Arch Microbiol (1990) 154:410-413 Archives of

Hicrnbiology �9 Springer-Verlag 1990

Selection of trichloroethene (TCE) degrading bacteria that resist inactivation by TCE Jens Ewers, Doris Freier-Schriider, and Hans-Joachim Knackmuss

Fraunhofer-Institut f/Jr Grenzfl/ichen und Bioverfahrenstechnik, Nobelstrasse 12, D-7000 Stuttgart 80, Federal Republic of Germany

Received February 20, 1990/Accepted April 26, 1990

Abstract. Two isoprene (2-methyl-1,3-butadiene) utilizing bacteria, Alcaligenes denitrificans ssp. xylosoxidans JE 75 and Rhodococcus erythropolis JE 77, were identified as highly efficient cooxidizers of TCE, cis- and trans- dichloroethene, 1,1-dichloroethene and vinylchloride. Isoprene grown cells eliminate chloride from TCE in stoi- chiometric amounts and tolerate high concentrations of TCE.

Key words: Isoprene - Cooxidation - Simultaneous break down

Trichloroethene is one of the most commonly used degreaser. Its widespread application in metal, glass and textile industry is also favoured due to its low acute toxicity and high votality. The biological persistence of TCE, however, makes it a widespread pollutant of soil and groundwater. Although the mechanism of persistence has not yet been investigated the high degree of chloro- substitution must be suspected as the cause of the impeded electrophilic attack of the molecule by oxy- genase of aerobic microorganisms. Furthermore TCE is unlikely to serve as sole source of carbon and energy so that productive breakdown cannot be expected.

In contrast methane or toluene degrading bacteria have been described as cooxidizers of TCE (Wilson and Wilson 1985; Fogel et at. 1986; Nelson et al. 1986, 1987, 1988; Fliermans et al. 1988; Little et al. 1988; Tsien et al. 1989; Uchiyama et al. 1989). Active enzymes in the methylotrophs are monooxygenases which display a rather broad substrate specificity and cooxidize a wide spectrum of chemicals including TCE. The degradation of TCE by toluene degrading systems have in part been correlated with unusual monooxygenase activities which hydroxylate toluene in 4-position (Winter et al. 1989) or in 2-position (Shield et al. 1989). The classical toluene dioxygenase has also been implicated in TCE oxidation which exhibits monooxygenase activity with certain sub-

strates (Wackett and Gibson 1988; Wackett and House- holder 1989; Zylstra et al. 1989). All these oxygenase mediated activities suffer from inactivation by the forma- tion of a highly reactive product. A TCE epoxide was proposed as a suicide metabolite. The mechanism of cytotoxicity of TCE in Pseudomonas putida F~ has re- cently been demonstrated by covalent modification of cellular components (Wackett and Householder 1989).

This observation prompted us to select for monooxy- genase activities in microorganisms which, in contrast to the hitherto described bacteria, are preadapted to detoxificy a TCE-epoxide. Suitable substrates for selec- tion of TCE degrading activities, that are not inactivated by the xenobiotic compound, must be structural ana- logues of TCE. This requires a readily degradable struc- ture which exhibits not only a TCE-like geometry but also a double bond that is attacked through epoxidation. Microorganisms which degrade these unsaturated com- pounds via epoxidation must harbour a potential to detoxify the epoxide by addition of cellular nucleophiles such as water to form trans-dihydrodiols (Habets- Criitzen and de Bont 1985).

Isoprene proved to fulfill some of these criteria be- cause it is readily utilized by a large number of bacteria from soil and water samples. From these sources 30 dif- ferent enrichment cultures were screened for oxidative activity towards TCE.

Cultures were grown on isoprene as sole carbon source in mineral medium (Dorn et al. 1974) using 100-ml Erlenmeyer flasks which contained 10 ml of medium. Isoprene was directly introduced as a liquid so that the initial concentration was 10 mM (all concentrations of volatile substrates were calculated as if being completely dissolved in the aqueous phase). Biomass from the enrich- ment cultures were washed twice and resuspended in 100 mM Tris-sulfate buffer pH 8.0. The cell suspensions (100 gt, optical density at 546 nm 15-20) were trans- ferred to wells of micro titer plates and supplemented with 100 ~tl TCE solution (2 mM). The plates were incu- bated at 30~ over night. According to the method of Weightman et al. (1985) the vials were treated with

