Abstract :
Inoculating specialized microorganisms directly into the mixed microbial flora in wastewater treatment processes is the most simple and economic way to use their abilities (Fujita and Ike, 1994). However, success or failure of a bioaugmentation depends on two conditions: the survival 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 suitable for use in this way, since the degradation rate can be enhanced even if the strain maintain relatively low populations in the processes. For that purpose, the survival of a naturally occurring microorganism, Comamonas testosteroni T-2, in activated sludge microcosms was investigated. This bacterium was able to mineralize completely p-toluenesulfonate (pTS), as a model xenobiotic compound.
Materials and methods
Comamonas testosteroni T-2, isolated by Thurnheer et al. (1986), was able to degrade, as a unique source of carbon and energy, p-toluenesulfonate, which is mainly used as hydrotropic agent in detergent formulation. Activated sludge was sampled from a domestic wastewater treatment plant. After three centrifugations (20000 × g, 4°C, 15 min) and washing with phosphate buffer, the sludges were directly inoculated without enrichment culture.
Aerobic growth studies were performed at 30°C in 2-litre Erlenmeyer flasks containing 500 ml of mineral medium with 6 mM of pTS as unique source of carbon and energy (Löcher, 1991). Six different concentrations of the specialized bacterial strain were inoculated: (i) 0%, 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 have each So/Xo ratios between 2.5 and 3 (Chudoba et al., 1992).
pTS was determined by HPLC (Grössenbacher et al., 1986). Dissolved organic carbon was measured by a TOC-500 analyser (Shimadzu, Duisburg, Germany). Sulfate was analyzed with a FIAstar® 5010 system (Tecator, Högönös, Sweden). Bacterial growth was determined by the measure of dry weight (dw) after collection of samples on membrane filters (porosity 0.45 μm) and by plating onto both selective pTS medium and plating count agar (PCA) medium for counts of pTS degrading microorganisms and total flora, respectively.
Results and discussion
pTS was removed by all inocula, even by the activated sludge alone (Fig. 1). Residual carbon accumulated 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 inocula were used. But, the higher the specialized inoculum size the faster the degradation occurred (Fig. 2). The time of adaptation to pTS elimination was also considerably shortened.
For each mixed inoculum (50, 25, 10 amd 5% of C. testosteroni), the proportion of pTS degrading microorganisms in the microbial population was determined during the culture. 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. When added in the concentration of 10–50%, pTS degraders even tend to dominate the final microbial population (Fig. 3). C. testosteroni composed about 95% of pTS degraders. Other strains like Rhodococcus sp., Bacillus sp. and Pseudomonas putida have also been isolated in small amounts.
Contrary to many other microorganisms able to degrade aromatic sulphates (Focht and Williams, 1970; Ripin et al., 1971; Bird and Cain, 1974; Hattendorf and Hempel, 1990), C. testosteroni metabolized completely and rapidly pTS even under unfavorable growth conditions with high concentrations of sulphate or natrium chloride or with little buffered 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 activated sludge, whatever the inoculum size (Fig. 2). Inamori et al. (1992) have described six different behaviours for an exogenous microorganism inoculated into a complex ecosystem. The development of the bacterium was mainly function of environmental conditions (i.e. temperature, oxygen, available substrates), predation, competition and the strain physiological characteristics (Goldstein et al., 1985; Atlas, 1992; Pipke et al., 1992). In the present study, growth conditions were optimal for the specialized microorganism, and no protozoa have been detected in the mixed cultures. Then, the only parameter which could modify 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 occurring pTS degrading microorganisms were present. In addition, the specialized inoculum size showed to have also an influence on the development of the exogenous bacterium (Fig. 3). The same phenomenon was previously described by Inamori et al. (1992) and Fujita et al. (1994). These authors pointed out the importance of the physiological state and biological characteristics on the survival of the inoculated strain. In the present experiments, the exogenous bacterium belonged to the genus Comamonas, often isolated of activated sludge and resistant to starvation (McClure et al., 1989; Pitter and Chudoba, 1990). C. testosteroni was inoculated during the exponential phase of growth, while activated sludge was directly added to the medium without an enrichment culture step. The addition of C. testosteroni enhanced the degradation of pTS. Indeed, the xenobiotic compound was the only source of carbon and energy and the growth could not be supported by more easily biodegradable substrates (Dwyer et al., 1988). Nevertheless, the pollutant removal was delayed in ecosystems containing high ratios of activated sludge. Corseuil and Weber (1994) observed that the delay for the degradation of benzene, toluene or xylene varied inversely with the initial number of microbes able to use these compounds, suggesting the need for development of some critical population of microorganisms.
In this study, the survival of C. testosteroni has been promoted by controlled environmental conditions. This specialized bacterial strain was able to enhance pTS degradation and dominate a natural complex microcosm rapidly.
