BIOLUMINESCENT BACTERIA AND BIOREMEDIATION

The increasing sensibility of both public and private institutions to the problem of contamination of industrial area, vacant sites, abusive tips has risen a new interest about decontamination of polluted sites. The choice of the best strategies of regeneration of an area must consider the characteristics of the site and the costs as regards to hazard/benefits, in particular, the costs of bioremediation and management of the area. One of the most promising and cheap strategies for hydrocarbons recovery is the acceleration of the natural process of biodegradation of these compounds in situ. Hydrocarbons can be degraded by various micro-organisms as bacteria and mushrooms. Bioluminescent bacteria (BLB) emit light when they find themselves in an optimal environment. If noxious substances are present, the luminescence decrease. Thus, the presence of toxic substances can be evaluated [1-2]. The bioluminescent bacteria toxicity test is simple and, compared to other bioassays, it is less time consuming, with a sensitivity within the same order of magnitude and allows to obtain results in a direct way. The BLB test was applied to samples of soil collected near a refinery at different depth levels and distances from the industrial plan. The microbial population of the soil was studied. The samples were then treated with different species of bacteria degrading hydrocarbons, both autochthonous (already present in the soil) or commercial, or with mixture of them. The bioluminescent bacteria were exposed to the samples for 12-20 hour. The acute and chronic biotoxicity was determined, following light emission for 60 minutes or for some hours (10-15), respectively. The acute toxicity is generally due to powerful or available toxicants, as heavy metal ions, while compounds with a slow penetration mechanism to enter bacterial cells or acting on bacteria reproduction are usually responsible for chronic biotoxicity. All the untreated samples proved to inhibit the luminescence as shown in Figure 1, where the perceptual inhibition at 60 minutes is reported (black values in figure). After the treatment with bacteria degrading hydrocarbons, only the samples collected at the deepest levels still inhibited the light emission (for example S2 720), while superficial specimens no longer inhibited the luminescence (for example S1C1). Figure 1: Inhibition (%) of the light emission of different soil samples before and after the treatment with hydrocarbons degrading bacteria. The BLB assay proved to be sensible at toxic substances and suitable to be applied to the control of bioremediation of contaminated area after treatment by bacteria degrading hydrocarbons. The use of a microplate assay allowed the simultaneous, rapid and at low cost analysis of numerous samples. The assay also allowed to optimise the mix of bacteria degrading hydrocarbons, starting from the species directly developed in real conditions in polluted grounds, selecting and improving them. References [1] Girotti S., Ferri E.N., Bolelli L., Sermasi G., Fini F. Applications of Bioluminescence in Analytical Chemistry. In: Chemiluminescence in Analytical Chemistry Garcìa-Campaña A.M.& Baeyens W.R.G. Eds., Marcel Dekker Inc., New York, 2001, 247-84. [2] Girotti S., Bolelli L., Roda A., Gentilomi G., Musiani M., Improved detection of toxic chemicals using bioluminescent bacteria”. Analytica Chimica Acta, 2002, 471, 113-120.