Environ. Sci. Technol. 1992,26, 1759-1764
Revzan, K. L.; Fisk, W. J.; Gadgil, A. J. Indoor Air 1991, 1 (2), 173-189. Forchheimer, P. H. Z. Ver. Dtsch. Ing. 1901,45,1782-1788. Patankar, S.V. Numerical heat transfer and fluid flow; Hemisphere Publishing: New York, 1980. Scheidegger, A. E. The physics of flow through porous media, 2nd ed.; Macmillan: New York, 1960. Received for review February 24, 1992. Revised manuscript received May 14,1992. Accepted May 15,1992. This research
was supported at Indoor Environment Program of Lawrence Berkeley Laboratory by Bonneville Power Administration (BPA) through Interagency Agreement DE-AI79-9OBPO6649, and by the Assistant Secretary for Conservation and Renewable Energy, Office of Building Technologies, Building Systems and Materials Division of the U.S. Department of Energy under Contract DE-AC03- 76SF00098. This research was also supported at Washington State Energy Office,Energy Extension Service, by BPA Coow Agreement DE-FC79-82BO34623 Modification A027 (WSE 6 0 4 f Partial support for Y.C.B.'sresearchparticipation was granted by ENTPE of Lyon, France.
Diones, Sulfoxides, and Sulfones from the Aerobic Cometabolism of Methylbenzothiophenes by Pseudomonas Strain BTI SanJa Saftiet and Phillip M. Fedorak"
Department of Microbiology, University of Alberta, Edmonton, Alberta T6G 2E9, Canada Jan T. Anderssonz
Department of Analytical and Environmental Chemistry, University of Uim, Postfach 4066, D-7900 Ulm, Germany Six methyl-substituted benzothiophenes were synthesized and used as substrates for Pseudomonas strain BT1. These compounds would not support growth but were cometabolized when the culture was grown on 1methylnaphthalene or glucose. Sulfur-containing metabolites were identified by GC-MS and GC-FTIR and, when possible, by comparison with authentic standards. 2,3Diones were the metabolites from 4-methylbenzothiophene, 5-methylbenzothiophene, and 6-methylbenzothiophene. Sulfoxides were the major products from 2methylbenzothiophene and 2,3-dimethylbenzothiophene. Sulfones were also detected as metabolites of the latter two compounds. Cometabolism of 7-methylbenzothiophene yielded the corresponding 2,3-dione,sulfoxide, sulfone, and several other unidentified compounds. Thus, the nature of the metabolites from these methylbenzothiophenes was dependent on the position of the methyl substitution. Introduction A wide variety of organosulfur compounds, specifically sulfur heterocycles, are present in many crude oils and coal tars. Their distribution and character depend on the source, maturity, and other alteration processes (1). However, although hundreds of the organosulfur compounds have been identified in petroleum, little is known about the implications of their release into the environment (2).This is an issue of increasing concern because they are recognized as persistent (3, 4 ) and potentially harmful environmental pollutants ( 5 , 6 ) . In comparison to the extensive investigations on the microbial metabolism of hydrocarbons and their removal from contaminated environments, little is known about the susceptibility of organosulfur compounds to microbial transformation. A recent review (2) showed that only a small number of known organic sulfur compounds have been used in biotransformation studies because most of the identified compounds are not commercially available. Laboratory experiments with petroleum-degrading bacterial cultures grown on crude oil have shown that the alkylbenzoPresent address: Institut fiir Biotechnologie 3, Forschungszentrum Jiilich GmbH, Postfach 1913, 5170 Jiilich, Germany. Present address: Analytical Chemistry Department, University of Miinskr, Wilhelm-Klemm-Straase8, D-4400 Mtinster, Germany. 0013-936X/92/0926-1759$03.00/0
