de H2SO4. Ácido acético foi o principal ácido orgânico volátil observado somente no reatores por tratamento de H2O2. Por meio da A análise da comunidade microbiana, para o domínio Bacteria, foi observado prevalência dos gêneros AUTHM297, Clostridium e Treponema nos reatores cujo substrato foi pré-tratado com H2O2. Gêneros relacionados à degradação de compostos aromáticos foram identificados e estiveram em maior abundância nos reatores cujo substrato foi pré-tratado com H2SO4. Methanolinea, Methanobacterium, e Methanosaeta foram os microrganismos do domínio Archaea mais abundantes e identificados em ambos os pré-tratamentos. O pré-tratamento hidrotérmico assistido com H2O2 foi a melhor opção em relação ao H2SO4, devido a maior solubilização de lignina, maior recuperação de glucano e baixa produção de compostos furânicos.

Anaerobic membrane bioreactor (AnMBR) serves as a more sustainable form of wastewater treatment. However, biofouling is particularly detrimental to the performanceof AnMBRs. This dissertation focuses on understanding more about the biofouling in nMBR, and to devise strategies to control or make use of these biofoulant layers. First, we aim to investigate the microbial community structure of sludge and biofilm from 13 different AnMBRs. Our findings indicate 20 sludge core genera and 12 biofilm core genera (occurrence ≥ 90% samples) could potentially account for the AnMBR performance. Sloan neutral model analysis indicates the anaerobic microbial consortium between sludge and biofilm is largely affected by stochastic dispersal and migration processes (i.e., neutral assembly), suggesting that the majority of these core genera are not selectively enriched for biofilm formation. Therefore, the second part of this dissertation aims to minimize the growth of the overall bacterial cells attached on the membranes. For this, membranes embedded with zinc oxide (ZnO) and copper oxide (CuO) nanoparticles were examined for their antifouling efficacies. Our findings indicate both CuO and ZnO nanoparticles embedded membranes could delay biofouling formation without significantly triggering the overall expression/abundance of antibiotic resistance genes (ARGs) and metal resistance genes (MRGs) in biofilm. Furthermore, CuO and ZnO nanoparticles could inhibit the expression of quorum sensing associated genes, resulting in lower quorum sensing signal molecules production. Despite the positive results demonstrated from this study as well as those from others, we recognize that no control strategies are likely to achieve total prevention of anaerobic biofouling. Therefore, the last part of this dissertation focuses on exploring the effects of different foulant layers on antibiotic-resistant bacteria (ARB) and ARGs removal. Our findings suggest both ARB and ARGs could be absorbed by membrane foulant. Transmembrane pressures and the foulant layer synergistically affected ARB removal, but the foulant layer is the main factor that contributed to ARG removal through adsorption. Overall, the collective findings could bring new insights to the anaerobic membrane biofouling phenomenon, and offer pragmatic approaches to minimize biofouling without compromising the post-AnMBR effluent quality.

Woody plant encroachment is a globally-prevalent vegetation change phenomenon that has shifted grass-dominated ecosystems to mixed grass and woody plant matrices over the last century. In the Rio Grande Plains of Texas, the introduction of N-fixing woody legumes has increased above- and belowground primary productivity and changed the litter chemistry of the system, accelerating rates of belowground biogeochemical processes. The purpose of this study was to assess the impact of grassland to woodland transition on i) P concentrations in soil physical fractions that differ in their organic matter turnover rates, ii) P availability within the soil over the course of woody encroachment and across the landscape, and iii) microbial community composition and diversity. Soil samples were collected in remnant grasslands and four woody landscape elements (clusters, groves, drainage woodlands, and playas) along a 135-yr chronosequence of woody plant encroachment. P was fractionated by the Hedley method and P concentrations were determined by alkaline oxidation and lithium fusion coupled with ascorbic acid colorimetry. Bacterial and fungal communities were characterized by molecular methods. Whole soil P concentrations were 2-5X greater in woody landscape elements than in grasslands, and nutrient concentrations increased linearly with time following woody plant invasion in all but the slowest-cycling physical fractions. Plant-available P and organic P increased dramatically with time following encroachment. Changes in P availability were more pronounced in drainages and playas than in upland clusters and groves. Analysis of the bacterial and fungal communities demonstrated that microbial communities in grasslands differ at both phylum and genus level from the flora of the wooded landscape elements. This study demonstrates that woody encroachment strongly influences the distribution and availability of soil P and indicates that nutrient cycles in the soil are closely linked and similarly affected by increased woody plant abundance. Microbial communities under woody species differ in composition from those of the grasslands, and are likely contributing to the observed changes in nutrient availability. Since N and P are generally the most limiting nutrients in terrestrial ecosystems, increased stores of P are likely to alter rates of microbial processes, plant-microbe and plant-plant interactions, and successional dynamics in this ecosystem and similar landscapes around the world.

