No description available.

Stichelton is a blue-veined raw milk cheese which is made following the Stilton cheese making process. In a previous study, a preliminary examination of its microflora during production was examined by traditional culture methods and initial PCR DGGE profiling. The aim of this study was to complete the profiling of Stichelton cheese and examine the contribution of its microbiota components to product characteristics. Stored samples of cheese production isolates were sequenced and in addition whole population DNA was extracted directly from a fully ripened Stichelton cheese (12 weeks) and from bulk cell suspensions collected on various media. The V3, V4V5 and V6V8 regions of 16S rDNA were amplified by PCR and separated by DGGE using 20 – 80 % urea formamide denaturing gradient. While Lactobacillus casei/paracasei, Staphylococcus equorum, Bacillus sp., Brevibacterium sp., Halomonas sp., Acinetobacter sp., Alkalibacterium sp. and Corynebacterium casei were only found by the molecular method, traditional culture detected a large number of potentially raw milk microbiota. Lactococcus lactis was detected in the raw milk sample and along the process by both methods. The L. lactis subsp. lactis which was detected in the core of matured Stichelton was shown by PFGE to be from the raw milk and not the starter culture used in Stichelton production. S. equorum was found in the crust of cheese pre-piercing and in all parts of the cheese post-piercing by the molecular approach only. This suggested this organism was introduced originally via handling. Five S. equorum isolated from Stilton, Danish Blue and Reblochon could grow up to 10% salt but did not tolerate low pH levels suggesting S. equorum in Stichelton must have been introduced by handling before or during ripening as if it was present in the early fermentation then it would die as fermentation progressed due to pH sensitivity. A model cheese system made with commercial UHT milk was used to examine the interaction between mixed Lc. lactis, P. roqueforti and S. equorum B2 isolated from the Stilton crust. S. equorum was either added 1.5 h after the addition of L. lactis or it was smeared on the surface of the cheese immediately after un-moulding. The viable counts and pH were analysed throughout the process, while texture, water activity and flavour volatiles using GCMS SPME were determined for one month ripened cheeses only. The results showed S. equorum survived in the cheese following either method of introduction and that in cheeses without P. roqueforti addition, the presence of incorporated and surface-spread S. equorum could inhibit the surface growth of a contaminant Penicillium. It also slowed the growth of starter P. roqueforti in cheeses made with this mould. A paler coloured crust, firmer textured cheese and a low amount of alcohols were shown in the model cheeses made with surface-smear S. equorum. Conversely, addition of S. equorum in the initial process made the cheese core softer and produced low amounts of acids. Ethanol, 3-methyl-1-butanol and 2-pentanone were the main flavour compounds in the model cheeses examined. The antifungal activity of the isolate was confirmed in laboratory media. Its ability to prevent Penicillium surface growth could be beneficial for white cheeses where this is an undesirable flaw. The results showed that the sporulation inhibitory effect on P. roqueforti was because of an antifungal agent produced by S. equorum, but it was not acid, bacteriocin or H2O2. Further study is needed to detect the antifungal agent. Overall, the study has expanded the understanding of the role non-starter bacteria may have in contributing to cheese ripening.

