ORIGINAL PAPER Zileena Zahir Æ Kimberley D. Seed Æ Jonathan J. Dennis Isolation and characterization of novel organic solvent-tolerant bacteria Received: 15 July 2005 / Accepted: 14 September 2005 / Published online: 20 October 2005 Ó Springer-Verlag 2005 Abstract Some organic solvents are extremely toxic to living organisms by virtue of their ability to partition into and disrupt the normal functioning of biological membranes. In recent years, several bacteria have been discovered that are more tolerant to these toxic solvents than most microorganisms. Using enrichment proce- dures, we have isolated new organic solvent-tolerant bacteria from both hyrdocarbon-contaminated and pristine soil samples. These organisms were character- ized by several different experimental procedures including description of their cellular physiology, 16S rDNA homology, organic solvent tolerance range, and survival after solvent exposure. The results indicate that gram-positive bacteria can be isolated from the envi- ronment that are as tolerant to toxic organic solvents, if not more so, than the most organic solvent-tolerant gram-negative bacteria. Keywords Solvent tolerance Æ Organic solvents Æ Bacterial cell envelope Æ Enrichment Introduction It is generally thought that organic solvents exhibit ex- treme toxicity toward living microorganisms because of their accumulation in hydrophobic biological mem- branes. Because of this toxicity, organic solvents have in the past been used as permeabilization agents, disinfec- tants, food preservatives, and industrial solvents (Davidson and Barnden 1981; de Bont 1998). The hydrophobicity of organic solvents can be expressed in terms of P ow , which represents the ability of a com- pound to partition over an octanol/water two-phase system (Sikkema et al. 1994). It has been established that log P ow is correlated with the toxicity of a specific or- ganic solvent and that log P ow values, especially between two and four, are highly toxic for microorganisms (Os- borne et al. 1990). Whether a solvent is toxic to a bac- terial cell depends upon its concentration in the membrane, which relates to its water solubility and its ability to partition from the water phase to the mem- brane (de Bont 1998). Solvents with log P ow s below two are generally too hydrophilic to partition into mem- branes well, and solvents with log P ow s above four are too hydophobic to have high water solubility (Kieboom and de Bont 2000). The mechanisms of membrane tox- icity by organic solvents have been well studied and comprehensively reviewed (Sikkema et al. 1995). In gram-negative cells, extensive permeabilization is caused in the cytoplasmic membrane, while the outer membrane remains relatively intact. This preferential partitioning of organic solvents into the cytoplasmic membrane prevents normal functioning of the membrane as a selective barrier, allowing the leakage of such macro- molecules as RNA, phospholipids, and proteins (Woldringh 1973). Perhaps more importantly, organic solvents produce a loss of ion gradients across the cytoplasmic membrane that destroys energy production and transduction via proton motive force (Sikkema et al. 1994). The accumulation of toxic organic solvents in the membrane increases membrane fluidity, increases mem- brane swelling, and reduces the normal functioning of membrane-associated proteins (Sikkema et al. 1995; Weber and de Bont 1996). The accumulation of organic solvents results in the disruption of bilayer stability and membrane structure, causing a loss of membrane func- tion and ultimately cell death. Despite this extreme toxicity, organic solvent-tolerant bacteria that are capable of growth in a two-phase wa- ter–toluene system have been isolated. Many of these tolerant bacterial species, including the first strain iso- lated, were gram-negative bacteria such as Pseudomonas Communicated by K. Horikoshi Z. Zahir Æ K. D. Seed Æ J. J. Dennis (&) Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada, T6G 2E9 E-mail: jon.dennis@ualberta.ca Tel.: +1-780-4922529 Fax: +1-780-4929234 Extremophiles (2006) 10:129–138 DOI 10.1007/s00792-005-0483-y