World Journal
of Microbiology
and Biotechnology
9, 648452
Degradation of Delor 103, a technical mixture of polychlorinated biphenyls, by selected bacteria K. Dercov&* s. BalBi, L’. HaluSka, E. Sturdik, K. VozArovh, J. KrupCik, E. Benickh and P. Bielek Ability to utilize a technical mixture of polychlorinated biphenyls (PCB), Delor 103, as the sole carbon source, has been tested in 14 bacterial strains. For the five best growing strains (Ah&genes latus, Alcaligenes eutrophus, Comamonas testosteroni, Micrococcus varians and Pseudomonas putida), the dependence of the degradation of individual PCB congeners on the number of chlorine substituents is discussed. I@/
WOT~S:
Alcaligenes, Comamonas, degradation,
Delor
103, Micrococcus,
Biodegradation of polychlorinated biphenyls (PCB) is of environmental interest because of their low degradability, toxicity and mutagenicity and the bioaccumulation of these persistent environmental pollutants (Abramowicz 1990; Hooper et al. 1990; Luotamo 1991; Rogan & Gladen 1992). The use of selected microorganisms, which are able to degrade PCB into less hydrophobic products, both aerobically and anaerobically (Furukawa 1982; Bedard et al. 1986; Quensen et al. 1990), represents one potential way to remove PCB from the environment. This communication reports the screening of 14 bacterial strains as potential PCB degraders, using Delor 103 as the only carbon source under aerobic conditions. Both liquid and solid media are used, the latter better simulating the conditions in soils. For the five best-growing microorganisms, the spectrum of degraded congeners is determined by GC with electron capture detection and its structural dependence discussed.
K. Dercova. S. Balat, L. Haluska, E. Sturdlk and K. Vozarova are with the Department of Biochemical Technology, Faculty of Chemical Technology, Slovak Technical University, 812 37 Bratislava, Slovakia; fax: + 427 49 31 98. J. KrupEik and E. Benicka are with the Department of Analytical Chemistry, Faculty of Chemical Technology, Slovak Technical University, 812 37 Bratislava. Slovakia. P. Bielek is with the Soil Fertility Research Institute, 827 13 Bratislava, Slovakia. ‘Corresponding author. @ 1993 Rapid Communications
648
of Oxford
Ltd
World ~ourml of Microbiology and Biotechnology, Vol 9, 1993
Materials
polychlorinated
biphenyls,
l?m&monas.
and Methods
Chemicals
A commercial mixture of PCB, Delor 103 (Chemko Straiske, Slovakia) containing 40 to 42% (w/v) of bound chlorine, biphenyl (Lachema, Czech Republic), and hexane UV (Sojuzchimexport, Commonwealth of Independent States) were used. For purification purposes, hexane was mixed (6: 1 v/v) several times with a mixture of H,SO, (3 M) and KMnO, (0.5 M), intensively shaken for I to 2 days, neutralized with 10% (w/v) Na,CO,, for 12 h, rectified and then dried with CaCO,. Microorganisms The following 14 bacterial strains were used in the experiments: Alcaligenes eutrophw CCM 3727, Batiks subtilis CCM 2722, Bacillus subtilis CCM 2216, Bacillus licheniformis CCM 2145, Bacillus stearothermophilas CCM 2183, Bacillw srrbfilis CCM 1718, Bacillus subtilis CCM 2267, Comamonas testosferoni CCM 1931, Micrococcw varians CCM 2353, Pseudomonas putida CCM 3423, Pseudomonas saccharophilla CCM 1980, Bacillus subtilis NA 64, Bacillus subtilis DPI (all from the Masaryk University, Bmo, Czech Republic; NA 64 and DPI originally from the Department of Biochemical Technology, Slovak Technical University, Bratislava, Slovakia, the rest from the Czech Collection of Microorganisms) and Alcaligenes lams DSM 1122 (from Deutsche Sammlung von Microorganismen, Braunschweig, Germany). Cultivation Conditions The synthetic medium used. This is composed