thiophenes were not found in the aromatic fraction of the oils (7-9). Similarly, analyses of petroleums that have undergone biodegradation in their reservoirs have shown the absence of alkylbenzothiophenes (9,IO). However, only a few biotransformation products of benzothiophenes have been identified. There have been several reports on the aerobic cometabolism of benzothiophene (2). The transformation products showed oxidation of the thiophene ring at different positions, but no cleavage of either aromatic ring. Benzothiophene and another commercially available compound, 3-methylbenzothiophene (3-MBT), were the focus of a study on the cometabolism of benzothiophenes in the presence of 1-methylnaphthalene (1-MN) (11). Cometabolism of benzothiophene yielded benzothiophene-2,3dione whereas that of 3-methylbenzothiophene yielded predominantly the sulfoxide of 3-methylbenzothiophene with a small amount of the corresponding sulfone. For this study, six methylbenzothiophenes were synthesized and used as substrates for a pure bacterial culture grown on 1-MN or glucose. The major focus of the study was the identification of the sulfur-containing biotransformation products. Experimental Section Chemicals. The methods for synthesis of 2-methylbenzothiophene (2-MBT), 2-methylbenzothiophene S,Sdioxide (8-MBT sulfone), 5-methylbenzothiophene (5MBT), 7-methylbenzothiophene (7-MBT), 2,3-dimethylbenzothiophene (2,3-DMBT), and a mixture of 4methylbenzothiophene (4-MBT) and 6-methylbenzothiophene (6-MBT) are given by Andersson (12). 4-MBT was prepared from 2-methylbenzaldehyde (13). Sulfones of 7-MBT and 2,3-MBT were synthesized (14). 5Methylbenzothiophene-2,3-dioneand 7-methylbenzothiophene-2,3-dione were also Synthesized (15). m-Tolyl methyl sulfoxide and m-tolyl methyl sulfone were prepared according to the procedures of Cerniani and Modena (16). Transformations of Methylbenzothiophenes with 1-MN as Growth Substrate. The bacterial strain used for these experiments was isolate BT1, a Pseudomonas sp. that can mineralize aromatic hydrocarbons but not aliphatic hydrocarbons (11). For each biotransformation study, 10 mL of a BT1 maintenance culture grown on 1-MN was used to inoculate 200 mL of liquid mineral
0 1992 American Chemical Society
Envlron. Sci. Technol., Vol. 26, No. 9, 1992 1759
medium (8)supplemented with a trace metal solution (11), 50 CLLof 1-MN, and a few milligrams of a methylbenzothiophene. 1-MN was chosen as the growth substrate because it is a convenient solvent for the methylbenzothiophenes and the intermediates of 1-MN metabolism have been identified (In,so these can be distinguished from possible metabolites from the methylbenzothiophenes. All cultures were incubated for 14 days at 28 "C with shaking. Appropriate sterile controls were established for each methylbenzothiophene. Transformations of Methylbenzothiophenes with Glucose as Growth Substrate. To determine whether the methylbenzothiophenes could be transformed by strain BT1 growing on a substrate other than an aromatic hydrocarbon, the culture was grown on 250 mg of glucose/L of mineral medium with trace metals in the presence of the appropriate sulfur heterocyclic compound. A suspension of BT1 grown in liquid glucose medium and washed with sterile phosphate buffer (0.03 M, pH 7.2) was used as inoculum. These 200-mL cultures were also incubated for 14 days before extraction. Analytical Methods. After incubation, the cultures were acidified with sulfuric acid to pH < 2 and extracted with methylene chloride (four times 20 mL) to recover substrates and products. The extracts were initially analyzed by capillary GC using a DB-5 column in an instrument equipped with a flame ionization detector (FID) and a sulfur-selective flame photometric detector (FPD) (11).
