PLASMID CONTENT EVALUATION OF ACTIVATED-SLUDGE

Plasmid content of activated sludge was evaluated by two different and complementary approaches. The first one consisted in the isolation of colonies from activated sludge samples, then isolated colonies were g rown in nutrient broth and the plasmids were extracted from these cult ures. The plasmid extraction procedure used a commercialized kit and t he corresponding protocol. By this first approach, 40% of isolated col onies harboring plasmids were found. The second approach was an adapta tion of the plasmid extraction kit directly on activated sludge sample s. This direct application required several washing steps of sludges s amples to avoid plasmid DNA digestion by DNases adsorbed within the sl udge. This direct and rapid technique was validated in terms of reprod ucibility, specificity for plasmid DNA and losses. It allows the isola tion of around 1 mu g of plasmid DNA per 10(11) cells in the sludge. T he two approaches presented in this paper indicated that the plasmid c ontent of activated sludge is important and its particular role within the wastewater epuration procedure should be investigated,