for bacteria described by Pirt (1967) was of five parts, A to E. Part A is K,HPO,
PCB degradation 84.5 g; B is MgS0,.7H,O, 20 g; C is CaCl,, 1.0 g; D is FeSO,. 7H,O, 5.0 g; ZnS0,7H,O, 5.0 g; MnS0,.5H,O, 5.0 g; CuSO,. 5H,O, 1.0 g; CoCl,.6H,O,l.O g; Na,B,O,, 1.0 g; NaMoO,H,O, 1.0 g; and E is NH&I, 20 g. Parts A, B, C and E were each dissolved in 500 ml of distilled water and autoclaved 20 min at 120 kPa. The first three ingredients of part D were each dissolved in 100 ml distilled water, and the other ingredients of part D in 1 1 each. These part D solutions were autoclaved separately (20 min at 20kPa), mixed 10:1:1:10:10:10:10 (by vol.) and 52ml then mixed with 448 ml distilled water to give the final part D solution. The solutions of parts A to E were then mixed 1O:l:l:l:lO (by vol.) and mixed with 876 ml to 124 ml, distilled water. The volume of samples and carbon source are specified in the description of each experiment. Cells were grown aerobically at 30°C on a rotary shaker (180 rev/min). inhibition of Groulth on Glucose Strains were incubated in 20-ml test tubes each containing 5 ml of the synthetic medium supplemented with 1% (w/v) glucose, 1 ml of vital inoculum (grown on medium with 1% (w/v) of glucose for 48 h; 0.05 g dry wt/l) and 1% (v/v) Delor 103 or 1% (w/v) of biphenyl. Biphenyl and PCB, which have limited solubility in aqueous media, formed an undissolved phase, at the bottom of the test tubes, which served as a depot. Growth was monitored over a period of 7 days by measuring absorbance (A) at 600 nm. Growth
on Biphenyl
and PCB in Liquid
after 0.25 min at 5O”C, raised to 190°C at 70°C/min, then to 260°C at 2.5”C/min and finally held at 260°C for 10 min. The detector response was calibrated using the method described by Krupcik et al. (1992). Delor 103 consists of about 40 congeners at detectable levels, with elution times ranging from 8 to 40 min. The reproducibility of the quantitative analysis was checked using a hexane solution of Delor 103 (at lop4 g/l) over 15 days. Relative deviations for congeners which did not interfere with background were around 3%. Growth
on Solid Medium
Petri dishes were filled with 2% (w/v) agar medium (Pirt 1967). Delor 103 (0.5 ml) was dissolved in 50 ml of hexane and 0.5 ml of the solution applied uniformly over the surface of the medium. Biphenyl was added from a stock solution (0.25 g/50 ml of hexane) in the same way. The final concentration of Delor 103 and biphenyl were 0.025% (v/v) and 0.025% (w/v), respectively. The plates were heated in a thermostat at 37°C for 48 h in order to evaporate the hexane. Plates were inoculated centrally with a loopful of the microorganisms, taken directly from a maintenance slope without adaptation, and incubated at 30°C for 21 days, Growth was assessed visually.
