Selection of resistant clones belonging to genera Klebsiella, Serratia and Pseudomonas in order to follow their implantation in a biofilter
Comparison with free tell system, fixed process applied for biological wastewater treatment have been shown to offer numerous advantages. The Biocarbone process, an aerobic down flow immersed bed reactor (ODA patent n° 78-30246), has been selected for many industrial and municipal wastewater treatment facilities. From this type of aerobic fixed-bed reactor, made of expanded schist as a granular support and fed with clarified domestic wastewater, eigthy-eigth strains were isolated (ZINEBI et al., 1992). Three of the bacterial strains were chosen for their abilities to express high levels of glucidolytic, proteolytic or lipolytic activities and to grow on the granular support as microcolonies which developed into a film of organisms over the whole surface. Our objective was to initiate biofilm formation by feeding the clean support with thon selected strains named : Klebsiella oxytoca, 501; Serratia marcescens, 532 and Pseudomonas putida, 601. In order to follow attachment kinetics of these selected strains of this biofilter, and to verify their perenity within the biofilm in non sterile conditions (mixed with indigeneous flora from the influent), a specific labelling method was required. As antibiotic-resistant mutants are easily isolated and the resistances can often serve as convenient genetic markers for use in characterizing bacterial strains, a direct selection of tells acquiring resistance to various antibiotics (ampicillin, streptomycin, nalidixic acid and rifampicin) bas been performed. Selected antibiotic-resistant strains were further incubated in presence of growth inhibitors or suicide substrates in order to select again spontaneous arising mutants well characterized by two distinct markers. From the two bacteria belonging to the Enterobacteriaceae family, mutants having lost the nitrate reductase have been isolated under anaerobic growth conditions in the presence of chlorate. In the case of Pseudomonas strain, mutants resistant towards substrate halogen analogues were obtained. Colonies resistant to antibiotics and resistant to lethal substrates were isolated : thus, colonies of Klebsiella resistant to streptomycin at 2 g/l, to rifampicin al 1 g/l and chlorate al 2 g/l ; colonies of Serratia resistant to streptomycin at 2 g/l or to rifampicine at 1 g/l and chlorate at 2 g/l and colonies of Pseudomonas resistant to nalidixic acid at 0.5 g/l and to bromoacetate at 2 g/l or to fluorouracil at 40 mg/l, were obtained. We have selected : trains showing the same doubling time as well as the same final population titan the parental strains when growths were performed with or without the markers. The three strains retained were : Klebsiella oxytoca, 501 R1S2Cl2 which grew on the Mac Conkey medium added with 1 g/l of rifampicin, 2 g/l at streptomycin and 2 g/l of chlorate; Serratia marcescens, 532 S2Cl2 (on Mac Conkey plus 2 g/l of streptomycin and 2 g/l of chlorate) and Pseudomonas putida, 601 NB2 (on King plus 0,5 g/l of nalidixic acid and 2 g/l of bromoacetate). These specific media for the detection of selectionned clones were selective toward a fixed indigenous flora since only 0,02 % of total heterotrophic population can grow. A column filled with grains of « Biodagen » either colonized by natural, microbial populations or with clean grains of « Biodagen » was fed with a population of the : train Klebsiella 501 R1S2Cl2. The strain colonized virgin « Biodagen » and maintained population of 4.106 CFU per grain for 9 days with new material and 105 CFU for 7 days with precolonized material. Experiment with a mixed population resulting from the three identified microbial species have been conducted with clean grains of « Biodagen », a whole population of 107 CFU per grain was obtained after two days and each identified strain corresponded to 1 % of the entire bacterial population. The relative concentrations of the three : trains did not decrease feeding the column with a mixture of the three : trains and of wastewater but slightly decreased when the column was fed with wastewater only.