Conventional disinfection and sterilization methods are often ineffective with biofilms, which are ubiquitous, hard-to-destroy microbial communities embedded in a matrix mostly composed of exopolysaccharides. The use of gas-discharge plasmas represents an alternative method, since plasmas contain a mixture of charged particles, chemically reactive species and UV radiation, whose decontamination potential for free-living, planktonic micro-organisms is well established. In this study, biofilms were produced using Chromobacterium violaceum, a Gram-negative bacterium present in soil and water and used in this study as a model organism. Biofilms were subjected to an atmospheric pressure plasma jet for different exposure times. Our results show that 99.6 % of culturable cells are inactivated after a 5 min treatment. The survivor curve shows double-slope kinetics with a rapid initial decline in c.f.u. ml "1 followed by a much slower declinewith D values that are longer than those for the inactivation of planktonic organisms, suggesting a more complex inactivation mechanism for biofilms. DNA and ATP determinations together with atomic force microscopy and fluorescence microscopy show that non-culturable cells are still alive after short plasma exposure times. These results indicate the potential of plasma for biofilm inactivation and suggest that cells go through a sequential set of physiological and morphological changes before inactivation.
INTRODUCTIONMost studies dealing with the growth and physiology of bacteria have been carried out using planktonic cells in batch cultures. Although these studies have provided extensive information regarding the basic molecular mechanisms that control the growth of individual bacteria, most bacteria live primarily in communities referred to as biofilms (Stoodley et al., 2002), where cooperative effects become important. Biofilms are microbial communities embedded in a matrix mostly composed of exopolysaccharides together with some proteins and nucleic acids. Biofilm formation can be considered as a developmental cycle that begins when planktonic bacteria attach to a surface, divide and recruit additional planktonic cells that attach to the cells already on the surface. In some cases, this process results in the development of a mature biofilm, in which cells cluster in pillar-and mushroom-like structures with water channels between them, forming a primitive circulatory system (Kolter & Losick, 1998). In other cases, more compact, flat and homogeneous biofilm layers (Heydorn et al., 2000;Picioreanu et al., 2000), spherical clusters (Matsumoto et al., 2007), discrete microcolony structures (Massol-Deyá et al., 1995), ball-shaped microcolonies (Tolker-Nielsen et al., 2000) and honeycomb-like structures (Marsh et al., 2003;Russo et al., 2006) have been reported. Although it is out of the scope of this paper to extensively review the different biofilm structures reported, it is generally accepted that biofilms are not a continuous monolayer surface deposit but instead a thin base film,...
