Non-thermal carbon dioxide-mediated inactivation process of Escherichia coli using bespoke system
  • Article Type: Research Article
  • Eurasian Journal of Biosciences, 2020 - Volume 14 Issue 2, pp. 4821-4827
  • Published Online: 31 Oct 2020
  • Open Access Full Text (PDF)


The reduction of the number of contaminated microbial cells in the biological and food solutions has desirably led to the development of many inactivation technologies aimed at the same goal--eradication of the microbial cells without harming the biological solution or generating toxic chemical agents. E.coli is known as food borne pathogen and cause serious problems in food industry and is used as a model organism in this study. The current CO2 mediated approach was used as a cheap and emerging approach to inactivate bacterial cells, E. coli. CO2 mediated approach is a non-thermal method, which has advantages over traditional methods such as autoclaving and γ -radiation. The results show that injecting CO2 into the system reduced the bacterial population by ~ 2.5-Log after 90 min and to 3.6 Log reduction after lactic acid addition. Bacterial cells were suffered from morphological changes and shape changes were obvious. Efficient energy consumption, avoidance of hazardous substances usage and can be applied in situ, are among its advantages, making it a promising and emerging inactivation technology.


  • Al-Mashhadani MKH, Bandulasena HCH and Zimmerman WB, (2012) CO2. mass transfer induced through an airlift loop by a microbubble cloud generated by fluidic oscillation, Ind Eng Chem Res, 51(4):1864–1877.
  • Blass, K.-F.R.Sheu.A.J.P., (1999) The α-Ketoglutarate Dehydrogenase Complex. Ann. N. Y. Acad. Sci 893:61–78.
  • Cabiscol E, Tamarit J and Ros J, (2000) Oxidative stress in bacteria and protein damage by reactive oxygen species, International microbiology 3(1): 3–8.
  • Carroll J, Slupsky J and Mather A, (1991) The solubility of carbon dioxide in water at low pressure, J. Phy. Chem. Ref. Data 20(6): 1201-1209.
  • Chatterjee A, Magee JL and Dey SK, (1983) The Role of Homogeneous Reactions in the Radionlisys of Water, Radiation Research 96(1):1–19.
  • Conway T, (1992) The Entner‐Doudoroff pathway: history, physiology and molecular biology, FEMS Microbiology Reviews 103: 4465–70.
  • Dillow A, Dehghani F, HRKACH J S, FOSTER N R and LANGER R,(1999) Bacterial inactivation by using near-and supercritical carbon dioxide. Proc. Natl. Acad. Sci. 96: 10344–10348.
  • Fraser D, (1951) Bursting Bacteria by Release of Gas Pressure, Nature 167: 33 – 34.
  • Garcia-Gonzalez L, Geeraerd A, Spilimbergo S, Elst K, Van Ginneken L, Debevere J, Van ImpeJ F and Devlieghere F, (2007) High pressure carbon dioxide inactivation of microorganisms in foods: the past, the present and the future. Int J food Micro 117:1–28.
  • Garrett BC, Dixon D a., Camaioni DM and et al, (2005) Role of water in electron-initiated processes and radical chemistry: Issues and scientific advances, Chem Rev 105(1):355–389.
  • Hallsworth, J.E., Heim, S., Timmis, K.N., (2003) Chaotropic solutes cause water stress in Pseudomonas putida Environ. Microbiol. 5:1270–1280.
  • Harley JP, (2002) Laboratory exercises in microbiology, 5th ed, The McGraw−Hill Companies, Boston, Mass, pp 1-12.
  • Heipieper HJ, Keweloh H and Rehm HJ, (1991) Influence of phenols on growth and membrane-permeability of free and immobilized Escherichia coli,Applied and Environmental Microbiology 57(11): 1213–1217.
  • Heipieper HJ, Weber FJ, Sikkema J, Keweloh H and De Bont JAM, (1994) Mechanisms of resistance of whole cells to toxic organic solvents,Trends in Biotechnology 12(10): 409–415 (1994).
  • Heipieper, H.J., Meulenbeld, G., Oirschot, Q. Van, Bont, J. De, (1996) Effect of Environmental Factors on the trans / cis Ratio of Unsaturated Fatty Acids in Pseudomonas putida S12. 62: 2773–2777.
  • Hong S and Pyun Y. (1999) Inactivation kinetics of Lactobacillus plantarum by high pressure carbon dioxide. J Food Sci 64(4): 728–733.
  • Hwang G, Park SR, Lee C-H, Ahn I-S, Yoon Y-J and Mhin BJ, (2009) Influence of naphthalene biodegradation on the adhesion of Pseudomonas putida NCIB 9816-4 to a naphthalene-contaminated soil, J Hazard Mater 172(1):491–493.
