Manfred van Afferden l, Sigrid Schacht l, Jiirgen Klein l, and Hans G. Triiper 2. 1 DMT-Gesellschafl ftir Forschung und Prtifung mbH, Franz-Fischer-Weg 61, ...
Arch Microbiol (1990) 153 : 324- 328
Archives of
Hicrnbinlngy 9 Sprmger-Verlag1990
Degradation of dibenzothiophene by Brevibacterium sp.DO Manfred van Afferden l, Sigrid Schacht l, Jiirgen Klein l, and Hans G. Triiper 2 1 DMT-Gesellschafl ftir Forschung und Prtifung mbH, Franz-Fischer-Weg 61, D-4300 Essen 13, Federal Republic of German~ 2 Instltut f/Jr Mikroblologie der Rheinischen Friedrich-Wilhelms-Universit~it,Meckenheimer Allee 168, D-5300 Bonn 1, Federal Republic of Germany Received July 26, 1989/Accepted November 11, 1989
Abstract. Dibenzothiophene, a polycyclic aromatic sulfur heterocycle, represents as a model compound the organic sulfur integrated in the macromolecular coal matrix. A pure culture of a Brevibacterium species was isolated, which is able to use dibenzothiophene as sole source of carbon, sulfur and energy for growth. During dibenzothiophene utilization sulfite was released in a stoichiometrical amount and was further oxidized to sulfate. Three metabolites of dibenzothiophene degradation were isolated and identified as dibenzothiophene-5-oxide, dibenzothiophene-5-dioxide and benzoate by cochromatography, UV spectroscopy and gas chromatographymass spectrometry analyses. Based on the identified metabolites a pathway for the degradation of dibenzothiophene by Brevibacterium sp.DO is proposed. Key words: Dibenzothiophene Mineralization rium sp.
Desulfurization Degradation pathway - Brevibacte-
The catabolism of dibenzothiophene (DBT) and other thiophenic compounds has received a lot of attention during the last years. The main interest has been focussed on the microbial desulfurization of DBT as a model compound representing an organic sulfur structure in coal and crude oil. Concerning coal more than 90% of the sulfur are presented as pyritic and organic sulfur. In order to avoid SO2 emissions during combustion both sulfur forms have to be removed. While the microbial removal of pyritic sulfur is well documented, only few examinations have been carried out on the biochemical capabilities of microOffprint requests to: M. van Afferden abbreviations. DBT, dibenzothiophene; PASH, polycyclic aromatic sulfur heterocycle: PAH, polycyclic aromatic hydrocarbons: GC-MS, gas chromatography-mass spectrometry; HPLC, high pressure liquid chromatography; IC, ion chromatography
Non-standard
organisms to attack the organic sulfur in coals (for reviews see Bos and Kuenen 1989; Klein et al. 1988; Couch 1988). The organic sulfur in coal is integrated in the macromolecular coal matrix in form of thiolic, sulfidic and thiophenic substructures (Boudou et al. 1987). This implies a cleavage of covalent C-S bonds for its elimination. Apart from the substructures of heterocyclic bound sulfur in coal, DBT belongs to a group of recalcitrant polycyclic aromatic sulfur heterocycles (PASHs) of environmental concern. These compounds are mainly introduced into the environment from coal gasification or liquefaction processes and crude oil contaminations (Ensley 1984). Although DBT is not a genotoxic compound (Krawiec and Montenecourt 1986; Pelroy et al. 1983), it has a high potential for biological accumulation (Ogata and Miyake 1980) and it is known to be among the most persistent compounds in the environment (Berthou and Vignier 1986; Gundlach et al. 1983). Compared to the detailed knowledge of the microbial degradation of PAHs, PASHs have not been investigated intensively. Concerning DBT so far no organism has been isolated that degrades it completely. However, a partial microbial degradation of DBT is described by several authors (Kodama et al. 1973; H o u and Laskin 1976: Laborde and Gibson 1977; Foght and Westlake 1988). A different microbial attack on DBT has been investigated by Holland et al. (1986) and Fortnagel et al. (1989), who show that microorganisms oxidize DBT to the corresponding sulfoxide and sulfone. Recently Mormile and Atlas (1988) have demonstrated that biodegradation products of DBT are metabolized further by soil and sediment enrichment cultures. However, no sulfite or sulfate accumulation has been detected in these experiments. Sulfate release due to a microbial attack on DBT was established by Kargi and Robinson (1984) and Isbister and Kobylinski (1985), but the biochemical mechanisms leading to a release of sulfate have not further been investigated. As previously reported (van Afferden et al. 1988) a mixed culture consisting of two species was isolated that utilizes DBT as sole sulfur source for growth. The mi-
325 crobial attack leading to a release o f sulfate was initiated by sulfoxidations to the c o r r e s p o n d i n g sulfoxide a n d sulfone. This p a p e r reports o n the total m i n e r a l i z a t i o n of D B T by a pure c u l t u r e of a Brevibacterium sp. u s i n g D B T as sole source o f sulfur, c a r b o n a n d energy for g r o w t h a n d o n the biochemical p a t h w a y involved.