Routine GC-MS analyses were done with a HewlettPackard Model 5890 GC fitted with a DB-1 column and coupled to a 5970 series mass selective detector. Highresolution and chemical ionization GC-MS were done in the Mass Spectrometry Laboratory, Chemistry Department, University of Alberta, using the conditions outlined elsewhere (18). Isobutane or ammonia was used as the reagent gas for chemical ionization GC-MS. GC-FTIR analyses (11)were done in the Spectral Services Laboratory, Chemistry Department, University of Alberta. The GC-MS was equipped with a 30-m DB-5 column; the GC-FTIR system used a 30-m Ultra 2 column (HewlettPackard). When sulfones were detected by GC analysis, their presence in the culture extract was verified by HPLC analysis because methylbenzothiophene S-oxides decompose in GC injection ports yielding the parent methylbenzothiophene and the corresponding sulfone (19). Details of the HPLC method are given elsewhere (19). Acetonitrile/water (5050) was used as the mobile phase and the retention times of the microbially-produced sulfones were matched to those of authentic standards.
Results Isolate BT1 would not grow on any of the methylbenzothiophenes tested; therefore, it was grown on 1-MN or glucose in the presence of various alkylbenzothiophenes to determine if these sulfur heterocyclic compounds would be cometabolized. On the basis of previous work with benzothiophene and 3-MBT (111, it was predicted that benzothiophenes with methyl groups on the thiophene ring would yield the corresponding sulfoxides and sulfones, whereas those with the methyl groups on the benzene ring would yield 2,3-diones. Although the syntheses of a few sulfoxides of methylbenzothiophenes have been reported (20), in general, these syntheses are difficult because most oxidation methods do not stop at the sulfoxide but yield the sulfone. Thus in this study, the identities of metabolites thought to be sulfoxideswere surmised from GC-MS and GC-FTIR analyses. 1780
Envlron. Scl. Technol., Vol. 26, No. 9, 1992
'1'
a 43
I
b
Wavenumbers (cm-')
Figure 1. Mass spectrum (a) and FTIR spectrum (b) of the major metabolite from 2-MBT.
A few sulfur-containing transformation products were detected in some of the sterile controls. However, the transformation products described below did not appear in the corresponding sterile controls and, therefore, were due to biotransformations. Only those products resulting from microbial cometabolism were identified, and no attempts have been made to identify the products of abiotic transformations. Cometabolism of 2-MBT. GC analysis of the methylene chloride extract from isolate BT1 grown on l-MN and 2-MBT showed two poorly resolved sulfur-containing products. The mass spectrum of the more abundant metabolite (Figure la) had a weak molecular ion of m/z 164. Chemical ionization GC-MS, using isobutane as the reagent gas, verified that the molecular weight of this compound was 164, suggesting that the metabolite was a sulfoxide. The base peak at m/z 147 would result from the loss of OH. Porter (21) suggested that the m/z 147 ion observed in the mass spectra of 2-MBT and 3-MBT was the benzothiopyrilium cation. This ion was also observed for 3-methylbenzothiophene S-oxide (3-MBT sulfoxide) (11).Porter (21)suggested the formula C7HSSfor the fragment at mlz 121. The culture extract was analyzed by GC-FTIR, and the spectrum of the more abundant metabolite showed a single strong absorption at 1080 cm-l (Figure lb) that is characteristic of a sulfoxide (22). This FTIR spectrum is very similar to that of 3-MBT sulfoxide (11).
GC-MS analysis of the culture extract gave no evidence of the presence of 2-MBT sulfone; however, GC-FTIR analysis detected a minor peak on the tail of the sulfoxide peak. The spectrum showed two strong absorptions at 1162 and 1336 cm-l, characteristic of a sulfone (22), and matched that of authentic 2-MBT sulfone. In addition, HPLC analysis of the culture extract showed a peak with the same retention time as the reference 2-MBT sulfone. Cometabolism of 5-MBT. Only one sulfur-containing metabolite was found in the extract of the culture grown in the presence of 5-MBT. GC-MS and GC-FTIR results
139
150
I
3
PI a
121
mlz
rnh 1
.
