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
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
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.