Materials and methods
Organisms The strain DO was isolated on the basis of being able to grow on dibenzothiophene (DBT) as sole source of carbon, sulfur and energy from the two membered bacterial consortium FODO (van Afferden et al. 1988). The strain DO was identified as a Brevibacterium species by the DSM, Braunschweig, FRG.
Chemicals Dlbenzothiophene and benzoate were obtained from Merck, Darmstadt, FRG, with a purity exceeding 99% as shown by GC analyses. DBT-sulfone was purchased from Aldrich-Chemie, Steinheim, FRG. DBT-5-oxide was prepared as described by Gilman and Esmay (1952) and purified as previously described (van Afferden et al. 1988). All other chemicals were of the highest purity commercially available.
Media and growth conditions All culture vessels used were thoroughly cleaned with 4 M HC1 to prevent sulfur contaminations. Brevibactermm sp. was grown in a mineral salt medium consisting of 0.2 g MgC12x 6 H20, 1.0 g KNO3, 0.1g CaC12x2 H20, 0.01g FeC13x6HzO and 3.5g K2HPO4 per liter of disUlled water. Additionally 1 ml per liter of a trace element solution was added (according to Pfennig and Lippert 1966). The mineral medmm was adjusted to pH 7.4 with HCI. 2.5 ~tg/ 1thiamine dichloride was added aseptically to the sterilized medium. DBT/DBT-5-dioxide routinely were added as described by van Afferden et al. (1988). A cultivation of larger scale was accomplished by using a 2.25 1 glass fermenter with 1.2 1culture volume. The culture was grown at 30~ aerated at 1.5 1 air/min and stirred at 450 rpm. In order to assure a homogeneous suspension of the Insoluble substrates DBT and DBT-5-dioxide respectively were added to the sterilized medium to a final concentration of 3 mM as follows: Substrates were finely grounded in a mortar and classified to a defined particle size by passing through a sieve (pore size 100 gm, DIN 4188). The powder of particle size _< 100 gm was applicated to the sterile medmm aseptically and suspended by ultrasonificatlon for 10 min. During incubation t0 ml samples were withdrawn from the culture with a sterile syringe. Using this method the DBT/DBT-5-dloxide concentrations (3 raM) determined in 20 samples differed within a deviation of +_2%, thus providing a representative sampling during growth experiments. Substrate oxidation with resting cells pregrown on DBT was carried out in 10 ml screw-cap culture tubes (3 ml culture volume) Cells were washed twice with 20 mM phosphate buffer and after centrifugation they were resuspended to a final cell density of AEsvs = 4.5 and incubated at 30~ in parallel.
Analytical methods Substrates and water soluble metabolites were estimated as described by van Afferden et al. (1988).
Sulfite and sulfate were determined by ion chromatography (IC) with a Biotronik IC 1000 ion chromatograph equipped with a 655 A-11 Liquid Chromatograph (Merck, Darmstadt, FRG), a BT 1 AN anion exchange column with cation suppressor technic (BT S AG column), a conductive detector BT 0331 (Biotronik, Maintal, FRG) and a C-R5A Chromatopac Integrator (Shimadzu, Kyoto, Japan). A solution of 3 mM Na2CO3 and 3.7 mM NaHCO3 was used as eluent. The UV spectra analyses were performed after HPLC separation m situ with a photodiode array detector using following equipment: Autosampter Waters 712 WISP, multisolventdelivery system Waters 600 E, photodiode array detector Waters 990, personal computer NEC Power Mate 2 (software: chromatogramm calculation and library search enhancement vers. 4.08); column: Cis PAH analysis cartridge, 5 mm x 100 mm, particle size 100 gm; solvent system: linear gradient of 35% acetonitrile (v/v), 65% water (v/v) containing 0.12% H2POa (v/v) over 15 rain to 100% acetomtrile containing 0.12% H2PO4 (v/v). GC-MS analyses were carried out according to van Afferden et al. (1988). The mineralization of DBT by Brevibacterium sp. was analysed by the following methods: a) In the stationary phase of growth possibly dissolved DBT degradation products were detected in the supernatant culture fluid by HPLC analyses, b) The quantification of the dissolved organic carbon in the supernatant culture fluid was carried out using a DOC-analyzer (Shimadzu TOC 500, Kyoto, Japan). c) Organic extractable metabolites of DBT were estimated after acidification and extraction of the bacterial culture with dichloromethane by GC analyses, d) In addition to GC analyses the organic phase was analysed by UV spectrometry (spectrophotometer: Shimadzu UV-210 A, Kyoto, Japan). The growth of the culture was determined by following the increase of protein (according to Lowry et al. 1951) after the interferlng aromatic substrates had been quantitatively removed by extraction with dichloromethane.