h
I
I I I
4000
3500
3000
2001
2500
Wavenumbers (cm-')
Flgure 2. Mass spectrum (a) and FTIR spectrum (b) of the metabolite from 5-MBT. Wavenumbers (crn")
Flgure 4. Mass spectrum (a) and FTIR spectrum (b) of 1 from a culture of Pseudomonas strain BT1 grown on 1-MN in the presence of a mixture of 4-MBT and 6-MBT. D
a LL
w 0. 0
d.. c
a, r
a
I
11
J
Ji
0
10
1L
"i
L
20
J 30
J
U 40
L 50
Time (min)
Flgure 3. GC analysis of extract from a culture of Pseudomnas strain BT1 grown on 1-MN in the presence of a mixture of 4-MBT and 6-MBT.
suggested that the metabolite was 5-methylbenzothiophene-2,3-dione. The mass spectrum (Figure 2a) shows a weak molecular ion at m/z 178. The base peak at m/z 150 arose from the loss of CO, the fragment at m/z 121 suggested a subsequent loss of CHO. The spectrum was virtually identical to that of authentic 5-methylbenzothiophene-2,3-dionewhich had the same GC retention time as the metabolite. The FTIR spectrum of the metabolite (Figure ab) showed a strong absorption at 1737 cm-l and matched the strong absorption at 1739 cm-l of authentic 5-methylbenzothiophene-2,3-dione Cometabolism of 4-MBT and 6-MBT. Three sulfurcontaining metabolites were observed in the extract of a BT1 culture grown on 1-MN and the mixture of 4-MBT and 6-MBT (Figure 3). Two of the metabolites, designated compounds 2 and 3, had virtually the same mass spectra and very similar FTIR spectra. Both mass spectra showed a weak molecular ion of m/z 178, a base peak at m/z 150, and a fragment peak at m/z 121, suggesting that these two metabolites were the 2,3-diones of 4-MBT and 6-MBT. This fragmentation pattern was similar to that of 5-methylbenzothiophene-2,3-dione (Figure 2b). The FTIR spectra show a strong absorption at 1728 cm-' with a shoulder at about 1760 cm-l for one compound and a.
-
strong absorption peak at 1738 cm-' with no shoulder for the other. For comparison, the FTIR spectrum of authentic 5-methylbenzothiophene-2,3-dione had a strong absorption at 1739 cm-l, and that of authentic benzothiophene-2,3-dione showed a strong absorption at 1736 cm-l with a shoulder at about 1770 crn-l(II). The GC-MS and GC-FTIR results indicate that 2 and 3 were isomers of methylbenzothiophene-2,3-dione. When the extract from the bacterial culture grown in the presence of the pure preparation of 4-MBT was analyzed, only 2 was detected in the chromatogram. Thus, 2 was 4-methylbenzothiophene-2,3-dione and 3 was 6methylbenzothiophene-2,3-dione. Because 1 was not found in cultures grown in the presence of pure 4-MBT, this metabolite must arise from 6-MBT. The mass spectrum of 1 (Figure 4a) showed a molecular ion of m/z 154, and high-resolution mass spectrometry gave an empirical formula of C8HloOS. The predominant ion observed from chemical ionization GCMS using isobutane as the reagent gas was (M + 1)" 155, confirming that the molecular weight of the metabolite was 154. The FTIR spectrum of this metabolite (Figure 4b) showed a strong absorption at 1099 cm-l, which is characteristic of a sulfoxide, an ether, or a secondary alcohol, although there k no strong 0-H stretch at 3200-3600 cm-'. A culture extract containing 1 was oxidized (14) to give the corresponding sulfone. Electron impact GC-MS showed the molecular weight of the sulfone of the metabolite was 170. This was confirmed by chemical ionization GC-MS using ammonia as the reagent, which gave the (M + 18)' ion at m/z 188. Thus the molecular weight of the sulfone obtained from oxidation of 1 was 16 mass units higher than that of 1. This indicated that 1 was not a sulfide-containing alcohol because the oxidation of a such an alcohol would yield a product with molecular weight of 32 mass units greater than the reactant. These results suggested that 1 was m-tolyl methyl sulfoxide. Indeed, 1 and its oxidation product had the same GC retention times and mass spectra as authentic m-tolyl Environ. Sci. Technol., Vol. 26, No. 9, 1992
1761
Table I. Products of the Cometabolism of Various Benzothiophenes by Pseudomonas Strain BT1 Grown on I-MN o r Glucose" benzothiophene
miz
3-MBTb 2-MBT 2,3-DMBT benzothiopheneb 4-MBT and 6-MBTc
Figure 5. Mass spectrum of the major metabolite from 2,3-DMBT.