Exposure of artificial surfaces such as ship hulls to a marine environment leads to the attachment of assorted biomolecules, single celled organisms and marine invertebrates such as barnacles or mussels. Together, they form a structure known as a biofilm. These films lead to higher fuel consumption and add considerable expense to the operation of ships used by industrial and naval organisations. The work presented in this thesis describes the surface analysis of a novel poly(dimethylsiloxane) (PDMS) based foul-release coating. The coating also contains poly(ethylene glycol) groups (PEG). The differing chemical properties between these two domains led to an observed surface modification effect in water, whereby contact angle measurements decreased from ~110o to ~65 o over a period of five minutes. This effect was rapidly reversible on drying. Time of Flight-Secondary Ion Mass Spectrometry cryogenic depth profiling experiments confirmed this change in surface chemistry where the frozen surface of the coating was shown to have a higher intensity of ions associated with PEG groups at the surface compared to that in the bulk. Water immersion also led to a swelling of the surface seen by a change in the surface topography by Atomic Force Microscopy investigations. When applied to glass surfaces the coatings were flat and generally defect free regardless of the application method used. On exposure to Pseudomonas aeruginosa the coatings were found to be ten times more effective at preventing bacterial adhesion in the first instance than a PDMS standard. The mechanism of action was shown to be non-toxic by live/dead staining and did not appear to affect the way in which bacteria move on a surface. A flow adhesion assay demonstrated that a flow rate of almost two orders of magnitude lower was required to remove fifty percent of bacteria from a coated surface than on a glass standard, demonstrating the foul-release ability of the switching coating. Sea trials in a French coastal region highlighted the importance of exposing candidate coatings to a true marine environment for a suitable duration in order to determine their potential for use. Ultimately we show that the coating presented is a candidate for use as an effective coating for preventing marine biofouling and surface analysis was deemed to be an appropriate methodology to analyse coatings that have changing properties on exposure to water.

There is an increasing need to produce fuels and chemical commodities from renewable resources. Current efforts have been mainly focused on liberating sugars from plant-derived lignocellulosic feedstocks. However, lignocellulosic materials have naturally evolved to resist their microbial and enzymatic degradation and this poses a major problem. Due to these difficulties, alternative feedstocks derived from biomass gasification have recently become a major focus of research. The gasification process generates mixtures of hydrogen, carbon monoxide and carbon dioxide, known as syngas, which can be utilised by some autotrophic bacteria as the sole sources of carbon and energy. Cupriavidus necator strain H16 was chosen as a chassis organism for the current investigation as it can grow to high cell densities on CO2/H2 and, under nutrient limiting conditions, stockpiles huge amounts poly[R-(–)-3-hydroxybutyrate] (PHB). The long term aim of the study was to employ metabolic engineering approaches to re-direct carbon flux so that desirable chemicals are produced instead of PHB. Following the establishment of defined growth media, genetic tools, and DNA delivery methods, the natural resistance of the bacterium to a range of desirable target chemicals was tested and 3-hydroxypropanoic acid (3-HP) identified as a suitable target. However, it was noted that C. necator was able to utilise this compound as the sole source of carbon and energy. Hence, several genes involved in the degradation of 3-HP were identified and inactivated through ORF deletion, resulting in strain CNCA13 unable to grow on this compound. However, this strain was still able to co metabolise 3-HP alongside other carbon sources such as fructose or gluconate, necessitating further investigation, including the introduction of additional gene deletions. Some of these deletions belonged to genes or pathways involved in a reductive route for the assimilation of the compound. The inactivation of one of these candidates over the strain CNCA13 led to prevent the co-assimilation of 3-HP alongside fructose. Following strain development, a heterologous pathway designed to produce 3-HP from actyl-CoA in two enzymatic steps was introduced into the organism. The first committed step in this pathway is the carboxylation of acetyl-CoA to malonyl-CoA, catalysed by the enzyme acetyl-CoA carboxylase (ACC). The second step is the reduction of malonyl-CoA to 3-HP, a conversion catalysed by the bifunctional enzyme malonyl-CoA reductase (MCR) or, in some archaea, by the combination of two monofunctional reductases. Genes encoding ACC subunits and MCRs from different bacteria and archaea were codon-optimised, assembled into functional operons and screened for efficient expression in C. necator H16. All genes were found to be expressed, but production of 3-HP could not be observed, even in strains lacking the ability to produce PHB or to consume 3-HP as the sole source of carbon. Thus, further work is needed to efficiently redirect carbon flux through the generated pathway.

No description available.

