Aims: To improve the thermotolerant properties (TTP) of acetic acid bacteria (AAB) cells for high temperature acetification. Methods and Results: At high temperature (361 degrees C), the acetification rate (ETA) is usually lower than at 301 degrees C. The addition of 015% calcium chloride (CaCl2) may decrease the negative effect of the increase of temperature from 30 +/- 1 degrees C to 36 +/- 1 degrees C on the ETA. The effect of CaCl2 on the thermotolerant properties of acetic acid bacteria cells was investigated. The CaCl2 increased the content of phospholipids (phosphotidylcholine and phosphatidylglycerol), fatty acids (cis-vaccenic acid, palmitic acid and myristic acid) and the activities of membrane-bound enzymes involved in acetification, alcohol dehydrogenase and aldehyde dehydrogenase. Transmission electron microscope images revealed a more compact cell wall with CaCl2. Process consistency at 36 +/- 1 degrees C was tested in nine sequential acetification cycles using 015% (w/v) CaCl2. High ETAs (933 +/- 06; 867 +/- 08 and 967 +/- 07gl(-1)day(-1)) were obtained during the last three cycles. Conclusions: The results confirm that changes of the content of lipid, activities of membrane-bound enzymes and cell-wall thickness occurred with added CaCl2. Significance and Impact of the Study: High temperature acetification (HTA) with additions of CaCl2 was investigated. Significant reductions in the overall production costs result from lower cooling costs associated with HTA.