methyl sulfoxide and m-tolyl methyl sulfone, respectively. Cometabolism of 7-MBT. GC analysis of this culture extract showed several sulfur-containingcompounds which did not appear in the sterile control. Because of the prevalence of diones, sulfoxides, and sulfones observed with other methylbenzothiophenes, these were the first compounds sought. The mass spectrum of one metabolite showed a weak molecular ion at m/z 178, the parent peak at m/z 150, and a major peak at m/z 121, similar to the spectrum in Figure 2a. The FTIR spectrum showed a strong absorption at 1735 cm-l, which was characteristic of other 2,3-diones observed in these studies. Indeed, this metabolite had the same GC retention time, mass spectrum, and FTIR spectrum as the synthesized 7-methylbenzothiophene2,3-dione. The identity of a second metabolite appeared to be 7-methylbenzothiopheneS-oxide (7-MBTsulfoxide),based on the results of the GC-MS and GC-M'IR analyses. The mass spectrum showed a weak molecular ion at m/z 164, with abundant fragment ions at m/z 147,135,136,148, and 121. The FTIR spectrum showed a strong absorption at 1079 cm-l, which is also consistent with the absorption of a sulfoxide. A third metabolite gave a mass spectrum with a molecular ion at m / 180 ~ and a base peak at m/z 151. The FTIR spectrum showed two strong absorptions at 1334 and 1164 cm-'. These data and the GC retention of the metabolite matched those of the authentic 7-methylbenzothiophene S,S-dioxide (7-MBT sulfone). The presence of the sulfone in the culture extract was confirmed by HPLC analysis. Cometabolism of 2,3-DMBT. Two sulfur-containing metabolites were observed in this culture extract. The major metabolite had a weak molecular ion of m/z 178 and fragment ions at m/z 163 and 145, corresponding to the loss of CH3 followed by the loss of HzO, respectively (Figure 5). The FTIR spectrum showed a single strong absorption at 1070 cm-l, suggesting that the metabolite was 2,3-dimethylbenzothiopheneS-oxide (2,3-DMBT sulfoxide). The mass spectrum of the minor metabolite showed a molecular ion at m/z 194 and a base peak at m/z 151. The F"IR spectrum showed two strong absorptions at 1170 and 1323 cm-l. GC-MS and GC-FTIR analyses of the synthesized 2,3-dimethylbenzothiopheneS,S-dioxide (2,3DMBT sulfone) yielded spectra that matched those of the metabolite. HPLC analysis of the culture extract verified the presence of the sulfone. Cometabolism of Methylbenzothiophenes with Glucose as Substrate. The diones, sulfoxides, and sulfones identified as metabolites in the cultures grown on 1-MN in the presence of the methylbenzothiophenes were also detected in the respective cultures grown on glucose. m-Tolyl methyl sulfoxide was also found in the cultures grown on glucose in the presence of the mixture of 4- and 6-MBT. Thus, the biotransformations of these methylbenzothiophenes by strain BT1 were not dependent on the 1762 Environ. Sci. Technot., Vol. 26, No. 9, 1992
4-MBT 5-MBT 7-MBTd
2,3-dione -
-
+++ +++and +++ +++ +++ +
sulfoxide
+++ +++ +++
sulfone 4-
+ 4-
-
-
-
-
-
+++
+++
'-, not detected; +++, major metabolite detected; +, small quantity detected. *From ref 11. 'One other metabolite, identified as rn-tolyl methyl sulfoxide, was found. dSeveral other sulfur-containing metabolites were detected but not identified.
