INTRODUCTION

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2-Mercaptobenzothiazole (MBT) and its derivatives are mainly used as rubber vulcanization accelerators. As a result of MBT manufacture, process waters originate which contain 2-hydroxybenzothiazole (OBT), benzothiazole (BT), and benzothiazole-2-sulfonate (BTSO₃), apart from MBT itself. A biological purification of such wastewaters is often considered to be problematic (1, 12, 13), probably due to the toxic effects ascribed to MBT (5, 6, 13-15). This might explain why only a few benzothiazole-degrading organisms are reported in the literature and why information on benzothiazole degradation pathways is scarce. Our own research led to the isolation of Rhodococcus rhodochrous OBT18, which is capable of OBT and BT degradation (4).

Recently, Gaja and Knapp (8) described a Rhodococcus strain PA, growing on BT as the sole source of carbon, nitrogen, and energy, and strain TA, growing on 2-aminobenzothiazole. For MBT, no degrading organisms have been isolated in pure culture so far, although many axenic bacterial soil and water isolates can transform MBT into the relatively stable methylthiobenzothiazole (7). As for BTSO₃, only mixed cultures which at high inoculum densities degraded this compound as the sole source of carbon and nitrogen have been obtained (11).

In this study we report for the first time on a pure culture, isolated on BTSO₃, and we propose the initial transformation steps in benzothiazole biodegradation.

	MATERIALS AND METHODS

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Materials. Benzothiazoles were kindly provided by Bayer Antwerpen N.V.

Media. Mineral medium MM1% was prepared as described earlier (4). In order to obtain a sulfate-free medium, sulfate salts were replaced with the respective chloride salts. For solid media, 18 g of agar was added per liter.

Isolation and identification of BTSO₃-degrading organisms. Activated sludge samples were obtained from a full-scale purification plant treating benzothiazole-containing wastewater or from laboratory fed-batch reactors treating MBT-BT substrate combinations. A 10-fold dilution series of the sludge samples in 1% (wt/vol) NaCl was inoculated in MM1% amended with 50 mg of BTSO₃ liter¹. The same procedure was repeated several times for the lowest dilution still showing BTSO₃ degradation. Stable BTSO₃-degrading cultures were obtained; these cultures were regularly transferred to fresh medium, using a 10% (vol/vol) inoculum. Finally, culture samples were plated on solid MM1% with 50 mg of BTSO₃ liter¹. Colonies were selected, purified on the same medium, and after growth on nutrient agar, transferred to liquid MM1% with 50 mg of BTSO₃ liter¹ to check for their BTSO₃ degradation capacity. Several BTSO₃-degrading isolates were then identified by standard Gram staining, catalase, and oxidase tests, by their BIOLOG metabolic profiles (Biolog, Inc., Hayward, California), and by their fatty acid methyl ester (FAME) profiles (Microbial ID, Inc., Newark, Delaware) (16).

Maintenance of isolates. Since the BTSO₃ degradation capacity was not lost after growth on nutrient agar, the isolates were routinely maintained on this medium. For the experiments with isolate BTSO₃1, cells were transferred to nutrient broth (NB) test tubes and subsequently to Erlenmeyer flasks. They were grown for 36 h at 28°C, centrifuged (30 min, 10,000 × g, 4°C), washed three times with salt solution (1% [wt/vol] NaCl), and diluted to an optical density at 595 nm (OD₅₉₅) of between 0.2 and 0.3. From this suspension, 2-ml amounts were used to inoculate 100 ml of appropriate experimental media. Strain OBT18, degrading OBT and BT (4), was maintained and cultured under the same conditions.

Growth of the isolates on benzothiazoles. MM1% was amended with 25 mg of BT liter¹ and 50 mg of MBT, OBT, or BTSO₃ liter¹ alone or in combinations. After inoculation with strain BTSO₃1, samples were taken at regular intervals and scored for OD₅₉₅ and BT concentrations. Noninoculated blanks were provided, and all incubations were done on a linear shaker at 28°C.

