Degradation of benzene 1,3-disulfonate by a mixed bacterial culture

ELSEVIER

FEMS Microbiology

Letters I36 (1996) 45-50

Degradation of benzene 1,3-disulfonate by a mixed bacterial culture Matthias Contzen a, Rolf-Michael Wittich b, Hans-Joachim Andreas Stolz a,* ” ’ Gesrllschqft.fiir

Institutfiir

Mikrohiologie

Biotechnologische

der fJnil,ersitiit

Forschung

Received 2 October

(GBF),

Stuttgart,

Allmandring

Bereich Mikrobiologie.

1995; revised 22 November

3/. 70569 Stuttgart,

Masrhemder

Knackmuss German!

Weg I, D-38124

1995: accepted 28 November

a,

Braunschweig.

Germany

1995

Abstract

A benzene I Sdisulfonate degrading mixed bacterial culture was isolated from the River Elbe downstream of Hamburg. The mixed culture was composed of five different bacterial strains. None of these strains grew in axenic culture with benzene l,3-disulfonate as sole source of carbon and energy. In the presence of 4-nitrocatechol, resting cells of the mixed culture converted benzene I ,3-disulfonate to catechol 4-sulfonate. Experiments with cell-free extracts demonstrated that catechol 4-sulfonate was further metabolized via 3-sulfomuconate and 4-carboxymethyl-4-sulfobut-2-en-4-olide. Keyords:

Biodegradation;

Benzene

I ,3-disulfonate;

Mixed bacterial culture; Catechol 4-sulfonate;

1. Introduction Aromatic substituents

compounds are generally

which

carry

recalcitrant

sulfonic against

acid

Protocatechuate-3.4-dioxygenase

production of resorcinol [8]. The biodegradability this compound by activated sludge has been scribed previously [2,9].

type II

of de-

micro-

[I ,Z]. Nevertheless, several bacterial have been isolated which utilize monosulfonated benzene or naphthalene derivatives as sole source of carbon and energy [3-51. In contrast, to the best of our knowledge, there has been no report about the bacterial degradation of disulfonated benzenes, and only two publications about the biodegradation of disulfonated naphthalenes [6,7]. Benzene 1,3-dismfonic acid is of technical importance because it is an intermediate in the industrial

bial degradation strains

* Corresponding (7

author. Tel.: +49 (711) 685 5489; Fax: +49

I I ) 6855725.

037%1097/96/$12.00 0 1996 Federation SSDl 0378-1097(95)00483-I

of European

Microbiological

2. Materials

and methods

2. I. Culture media and preliminary

tanonomic char-

acterization

The standard medium consisted of a mineral medium [3] supplemented with a vitamin solution [IO]. In most experiments benzene 1,3-disulfonate was supplied as source of carbon and energy (5-10 mM). For the separation of single strains the mixed culture was spread on nutrient broth or a mixture of Societies.

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46

M. Contren et al. / FEMS Microhiolog?:

different carbon sources (glucose, fructose, succinate, 5 mM each, plus 2 g/l tryptone, 5 g/l yeast extract, 5 g/l beef extract, and 4 g/l peptone). For a preliminary taxonomic characterization of the strains the API 20 NE test system and the ‘APILAB PC (Version 2.0)’ software (bioMCrieux SA; Marcy l’Etoile, France) and the Biolog identification system (GN MicroPlates, Biolog Inc., Hayward, CA, USA) were used. 2.2. Preparation

