SURVIVAL AND ACTIVITY OF COMAMONAS-TESTOS TERONI IN MIXED POPULATION

Inoculating specialized microorganisms directly into the mixed microbi al flora in wastewater treatment processes is the most simple and econ omic way to use their abilities (Fujita and Ike, 1994). However, succe ss or failure of a bioaugmentation depends on two conditions: the surv ival of the microbe and the demonstration of its degradative activity (Stephenson and Stephenson, 1992). Specialized bacteria which exhibit extremely high degradation activities of xenobiotic compounds may be s uitable for use in this way, since the degradation rate can be enhance d even if the strain maintain relatively low populations in the proces ses. For that purpose, the survival of a naturally occurring microorga nism, Comamonas testosteroni T-2, in activated sludge microcosms was i nvestigated. This bacterium was able to mineralize completely p-toluen esulfonate (pTS), as a model xenobiotic compound. Materials and method s Comamonas testosteroni T-2, isolated by Thurnheer et al. (1986), was able to degrade, as a unique source of carbon and energy, p-toluenesu lfonate, which is mainly used as hydrotropic agent in detergent formul ation. Activated sludge was sampled from a domestic wastewater treatme nt plant. After three centrifugations (20000 x g, 4 degrees C, 15 min) and washing with phosphate buffer, the sludges were directly inoculat ed without enrichment culture. Aerobic growth studies were performed a t 30 degrees C in 2-litre Erlenmeyer flasks containing 500 mi of miner al medium with 6 mM of pTS as unique source of carbon and energy (Loch er, 1991). Six different concentrations of the specialized bacterial s train were inoculated: (i) O%, containing only mixed activated sludge; (ii) 100%, i.e. pure culture of C. testosteroni; (iii) 50%; (iv) 25%; (v) 10%; and (vi) 5% of C. testosteroni in the total inoculum. In all inocula, the total initial biomass concentration was calculated to ha ve each So/Xo ratios between 2.5 and 3 (Chudoba et al., 1992). pTS was determined by HPLC (Grossenbacher et al., 1986). Dissolved organic ca rbon was measured by a TOC-500 analyser (Shimadzu, Duisburg, Germany). Sulfate was analyzed with a FIAstar(R) 5010 system (Tecator, Hogonos, Sweden). Bacterial growth was determined by the measure of dry weight (dw) after collection of samples on membrane filters (porosity 0.45 m u m) and by plating onto both selective pTS medium and plating count a gar (PCA) medium for counts of pTS degrading microorganisms and total flora, respectively. Results and discussion pTS was removed by all ino cula, even by the activated sludge alone (Fig. 1). Residual carbon acc umulated in cultures containing high concentration of activated sludge . The increase of sulphate concentrations showed that this ion was one of the final product of degradation with all inocula. As can be seen in Fig. 2, the xenobiotic compound removal was enhanced when mixed ino cula were used. But, the higher the specialized inoculum size the fast er the degradation occurred (Fig. 2). The time of adaptation to pTS el imination was also considerably shortened. For each mixed inoculum (50 , 25, 10 and 5% of C. testosteroni), the proportion of pTS degrading m icroorganisms in the microbial population was determined during the cu lture. As is shown in Fig. 3, the number of pTS degrading bacteria at the end of culture. was a function of the specialized inoculum size. W hen added in the concentration of 10-50%, pTS degraders even tend to d ominate the final microbial population (Fig. 3). C. testosteroni compo sed about 95% of pTS degraders. Other strains like Rhodococcus sp., Ba cillus sp. and Pseudomonas putida have also been isolated in small amo unts. Contrary to many other microorganisms able to degrade aromatic s ulphates (Focht and Williams, 1970; Ripin et al., 1971; Bird and Cain, 1974; Hattendorf and Hempel, 1990), C. testosteroni metabolized compl etely and rapidly pTS even under unfavorable growth conditions with hi gh concentrations of sulphate or natrium chloride or with little buffe red medium, conditions that are often found in industrial wastewaters as those from dyestuffs production (Fresenius et al., 1990). Moreover, C. testosteroni has been shown to be able to persist and to grow in a ctivated sludge, whatever the inoculum size (Fig. 2). Inamori et al. ( 1992) have described six different behaviours for an exogenous microor ganism inoculated into a complex ecosystem. The development of the bac terium was mainly function of environmental conditions (i.e. temperatu re, oxygen, available substrates), predation, competition and the stra in physiological characteristics (Goldstein et al., 1985; Atlas, 1992; Pipke et al., 1992). In the present study, growth conditions were opt imal for the specialized microorganism, and no protozoa have been dete cted in the mixed cultures. Then, the only parameter which could modif y the survival of C. testosteroni, in the systems was competition with natural flora. This factor was also minimized: activated sludges were taken from domestic wastewater treatment plant and few naturally occu rring pTS degrading microorganisms were present. In addition, the spec ialized inoculum size showed to have also an influence on the developm ent of the exogenous bacterium (Fig. 3). The same phenomenon was previ ously described by Inamori et al. (1992) and Fujita et al. (1994). The se authors pointed out the importance of the physiological state and b iological characteristics on the survival of the inoculated strain. In the present experiments, the exogenous bacterium belonged to the genu s Comamonas, often isolated of activated sludge and resistant to starv ation (McClure et al., 1989; Fitter and Chudoba, 1990). C. testosteron i was inoculated during the exponential phase of growth, while activat ed sludge was directly added to the medium without an enrichment cultu re step. The addition of C. testosteroni enhanced the degradation of p TS. Indeed, the xenobiotic compound was the only source of carbon and energy and the growth could not be supported by more easily biodegrada ble substrates (Dwyer et al., 1988). Nevertheless, the pollutant remov al was delayed in ecosystems containing high ratios of activated sludg e. Corseuil and Weber (1994) observed that the delay for the degradati on of benzene, toluene or xylene varied inversely with the initial num ber of microbes able to use these compounds, suggesting the need for d evelopment of some critical population of microorganisms. In this stud y, the survival of C. testosteroni has been promoted by cont