Results
Determination of nutritive conditions for the Brevibacterium sp. As described in a previous r e p o r t a mixed culture, consisting o f a n Alcaligenes denitrificans subspecies a n d a Brevibacterium species, was isolated that utilizes D B T as sole source of sulfur for g r o w t h (van A f f e r d e n et al. 1988). The pure cultures o f each species did n o t show a n y significant D B T m e t a b o l i s m . F u r t h e r i n v e s t i g a t i o n o f the m o d e of i n t e r a c t i o n within the two m e m b e r e d mixed culture showed t h a t the growth of Brevibacterium sp. o n solid c o m p l e x m e d i u m was s t i m u l a t e d in the n e a r s u r r o u n d i n g s o f a c o l o n y o f the Alcaligenes strain, This implies that at least one growth factor was excreted into the m e d i u m b y Alcaligenes denitrificans. The same effect of s t i m u l a t i o n was observed replacing the Alcaligenes species b y 10 ~tl o f a v i t a m i n s o l u t i o n (according to K i r k et al. 1978) centered o n the n u t r i e n t b r o t h agar plate. I n a v i t a m i n s u p p l e m e n t e d m i n e r a l m e d i u m with D B T as sole source o f c a r b o n , sulfur a n d energy for growth, Alcaligenes denitrificans showed n o ability to degrade D B T , whereas the pure culture of Brevibacterium sp. grew o n D B T . By a d d i n g single vitamines to the culture med i u m t h i a m i n e was identified to be the growth factor needed by Brevibacterium sp.
326 Table 1. Characterization of products of dibenzothiophene (DBT) degradation by Brevibacteriumsp.
a
.a
~.16o', '~ 12o
Retention time a (min)
.2~
DBT-5-oxide DBT-5-dioxide Benzoate Sulfite Sulfate
80,
40, 0
0 b
v
Products
HPLC
IC
5.1 7.1 4.1 -
16.9 19.7
UV-Spectroscopy maxima a (rim)
220,246,282,326 208,234,240,278,288,320 230,272 -
" Identical results were obtained with authentic chemicals
3
15.
F
10,
~
~ IB0:
.
.
.
.
.
o12O-
a~
'k ~
.2~
1
i .
.
.
.
.
.
.
.
100
.
,
200
0
300
Time (h) Fig. I. a Growth (~) of Brevibacterium sp. on dibenzothiophene (DBT) (9 b formation of sulfate(@), DBT-5-oxidc (A) and DBT5-dloxide(4) during growth
0
0
9
"
40
9
"
~
80 t20 Time (h)
"q~"
160
'~
0
Fig. 2. Growth (FT) of Brevibactermm sp. on dibenzothiophene-5dioxide ( 9 and formation of benzoate ( 9 during growth
Degradation of DBT by Brevibacterium sp. The pure culture of Brevibacterium sp. was grown on DBT (3 raM). After a lag phase of 162 h DBT was degraded concomitantly with the growth of the culture within 105 h. The culture grew with a doubling time of 22 h to a final protein concentration of 177 rag/1 (Fig. i a). In sterile controls incubated in parallel no abiotic degradation of DBT was observed. During the degradation of DBT a stoichiometrical amount of sulfate was released into the culture fluid (Fig. 1 b). Sulfate was identified by IC cochromatography (Table 1) and accumulated to a final concentration of 2.95 raM. No significant amount of sulfite as a probable intermediate in desulfurization was detected. In addition to the accumulation of sulfate the formation of two metabolites was observed by HPLC analyses. They accumulated in small amounts during the lag phase of growth and were successively degraded during the logarithmic phase of growth. The metabolites were identified by GC-MS analyses of dichloromethane extracts of the culture supernatants. The obtained fragmentation pattern of the metabolite occuring first, m/z (%): 200 (9), 184 (100), 171 (7), 152 (9), 139 (17) and 92 (14) was identical with that of synthetic DBT-5-oxide. For the other metabolite a M + was detected at m/z 216 with minor ion fragments at m/z (%): 187 (41), 171 (14), 168 (36), 160 (34), 150 (18), 139 (24) and 136 (34). The ion fragments fit well with that of
authentic DBT-5-dioxide. Additionally these results were confirmed by HPLC cochromatography and comparison of the UV absorption spectra with authentic chemicals (Table 1). To determine the degree of DBT degradation the culture was investigated in the stationary phase of growth. No water soluble metabolites were detected in the supernatant culture fluid by HPLC analyses. The dissolved organic carbon was quantified to 39 rag/1. No extractable organic metabolites were detected in the culture fluid.