of cell-free

extracts

Cell suspensions in 50 mM Na,HPO,/KH,PO, buffer, pH 7.4, were disrupted by using a French press (SLM Aminco; SLM Instruments Inc., Urbana, IL, USA) at 1.1 X 10’ Pa. Cells and cell debris were removed by centrifugation at 100000 X g for 30 min at 4°C. 2.3. Protein estimation and enzyme assays Protein content of cell-free extracts was determined by the method of Bradford [ 111. Bovine serum albumin was used as a standard. The protein content of whole cells was determined by the method of Schmidt et al. [12]. An optical density (OD546nml of 1 corresponded to a protein content of 0.2 mg ml-i during the exponential growth of the mixed culture with benzene 1,3-disulfonate. One unit of enzyme activity is defined as the amount of enzyme that converts 1 pmol of substrate per min. Protocatechuate-3,6dioxygenase (P340 type I enzyme) was measured by the procedure of Stanier and Ingraham [13], but using Tris . HCl buffer (50 mM, pH 8.0). For the assay of protocatechuate-3,4-dioxygenase type II activity, protocatechuate was replaced by catechol 4-sulfonate as substrate [ 141. For the assay of the 3-sulfomuconate converting enzyme ( = ‘carboxymuconate cycloisomerase type II’ [14]), the cuvettes contained, in 1 ml, 50 pmol Tris . HCl, pH 8.0, 1 pmol MnCl,, and 0.1 pmol 3-sulfomuconate. The reaction was started by the addition of cell-free extract and the decrease in absorbance recorded at h = 260 nm (eZeOn,,,= 2.3 mM-‘cm _ ’ [ 151). The 3-sulfomuconate for this assay was prepared enzymatically from catechol 4-

Letters

136 (1996) 45-50

sulfonate. Protocatechuate-3,4-dioxygenase type II was partially purified from cell-free extract of the mixed culture by anion-exchange chromatography. The active fractions were pooled (4 ml), catechol 4-sulfonate added (0.74 mM) and the solution stirred for I h. The formation of 3-sulfomuconate from catechol 4-sulfonate was analyzed by HPLC. Only one signal for the formation of a product was found during chromatography. Therefore, it was assumed that catechol 4-sulfonate was converted to stoichiometric amounts of 3-sulfomuconate. Because 3sulfomuconate was unstable under acid conditions [ 141 the product solution was not further worked up, but used directly for the assay of the 3-sulfomuconate converting enzyme. 2.4. Enzyme separation Protein was purified at room temperature by use of a fast-performance liquid chromatography system consisting of a LCC 500 controller, pump 500, UV- 1 monitor, REC-482 recorder, and FRAC autosampler from Pharmacia (Uppsala, Sweden). Crude extract was applied to a Mono Q column (HR 5/5; Pharmacia). Protein was eluted with 40 ml of a linear gradient of Tris . HCl (50 mM, pH 8.0) into Tris . HCl (50 mM, pH 8.0) plus 0.4 M NaCl at a flow rate of 0.6 ml/min. Fractions (0.75 ml) were collected and enzyme activity determined spectrophotometrically. The protocatechuate-3,4-dioxygenase type II and the 3-sulfomuconate converting enzyme eluted at 0.12 M and 0.28 M NaCl, respectively. 2.5. Comersion

of benzene

chol I-sulfonate

by resting cells

1,3-disulfonate

to cate-

The mixed culture was grown with benzene 1,3disulfonate and exponentially growing cells harvested by centrifugation and resuspended into 50 mM Na,HPO,/KH,PO, buffer, pH 7.4, to an optical density (OD 546nm)= 8. These cell suspensions (5 ml> were shaken (3O”C, 100 rpm) and benzene 1,3disulfonate (0.5 mM) and 4-nitrocatechol (0.5 mM1 were added. Every 10 min samples were taken, cells removed by centrifugation and the supematants analyzed by HPLC.

