Influence of cations on growth of thermophilic Geobacillus spp. and Anoxybacillus flavithermus in planktonic culture

Abstract

Free ions of Na(+), K(+), Ca(2+), and Mg(2+) influenced the optical density of planktonic cultures of thermophilic bacilli. Anoxybacillus flavithermus E16 and Geobacillus sp. strain F75 (milk powder manufacturing plant isolates) and A. flavithermus DSM 2641 and G. thermoleovorans DSM 5366 were studied. Ca(2+) and Mg(2+) were associated with increases in optical density more so than Na(+) and K(+). Overall, it appeared that Ca(2+) and/or Mg(2+) was required for the production of protein in thermophilic bacilli, as shown by results obtained with A. flavithermus E16, which was selected for further study.

Fig 1

Optical densities of A. flavithermus E16 (A), A. flavithermus DSM 2641 (B), Geobacillus sp. F75 (C), and G. thermoleovorans DSM 5336 (D) planktonic cultures grown in casein digest medium (1 g/liter) supplemented with a total cation concentration of 2 mM with varied proportions of Na⁺, K⁺, Ca²⁺, and Mg²⁺, relative to the optical density of the respective bacterial isolate grown in casein digest medium (1 g/liter) unsupplemented with cations (baseline control). N, K, C, and M designate free Na⁺, K⁺, Ca²⁺, and Mg²⁺, respectively. CM1:5 refers to a Ca²⁺-Mg²⁺ ratio of 1:5, NKC refers to a Na⁺-K⁺-Ca²⁺ ratio of 1:1:2, and all other treatments have equal proportions of each supplemented cation. The cultures were incubated at 55°C for 10 h. Two replicates were done, and each replicate consisted of an average of triplicate cultures. The error bars represent 95% confidence intervals (P ≤ 0.05), which were determined using SAS statistical analysis software.

Fig 2

Optical densities of A. flavithermus E16 (A), A. flavithermus DSM 2641 (B), Geobacillus sp. F75 (C), and G. thermoleovorans DSM 5336 (D) planktonic cultures grown in casein digest medium (1 g/liter) supplemented with equal proportions of Na⁺, K⁺, Ca²⁺, and Mg²⁺ at total cation concentrations of between 2 and 250 mM, relative to the optical density of the respective bacterial isolate grown in casein digest medium (1 g/liter) unsupplemented with cations (baseline control). The cultures were incubated at 55°C for 10 h. Two replicates were done, and each replicate consisted of an average of triplicate cultures. The error bars represent 95% confidence intervals (P ≤ 0.05), which were determined using SAS statistical analysis software.

Fig 3

Amounts of polysaccharide (A) and protein (B), associated with the pellet after centrifugation at 11,800 × g, per CFU of A. flavithermus E16 culture after a 10-h incubation at 55°C, grown in casein digest medium (1 g/liter) supplemented with a total cation concentration of 0, 2, or 125 mM (consisting of equal proportions of Na⁺, K⁺, Ca²⁺, and Mg²⁺) (n = 3). Error bars represent ±1 standard deviation (σ_n−1).