Results
and Discussion
Mediwn
Growth of microorganisms on Delor 103 (1% v/v) or biphenyl (1% w/v) as the sole carbon source was monitored in 20-ml test tubes each containing 3 ml of the synthetic medium and 3 ml of bacterial suspension (0.05 g dry wt/l) pre-adapted for 7 days to Delor 103 or biphenyl. Determination of Degraded Congeners Cultures were incubated for 13 days in 500-ml flasks, each with 150 ml of synthetic medium saturated with Delor 103 as the sole carbon source (the surplus was removed) and 10% (v/v) of 48-h inoculum (0.25 g dry wt/l) pre-adapted to Delor 103 for 2 months. Extracfion. Following incubation, PCB were extracted from 10 ml of the cultivation medium with 3 x 3 ml hexane. The samples were intensively shaken for 1 min and the phases separated. The extracts were concentrated to 1 ml by blowing N, above the surface. The discrimination degree of each of the various congeners was tested for the Delor 103 solution (at 10C4 g/l) and an appropriate correction was includ’ed in the calculation of the mass concentration. Extraction efficiency, assessed by comparison with the Delor 103 dissolved in medium, was better than 99%. Extraction efficiency was also checked in the experiments, after the cells were lysed by increasing the pH to 12 with 5 M NaOH and heating at 70°C for 60 min. In all other aspects, the cell-free controls were treated in the same way as the experimental samples. The presence of microorganisms did not decrease extraction efficiency. PCB
Gas Chromatography. The degradation kinetics of the individual PCB congeners of Delor 103 were monitored by GC of 1~1 samples of the hexane extracts with H, as carrier gas, using an electron capture detector (ECD) (300°C, make-up gas N,, at 40 mbmin), fitted with a column injector and using a 40 m x 0.25 mm I.D. fused-silica capillary column with a non-polar (100% dimethylpolysiloxane gum; stationary phase SPB -I thickness 1 pm). The pressure was held for 0.2 min at 40 kPa and then increased to 220 kPa at 70 kPa/min. The temperature was,
Inhibition of Growth by Biphenyl and Delor 103 PCB-degrading microorganisms can use PCB, the less chlorinated congeners preferentially, as sole carbon source. If PCB are present in sufficient amounts (0.1 to 1% w/v), growth can be easily monitored. Such concentrations of many xenobiotics would be toxic to the majority of microorganisms. Fortunately, PCB have limited solubility in aqueous-media and so their addition in these amounts results in undissolved PCB droplets, at the bottom of the test-tubes which serve as depots continually supplying the carbon source. Simultaneously, the PCB concentration in the medium is kept low. Since PCB are toxic to a variety of microorganisms, it is necessary to determine their non-toxic concentrations prior to any study of their biodegradability. Only the strains with weak to no inhibition can be expected to grow on Delor 103 as sole carbon source. Since the toxicity tests are shorter than growth experiments, they serve as a useful preliminary screening. This kind of pre-selection can also be used for other compounds. The semi-quantitative results of the tests of the inhibition by biphenyl and Delor 103 of growth after 7 days’ incubation are summarized in Table I. Alcaligenes lafus, Alcaligenes eutrophus, Bacillus stearothermophilus, Comamonas testosferoni, Micrococcus varians, Pseudomonas putida and Pseudomonas saccharophilla appeared not to be inhibited. Growth on Biphenyl and Delor 103 The ability of 14 bacterial strains to grow on a synthetic medium with either Delor 103 or biphenyl (possible inducer) as the only carbon source was tested using both liquid and
World Journal of Microbiology and Biotechnology. Vol 9. 1993
649
K. Dercovh
Table
et
al.
1. The growth
of bacteria
on biphenyl
(6) and Delor
Mlcroorganlsm*
103 (D).
lnhlbltlont
Utlllzatlon
0
A. latus DSM 1122 A. eutrophus CCM 3727 B. licheniformis CCM 2145 B. stearothermophilus CCM 2183 B.subtiius DP 1 B.subtilus NA 64 B. subtilis CCM 1718 8. subtilis CCM 2218 B. subtilis CCM 2267 B. subtilis CCM 2722 C. testosteroni CCM 1931 M. varians CCM 3253 Ps. putida CCM 3423 Ps. saccharophilla CCM 1980
D
+
+
+ -
+ ++ ++ ++ ++ ++
-
-
Liquid
medlumz
Solid
B
D
++ ++ + + ++ + + + + -t ++ ++ ++ -
+ + + + + + + + + + + -
Full names of strains and origins are given in Materials and Methods. t + +-Intense (AA < 0.1); +-weak (AA < 0.4); --none (AA > 0.4). $ Growth after 14 days: + +-intense (AA > 0.2); +-weak (AA > 0.1); --none 8 Growth after 7 days, assessed visually: + +-intense; +-weak; --none.
B
mediums D
l
(AA < 0.05).
28
2'0
10
Retention Flgure 1. Degradation saturated with Delor degradation for each
time
2"
(min)
kinetics of the individual congeners of Delor 103 by Alcaligenes eutrophus CCM 3727 (in mineral medium 103, pH 6.5, rotary shaker, 30°C) after 0, 6 and 13 days. Congener assignments and percentages of Delor 103 peak are presented in Figure 2.