presence of an aromatic hydrocarbon. Discussion Diones, sulfoxides, and sulfones have been observed as bacterial transformation products of thiophenes in previous studies. Benzothiophene S-oxide and benzothiophene2,3-dione were identified as metabolites of the cometabolism of benzothiophene (23). Similarly, dibenzothiophene S-oxide (24),dibenzothiophene S,S-dioxide (28, and benzothiophene-2,3-dione(23) were produced from dibenzothiophene. Enzymatic oxidation of thiaarenes by cytochrome P450-dependent mixed-function oxidases results predominantly in the formation of sulfoxides and sulfones rather than ring-oxidation products such as phenols and dihydrodiols (6). Table I summarizes the products of the cometabolism of methylbenzothiophenes by Pseudomonas strain BT1 found in this and a previous study (11). With the exception of 7-MBT, the results show that the cometabolism of those methylbenzothiophenes with methyl groups on the thiophene ring yielded the corresponding sulfoxides and sulfones, whereas those with the methyl groups on the benzene ring yielded 2,3-diones. 7-MBT yielded a variety of metabolites including the corresponding sulfoxide, sulfone, and dione. The identities of the other metabolites have not been determined. Because of the limited amounts of the synthesized methylbenzothiophenes available, the aim of this study was to identify products of their cometabolism by strain BT1 grown under standard conditions (i.e., 28 OC for 14 days). Interestingly, recent results with benzothiophene and 3MBT indicate that the biotransformaticms occur within 2-4 days (unpublished data). Experiments to obtain quantitative results on the amounts of the methylbenzothiophenes biotransformed were not done because of the relatively large quantities of methylbenzothiophenes required for sterile controls to account for any evaporation of the sulfur compounds and for replicate cultures. In addition, quantitative studies were hampered by the lack of the authentic sulfoxides and some of the 2,3-diones. After the 14-day incubation period, little 5-MBT was detected in the extract of the culture grown in the presence of that compound. The corresponding 2,3-dione was the predominant sulfur-containing compound found. In contrast, relatively small amounts of the oxidized metabolites were detected in the extracts of cultures that received the other organosulfur compounds. In these cases, the parent methylbenzothiophenes were the major sulfur-containing compounds found. For example, the chromatogram in Figure 3 shows relatively small amounts of the biotransformation products (1,2, and 3) compared to the amount
of the 4-MBT that remained in the culture. Similar results were obtained for 2-MBT, 7-MBT, and 2,3-DMBT. Studies with benzothiophenes (11, 23) and dibenzothiophenes (24, 26) indicate that both the benzene and thiophene rings in unsubstituted and substituted benzothiophenes appear to be resistant to cleavage by aerobic microorganisms. Only one report (27) indicated that the substituted benzothiophene, 3-hydroxy-2-formylbenzothiophene, a common metabolite from dibenzothiophene (24, 26), was subject to further biotransformation. The appearance of rn-tolyl methyl sulfoxide in cultures grown in the presence of 4-MBT and 6-MBT indicated that the benzothiophene ring in the latter compound was broken. However, the absence of any C-0 bonds in the ringcleavage product is unusual, and the mechanism by which this compound was formed is unknown. Although the photochemical transformation of benzothiophene to benzothiophene-2,3-dionein aqueous solution has been demonstrated (28), no 2,3-diones of the methylbenzothiophenes were formed in our sterile controls under the incubation conditions used. Abiotically, benzothiophene-2,3-dione hydrolyzes to (2-mercaptopheny1)glyoxylic acid and subsequently oxidizes photochemically to 2-sulfobenzoic acid, which is too polar to be recovered by solvent extraction (28). If the same aboitic reactions occurred with the 2,3-diones formed from biotransformations summarized in Table I, the extraction methods used in these experiments would not have recovered the corresponding sulfobenzoic acids. The analytical methods and culture incubation conditions used in this investigation influenced the metabolites that were detected and identified. For example, only those metabolites that could be extracted into methylene chloride from acidified 200-mL cultures and only those that were amenable