Physiology of dairy-associated Bacillus spp. over a wide pH range

Bacillus species isolated from alkaline wash solutions used for cleaning in place in South African dairy factories have been suggested to contaminate product contact surfaces of dairy processing equipment and result in post-pasteurization spoilage of milk and milk products. Growth and attachment of such Bacillus isolates under alkaline and acidic conditions have not been previously described. Therefore, the in vitro growth temperature and pH ranges, attachment abilities and hydrophobicity, and enzyme production capabilities of four Bacillus isolates (tentatively identified as B. subtilis115, B. pumilus122, B. licheniformis137 and B. cereus144) previously isolated from the alkaline wash solutions in a South African dairy were examined. Growth pH ranges were determined in buffered Standard One-like Nutrient Broth and in unbuffered 1% Milk Medium at pH values ranging from 3 to 12. Growth and attachment to stainless steel surfaces and production of protease and lipase enzymes were determined in 1% Milk Medium at pH 4, 7 and 10. Colony hydrophobicity of each isolate by the Direction of Spreading Method (DOS) was also determined at pH 4, 7 and 10. In addition, Arrhenius plots were used to examine the growth temperature ranges of the isolates. All isolates grew at pH values ranging from 4.5 to 9.5 in buffered Standard One-like Nutrient Broth, and from pH 4 to 10 in 1% Milk Medium. All isolates also attached to stainless steel at pH 4, 7 and 10 in 1% Milk Medium. Generally the attachment of B. subtilis115, B. pumilus122 and B. licheniformis137 to stainless steel surfaces was enhanced at pH 4 and 10, compared to pH 7. By contrast, the best attachment of B. cereus144 cells to stainless steel surfaces was at pH 7. Planktonic and attached cells of all isolates produced proteolytic enzymes at pH 7 and 10, but not at pH 4. Similarly, planktonic and attached cells of B. subtilis115, B. pumilus122 and B. licheniformis137 produced lipolytic enzymes at pH 7 and 10, and weak lipolysis was observed at pH 4. The Bacillus cereus144 isolate showed no lipolytic activity at pH 10. All isolates exhibited low hydrophobic properties at all pH values even though attachment to stainless steel at the same pH values occurred. None of the isolates grew below 11°C or above 56°C, and optimum growth temperatures were in the high mesophilic range (36–44°C).