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published_or_final_version / abstract / Ecology and Biodiversity / Doctoral / Doctor of Philosophy

Spores of thermophilic bacilli are a common concern during the manufacture of milk powder. Spores are believed to occur in high numbers in milk powder due to their ability to survive pasteurisation, attach to stainless steel surfaces, germinate, grow as biofilms and subsequently enter the product stream and thereby contaminate the final product. In this study, thirty one thermophilic bacilli isolates were obtained from a New Zealand milk powder production line and identified as either Anoxybacillus flavithermus or Geobacillus spp. using random amplified polymorphic DNA (RAPD) and species-specific PCR. Sporulation media and a polyethylene glycol two-phase separation system were modified to produce high yields of spores free from debris. The spores of four Geobacillus spp. isolates (CGT-8, D4, E7 and E11) were characterised in terms of structure (electron microscopy), surface charge (zeta potential), hydrophobicity (contact angle and microbial adhesion to hexadecane) and attenuated total reflectance infrared spectroscopy (ATR-IR). Spores from three of the four isolates possessed an exosporium while the fourth did not. However the integrity of the exosporium varied over time. The spores were negatively charged (-10 to -20 mV) at neutral pH and high ionic strength (0.1 M KC1). Both hydrophobicity assays revealed that the spores of the four isolates were relatively hydrophilic while ATR-IR revealed the spores' surfaces consisted of protein and polysaccharides. The influence of these spore characteristics on adhesion to a variety of substrata under high flow rates was examined using the extended Derjaguin, Landau, Verwey and Overbeek (XDLVO) theory. Spores generally attached in higher numbers to hydrophobic surfaces compared to hydrophilic surfaces, however this observation was more prevalent for isolate D4. This result indicated that a single mechanism could not describe the adhesion of spores from different strains. A series of glass surfaces with modified characteristics were produced in order to test the antifouling properties on the adhesion of D4 spores. Spores suspended in a high ionic strength medium (0.1 M KC1) attached in greater numbers (1 Log₁₀ CFU cm⁻�) to positively charged and hydrophobic surfaces compared with negatively charged and hydrophilic surfaces. A clean in place (CIP) procedure, reduced spore numbers on hydrophobic and hydrophilic surfaces by 1.5 and by 2.0 Log₁₀ CFU cm⁻�, respectively. When spores were suspended in milk, there was little difference in the number of spores attaching to the different surfaces (ie. 3.5 to 3.8 Log₁₀ CFU cm⁻�), and spore removal from surfaces via a CIP regime was unchanged (1.5 to 2.0 Log₁₀ CFU cm⁻� reduction) compared with spores that attached in simple 1:1 electrolyte media. The effects of a caustic wash on spore surface characteristics and adhesion was determined. There was a significant reduction in spore viability (2 Log₁₀ CFU mL⁻�) after a 30 min caustic wash at 65 �C in the current study, however surviving spores displayed a greater propensity to attach to stainless steel. Surface characterisation results revealed an increase in hydrophobicity and a greater negative charge on the spores' surface after treatment with NaOH. Surviving spores could potentially recontaminate sections of the plant which are cleaned with this recycled caustic wash solution, thereby seeding surfaces with spores at the beginning of the next processing run. In conclusion, while surfaces that reduce spore adhesion and enhance removal can be produced, exposure to complex solutions such as milk can reduce the anti-fouling effectiveness of such surfaces to spore adhesion.