Resting cell experiments. Strain OBT18 or BTSO₃1 was grown in a final volume of 800 ml of NB in 2-liter Erlenmeyer flasks. After 24 h of growth, cells were harvested (30 min, 10,000 × g, 4°C) and resuspended to an OD₅₉₅ of 5 in MM1% with 1 mM concentrations of different benzothiazole substrates. As controls, a cell suspension in MM1% without any benzothiazoles and noninoculated blanks were included. In aerobic conditions, incubations were in shaken Erlenmeyer flasks. For anaerobic incubations, vials were closed with rubber stoppers and made anaerobic by flushing with N₂ for 20 min. At regular intervals, 1-ml samples were withdrawn, centrifuged for 5 min at 10,000 × g, and analyzed for remaining benzothiazole concentrations.

Isolation of an unknown intermediate compound. For the isolation of the unknown metabolite, a resting cell experiment was performed in a similar way as described above but on a larger scale. Strain OBT18 cells, harvested from 3 liters of NB, were resuspended in 600 ml of MM1% amended with 1 mM OBT. After 4 h of aerobic incubation, cells were removed by centrifugation (30 min, 10,000 × g) and the supernatant was analyzed by high-pressure liquid chromatography (HPLC) to check for the disappearance of OBT and the presence of the intermediate compound. The supernatant was then acidified to pH 1 with concentrated HCl, and after the addition of 10 g of NaCl, a 50-ml aliquot was extracted four times with 25 ml of diethyl ether. The organic phases were combined and concentrated by evaporation in vacuo. Thin-layer chromatography (TLC) on silica gel plates (20 by 20 cm; granule diameter, 200 µm; Merck, Overijse, Belgium) with a 70:30 (vol/vol) ethyl acetate-chloroform solvent system showed the presence of four spots under UV light. After preparative TLC under similar conditions, the four separated bands were removed from the TLC plate. The adsorbed compounds were eluted with a 70:30 (vol/vol) ethyl acetate-acetonitrile mixture, and each fraction was then evaporated to dryness in vacuo.

Analytical methods. Benzothiazoles were determined by HPLC analysis by the method of De Vos et al. (3). Alternatively, HPLC analyses were performed in similar conditions on a Lichrosorb RP18 column with a mobile phase of 35:65 (vol/vol) acetonitrile-water to which the commercially available ion pair reagent Pic A (Fluka) was added.

	RESULTS

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Isolation and characterization of a BTSO₃-degrading isolate. The starting material for the isolation of benzothiazole-degrading organisms, was activated sludge from a full-scale wastewater treatment plant and from lab-scale reactors fed with benzothiazoles. After several unsuccessful isolation attempts, dilution to extinction in mineral medium with 50 mg of BTSO₃ liter¹ proved to be a suitable method for the isolation of BTSO₃-degrading organisms. Several axenic cultures were obtained, using BTSO₃ as the sole source of carbon, nitrogen, and energy. They were all gram-positive, oxidase-negative, and catalase-positive bacteria, and all had similar morphological characteristics. By using BIOLOG data (similarity index of >0.86) and FAME analysis (similarity index of >0.50), they were tentatively identified as R. erythropolis strains.

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View larger version (18K): [in this window] [in a new window]   FIG. 1.   Biodegradation of 600 mg of BTSO3 liter1 (2.8 mM) by R. erythropolis BTSO31 in a sulfate- and ammonium-free mineral medium. Growth was measured as OD595. Symbols: , BTSO3; , sulfite; +, sulfate; *, ammonium; , growth expressed as OD595.

Biotransformation of other benzothiazole substrates by R. erythropolis BTSO₃1. Strain BTSO₃1 was inoculated in MM1% with 50-mg/liter concentrations of various benzothiazoles as the sole sources of carbon, nitrogen, and energy. Growth and concomitant substrate disappearance were observed on OBT, BT, and BTSO₃. However, the lag phase for BT degradation was generally longer than for OBT or BTSO₃ degradation, and in some experiments, no BT disappearance was observed at all. During incubations on MBT, the medium turned yellow, and later, a reddish precipitate was formed, although no MBT turnover could be seen by HPLC analysis.

View larger version (17K): [in this window] [in a new window]   FIG. 2.   Growth of strain BTSO31 on a mixture of benzothiazole substrates. The initial OBT, BT, and BTSO3 concentrations were 0.33, 0.19, and 0.23 mM, respectively. Growth was measured as OD595. Symbols: , BT; , OBT; , BTSO3; , growth expressed as OD595.