Floating sulphur biofilms have been observed to occur on sulphate-containing natural systems and waste stabilization ponds. It has been postulated that these biofilms form on the surface of the water because sulphate reducing bacteria present in the bottom layers of the water body reduce sulphate to sulphide which then diffuses upwards and is oxidized under the correct redox conditions to sulphur by sulphide oxidizing bacteria. Very little information exists on these complex floating systems and in order to study them further, model systems were designed. The Baffle Reactor was successfully used to cultivate floating sulphur biofilms. Conditions within the reactor could be closely scrutinized in the laboratory and it was found that sulphate levels decreased, sulphide levels increased and that sulphur was produced over a period of 2 weeks. The success of this system led to it being scaled-up and currently a method to harvest sulphur from the biofilm is under development. It is thought that biofilms are highly complex, heterogeneous structures with different bacteria distributed in different layers. Preliminary work suggested that bacteria were differentially distributed along nutrient and oxygen gradients within the biofilm. Biofilms are very thin structures and therefore difficult to study and Gradient systems were developed in an attempt to spatially separate the biofilm species into functional layers. Gradient Tubes were designed; these provided a gradient of high-sulphide, low oxygen conditions to high-oxygen, low-sulphide conditions. Bacteria were observed to grow in different layers of these systems. The Gradient Tubes could be sectioned and the chemical characteristics of each section as well as the species present could be determined. Silicon Tubular Bioreactors were also developed and these were very efficient at producing large amounts of sulphur under strictly controlled redox conditions. Microscopy and molecular methods including the amplification of a section of Ribosomal Ribonucleic acid by Polymerase Chain Reaction were used in an attempt to characterize the populations present in these biofilm systems. Denaturing Gradient Gel Electrophoresis was used to create band profiles of the populations; individual bands were excised from the gels and sequenced. Identified species included Ectothiorhodospira sp., Dethiosulfovibrio russensis, Pseudomonas geniculata, Thiobacillus baregensis and Halothiobacillus kellyi.

No description available.

Biofertilizers are broths containing beneficial microorganisms that are applied to soils to enhance crop production and soil fertility. The microbes in a biofertilizer enhance and drive natural processes such as nutrient transformation and cycling, organic matter decomposition, and gas emission. Environoc 401 manufactured by Biodyne-USA is described as an agricultural soil enhancer that is comprised of a consortium of beneficial microorganisms. Production of Environoc 401 is achieved by an incubation that begins with a concentrated lyophilized microbial consortium. The focus of this study is to try to understand the community dynamics that occur during the incubation process to help predict the proportions of individual strains and the overall metabolic activity of the microbial community in Environoc 401 under different conditions. In order to quantify individual strains in Environoc 401, species-specific primers were developed for use in quantitative-PCR. These primers were then used to quantify target strains in Environoc 401 broth stored at 22 °C and 27 °C for 1 month and sampled at time 0, 1 week, and 1 month to evaluate the effect of storage conditions on the microbial community. In general, Environoc 401 stored at 22 °C had greater substrate utilization richness compared to Environoc 401 stored at 27 °C, but only after 1 month. The microbial community within Environoc 401 stored at 27 °C after 1 month did not utilize any amines or phenolic compounds, while the communities stored at 22 °C did use these substrates. To evaluate the overall effect of Environoc 401 on plants and on the microbial activity in potting medium, the product was used in the potting soil of soybean plants grown in an environmental growth chamber. This study contained five treatments upon unifoliate emergence: a no treatment control, pesticide and chemical fertilizer, pesticide and biofertilizer (as Environoc 401), biofertilizer only, and chemical fertilizer only. Soil medium samples were collected from each treatment at the time of seed planting, 24 hrs before application, 24 hrs after application, 2 weeks after application, and 1 month after application. The soybean plants treated with Environoc 401 generally had the highest average total plant height, average number of leaves, average dry weight of leaves, stems, and roots, and the least acidic pH. Samples from both studies were also used to inoculate Biolog EcoPlates to assess changes in carbon-source utilization patterns for each condition and to generate Community-Level Physiological Profiles (CLPPs). Principle Component Analysis was performed on the CLPPs and diversity was also assessed using Shannon’s diversity indices for samples from both studies. The CLPPs for the storage samples clustered tightly after 1 week of storage, however, after 1 month of storage the two temperatures diverged greatly. The CLPPS for the soybean plant treatment samples clustered tightly 24 hours prior to treatment but varied greatly after treatment application. <a>These results indicate that treatment application, storage time, and temperature affect carbon utilization within the microbial communities. These results are a reflection on the activity and health of the microbial community and future studies should explore changes taking place on a finer scale by targeting specific carbon sources or conditions.</a>