Northern peatlands are large repositories of carbon and little is known about the effect the microbial community has on carbon mineralization rates, and there is concern that a loss of microbial diversity due to environmental change may lead to reduced ecosystem functioning. Microbial communities vary among peatland types and abiotic variables such as temperature and pH have a large influence on carbon dioxide production, but distinguishing between abiotic controls and the role of microbial community structure has proved challenging. Microbial activity and community composition was characterized in three peatlands within the James Bay Lowlands, Ontario. Similar dominant microbial taxa were observed at all three peatlands despite differences in nutrient content and substrate quality and geographic location. In contrast, microbial activity differed among the sites, indicating that it is influenced by the quality of the peat substrate and the presence of microbial inhibitors. A series of reciprocal field and laboratory transplant experiments were conducted at a rich and poor fen near White River, Ontario to more explicitly distinguish between the abiotic and microbial controls on carbon mineralization. The effect of transplantation differed between the laboratory and field studies and when viewed individually could lead to different interpretations of the effect of substrate change. Surprisingly, intensive sampling within both fens was unable to reveal a difference between the rich and poor fen microbial community due to high within site temporal and spatial variation. Thus studies with small sampling effort will have a very incomplete understanding of microbial community structure and thus microbial ecology. A reciprocal sterilization transplant experiment was also conducted to examine how different microbial communities adapted to various peat substrates influenced C-mineralization patterns. Post-inoculation/incubation bacterial communities across peatlands converged towards a similar community structure, suggesting that abiotic variables are the dominant control on peatland microbial activity and community composition. The studies presented in this thesis collectively show that across a broad range of temperate and sub-arctic peatland types dominant members of the microbial community are generally similar, and decomposition rates can be predicted by broader controlling environmental factors rather than temporal niche or distributional constraints of the microbial community.

Northern peatlands are large repositories of carbon and little is known about the effect the microbial community has on carbon mineralization rates, and there is concern that a loss of microbial diversity due to environmental change may lead to reduced ecosystem functioning. Microbial communities vary among peatland types and abiotic variables such as temperature and pH have a large influence on carbon dioxide production, but distinguishing between abiotic controls and the role of microbial community structure has proved challenging. Microbial activity and community composition was characterized in three peatlands within the James Bay Lowlands, Ontario. Similar dominant microbial taxa were observed at all three peatlands despite differences in nutrient content and substrate quality and geographic location. In contrast, microbial activity differed among the sites, indicating that it is influenced by the quality of the peat substrate and the presence of microbial inhibitors. A series of reciprocal field and laboratory transplant experiments were conducted at a rich and poor fen near White River, Ontario to more explicitly distinguish between the abiotic and microbial controls on carbon mineralization. The effect of transplantation differed between the laboratory and field studies and when viewed individually could lead to different interpretations of the effect of substrate change. Surprisingly, intensive sampling within both fens was unable to reveal a difference between the rich and poor fen microbial community due to high within site temporal and spatial variation. Thus studies with small sampling effort will have a very incomplete understanding of microbial community structure and thus microbial ecology. A reciprocal sterilization transplant experiment was also conducted to examine how different microbial communities adapted to various peat substrates influenced C-mineralization patterns. Post-inoculation/incubation bacterial communities across peatlands converged towards a similar community structure, suggesting that abiotic variables are the dominant control on peatland microbial activity and community composition. The studies presented in this thesis collectively show that across a broad range of temperate and sub-arctic peatland types dominant members of the microbial community are generally similar, and decomposition rates can be predicted by broader controlling environmental factors rather than temporal niche or distributional constraints of the microbial community.