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