M. Contren et al. / FEMS Microbiology

2.6. Analytical

methods

Benzene I ,3-disulfonate and metabolites were analyzed by reverse-phase high-pressure liquid chromatography (HPLC) (HPLC Millenium Chromatography Manager 2.0 or Maxima software, equipped with a programmable multi-wavelength detector model 486 or a photodiode array detector 994 or 996 and HPLC pump 5 10; Waters Associates, Milford, MA, USA). The separated compounds were detected at 210 nm. A reverse-phase column (250 X 4 mm, internal diameter; Grom, Herrenberg, Germany) with LiChrosorb RP 18 5pm particles (Merck, Darmstadt, Germany) was used. For the separation of benzene 1,3-disulfonate and catechol 4-sulfonate the mobile phase consisted of 25% (v/v> methanol, 72% (v/v> water and 3% (v/v) of a commercially available solution of an ion-pair reagent (PicA; Waters). The pH of this solvent system was lowered by the addition of H 3P0, to pH 2.5. The flow rate was 0.7 ml/min. Under these conditions the retention times (R,) of benzene 1,3_disulfonate, catechol 4-sulfonate and 4-nitrocatech01 were 15.3 min, 5.9 min, and 30.5 min, respectively. Catechol 4-sulfonate (R, = 5.0 min) was separated from 3-sulfomuconate (R, = 4.2 min) and 3sulfomuconate was distinguished from 4-carboxymethyl-4-sulfobut-2-en-4-olide (R, = 5.1 min) with a solvent system composed of 98.9% (v/v> water, 1% (v/v) methanol and 0.1% (v/v) H,PO,. Sulfate was determined by a modification of the barium chloroanilate method as described before [3]. The sulfite concentration in the culture fluid was measured by the method of Johnston et al. [ 161. 2.7. Chemicals Benzene 1,3-disulfonic acid was supplied by Sigma (Deisenhofen, Germany). 4_Methylbenzenesulfonic acid was purchased from Fluka (Buchs, Switzerland), and 3-methylbenzenesulfonic acid from Lancaster Chemicals (Miihlheim/Main, Germany). Benzenesulfonic acid was obtained from Riedel-DeHaen (Seelze-Hannover, Germany). Catechol 3sulfonate and catechol 4-sulfonate were kindly provided by Dr. F. Junker (Zurich) and Dr. A. Hammer (Stuttgart), respectively. All other chemicals used for

Letters 136 (19%)

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mineral salts media and buffer solutions were obtained from E. Merck AG (Darmstadt, Germany).

3. Results and discussion 3.1. Characterization

of bacterial strains

A mixed bacterial culture (RW2) was enriched with benzene 1,3-disulfonate (5 mM) from an aerobic sediment taken from the Elbe river downstream of Hamburg by standard enrichment techniques. Five different types of bacterial colonies (RW21 -RW25) were differentiated on agar plates with NB or the mixture of carbon sources described above. The composition of the mixed culture did not change after a series of transfers in benzene 1,3_disulfonate medium and the five strains could always be identified as sole members of the culture. The five strains were purified by standard techniques on complex media. No growth of single colonies was found on benzene 1,3-disulfonate agar plates. There was also no growth of pure cultures observed when the benzene 1,3-disulfonate plates were supplemented with NB (20 mg/l) or a vitamin solution [lo]. All five strains grew in liquid culture as short rods which were not motile. The cells were Gram-negative and oxidase positive. A preliminary taxonomic characterization of the strains gave only unsatisfactory results. Only strain RW21 was identified by both test systems with high probability as Alcaligenes nylosoxidans. Strain RW22 was identified as Xanthomonas maltophilia by the Biolog and API systems with high and low probability, respectively. The five axenic cultures were mixed and then transferred to a liquid culture with benzene 1,3-disulfonate as sole source of carbon and energy, but no growth with benzene 1,3-disulfonate was obtained. Therefore, the metabolism of benzene 1,3-disulfonate was studied with the original mixed culture that was enriched and grown on benzene 1.3-disulfonate. The ability to degrade benzene 1,3-disulfonate was also lost when the complete benzene 1,3-disulfonate degrading mixed culture was grown for 12 days in liquid culture with NB in the presence or absence of benzene 1,3-disulfonate (1 mM).

