Evaluation of viability and growth of Acetobacter senegalensis under different stress conditions Rasoul Shafiei a, b, ⁎, Frank Delvigne c, 1 , Manoochehr Babanezhad d , Philippe Thonart a, c, 2 a Walloon Center of Industrial Biology, University of Liège, Sart-Tilman, 4000 Liège, Belgium b Department of Biology, Faculty of Sciences, University of Isfahan, Isfahan, Iran c Walloon Center of Industrial Biology, Bio-industry unit, Gembloux Agro-Bio Tech, 5030 Gembloux, Belgium d Department of Statistics, Faculty of Sciences, Golestan University, Gorgan, Golestan, Iran abstract article info Article history: Received 8 October 2012 Received in revised form 25 February 2013 Accepted 10 March 2013 Available online 20 March 2013 Keywords: Stress Respiration Cell envelope integrity Dehydrogenase Preadaptation Acetic acid bacteria (AAB) are used in production of vinegars. During acetic acid fermentation, AAB encounter various aggressive conditions which may lead to a variety of cellular disorders. Previous researches mainly studied the influences of different carbon sources on tolerance of AAB to ethanol and acetic acid. In this study, different techniques were used comparatively to investigate the effects of preadaptation on the ability of A. senegalensis to tolerate ethanol and acetic acid. In general, the carbon sources used for preadaptation of A. senegalensis exhibited significant effects on the tolerance of cells to stressors. Flow-cytometric assessments of preadapted cells in ethanol showed that 87.3% of the cells perform respiration after exposure to a stress me- dium containing 5% (v/v) ethanol and 3% (w/v) acetic acid. However, 58.4% of these preadapted cells could keep their envelope integrity under the stress condition. They could also grow rapidly (μ max = 0.39/h) in the stress medium (E5A3) with a high yield (> 80%). A. senegalensis grown in glucose exhibited a low tolerance to acetic acid. Analysis of their respiration capacity, membrane integrity and culturability revealed that almost all the population were dead after exposure to 5% (v/v) ethanol and 3% (w/v) acetic acid. In contrast, exposure of A. senegalensis preadapted in a mixture of glucose and acetic acid to a stress medium containing 5% (v/v) ethanol and 3% (w/v) acetic acid, exhibited an intact respiration system and cellular membrane integrity in 80.3% and 50.01% of cells, respectively. Moreover, just 24% of these cells could keep their culturability under that stress condition. In summary, cell envelope integrity, growth and culturability are more susceptible to pH and acetic acid stresses whereas respiration system is less subjected to damages under stress condition. In addition, pread- aptation of A. senegalensis in a mixture of glucose and acetic acid enables it to tolerate and grow in ethanol and acetic acid. © 2013 Elsevier B.V. All rights reserved. 1. Introduction Acetic acid bacteria (AAB) play important roles in the production of fermented foods. They are also used for production of some bio- technologically important products, such as sorbose and gluconic acids (Raspor and Goranovic, 2008; Trcek, 2005). Different species of AAB are involved in production of industrial and traditional vinegars, however it is still state of the art to use a frac- tion of fermented mash as a suspension containing moderate number of viable cells for inoculation of Acetator® (Sokollek et al., 1998). AAB, especially Acetobacter sp. and Gluconacetobacter sp. tolerate high concentrations of acetic acid and thus both genera are used industrially for production of vinegar (Kanchanarach et al., 2010). Acetobacter sp. gradually acquires resistance against higher concentra- tions of acetic acid when properly adapted to the conditions (Azuma et al., 2009). The stability of important traits has been widely studied within AAB, showing high frequency of phenotypic changes, mainly regarding ethanol oxidation and acetic acid resistance (Gullo et al., 2012). Thus, any process used for cultivation and preservation of AAB should consider the possibility of changes in important traits of AAB. In tropical regions, mesophilic AAB cannot be used efficiently in acetic acid production since the culture medium must be cooled down regularly during fermentation. Running a process under contin- uous cooling, increases the cost of production. Therefore, usage of thermotolerant strains is inevitable. Ndoye and coworkers, isolated a novel thermotolerant acetic acid bacterium, A. senegalensis, from International Journal of Food Microbiology 163 (2013) 204–213 ⁎ Corresponding author at: Walloon Center of Industrial Biology, University of Liège, Sart-Tilman, 4000 Liège, Belgium. Tel.: +32 43663994; fax: +32 3662862. E-mail addresses: R.shafiei@doct.ulg.ac.be, Ra.shafiei@gmail.com (R. Shafiei), F.delvigne@ulg.ac.be (F. Delvigne), M.babanezhad@gu.ac.ir (M. Babanezhad), P.thonart@ulg.ac.be (P. Thonart). 1 Tel.: +32 81622311. 2 Tel.: +32 43662861. 0168-1605/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijfoodmicro.2013.03.011 Contents lists available at SciVerse ScienceDirect International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro