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    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
    (Massey University, 2013) Somerton, Benjamin Thomas
    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.
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    Insights into the Geobacillus stearothermophilus species based on phylogenomic principles.
    (26/06/2017) Burgess SA; Flint SH; Lindsay D; Cox MP; Biggs PJ
    BACKGROUND: The genus Geobacillus comprises bacteria that are Gram positive, thermophilic spore-formers, which are found in a variety of environments from hot-springs, cool soils, to food manufacturing plants, including dairy manufacturing plants. Despite considerable interest in the use of Geobacillus spp. for biotechnological applications, the taxonomy of this genus is unclear, in part because of differences in DNA-DNA hybridization (DDH) similarity values between studies. In addition, it is also difficult to use phenotypic characteristics to define a bacterial species. For example, G. stearothermophilus was traditionally defined as a species that does not utilise lactose, but the ability of dairy strains of G. stearothermophilus to use lactose has now been well established. RESULTS: This study compared the genome sequences of 63 Geobacillus isolates and showed that based on two different genomic approaches (core genome comparisons and average nucleotide identity) the Geobacillus genus could be divided into sixteen taxa for those Geobacillus strains that have genome sequences available thus far. In addition, using Geobacillus stearothermophilus as an example, we show that inclusion of the accessory genome, as well as phenotypic characteristics, is not suitable for defining this species. For example, this is the first study to provide evidence of dairy adaptation in G. stearothermophilus - a phenotypic feature not typically considered standard in this species - by identifying the presence of a putative lac operon in four dairy strains. CONCLUSIONS: The traditional polyphasic approach of combining both genotypic and phenotypic characteristics to define a bacterial species could not be used for G. stearothermophilus where many phenotypic characteristics vary within this taxon. Further evidence of this discordant use of phenotypic traits was provided by analysis of the accessory genome, where the dairy strains contained a putative lac operon. Based on the findings from this study, we recommend that novel bacterial species should be defined using a core genome approach.