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M. Comen

et al. / FEMS Miuohiolog?:

Letters

136

(19%)45-50

3.3. Catechol 4-.vdfonnte

L 30

2.0

5 :

-25

-

-20

;

-15

c 5 e 6

B 185

8

14 - IO

c

OS

5

o,o 0

50

100

150

200

250

300

350

400

450

500

Time (h)

Fig. 1. Growth of the mixed culture with benzene 1,3-disulfonate. The mineral medium contained benzene 1,3-disulfonate (IO mMJ and a vitamin solution according to Sharak-Genthner et al. [IO]. The medium was inoculated with an exponentially growing precuhure. Increase in cell density (0) was determined photometrically at 546 nm. The concentration of benzene 1,3-disulfonate ( n ) was determined by HPLC. The concentrations of sulfate f A J and sulfite (v) were quantified according to Niirtemann et al. [3] and Johnston et al. [ 161, respectively. At the point indicated by the arrow, 5 mM benzene 1,3-disulfonate were again added.

The benzene 1,3-disulfonate degrading mixed culture grew also with benzenesulfonate, 3- and 4-methylbenzenesulfonate as sole source of carbon and energy. 3.2. Growth of the mixed culture with benzene 1,3disulfonate and complete desulfonation of the substrate To ensure that benzene 1,3-disulfonate was indeed completely desulfonated, the mixed culture was grown with benzene 1,3-disulfonate (15 mM) and the concentrations of sulfite and sulfate released were quantified. Sulfate was excreted into the medium in almost stoichiometric amounts. The final concentration of sulfate in the culture medium was 3 1 mM and only small amounts of sulfite ( < 5 PM) accumulated transiently in the culture fluid (Fig. 1). Thus benzene 1,3-disulfonate was quantitatively desulfonated by the mixed culture. The shortest doubling time observed was t, = 18 h.

as intermediate

No metabolites were found by HPLC-analysis during growth of the mixed culture with benzene 1,3-disulfonate. According to the metabolic pathways of other sulfonated aromatic compounds an initial dioxygenolytic desulfonation of benzene 1,3disulfonate was expected. This would result in the formation of catechol 3-sulfonate or catechol 4sulfonate from benzene 1,3_disulfonate, which have been identified as metabolites in the degradative pathways of 2-aminobenzenesulfonate (orthanilate) and 4-aminobenzenesulfonate (sulfanilate), respectively [5,17]. Both sulfonated catechols are presumably oxidized by iron containing dioxygenases [ 14,181. If resting cells of the mixed culture were supplied with benzene 1,3-disulfonate and 4-nitrocatechol, an inhibitor of several dioxygenases [19], a metabolite accumulated which was identified by HPLC-analysis by comparison with an authentic standard according to its retention time and in situ recorded UV/Vis spectrum as catechol 4-sulfonate. No turnover of 4-nitrocatechol occurred. Under the chromatographic conditions applied catechol 4sulfonate (R, = 5.9 min) was clearly separated from catechol 3-sulfonate (R, = 9 min). 3.4. Oxidation qf catechol I-suifonate The oxidation of catechol 4-sulfonate by a modified type of protocatechuate-3,4-dioxygenase (type II enzyme) has been described before [14]. Therefore, the turnover of protocatechuate or catechol 4sulfonate by cell-free extracts from the benzene 1,3disulfonate degrading mixed culture was analyzed using UV/Vis overlay spectra. The spectral changes observed during both reactions were characteristic for the ortho-cleavage of both substrates [14,20]. No indications for an extradiol cleavage mechanism ( = increase in absorbance at h > 350 nm) was found [2 1,221. The specific activities with protocatechuate and catechol 4-sulfonate were 0.3 1 and 0.10 U/mg of protein, respectively. The proteins of the cell-free extract were separated by anion-exchange chromatography using FPLC. Only one enzyme activity for catechol 4sulfonate eluted from the column. This active fraction converted also protocatechuate. The oxidation of

M. Contzen et al. / FEMS Microbiology

Letters

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new signal which showed an in situ recorded UVspectrum identical to the spectrum described previously for 3-sulfomuconate [ 14,151. 3.5. Further metabolism

OH

VII

F I

v

coo.

00’

ols

00.