411

1 4 - - ~ 4 o [

= - ~- I X / - - . A . . . ~ /

4 ~ lo

V \o/ o o l ,

0 0 10 20 30 40 50

Time (h)

Fig. 1. Growth of Rhodococcus erythropolis JE77 with isoprene (5 raM) in the presence of TCE (total concentration 1.1 mM) in mineral salts medium. Samples from the gasphase were analyzed for isoprene (A) and TCE (A) by GC (Gaschromatograph 438 A equipped with Pora Plot Q column 25 m x 0.32 mm, FID or ECD from Chrompack BV, Delft, Netherlands; the oven temperature

1 E 1 , 2 ~

" E '~" 1 |

"~ 0.8 e- " 0 Q

0.6

0 0.4

0.2

-0.1 0

60 60

was 200~ Chloride concentration ([1) in the culture fluid was determined by ion chromatography (hplc pump S 1000, conductivity detector S 3/10, separation column LCA AO1 and suppressor column RA 03 from Sykam GmbH, Gauting, FRG; molile phase: 1 mM NazCO3, 2 mM NaHCO3; flowrate 2 ml min-1). Growth ( t ) was followed spectrophotometrically at 546 nm

7 O

._ 60 I-" .c 0~ 5 0

e - J

40- 121

30

/ 20 t " l " J I0 m ~mm~mm'-''-'~m/,

o ) i i

0 200 400 600 TCE ( rag / I )

I I 800 1000 1200

Fig. 2. Doubling times of strain 77 during growth with 10 mM isoprene in the presence of increasing concentrations of TCE. For turbidity measurements fluted Erlenmeyer flasks were equipped with a side tube which allowed estimation of doubling times by use of a Klett-Summerson Photoelectric Colormeter (Klett Manufacturing Co., New York, USA)

AgNO3 solution (10 gl, 0.1 M) and exposed to 254 nm UV-light (UV-lamp Min UVIS from Desaga, Heidelberg, FRG).

Cultures with the highest activities of dechlorination were identified by precipitation of silver. Single isolates were obtained by growth (30 ~ C) on selective agar plates in an isoprene atmosphere (10% v/v) and subsequently on nutrient broth (8 g/l). The isolates were screened more quantitatively for maximum dechlorinating activity in micro titer plates by the following procedure: Cells were grown in Erlenmeyer flasks and washed twice with 0.25 mM phosphate buffer pH 6.9 which contained so- dium chloride (9 g/l). Cells were suspended in the buffer solution (100 gl each, OD54e = 10) and transferred to a series of 7 wells containing NaC1/phosphate buffer (100 gl) with increasing phosphate concentrations (0.25, 0.5, 1.0, 1.5, 2.5 and 5.0 mM). Bromocresol purple

(24 mg/1) and TCE (260 mg/1) were added to the wells. Cells in the seventh well (0.25 mM phosphate) were inac- tivated by NaN3 (20 g/l) and served as a control. Relative dechlorinating activities were judged after incubation over night by comparing indicator shifts at different buffer concentrations. Two of the isoprene utilizing iso- lates noticeably exhibited high activities and caused a colour change from violet to yellow even at 5 mM phos- phate buffer. They were identified as Alcaligenes denitri- ficans ssp. xylosoxidans JE 75 and Rhodococcus erythropolis JE 77 by the German Collection of Microor- ganisms and Cell Cultures DSM, Mascheroder Weg 1 B, 3300 Braunschweig, FRG.

Growth of the strain JE 77 in the presence of TCE is described by Fig. 1. Obviously TCE is mineralized with high velocity only after complete consumption of the growth substrate isoprene. However during growth on isoprene TCE is still dechlorinated even at a concen- tration as high as 1000 mg/1 of medium.

Degradation of TCE by the present organisms can be explained by epoxidation as the initial catabolic reaction. This corresponds to the degradation of isoprene by a Norcardia strain, which attacks this substrate by a mono- oxygenase (van Ginkel et al. 1987). The hypothesis of epoxidation is supported by identification of propene oxide as the product if oxidation of propene by resting cells of strain JE 77 (GC analysis as described in Fig. 1, the oven temperature was 110~ The TCE epoxide, when generated by methane or toluene degrading organisms, is highly reactive and, as shown by a recent publication (Wackett and Householder 1989), inactivates the cells by covalent modification of cellular components.

