A study of the abiotic factors influencing the biofilm and spore formation of dairy isolates of Geobacillus stearothermophilus and characterisation of spores based on their heat resistance : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Safety & Microbiology at Massey University, Palmerston North, New Zealand

Figures 2.1 (=Setlow et al., 2003 Fig 2) & 2.4 (=Setlow et al., 2014 Fig 2) were removed for copyright reasons. Other possibly copyrighted Figures remain for the sake of clarity.Geobacillus stearothermophilus, a Gram-positive thermophilic bacterium is an important contaminant in the dairy industry during milk powder manufacture. This bacterium is capable of survival and growth in sections of the milk powder manufacturing plant where higher temperatures (40-65 °C) prevail. They form biofilms on industrial processing surfaces and their spores are heat resistant. Although, these bacteria are not pathogenic, their presence is an indicator of poor plant hygiene and diminishes the quality of end products thereby incurring economic loss. Previous studies have focused on designing control strategies to prevent the biofilm and spore formation during milk powder manufacture but with limited success. This is largely attributed to the limited knowledge we possess on the biofilm and spore forming capacity of these thermophiles and the impact of abiotic factors. This study focussed on the role of abiotic factors viz. incubation temperature and total dissolved solids on the biofilm and spore formation of Geobacillus stearothermophilus A1, D1, P3 and ATCC 12980. The heat resistance of the spores was characterized and the effect of phosphate on the heat resistance of spores was discussed. The effect of temperature and total dissolved solids on the biofilm and spore formation was evaluated using a Centre for Disease Control (CDC) reactor in a milk environment. The results from the CDC study demonstrated that 65 °C is the most preferred temperature for biofilm and spore formation. Milk proteins influence the attachment of bacteria to the stainless steel surface with caseins having the greatest effect. The three strains were characterized based on their heat resistance and the effect of phosphate on the heat resistance of these spores was determined using atomic absorption spectroscopy (AAS). AAS demonstrated that phosphate when present in the heating medium causes release of cations from within the spore thereby lowering their heat resistance. In addition, the Calcium and Dipicolinic acid (DPA) content of these spores was determined and the DPA content was found to be associated with the heat resistance of spores. The heat resistance of spores obtained by a milk biofilm system under continuous flow was determined and compared with previous values obtained from spores prepared in a static laboratory medium. There is no significant difference (p ≤ 0.05) in the heat resistance of spores obtained by the biofilm in milk and by a sporulation medium. The results from spore formation under different temperatures and total solids will aid in the design of thermal processing steps aimed towards controlling the biofilm and spore formation of G. stearothermophilus during milk powder manufacture. The effect of phosphate on the heat resistance of spores, will aid in the design of a phosphate based cleaning agent to minimise product spoilage caused by the presence of spores of G. stearothermophilus. The results showing the similarity between the heat sensitivity of spores prepared in sporulating medium and a continuous flow CDC reactor using milk provide confidence in results on heat resistance taken from laboratory sporulating medium in terms of their relevance to the dairy industry