Spores of thermophilic bacilli are a common concern during the manufacture of milk powder. Spores are believed to occur in high numbers in milk powder due to their ability to survive pasteurisation, attach to stainless steel surfaces, germinate, grow as biofilms and subsequently enter the product stream and thereby contaminate the final product. In this study, thirty one thermophilic bacilli isolates were obtained from a New Zealand milk powder production line and identified as either Anoxybacillus flavithermus or Geobacillus spp. using random amplified polymorphic DNA (RAPD) and species-specific PCR. Sporulation media and a polyethylene glycol two-phase separation system were modified to produce high yields of spores free from debris. The spores of four Geobacillus spp. isolates (CGT-8, D4, E7 and E11) were characterised in terms of structure (electron microscopy), surface charge (zeta potential), hydrophobicity (contact angle and microbial adhesion to hexadecane) and attenuated total reflectance infrared spectroscopy (ATR-IR). Spores from three of the four isolates possessed an exosporium while the fourth did not. However the integrity of the exosporium varied over time. The spores were negatively charged (-10 to -20 mV) at neutral pH and high ionic strength (0.1 M KC1). Both hydrophobicity assays revealed that the spores of the four isolates were relatively hydrophilic while ATR-IR revealed the spores' surfaces consisted of protein and polysaccharides. The influence of these spore characteristics on adhesion to a variety of substrata under high flow rates was examined using the extended Derjaguin, Landau, Verwey and Overbeek (XDLVO) theory. Spores generally attached in higher numbers to hydrophobic surfaces compared to hydrophilic surfaces, however this observation was more prevalent for isolate D4. This result indicated that a single mechanism could not describe the adhesion of spores from different strains. A series of glass surfaces with modified characteristics were produced in order to test the antifouling properties on the adhesion of D4 spores. Spores suspended in a high ionic strength medium (0.1 M KC1) attached in greater numbers (1 Log₁₀ CFU cm⁻�) to positively charged and hydrophobic surfaces compared with negatively charged and hydrophilic surfaces. A clean in place (CIP) procedure, reduced spore numbers on hydrophobic and hydrophilic surfaces by 1.5 and by 2.0 Log₁₀ CFU cm⁻�, respectively. When spores were suspended in milk, there was little difference in the number of spores attaching to the different surfaces (ie. 3.5 to 3.8 Log₁₀ CFU cm⁻�), and spore removal from surfaces via a CIP regime was unchanged (1.5 to 2.0 Log₁₀ CFU cm⁻� reduction) compared with spores that attached in simple 1:1 electrolyte media. The effects of a caustic wash on spore surface characteristics and adhesion was determined. There was a significant reduction in spore viability (2 Log₁₀ CFU mL⁻�) after a 30 min caustic wash at 65 �C in the current study, however surviving spores displayed a greater propensity to attach to stainless steel. Surface characterisation results revealed an increase in hydrophobicity and a greater negative charge on the spores' surface after treatment with NaOH. Surviving spores could potentially recontaminate sections of the plant which are cleaned with this recycled caustic wash solution, thereby seeding surfaces with spores at the beginning of the next processing run. In conclusion, while surfaces that reduce spore adhesion and enhance removal can be produced, exposure to complex solutions such as milk can reduce the anti-fouling effectiveness of such surfaces to spore adhesion.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniversitet, 2005. / Härtill 5 uppsatser.

Thesis submitted in fulfilment of Master's Degree in Technology: Biotechnology, Durban Institute of Technology, 2004. / M

This diploma thesis studies the utilization of whey using thermophilic bacteria of the genus Thermus and Geobacillus. The whey stripped off proteins was used as a cultivation medium during experiments. The cultivation took place in the Erlenmeyer flasks, to assess the optimal conditions for microrganism’s growth. During the cultivation in the bioreactor, growth curves were established. The amount of whey utilization was assessed by analytical methods that determine the concentration of reducing saccharides (lactose) and chemical oxygen demand (COD) while studying the optimal conditions and a method determining the concentration of reducing saccharides during growth curves analysis.

This diploma thesis deals with selected thermophilic representatives of genera Geobacillus, Saccharococcus and Bacillus, taking screening of its biotechnological potential into account. Bacteria from the first two genera came from Czech and German collection of microorganisms, while bacteria of genus Bacillus were natural isolates. Researched strains were examined from a viewpoint of carbon source utilization and furthermore, production of biosurfactants, extracellular hydrolytic enzymes (protease, amylase, lipase, cellulase, xylanase), organic acids, antimicrobial agents and microbial plastics – polyhydroxyalkanoates was also tested. Bacteria S. thermophilus, G. uzenensis and G. zalihae evinced a substantial ability of biosurfactant production. Strains G. jurassicus, G. uzenensis, G. gargensis and G. lituanicus were capable of intensive production of all tested, technologically significant enzymes. Highest antimicrobial effects were reached with bacteria G. stearothermophilus and G. thermocatenulatus. Largest production of acetic acid was achieved with G. jurassicus and lactic acid with G. thermodenitrificans. Ability to produce polyhydroxyalkanoates was proved at genotype level by some cultures only, however at fenotype level, response was negative. On the contrary, bacteria genus Bacillus were able to produce polyhydroxyalkanoates, although in small amounts under given circumstances. With remaining researched metabolites, production ability was considerably lower, compared to genera Geobacillus and Saccharococcus.