Antibiotic use in agricultural ecosystems has the potential to increase resistance to antibiotics in soil microbial communities since 40-95% of an antibiotic dose administered to livestock is excreted intact or as metabolites. Exposure to antibiotics is also known to alter microbial community composition, biomass, and physiology, but the potential influences of antibiotic residues on the essential ecosystem processes that microbes regulate, e.g., carbon and nitrogen cycling are not well understood. I investigated the effects of antibiotic residues associated with dairy cattle operations on soil microbial communities and the ecosystem processes they regulate. I examined the effects of antibiotic exposure on the biogeochemical functioning of soil microbial communities by measuring the activity of extracellular enzymes associated with organic matter processing and nutrient mineralization in soils collected from dairy cattle operations across the United States. At each experimental station paired sites were identified by local managers that represented sites with high and low stocking rates of dairy cows who had been treated prophylactically with antibiotics to prevent mastitis. Responses varied among individual enzymes, but I found an overall significant decrease in total hydrolytic enzyme activity under high cattle stocking rates indicating a change in the functioning of the microbial community in soils exposed to antibiotic laden manure. Principle components analysis suggest that while some of the variation in enzyme activities are associated with the abundance of antibiotic resistance genes, soil organic matter (total organic, mineralizable, and particulate organic carbon) was the most significant variable accounting for differences in enzyme activities. This reflects an inherent challenge in studies of antibiotic exposure in agricultural landscapes: the difficulty of distinguishing direct effects of antibiotic residues from the organic matter and nutrient subsidy associated with manure applications. To address this concern I conducted a series of incubation experiments manipulating soils to isolate the influences of antibiotics, manure resource subsidies, and bovine microbiome inoculants into soils. Specifically, I examined soil respiration and antibiotic resistance gene counts using qPCR following treatment with cephapirin, pirilimycin and a positive and negative control. I found that pre-exposure to antibiotics and manure is important in modulating the response of microbial communities (soil respiration, and gene copy numbers of AmpC and TetO) to further antibiotic exposure. I conclude that antibiotics themselves have a direct effect on soil communities and their functioning that is additive to the effect of manure (i.e., as a resource subsidy). This effect is mediated by the history of previous exposure to antibiotics, i.e., cattle stocking density. These results suggest that antibiotic residues from dairy cattle operation may have significant effects on microbial communities and the biogeochemical cycling they regulate in agricultural ecosystems. / Master of Science