Degradation of DBT-5-dioxide by Brevibacterium sp. DBT-5-dioxide (3 mM) served as sole carbon, sulfur and energy source for the growth of a pure culture of Brevibacteriurn sp. As shown in Fig. 2 DBT-5-dioxide was degraded concomitantly with the growth of the culture. After a lag phase of 65 h the culture grew with a doubling time of 12 h and yielded 182 mg of protein/1 in the stationary phase of growth. During growth of the culture sulfate was released into the culture fluid and accumulated to a final concentration of 2.96 mM. During the exponential phase of growth the formation and successive degradation of a metabolite was detected by HPLC analyses (Fig. 2). This metabolite was identified as benzoate by GC-MS analyses [m/z (%): 122 (83), 105 (100), 77 (71),
327
DBT
~
0 , 8 ,
~
~
DBT- 5- oxide II 0
:
m
0
i
-
B 3 Time (h)
4
DBT
5
Fig. 3. Degradation of dibenzothiophene ( E ) and formation of sulfite ({3) and sulfate ( 0 ) by DBT grown resting cells of Brevibacterium sp. (cell density: AEsv8 = 4.5)
-
5 - dioxide
I
sou-- so 51 (31)]. Furthermore the HPLC retention time and the obtained UV spectra confirm this result (Table 1). The culture of Brevibacterhml sp. mineralized DBT-5-dioxide completely. No water soluble or organic extractable metabolites were detected in the stationary phase of growth. The same results were obtained using benzoate (5 mM) as carbon source (sulfur source: 200 gM MgSO4). The residual amount of dissolved organic carbon in the supernatant culture fluid was 37 mg/1 in the case of DBT-5-dioxide and 32 rag/1 in the case of benzoate grown cells.
Formation of sulfite by resting cells of Brevibacterium sp. DBT pregrown cells ofBrevibacterium sp. were incubated in parallel at high cell densities (AEsv8 = 4.5) with a concentration of 1 mM DBT. Within 290 rain a concentration of 614 gM sulfite and 53 gM sulfate accumulated concomitantly with the utilization of DBT (718 gM) as shown in Fig. 3. Sulfite was identified by IC cochromatography with authentic sulfite (NazSO3) (Table 1). Comparable results were obtained using DBT-5-dioxide as substrate for DBT pregrown cells. In control experiments it was shown that the abiotic oxidation of sulfite (NazSO3) to sulfate was more rapid in buffer than in high density cell suspensions of Brevibacterium sp. This could be due to the 02 consumption of the resting cells, which decrease chemical oxidation of sulfite and may explain the accumulation of sulfite in high density cell suspensions of Brevibacterium sp.
Discussion
In the present study it is shown that in the presence of thiamine the Brevibacterium species grows on DBT as sole sulfur, carbon and energy source for growth. DBT utilization by Brevibacterium sp. is initiated by an oxidative attack on the sulfur heteroatom. These initial reactions lead to the formation of DBT-5-oxide and DBT-5-dioxide and may be catalyzed by sulfoxidases
@
C00- Benzoate
I I H20+C02 Fig. 4. Proposed metabolic pathway for the degradation ofdibenzothiophene by Brevibacterium sp.