In contrast the present organisms, particularly strain JE 77, are not only highly efficient cooxidizers of TCE but also extremely insensitive against high concentra- tions of the xenobiotic. Thus, growth with isoprene is largely unaffected by TCE concentrations _< 200 mg/1 [< 1.5 mM]. At > 600 mg TCE/1 [> 4.6 raM], however, the doubling time increased drastically (Fig. 2). Never-

412

800 . L

2~ I I I

0 50 100 150 200

Time (rain)

Fig. 3. Degradation of TCE by isoprene grown cells of Rhodococeus erythropolis JE 77. The head space vials (20 ml) contained 5 ml of resting cells of different cell densities, corresponding to 116 (I~), 83 (+), 68 (2x), 34 (11) I.tg protein/ml. They were incubated with 800 gg/1 of TCE at 30~ and shaken at 130 rpm. Samples were taken at intervals and residual TCE concentrations were determined gaschromatographically after multiple head space extraction (Kolb and Pospisil 1985; Kuch 1980) as described in Fig. i (head space sampler HSS 3950 from Dani S.p.A., Monza, Italy)

Table 1. Specific rates" of conversion of isoprene and chloro- substituted ethenes by isoprene grown cells b of Alcaligenes denitri- ficans ssp. xylosoxidans JE 75 and Rhodococcus erythropolis JE 77

Initial Strain Strain concentration JE 75 JE 77 [mg/l]

Perchlorethene 0.4 _< 1 x 10 -5 _< 1 x 10 -s Trichlorethene 0.8 1.5 x 10 -* 1.8 x 10 -4 cis-Dichloroethene 9 7.0 x 10 .3 3.8 x 10 .3 trans-Dichloroethene 9 8.2 x 10 -4 8.8 x 10 .4 1,l-Dichloroethene 6 2.2 x 10 .2 1.2 x 10 -2 Vinylchloride 40 2.6 x 10 -2 1.6 x 10 -2 Isoprene 60 7.1 x 10 - 2 6.9 x 10- 2

a Spec. activities are given in micromoles substrate oxidized per minute per mg protein. Different initial substrate concentrations were used because of major differences in the response of the detec- tor system of the gas chromatograph towards different ethenes. After solubilization of the cell protein (1 N NaOH, 90~ 10 rain), the determination was carried out by the method of Lowry (Hartree 1972) b Cells were grown and processed as described in Fig. 3

theless under these conditions TCE dechlorination still occurs during growth with isoprene. Reaction mixtures with 63 rag/1 or 147 rag/1 [0.48 mM or 1.1 raM] TCE, when analyzed after 18 days of incubation, contained 1.2 mM or 1.9 mM chloride. This corresponds to 83 and 57% of dechlorination of TCE respectively. At TCE con- centrations higher than 200 rag/1 the maximum concen- tration of chloride in the culture fluid was 2.1 - 2 . 2 raM. Obviously, in the absence of an energy substrate resting cells (360-400 lag protein/ml from 10 mM isoprene) can maximally mineralize 90 rag/1 [0.68 mM] TCE.

Turnover of TCE at low concentrations (< 800 lag/l) by isoprene grown resting cells (taken from the ex- ponential growth phase, washed and resuspended in

25 mM phosphate buffer, pH 6.9) was strictly pro- portional to the amount of biomass in the reaction mix- ture and was constant over a period of at least 2.5 h (Fig. 3). Specific turnover rates of isoprene and differ- ently chlorinated ethenes were similar in both organisms, A. denitrificans ssp. xylosoxidans JE 75 and Rhodococcus erythropolis JE 77 (Table 1). High initial activities were measured with 1,1-dichlorethene. This compound, how- ever, is the only substrate which caused a 80% reduction of activity within 20 rain. The epoxide of this compounds was shown to be extremly reactive and decomposes under physiological conditions with a half life time of 2 s (Liebler and Guengerich 1983). Therefore, in its cytotoxic effect, l,l-dichloroethene differs from all other chloro- ethenes.

Obviously the selection with isoprene as a structural analogue reveals a naturally existing catabolic potential towards chloroethenes, particularly TCE, which out- matches the hitherto described microorganisms with respect to its extraordinary tolerance towards high sub- strate concentrations. Nevertheless the existing isolates do not efficiently degrade tetrachloroethene. This re- quires the improvement of structural analogy between the enrichment substrate and the higher chlorinated ethenes which takes into consideration also the electrophilic character of these compounds. Furthermore, high struc- tural analogy would make both the chloroethene and the energy supplying, inducing chemical competitive sub- strates and allow simultaneous degradation of these xenobiotics in a continuous process.

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