Every year, about 550,000 patients receive medical attention for minor and major burns in the United States.1 In 2020, it was estimated that 11 million people worldwide suffered from burn injuries, with 150,000 of those burns being fatal.2 Burns are among the most painful and debilitating recalcitrant wounds that can often turn terminal when infection occurs. The different grades for burns that we aim to treat are first, second, and third degrees.2 Each burn type is susceptible to secondary infection that can be life threatening, and as a result, are extensively treated with antimicrobial agents.2 At present, only a handful of FDA-approved products are available in the market that can successfully treat second and third degree burn wounds and scars.3 Topical agents such as sodium hypochlorite, iodine, H2O2, silver etc. are used to combat burn wound infections.3 However, the relentless emergence of antibiotic resistant strains of pathogens, often with multiple antibiotic resistances together with the discovery of novel antibiotics, has necessitated investigating and developing better alternative treatments. In this effort, a cost-effective approach to engineer a microbe-resistant bandage system utilizing clay was undertaken as the research project. This unique microbe-resistant material has been developed using organo-modification and metal-ion exchanged clay scaffolds, and has been fully characterized using analytical techniques such as powder XRD, ATR-FTIR, XPS, ICP-OES etc. The hybrid clay samples have also been tested for their antimicrobial efficacy against Escherichia coli (gram-negative) and Staphylococcus aureus (gram-positive) bacteria in promoting the process of wound healing to serve as a representative of the ESKAPE group of bacteria, which includes Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species. By leveraging the excellent hydrophilic and moisture retention properties of clay, we can postulate achieving optimal moisture transport from the dressing through the wound area to accelerate the healing cascade by hassle-free self-application at the point of injury.4 The proposed research deals with the antimicrobial effects associated with metal cations within a clay matrix that can be used for the treatment of burn injuries and scars. Currently, most burn treatments involve high doses of silver-based products that make them costly.3 Moreover, most of these treatments are ointment-based, which exposes the wounds to cross-contamination when in contact with dust, debris, moisture, water, liquids, particulates etc. The goal is to develop a free-standing film composed of controlled amounts of silver ions tethered to the clay scaffolds that can be used to treat severe burns and scars. Performing animal studies using in vivo models for first or second degree burn injuries and scars exceeded the budget for this project, hence, antimicrobial efficacy against ESKAPE pathogens, viz. gram-negative and gram-positive bacteria using the engineered hybrid films was the focal point for this project. This unique and cost-effective system is much cheaper compared to ointments and other bandage systems. Moreover, the meso- and micro-porosities present within the clay can be easily leveraged for easy moisture and oxygen transport from bandage to skin, which is essential for natural healing of the wounds and burn injuries.4 Additionally, the antimicrobial/antibacterial efficacy of this unique bandage system can be suitable for prolonged use, thereby minimizing the inconveniences of frequent changing and reapplication. This helps to reduce the risk of infection and contamination, drastically. Clay has the well-known property of retaining moisture and has been used as a promoter for hemostasis, thereby, helping the composite films to serve multiple purposes in the burn and scar healing process.8 The hydroxyl groups in the clay used will be functionalized with trimethyl glycine (Betaine), expanding the clay galleries through intercalation, while Group II metal ions and silver ions can be easily exchanged with the sodium cations present in clay within the interstitial space. The metal ions (Ag+) exchanged organo-clay gallery is the main driving force for eliminating the microbes or bacteria. Thus, one of the prime goals for this effort is to develop an organo-modified Betaine-composite film that can be conformable to various shapes and sizes and will garner anti-microbial/ bacterial/ fungal properties. Other future goals include developing films with optimal metal ion concentrations in the clay scaffolds to reduce the cost (by replacing Ag+ ions with group II metal ions in the silicate scaffolds) without compromising the efficacy of the product. This research exhibits a novel, cost-effective solution to engineer microbe-resistant “hybrid” clay membranes by chemical modification, metal incorporation, intercalation, and exfoliation of clay-silicate galleries to prevent infections from ESKAPE pathogens. Results from the physico-chemical analyses have shown mechanical durability of the films. Antimicrobial efficacy tests using Escherichia coli (gram-negative) and Staphylococcus aureus (gram-positive) showed a significant reduction in bacterial growth, which indicates the antimicrobial efficacy of the clay films. In typical bacterial kill study experiments, the zone of inhibition was at or above 1 cm for both the gram-positive and gram-negative bacteria, with four samples tested with three 0.6 cm diameter discs against a clay control. Evidently, these matrices are effective at preventing the growth of bacteria that can prove to be infectious. This unique “hybrid” bandage system promotes: (a) prevention and control of both gram-negative and gram-positive bacteria, (b) nontoxic and biodegradable features, (and c) easy application on wounds. Beyond the antimicrobial efficacy, physical tests have been used to analyze the resulting clay films. X-Ray photoelectron spectroscopy is used to determine the quantitative elemental analysis, and binding energies and oxidation states of the elements. Powder X-Ray diffraction, ATR-FTIR, X-Ray fluorescence spectroscopy and viscosity have been used to determine physical properties, structures, and mechanical durability of the films.