Viruses are the most abundant organisms on Earth, yet their collective evolutionary history, biodiversity and functional capacity is not well understood. Viral metagenomics offers a potential means of establishing a more comprehensive view of virus diversity and evolution, as vast amounts of new sequence data becomes available for comparative analysis.Metagenomic DNA from virus-sized particles (smaller than 0.2 microns in diameter) was isolated from approximately 20 liters of sediment obtained from Boiling Springs Lake (BSL) and sequenced. BSL is a large, acidic hot-spring (with a pH of 2.2, and temperatures ranging from 50°C to 96°C) located in Lassen Volcanic National Park, USA. BSL supports a purely microbial ecosystem comprised largely of Archaea and Bacteria, however, the lower temperature regions permit the growth of acid- and thermo-tolerant Eukarya. This distinctive feature of the BSL microbial ecosystem ensures that virus types infecting all domains of life will be present. The metagenomic sequence data was used to characterize the types of viruses present within the microbial ecosystem, to ascertain the extent of genetic diversity and novelty comprising the BSL virus assemblage, and to explore the genomic and structural modalities of virus evolution.Metagenomic surveys of natural virus assemblages, including the survey of BSL, have revealed that the diversity within the virosphere far exceeds what has currently been determined through the detailed study of viruses that are relevant to human health and agriculture. The number of as-yet-uncharacterized virus protein families present in the BSL assemblage was estimated by clustering analysis. Genomic context analysis of the predicted viral protein sequences in the BSL dataset indicates that most of the putative uncharacterized proteins are endemic or unique to BSL, and are largely harbored by known virus types. A comparative metagenomic analysis approach identified a set of conserved, yet uncharacterized BSL protein sequences that are commonly found in other similar and dissimilar environments.New sequence data from metagenomic surveys of natural virus assemblages was also used to better characterize and define known virus protein families, as some of the viruses found in the BSL environment represent distant relatives of well-characterized isolates. By comparing viral genes and protein sequences from these highly divergent species, it is possible to better understand the dynamics of adaptation and evolution in the virosphere. Additionally, as structures of virus proteins continue to be experimentally determined by X-ray crystallography and cryo-electron microscopy, a merger of structural and metagenomic sequence data allows the opportunity to observe the structural dynamics underlying virus protein evolution.Capsid (structural) proteins from two distinct Microviridae strains; a globally ubiquitous and highly sequence-diverse virus family, were identified in, and isolated from the BSL metagenomic DNA sample. These BSL capsid protein sequences, along with several other homologous sequences derived from metagenomic surveys and laboratory isolates, were mapped to the solved structure of a closely related capsid protein from the Spiroplasma phage-4 microvirus. Patterns of amino acid sequence conservation, unveiled by structure-based homology modeling analysis, revealed that the protein sequences within this family exhibit a remarkable level of plasticity, while remaining structurally and functionally congruent.Lateral gene transfer is thought to have had a significant impact on the genomic evolution and adaptation of virus families. Genomic context analysis was also utilized to identify interviral gene transfer within the BSL virus assemblage. An ostensibly rare interviral gene transfer event, having transpired between single-stranded RNA and DNA virus types, was detected in the BSL metagenome. Similar genomes were subsequently detected in other ecosystems around the globe. The discovery of this new virus genome dramatically underscores the scope and importance of genetic mobility and genomic mosaicism as major forces driving the evolution of viruses.The analyses conducted herein demonstrate the many ways in which viral metagenomic sequence data may be utilized to not only evaluate the composition of a natural virus assemblage, but to discover new viral genes, and to better understand the dynamics of both genomic and structural evolution within the virosphere.