PCB degradation solid media. During 14 days’ cultivation, growth was assessed spectrophotometrically in liquid media and visually on solid media (Table I). An increase in A of at least 0.1 was considered evidence of growth. The best results were achieved using Alcaligenes latus, Alcaligenes eutrophus, Comamonas testosteroni, Micrococcus varians and Pseudomonas pufida, for which the spectra of degraded congeners were determined in subsequent experiments. Determination of Degraded Congeners The hexane extracts of the cultivation media of the five selected strains, initially containing Delor 103 at saturation concentration (i.e. no undissolved Delor 103 present), were analysed by GC-ECD after 13 days’ cultivation. It is generally accepted that there is a common major catabolic pathway for chlorinated biphenyls (Mass6 et al. 1984; Bedard et al. 1987), the pattern and number of chlorine substitutions affecting the biodegradability of individual congeners (Furukawa el al. 1979; Bedard & Haberl 1990). The relationship between the biodegradation rate constants of a number of polychlorinated biphenyls and their hydrophobic and electronic structural parameters have been studied (Parsons et al. 1991). The results suggest that reactivity and possibly the enzyme binding of PCB, rather than their permeation through the bacterial membranes, control their biodegradation rates. In the present study, the most even degradation of individual congeners was observed with Alcaligenes eutrophus (Figure I). Micrococcus varians, on the other hand, gave the highest degree of congener specificity (Figure 2). Congener 118 (2,3’,4,4’,5-CB) was the most resistant compound in the mixture; except for Pseudomonas putida, all strains degraded 42 to 53% of the initial amount of this compound. Since congener 118 does not contain two ortho-chlorines, our data do not seem to support the Furukawa (1982) postulate that PCB congeners containing two chlorines in the orthopositions of the single ring or both rings are the ones that
show striking resistance to degradation. The results (Figure 2) indicate that congeners 4 (except with Pseudomonas putida, Alcaligenes eutrophus and Alcaligenes latus), 18, 45, 52, 49, 47 + 48 (52, 49 and 47 + 48 except with Pseudomonas putida and Alcaligenes lab), 44 (except with Alcaligenes lab), and 40 (except with Alcaligenes latus and Comamonas teslosteroni) are well degraded (more than 80% after 13 days‘ incubation). The other ortho-substituted congeners were either coeluted or not detected. Degradation Speclrum of Delor 103 by Bacterial Strains To compare the ability of bacterial strains to degrade di-, tri-, tetra- and penta-chlorobiphenyls in the PCB mixture (Delor 103), the percentage of degradation was calculated for individual groups of congeners, differing in the number of chlorine atoms (Figure 3). More than 85% of the di-CB were degraded by all the strains except Alcaligenes eutrophus (77%). The small decrease in the concentrations of tri-, tetra- and penta-CB were observed with Micrococcus varians, which gave < 77% degradation. Degradations by Pseudomonas putida, Comamonas testosteroni, Alcaligenes lafus and Alcaligenes eutrophus were in the range of 78 to 96%. There are apparent differences in microbial selectivity for individual congeners among the 30 detectable congeners
Alcaligenes latus
Comamonas testosteroni
Micrococcus varians
Micrococcus
varians Comamonas festostefoni
Pseudomonas putida 100
-
Alcaligenes la tus Alcaligenes eutrophus di-CB
Pseudomonas putida
Figure Delor
2. Degradation 103 by bacterial
not detected.
congeners profiles strains
of individual PCB congeners after 13 days. --Congener
tetra-CB
penta-CB
congeners
Figure 3. Degradation profiles of di-, tri-, tetraand pentachlorobiphenyls of Delor 103 by bacterial strains after 13 days. PCB congeners (listed in order of increasing GC retention time)
:
PCB
tri-CB PCB
of
were di-CB: 4.7, 9, 6, 5, 8, 15; tri-CB: 19, 18, 17, 27, 16, 32, 26, 31, 28, 33, 22, 37; tetra-CB: 53, 51, 45, 46, 52, 49, 40, 47, 44, 42, 37, 71, 72, 64, 41, 40, 74, 94, 70, 76, 66, 56, 60, 77; and penta-CB: 101, 110, 118.