  • Ingram LO, (1975) Adaption of membrane lipids to alcohols, J. Bacteriol 125(2): 670–678.
  • Ingram LO, (1977) Changes in lipid composition of Escherichia coli resulting from growth with organic solvents and with food Changes in Lipid Composition of Escherichia coli Resulting from Growth with Organic Solvents and with Food Additives, Microbiology 33(5): 1233–1236.
  • Isenschmid A, Marison I and Stockar U Von, (1995) The influence of pressure and temperature of compressed CO2 on the survival of yeast cells, J Biotechnol 39: 229-237.
  • Kim J and Park W, Oxidative stress response in Pseudomonas putida, Applied Microbiology and Biotechnology 98(16): 6933–6946 (2014).
  • Mrozik, A., Piotrowska-Seget, Z., Łabuzek, S., (2004) Cytoplasmatic bacterial membrane responses to environmental perturbations, Polish J. Environ. Stud. 13: 487–494.
  • Mulakhudair, A. R., J. Hanotu, W. Zimmerman, and A. Mulakhudair, (2016) “Exploiting ozonolysis-microbe synergy for biomass processing : lignocellulosic biomass pretreatment,”, 93:187–193.
  • Mulakhudair, A. R., M. Al-Mashhadani, J. Hanotu, and W. Zimmerman,(2017) “Inactivation combined with cell lysis of Pseudomonas putida using a low pressure carbon dioxide microbubble technology,” J. Chem. Technol. Biotechnol., 92,: 1961–1969.
  • Nair P. (1995). Currently practised sterilization methods-some inadvertent consequences. J Biomater Appl 10: 121-135.
  • Nikolajeva V, Griba T and Petriņa Z,Factors (2012).influencing adhesion of Pseudomonas putida on porous clay ceramic granules, Environmental and Experimental Biology 10: 77–80
  • Nwadibe EC, Aniebonam E E, Jude OU. (2020). Effect of crude oil pollution on soil and aquatic bacteria and fungi. Journal of Experimental Biology and Agricultural Sciences, 8(2) : 176– 184. DOI:
  • Oulé M, Tano K, Bernier A-M and Arul J, (2006) Escherichia coli inactivation mechanism by pressurized CO2, Can. J. Microbiol 52:1208–1217.
  • Premnath V, Harris W, Jasty M and Merrill E. (1996) Gamma sterilization of UHMWPE articular implants: an analysis of the oxidation problem. Biomaterials 17: 1741–1753.
  • Rochex A, Lecouturier D, Pezron I and Lebeault JM. (2004) Adhesion of a Pseudomonas putida strain isolated from a paper machine to cellulose fibres, Appl Microbiol Biotechnol 65(6):727–33.
  • Schäfer, A., H. Harms, and A. J. B. Zehnder,(1998) “Bacterial accumulation at the air-water interface,” Environ. Sci. Technol., 32(23): 3704–3712.
  • Singh M, Saurabh Gupta1, Kundan Kumar Chaubey, Pravin Kumar Singh, Vishal Khandelwal, Pradeep Choudhary, Gaurav Pant, Shoor Vir Singh, Jagdip Singh Sohal. BIO-safety of milk products and mycobacterium avium subspecies paratuberculosis as major microbial contaminant using multiple tests including culture and sybr green real-time assay. Journal of Experimental Biology and Agricultural Sciences, 8(4): 508 – 523. DOI:
  • Spilimbergo S and Bertucco A, (2003) Non-thermal bacterial inactivation with dense CO2. Biotechnol Bioeng 84(6): 627–638(2003).
  • Sudiarso and Prihandarini R (2020). Study of the utilization of rim (refresh microorganism) in sugarcane. Online Journal of Animal and Feed Research, 10(5): 210-215
  • Trček J, Mira NP and Jarboe LR, (2015) Adaptation and tolerance of bacteria against acetic acid, Applied Microbiology and Biotechnology 99:.6215–6229.
  • Uday, K.V. and Pramod.A., (1986) The effect of CO2 ventilation on kinetics and yields of cell mass and ethanol in the batch cultures of Zymomonas mobilis, Biotechnol. Lett. 8: 811–816.
  • VanBogelen, R. a., Kelley, P.M., Neidhardt, F.C.,(1987) Differential induction of heat shock, SOS, and oxidation stress regulons and accumulation of nucleotides in Escherichia coli. J. Bacteriol 169: 26–32.
  • Walls, P. L. L., Bird, J. C., and Bourouiba, L., (2014) “Moving with bubbles: a review of the interactions between bubbles and the microorganisms that surround them,” Integr. Comp. Biol., vol. 54, no. 6, pp. 1014–1025.
  • Watson, K., (1990) Microbial stress proteins” Adv. Microb. Physiol. 31:184– 223.
  • Zhang X and Houk KN, (2005) Acid / Base Catalysis by Pure Water : The Aldol Reaction, J Org Chem, 70(24):9712–9716.
  • Zhang Y, (2008) Geochemical kinetics, Princeton University press, Princeton and oxford.


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