Résumés
Résumé
Des souches appartenant aux espèces : Klebsiella oxytoca, Serratia marcescens et Pseudomonas putida, isolées d'un biofiltre utilisé pour le traitement d'effluents urbains ont été choisies parmi une centaine d'autres pour être réimplantées dans un réacteur du même type. Dans le but de suivre leur fixation en réacteur ouvert, une méthode spécifique de sélection a été développée. Des clones de ces souches résistant naturellement à des antibiotiques (rifampicine, streptomycine, acide nalidixique) et à des substrats suicides (chlorate, bromoacétate, fluorouracile) ont été recherchés. Cette sélection a permis d'obtenir des clones de Klebsiella et de Serratia résistants à 2 g/l de streptomycine, 1 g/l de rifampicine et à 2 g/l de chlorate ainsi que des clones de Pseudomonas résistants à 0,5 g/l d'acide nalidixique et à 2 g/l de bromoacétate ou à 40 mgll de fluorouracile.
Les clones résistants dont les caractéristiques de croissance et les activités enzymatiques sont identiques à celles de la souche sauvage et dont la stabilité génétique a été maintenue après de nombreux repiquages ont été retenus. Afin de valider notre méthode de reconnaissance, une numération de la flore indigène d'un effluent urbain a été réalisée sur les milieux spécifiques des clones résistants : seule une faible proportion de cette flore, à savoir 0,02 % est capable de s'y développer. Des essais préliminaires d'ensemencement du biofiltre avec les souches sélectionnées ont été réalisés, ils montrent que celles-ci s'implantent puisqu'elles sont retrouvées sur les grains de matériau de garnissage et que chacune d'elle représente 1 % de la flore totale.
Mots-clés:
- Filtre biologique,
- biomasse fixée,
- sélection,
- résistance aux antibiotiques,
- résistance aux substrats suicides,
- implantation
Abstract
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 : Klebsiellaoxytoca, 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.
Keywords:
- Biofilter,
- fixed-biomass,
- selection,
- resistance to lethal substrates,
- resistance to antibiotics,
- implantation