(ii I

III

so,

VIII

oo

1 c

IV

-O”* so,kr

-I

The fractions obtained by anion-exchange chromatography of the cell-free extract were incubated with 3-sulfomuconate and the reaction analyzed by HPLC. Thus one activity maximum was identified which converted 3-sulfomuconate CR, = 4.2 min) to a product with a slightly longer retention time (R, = 5.1 min). The in situ recorded UV-spectrum showed an absorbance maximum at A = 216 nm. The conversion of 3-sulfomuconate to the same product was catalysed by the addition of H,PO, (5 ~1, 85%) to a solution of 3-sulfomuconate (100 ~1, 0.74 mM). The same UV-spectrum and the formation by acid catalysis from 3-sulfomuconate have been described previously for 4-carboxymethyl-4-sulfobut-2-en-4-olide [14,15]. 3.6. Proposed disulfonate

D

of 3-sdfomuconate

degradatice

pathway ,for benzene

1,3-

coo

V 0

IJ

00.

VI

Fig. 2. Proposed pathway for the degradation of benzene 1,3-disulfonate by the mixed culture in comparison to the degradation of protocatechuate via the l3-ketoadipate pathway. Key to enzymes: A, ‘benzene- I ,3-disulfonate I ,2-dioxygenase’; B, protocatechuate3,4-dioxygenase type II; C, 3-carboxymuconate cycloisomerase type Ii; D. sulfolactone hydrolase; E, maleylacetate reductase; F, protocatechuate-3,4-dioxygenase; G, 3-carboxymuconate cycloisomerase; H, 4carboxymuconolactone decarboxylase; I, 4-oxoadipate enol-lactone hydrolase. Key to compounds: I, benzene 1,3disulfonate; II, catechol 4sulfonate; III, 3-sulfomuconate; IV, 4-sulfomuconolactone (4-carboxymethyl-4-sulfobut-2-en-4-elide); V maleyiacetate; VI 3-oxoadipate; VII protocatechuate; VIII, 3carboxymuconate; IX, 4carboxymuconolactone; X, 4-oxoadipate enol-lactone.

The initial step in the biodegradation of benzene 1,3-disulfonate is probably a dioxygenolytic desulfonation to catechol 4-sulfonate (Fig. 2). The further metabolism of catechol 4-sulfonate is obviously identical with the modified protocatechuate branch of the fl-ketoadipate pathway previously described for the metabolism of 4-aminobenzenesulfonate (sulfanilate) [14]. Recently, the formation of 3sulfocatechol from 2-aminobenzenesulfonate and the subsequent oxidation of 3-sulfocatechol to a nonaromatic product has been shown [ 17,181. These results together with the present study indicate that the oxidative ring-cleavage of sulfocatechols is a common reaction involved in the metabolism of sulfonated benzenes and thus of great importance for the biodegradation of this class of xenobiotics.

References catechol 4-sulfonate by the active fraction was analyzed by HPLC. The decrease in the signal for catechol 4-sulfonate resulted in the formation of a

[l] Alexander, M. and Lustigman, B.K. (1966) Effect of chemical structure on microbial degradation of substituted benzenes. J. Agr. Food Chem. 14, 410-413.