Benzothiazole biodegradation pathways. The newly isolated strain BTSO₃1 degrades OBT, BT, and BTSO₃. Earlier we characterized the isolate OBT18, capable of BT and OBT degradation only (4). We now wanted to study and compare the biodegradation pathways of the different benzothiazole substrates in these two strains. To this end, either strain BTSO₃1 or OBT18 was grown on NB and used for resting cell experiments. For incubations with strain BTSO₃1 under aerobic conditions, the following observations were made. MBT concentrations did not change, although a yellow compound was formed in the medium. Incubations with BT led to the transient accumulation of OBT (determined in two different chromatographic systems with coelution of OBT standards), which reached a maximum concentration right before all BT had disappeared from the medium (Fig. 3). During incubations with OBT or BT, an unknown compound reached a maximal concentration when the original substrate was almost exhausted and was subsequently degraded as well (Fig. 4). Based on its retention time in the chromatographic system, this intermediate must be of a rather high polarity, like BTSO₃. In anaerobic resting cell assays, BT, MBT, and OBT concentrations remained constant throughout the test period, whereas BTSO₃ was transformed into OBT in nearly equimolar amounts (Fig. 5).

View larger version (16K): [in this window] [in a new window]   FIG. 3.   Aerobic transformation of 1 mM BT by resting cells of strain BTSO31. Similar results were obtained in three independent experiments.

View larger version (18K): [in this window] [in a new window]   FIG. 4.   Transient accumulation of an unknown intermediate during the aerobic transformation of 1 mM OBT by resting cells of strain BTSO31. The concentration of the intermediate was calculated by using the response factor of BTSO3 in the chromatographic system. Similar results were obtained in three independent experiments.

View larger version (15K): [in this window] [in a new window]   FIG. 5.   Anaerobic transformation of 1 mM BTSO3 by resting cells of strain BTSO31. Similar results were obtained in three independent experiments.

View larger version (11K): [in this window] [in a new window]   FIG. 6.   Mass spectrum of substance 2 (see Results). Based on the mass of the molecular ion, a dihydroxybenzothiazole structure was proposed. After an acetylation reaction, substance 2 was acetylated twice, which confirms this structure.

	DISCUSSION

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As opposed to BTSO₃ and MBT, OBT and BT have generally been considered to be easily biodegradable (1) and axenic cultures degrading these compounds are available. R. rhodochrous OBT18 was isolated on OBT (4) and Rhodococcus strain PA was isolated on BT (8). They both degrade BT and OBT, but not MBT. BTSO₃ breakdown was not tested for strain PA and was not observed for strain OBT18. Thus, it seemed that at least the OBT and BT degradation pathways are closely related. In view of a comprehensive study of benzothiazole-biodegradative pathways, we also wanted to include a MBT degrader and a BTSO₃ degrader in the biodegradation studies. Although MBT is degraded in full- and lab-scale activated sludge systems, mixed or pure cultures with stable degradation properties have not yet been obtained. However, after several unsuccessful attempts, we have now succeeded in the isolation of a BTSO₃ degrader. The isolate BTSO₃1 was identified as an R. erythropolis strain and is also capable of OBT and BT mineralization. These results strongly suggest that the BTSO₃ degradation pathway overlaps or converges with the OBT and BT breakdown route. Compared to strain OBT18, strain BTSO₃1 has more enzymes, enabling the transformation of BTSO₃ into an intermediate that is probably common for OBT, BT, and BTSO₃ degradation. During incubations on MBT, colored products were formed, although the MBT concentrations hardly changed. Similar observations were made for strains OBT18 (4) and PA (8), which do not degrade BTSO₃. These data probably indicate that the specific enzymes involved in OBT and BT degradation can attack MBT but that either MBT itself or a toxic intermediate product prevents its further degradation. This might also explain the observed inhibitory effect of MBT on OBT and BT degradation by strains OBT18 (4) and on OBT, BT, and BTSO₃ degradation by strain BTSO₃1.

