Effect of cations on biofilm formation by Geobacillus species and Anoxybacillus flavithermus dairy isolates : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Technology at Massey University, Palmerston North, New Zealand

The concentration of free cations is one factor that may influence biofilm formation and consequent contamination of milk formulations by Geobacillus spp. and Anoxybacillus flavithermus during the manufacture of milk powders. Culture optical densities were measured to show that Ca2+ and Mg2+ predominantly increased the planktonic growth of Geobacillus spp. and A. flavithermus cultures. Culture cell numbers were enumerated, and a protein quantification assay was used to indicate that increases in optical density elicited by Ca2+ and Mg2+ supplementation was due to increased production of bacterial surface protein rather than an increase in cell numbers. High individual concentrations of Na+, K+ or Ca2+ (63 – 250 mM) inhibited the planktonic growth of Geobacillus spp., and Mg2+ protected Geobacillus spp. from high, inhibitory concentrations of Na+, K+ or Ca2+. The number of viable cells attached to stainless steel coupons was enumerated to show that cation concentrations or the monovalent to divalent cation ratio (2:1 compared to 10:1) did not influence the transition of bacteria from a planktonic to surface-attached form, or the subsequent formation of an established biofilm. However, preconditioning of the bacteria with cations increased their subsequent attachment. It was proposed that the transition of bacteria from a planktonic to surface-attached form is primarily mediated by the expression of bacterial surface proteins, as induced by cation preconditioning. The number of attached Geobacillus spp. was up to 4 log CFU cm-2 lower, for up to 18 h of biofilm formation, in a milk formulation that had a high monovalent to divalent cation ratio (greater than 10:1) relative to a milk formulation that had a monovalent to divalent cation ratio that resembled that found in unprocessed milk. Supplementation of a milk formulation that had a high monovalent to divalent cation ratio with Ca2+ or Mg2+ fully alleviated the inhibitory effect of the milk formulation on biofilm formation by Geobacillus spp. It was concluded that there is potential for the total thermophile count in milk powders that have high monovalent to divalent cation ratios to be markedly reduced. This would increase the quality and selling price of the milk powders