Physiological and molecular characterisation of an alkaliphilic bacillus isolated from a moderately alkaline environment.

Two strains of alkaliphilic, strictly aerobic, Gram-positive, non-motile bacteria were isolated. First, an alkaliphilic bacterium (optimum growth at pH 10) designated as MAK7 was isolated from a water and sediment sample obtained from a non-extreme environment (River Lathkill in the Derbyshire Peak District). Second, an alkaliphilic Bacillus sp. was isolated as a laboratory contaminant from a culture of MAK7 grown at pH 10. Phylogenetic analysis, based on 16S rRNA gene sequences, showed that isolate MAK7 was an unclassified strain of Bacillus, most closely related to a Bacillus isolate associated with marine sponges (99.4% sequence identity). The laboratory contaminant Bacillus sp. was most closely related to the species Bacillus cereus (100% sequence similarity). Generation times of 55 and 40 minutes were observed at external pH lOin Horikoshi medium for alkaliphilic Bacillus MAK7 and B. cereus respectively. The internal pH of Bacillus MAK7 and B. cereus at pH 10 was 9.00 ± 0.08 and 8.76 ± 0.28 respectively. To cope with the reversed ~pH at pH 10, the membrane potential of both strains increased significantly. However, there was a significant overall drop in the proton motive force at pH 10 to -112 m V for Bacillus MAK7 and -97 m V for B. cereus. The optimum salinity for growth was determined to be 100 mM NaCI for Bacillus MAK7 and 400 mM NaCI for B. cereus. However, neither strain showed a Na+ or K+ requirement for growth or optimal respiration rates, which is unusual for alkaliphilic bacteria. Increasing concentrations of NaCI were inhibitory to the respiration rate of both strains, but KCl concentrations up to 400 mM did not inhibit respiration. Enzyme activities (malate dehydrogenase, fumarase and hexokinase) in crude cell-free extracts were measured to investigate the effects of alkaline pH on metabolic pathways.