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[2] Wellens, H. (1990) Zur biologischen Abbaubarkeit monound disubstituierter Benzolderivate. Z. Wasser-Abwasser Forsch. 23, 85-98. [3] Niirtemann, B., Baumgarten, I., Rast, H.G. and Knackmuss, H.-J. (1986) Bacterial communities degrading amino- and hydroxynaphthalene-2-sulfonates. Appl. Environ. Microbial. 52, 1195-1202. [4] Thumheer, T., Kijhler, T., Cook, A.M. and Leisinger, T. (1986) Orthanilic acid and analogues as carbon sources for bacteria: Growth physiology and enzymic desulfonation. J. Gen. Microbial. 132, 1215-1220. [5] Feigel, B.J. and Knackmuss, H.-J. (1988) Bacterial catabolism of sulfanilic acid via catechol 4-sulfonic acid. FEMS Microbiol. Lett. 55, 113-l 18. [6] Wittich, R.M., Rast, H.G. and Knackmuss, H.-J. (1988) Degradation of naphthalene-2,6and naphthalene-1,6-disulfonic acid by Moraxellu sp. Appl. Environ. Microbial. 54, 1842- 1847. [7] Ohe, T. and Watanabe, Y. (1988) Microbial degradation of 1,6- and 2,6-naphthalene-disulfonic acid by Pseudomonas sp. DS-1. Agric. Biol. Chem. 52, 2409-2414. [8] Lindner, 0. (19851 Benzenesulfonic acids and their derivatives. In: Ullmann’s Encyclopedia of Industrial Chemistry, 5th edn. (Gerhartz, W., Ed.), Vol. A3, pp. 507-537, VCH Verlagsgesellschaft, Weinheim, Germany. [9] Pitter, P. and Chudoba, J. (1990) Biodegradability of Organic Substances in the Aquatic Environment. CRC Press Inc., Boca Raton, Florida, USA. [lo] Sharak-Genthner, B.R., Davies, C.L. and Bryant, M.P. (19811 Features of rumen and sewage sludge strains of Eubacterium limosum, a methanol- and Ha-CO,-utilizing species. Appl. Environ. Microbial. 42, I2- 19. [I 11 Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of protein utilizing the principle of protein dye binding. Anal. Biochem. 72, 248-254. [12] Schmidt, K., Liaaen-Jensen, S. and Schlegel, H.G. (1963) Die Carotinoide der Thiorhodaceae. Arch. Mikrobiol. 46, 117-126. [13] Stanier, R.Y. and Ingraham, J.I. (1954) Protocatechuic acid oxidase. J. Biol. Chem. 210. 799-808.

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[14] Feigel, B.J. and Knackmuss, H.-J. (1993) Syntrophic interactions during degradation of 4-aminobenzenesulfonic acid by a two species bacterial culture. Arch. Microbial. 159, 124130. [15] Feigel, B.J. (19901 Synergistischer Abbau von 4-Aminobenzolsulfonat durch Hydrogenophaga palleronii und Agrobacterium radiobarter. Thesis, University of Stuttgart. [16] Johnston, J.B., Murray, K. and Cain, R.B. (1975) Microbial metabolism of aryl sulphonates. A re-assessment of colorimetric methods for the determination of sulphite and their use in measuring desulphonation of aryl and alkylbenzene sulphonates. Antonie van Leeuwenhoek 41, 493-5 11. [17] Junker, F., Fields, J.A., Bangerter, F., Ramsteiner. K., Kohler, H.-P., Joannou, C.L., Mason, J.R., Leisinger, T. and Cook, A.M. (1994) Oxygenation and spontaneous deamination of 2-aminobenzenesulphonic acid in Alcaligenes sp. strain 0- 1 with subsequent meto ring cleavage and spontaneous desulphonation to 2- hydroxymuconic acid. Biochem J. 300. 429-436. [I81 Junker, F., Leisinger, T. and Cook, A.M. (19941 3Sulphocatechol 2,3-dioxygenase and other dioxygenases (E.C.l.13.11.2 and EC 1.14.12:) in the degradative pathways of 2-aminobenzenesulphonic, benzenesulphonic and 4toluenebenzenesulphonic acids in Alcaligenes sp. strain 0- 1. Microbiology 140, 1713-1722. [l9] Tyson, CA. (1975) 4-Nitrocatechol as a calorimetric probe for non-heme iron dioxygenases. J. Biol. Chem. 250, 17651770. [20] MacDonald, D.L., Stanier, R.Y. and Ingraham, J.L. (19541 The enzymatic formation of /%carboxymuconic acid. J. Biol. Chem. 210, 809-819. [21] Crawford, R.L. (1975) Novel pathway for degradation of protocatechuic acid in Bacillus species. J. Bacterial. 121, 53 l-536. 1221 Dagley, S., Geary, P.J. and Wood, J.M. (19681 The metabolism of protocatechuate by Pseudomonas testosteroni. Biochem. J. 109, 559-568.