BACKGROUND: Inactivation processes can be classified into non-thermal inactivation methods such as ethylene oxide and gamma-radiation, and thermal methods such as autoclaving. The ability of carbon dioxide enriched microbubbles to inactivate Pseudomonas putida suspended in physiological saline, as a non-thermal sterilisation method, was investigated in this study with many operational advantages over both traditional thermal and non-thermal sterilisationmethods. RESULTS: Introducing carbon dioxide enriched microbubbles can achieve similar to 2-Log reduction in the bacterial population after 90 min of treatment, addition of ethanol to the inactivation solution further enhanced the inactivation process to achieve 3, 2.5 and 3.5-Log reduction for 2%, 5% and 10%(v/v) ethanol, respectively. A range of morphological changes was observed on Pseudomonas cells after each treatment, and these changes extended fromchanging cell shape fromrod shape to coccus shape to severe lesions and cell death. Pseudomonas putida KT 2440 was used as a model of gram-negative bacteria. CONCLUSION: Using CO2 enriched microbubbles technology has many advantages such as efficient energy consumption (no heat source), avoidance of toxic and corrosive reagents, and in situ treatment. In addition, many findings from this study could apply to other gram-negative bacteria. (C) 2017 The Authors. Journal of Chemical Technology & Biotechnology published by JohnWiley & Sons Ltd on behalf of Society of Chemical Industry.