similar to those of mammalian or microbial origin as described by Holland (1988). During metabolization DBT is desulfurized accompanied by a release of a stoichiometrical amount of sulfite which is further oxidized to sulfate probably by abiotic oxidation. In addition the carbon skeleton of DBT is degraded via benzoate. Due to these results a pathway for the degradation of DBT by Brevibacterium sp. is proposed (Fig. 4). After growth ofBrevibacterium sp. on DBT, on DBT5-dioxide or on benzoate nor water soluble neither organic extractable metabolites were detected in the culture fluid. This indicates a complete mineralization of the substrates. The remaining amount of dissolved organic carbon of 8 . 0 - 9.0 % of the initial substrate carbon could be explained by an accumulation of lysis products of cells or biodetergents into the culture fluid. Moreover, the release of sulfate in stoichiometrical amounts indicates that the thiophene ring is cleaved and DBT is mineralized completely to H z O , C O 2 and SO~- by Brevibacterium sp. This proposed metabolic pathway for the total mineralization of DBT by Brevibacterium sp. represents an alternative to the "classical" pathway of partial degradation of DBT established by several authors (Kodama et al. 1973; Hou and Laskin 1976; Laborde and Gibson 1977). The "classical" reaction sequences of initial dioxygenation at the peripheric aromatic ring of DBT, followed by a meta cleavage analogous to the naphtha-
328 lene m e t a b o l i s m d e s c r i b e d b y G i b s o n a n d S u b r a m a n i a n (1984), l e a d s to the a c c u m u l a t i o n o f 3 - h y d r o x y - 2 - f o r m y l b e n z o t h i o p h e n e as a d e a d e n d m e t a b o l i t e . This implies t h a t o n l y three c a r b o n a t o m s are a v a i l a b l e for the g r o w t h o f m i c r o o r g a n i s m s a n d t h a t no d e s u l f u r i z a t i o n o f the o r g a n i c s u b s t r a t e occurs. In c o n t r a s t to this p a t h w a y D B T is d e s u l f u r i z e d b y Brevibacteriurn sp. i n i t i a t e d b y o x i d a t i o n o f the sulfur h e t e r o a t o m ; the release o f sulfite c o u l d i m p l y a n o r g a n i c sulfinic o r sulfonic a c i d as a n i n t e r m e d i a t e in D B T d e g r a d a t i o n . B e n z o a t e , w h i c h occurs as a m e t a b o l i t e in the d e g r a d a t i o n o f D B T b y Brevibacterium sp. suggests t h a t the c a r b o n skeleton c o u l d be d e g r a d e d a n a l o g o u s to the b a c t e r i a l d e g r a d a t i o n o f b i p h e n y l as d e s c r i b e d b y C a t e l a n i et al. (1971) a n d G i b s o n et al. (1973). H o w e v e r , the p o s i t i o n o f o x y g e n a t i o n s w h i c h lead to the cleavage o f the p e r i p h e r i c a r o m a t i c ring still r e m a i n s unclear. C o n s i d e r i n g the t o t a l m i n e r a l i z a t i o n o f D B T the o x i d a t i o n o f the sulfur h e t e r o a t o m a n d the s u b s e q u e n t cleavage o f the C-S b o n d s c o u l d be r e g a r d e d as the crucial steps w h i c h increase the a v a i l a b i l i t y o f the c a r b o n s k e l e t o n for a m i c r o b i a l a t t a c k . I n v e s t i g a t i o n s on the m i c r o b i a l d e g r a d a t i o n o f P A S H s a n d a r o m a t i c t h i o e t h e r s are rare. K a u f m a n n a n d K e a r n e y (1976) r e p o r t e d o n the b a c t e r i a l cleavage o f the t h i o e t h e r p r o m e t r y n e w h i c h p r o c e e d s t h r o u g h the sulfoxide a n d sulfone. T h e d e s u l f u r i z a t i o n resulted in the f o r m a t i o n o f the c o r r e s p o n d i n g h y d r o x y c o m p o u n d , while m e t h y l s u l f o n a t e was suspected to be a n i n t e r m e d i ate. A c o m p a r a b l e m e c h a n i s m for the d e s u l f u r i z a t i o n o f D B T b y c o o x i d a t i o n was p r o p o s e d b y Isbister a n d K o b y l i n s k i (1985), w h o suggested 2 , 2 ' - d i h y d r o x y b i p h e n y l to be a d e a d e n d p r o d u c t o f the m i c r o b i a l a t t a c k . T h e p r o p o s e d p a t h w a y for the d e g r a d a t i o n o f D B T b y Brevibacterium sp. does n o t s u p p o r t this m e c h a n i s m , for b e n z o a t e b u t n o t 2 - h y d r o x y b e n z o a t e was identified as an i n t e r m e d i a t e . C o n s i d e r i n g the m o d e l c h a r a c t e r o f D B T as a n o r g a n i c sulfur s t r u c t u r e in c o a l the p r o p o s e d r e a c t i o n s i n d i c a t e a p o s s i b l e m e c h a n i s m o f d e s u l f u r i z a t i o n o f the a r o m a t i c o r g a n i c sulfur c o m p o u n d s . H o w e v e r , it r e m a i n s to be s h o w n w h e t h e r this m e c h a n i s m is a p p l i c a b l e to coal.
Acknowledgements. We thank Dr. M. Beyer for valuable support and the department of coal environmental analytics (Bergbau-Forschung GmbH) for performing mass spectrometry. A part of this work was supported financially by the Bundesministerium ffir Forschung und Technotogie (BMFT, 03E-6215-A).
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