to the GC method used were detected. If sulfobenzoic acids (mentioned above) were formed, these would not be detected. In addition, any sulfur-free metabolites that might have been produced would not have been identified because this work focused on compounds detected by the sulfur-selective FPD. However, because the thiophene ring in substituted benzothiophenes is quite persistent in microbial cultures (11,23,24,26),its seems unlikely that the cometabolism of the methylbenzothiophenes would lead to sulfur-free products. Studies with indole (29-32) and indene (33,34), which are analogs of benzothiophene, showed that the fivemembered ring is more susceptible to oxidation than the benzene ring. The 2,3-diones, sulfoxides, and sulfones identified in our study illustrate that the five-membered ring in the methylbenzothiophenesis also more susceptible to microbial oxidation than the benzene ring. When indole served as a growth substrate, indole-2,3-dione (isatin) was found in denitrifying (31)and methanogenic cultures (35) but not in aerobic cultures (30). However, in the latter case, the Alcaligenes sp. which used indole as its sole source of carbon and nitrogen was able to be grown on indole-2,3-dione, leading to the conclusion that it was an intermediate that did not accumulate to a detectable concentration (30). Investigations with the benzothiophene analogs have shown or implied that hydroxylated and monoketones are oxidation products. For example, 3-hydroxyindole (indoxyl) was detected in a culture of Aspergillus niger that cometabolized indole when grown on glucose (32) and 3-hydroxyindole was readily oxidized by an indole-grown Alcaligenes sp.; thus 3-hydroxyindolewas presumed to be an early intermediate. 2,3-Dihydroxy-2,3-dihydroindole was inferred as an intermediate in the conversion of indole
to indigo by a recombinant Escherichia coli strain that contained the naphthalene dioxygenase gene from Pseudomonas putida (36). 2,3-Dihydroxyindole was hypothesized to be an intermediate because it wm rapidly oxidized by indole-grown, Gram-positive coccus (29). Diols were also detected during the oxidation of indene: a rat liver microsomal preparation yielded trans-1,2-indandiol (33) whereas P. putida strain F39/D yielded l-indenol and cis-1,2-indandiol(34). When strain F39/D was incubated with indene for longer than 1h, l-indanone was found in the culture. Although none was detected, it is presumed that hydroxylated methylbenzothiopheneswere precursors to the 2,3-diones identified in our study. However, the long 14-day incubation period would have precluded the detection of these transient intermediates. The metabolites of methylbenzothiophenes identified in this study are more polar and more water-soluble than their parent compounds. If the products of alkylbenzothiophene biotransformations prove to be more toxic than the parent compounds, the increased water solubility could pose an environmental problem because water-soluble compounds are more likely to migrate from a contaminated site and enter groundwater systems. Alternatively, if these metabolites can be mineralized by microorganisms, their increased solubility would be advantageous because they would be more readily available to the microbial population. To date, mineralization of these methylbenzothiophene-2,3-dionesand their sulfoxides and sulfones has not been demonstrated. Future studies must determine if the biotransformation products of methylbenzothiophenes reported herein are commonly produced by other microorganisms or whether they are unique to Pseudomonas strain BT1. If these are commonly produced metabolites that are not readily mineralized, the toxicity of alkylbenzothiophene metabolites should be investigated. Because of the difficulties involved in the chemical syntheses of methylbenzothiophene S-oxides, microbial cometabolism of appropriate methylbenzothiophenes may be an alternative method to produce the sulfoxides for toxicity studies. Acknowledgments We thank Debbi Coy and Jeff Gerard for technical assistance and s.E. Hrudey for access to his GC with a mass selective detector. Registry No. 4-MBT, 14315-11-8;5-MBT, 14315-14-1;&MBT, 16587-47-6; 7-MBT, 14315-15-2; 3-MBT, 1455-18-1; 2-MBT, 1195-14-8; 2,3-DMBT, 4923-91-5; 1-MN, 9014-10-2; 2-MBT sulfide, 33945-86-7; 2,3-DMBT sulfoxide, 70445-88-4; 7-MBT 2,3-dione, 57780-58-2; 5-MBT 2,3-dione, 50891-89-9; m-tolyl methyl sulfoxide, 13150-71-5; glucose, 50-99-7.