BTSO₃ degradation by R. erythropolis BTSO₃1 was not affected by changes in substrate concentration, pH, or salinity within a certain range and should therefore not pose problems in the environment. When strain BTSO₃1 was grown in a sulfate- and ammonium-free medium amended with BTSO₃, the substrate was completely mineralized. BTSO₃ can therefore be used as the sole source of carbon, nitrogen, sulfur, and energy. Nitrogen was found as ammonium and sulfur mainly as sulfate (Fig. 1). However, since sulfite was temporarily detected during growth, the sulfonate- and/or thiazole ring sulfur is presumably released as sulfite and subsequently oxidized to sulfate. In the stationary phase of growth, the molar ratio of BTSO₃ degraded/ammonium produced/sulfate produced was 1:0.5:0.6. Compared to the theoretical maximum ratio of 1:1:2, 50% of the nitrogen and 70% of the sulfur are not accounted for. This might be due to assimilation or release of nitrogen and sulfur in an unknown form. In contrast, Mainprize et al. (11) measured the expected stoichiometric amounts of ammonium and sulfate after growth of a mixed culture on BTSO₃.

The metabolism of heterocyclic compounds very often involves ring hydroxylations, followed by ring cleavage. When attached to six-membered rings, five-membered rings are usually cleaved first, with or without initial hydroxylation (9). In addition, for sulfonated aromatic compounds, at least four desulfonation mechanisms have been elucidated (reference 10 and references therein): an NADH-linked dioxygenation, a monooxygenation, a hydrolytic desulfonation, and a meta ring cleavage-associated desulfonation. All of these involve (in)direct oxygenation. By analogy with the degradative pathways mentioned, it was assumed that the hydroxylated benzothiazole OBT might be an intermediate in BT and BTSO₃ degradation. From sequential induction experiments and respirometry, no decisive evidence was obtained in (dis)agreement with this assumption (results not shown). However, in resting cell assays, OBT accumulation was observed during incubations on BT under aerobic conditions (Fig. 3). Since BT and OBT were not degraded in anaerobic assays, the transformation of BT into OBT definitely requires molecular oxygen and is probably catalyzed by an oxidase or a monooxygenase enzyme. BTSO₃ was mineralized under aerobic conditions and was transformed into equimolar amounts of OBT during anaerobic assays (Fig. 5). OBT therefore is an intermediate in BTSO₃ degradation as well, and in this case the hydroxyl group probably originates from water. Moreover, it seems that the introduction of the hydroxyl function occurs simultaneously with the elimination of the sulfonate group. Otherwise, BT, rather than OBT, would have accumulated during anaerobic incubations on BTSO₃. Both on OBT and BT and in aerobic conditions only, the transient accumulation of an unknown intermediate could be detected. It was identified as a diOHBT (Fig. 6), but the exact position of the second hydroxyl function could not be determined by mass spectroscopy. So far, the presence of a diOHBT product with the second hydroxyl group on the benzene nucleus has been clearly demonstrated. Preliminary evidence indicates that a second intermediate with a labile N-O or S-O was also present. In the future, larger amounts of both products will have to be isolated and they will have to be separated to allow for infrared and nuclear magnetic resonance spectroscopic analyses and to determine their exact chemical structure.

In summary, Figure 7 shows the initial transformations during benzothiazole biodegradation, as described above. All pathways converge into OBT. OBT is hydroxylated into diOHBT, which is in turn further degraded. This scheme is consistent with the observation that OBT degradation by strain BTSO₃1 never posed problems, whereas BT and BTSO₃ degradation sometimes did not take place. This might have been due to the malfunction of one or more initial enzymes operational in BT and BTSO₃ transformation into OBT or to a low level of expression or the loss of genes encoding for these enzymes. The biodegradation scheme also confirms our earlier assumption that R. erythropolis BTSO₃1 has more genetic information than R. rhodochrous OBT18 does. Except for the BTSO₃ branch, the whole degradation scheme proposed for strain BTSO₃1, also applies for OBT18, but it remains to be investigated whether identical enzymes are involved in both rhodococci.

View larger version (9K): [in this window] [in a new window]   FIG. 7.   Initial transformations in benzothiazole biodegradation by R. erythropolis BTSO31, as derived from the experimental results.

For the most problematic compound, MBT, no degradation occurs with the presented enzymatic potential. Whether this is due to high substrate specificities of the enzymes involved is hard to tell, but since OBT, BT, and BTSO₃ degradation were inhibited by MBT, it is more likely that MBT itself or a transformation product inhibits a common enzyme in the reaction sequence.