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Received for review February 20, 1992. Revised manuscript received M a y 15, 1992. Accepted M a y 18, 1992. Financial support was provided by Canada Centre for Mineral and Energy Technology, Energy Mines and Resources Contract 23440-09163101-SS and by the Natural Sciences and Engineering Research Council of Canada.
Accumulation of Cobalt, Zinc and Manganese by the Estuarine Green Microalga Chlorella salina Immobilized in Alginate Microbeads Geoffrey W. Garnham, Geoffrey A. Codd, and Geoffrey M. Gadd"
Department of Biological Sciences, University of Dundee, Dundee, DD1 4HN, Scotland, U.K. This paper describes cobalt, zinc, and manganese accumulation by Chlorella salina immobilized in calcium alginate microbeads, investigated by use of the radioisotopes 6oCo, 54Mn,and 65Zn. A rapid biosorption of the metals to C. salina cell walls and the alginate matrix, which was independent of light, temperature, or the metabolic inhibitor CCCP, was followed by a slower energy-dependent phase of uptake. Under similar conditions, immobilized cells accumulated greater amounts of Co,Zn, or Mn than free cells due to an increased active phase of uptake. Accumulation was also dependent on cell density in the alginate beads, with a greater uptake of cobalt at the highest cell densities. Desorption of cobalt from loaded beads was increased by decreasing pH and increasing concentrations of the cations, probably due to exchange of cobalt bound to the cell wall/alginate matrix for H+ or cations. H
Introduction Studies with immobilized microalgae and other photosynthetic microorganisms have mainly focused on the use of immobilized cells for biotransformations and biosynthesis of organic materials (1,2).However, several groups have investigated whether living immobilized microor1764 Environ. Scl. Technol., Vol. 26, No. 9, 1992
ganisms could be useful for the removal of metals and radionuclides from contaminated wastewaters and effluents (3, 4). Immobilized cell systems possess several advantages over freely suspended cells in both batch and continuous-flow systems, including better capability of reuse and regeneration of the biomass, easy separation of cells from the reaction mixture, high biomass loadings within a given bioreactor, manipulation of biomass independent of dilution rate, and minimal clogging in continuous-flow systems (5). Work involving the use of immobilized algae for removal of metals from solution has mainly concentrated on dead cells. For example Nakajima et al. (6) used polyacrylamide-entrappedChlorella vulgaris to remove UO$+,Au3+,Cu2+,Hg2+,and Zn2+,while Darnall et al. (7, 8) used C.vulgaris cells immobilized in a silica gel matrix for removal of A13+,Be2+,Cu2+,Pb2+,Ni2+,Zn2+, Cr3+,Co2+,Fe2+,U022+,Ag+, Hg2+,and Mn2+from solution. Such studies have shown that immobilized dead algae can accumulate at least as much metal as freely suspended dead cells. However, relatively few studies have used living algal cells in immobilized systems for metal removal, and to our knowledge, none has examined active uptake, although there are several examples where greater metal uptake by immobilized, as compared with freely suspended, cells has been demonstrated (9-11).
0013-936X/92/0926-1764$03.00/0
0 1992 American Chemical Society