World Journal
of
Microbiology and Biotechnology. Vol 9. 1993
651
K. Dercovh et al. (Figure 2). A tendency to degrade less-chlorinated congeners (di- and tri-CB) rather than high-chlorinated congeners (tetra- and penta-CB) was observed in Micrococctrs variant and Ahligenes lattrs (Figure 3). Degradation of di-, tri-, tetra- and penta-CB congeners by these two strains appears to be related to the degree of chlorination and confirms the observation made by Furukawa (1982): that degradation decreased as chlorine substitution increased, dependent on the chlorine position. For other microorganisms, the influence of chlorination degree on degradation rate is not so straightforward.
Acknowledgement This work was made possible by Grant No. l/99096/92 from the Slovak Grant Agency.
ally transformed eutrophus H850.
polychlorinated biphenyls Applied und Environmenful
by Alculigenes Microbiology 53,
1094-1102. Furukawa, K. 1982 Microbial degradation of polychlorinated biphenyls (PCBs). In Biodegrudution and Detoxificution of Environmenful Pollutants, ed Chakrabarty, A.M. pp. 33-57. Boca Raton: CRC Press. Furukawa, K., Tomizuka, N. & Kamibayashi, A. 1979 Effect of chlorine substitution on the bacterial metabolism of various polychlorinated biphenyls. Applied and Environmentul Microbiology 38, 301-310. Hooper, SW., Pettigrew, C.A. & Sayler, G.S. 1990 Ecological fate, effects and prospects for the elimination of environmental polychlorinated biphenyls (PCBs). Environmental Toxicology and Chemistry 9, 655-667. Krupak, J., Koran, A., Petrik, J., Leclercq, P. A., Ballschmiter, K. 1992 On the use of reference standards for quantitative trace analysis of PCBs by HRGC analyses of technical PCB formulations by HRGC/FID. Chromutogruphiu 33, 514-552. Luotamo, M. 1991 Congener specific assessment of human exposure to polychlorinated biphenyls. Chemosphere 23,
1685-1698. Mass&
References Abramowicz, D.A. 1990 Aerobic and anaerobic biodegradation of PCB: a review. Critical Reviews in Biotechnology 10, 141-251. Bedard, D.L. & Haberl, M.L. 1990 Influence of chlorine substitution pattern on the degradation of polychlorinated biphenyl by eight bacterial strains. Microbial Ecology 20, 87-102. Bedard, D.L., Haberl, M.L., May, RJ. & Brennan, M.J. 1987 Evidence for novel mechanisms of polychlorinated biphenyl metabolism in Alculigenes eufruphus H850. Applied and Environmental Microbiology 53, 1103-1112. Bedard, D.L., Unterman, R., Bopp, L.H., Brennan, M.J., Haberl, M.L. &Johnson, C. 1986 Rapid assay for screening and characterizing microorganisms for their ability to degrade polychlorinated biphenyls. Applied and Environmental Microbiology 5 1, 761-768. Bedard, D.L., Wagner, R.E., Brennan, MJ., Haberl, M.L. & Brown, J.F.Jr, 1987 Extensive degradation of Aroclors and environment-
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F., PCloquin, L., Ayotte, C. & Sylvestre, M. biodegradation of 4-chlorobiphenyl, a model compound of chlorinated biphenyls. Applied and Environmental Microbiology 4 7, 947-95 1. Parsons, J.R., Commandeur, L.C.M., Van Eyseren, H.E. & Govers, H.A.J. 1991 QSAR and PARS for biodegradation of PCBs. The Science of Toatu/ Environment 109/110, 275-281. Pirt, S.J. 1967 A kinetic study of the mode of growth of surface colonies of bacteria and fungi. Journal of Generul Microbiology
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47, 181-197. Quensen, J.F., Boyd, S.A. & Tiedje, J.M. 1990 Dechlorination of four commercial polychlorinated biphenyl mixtures (Aroclors) by anaerobic microorganism from sediments. Applied and Environmental Microbiology 56, 2360-2369. Rogan, WJ. & Gladen, B.C. 1992 Neurotoxicology of PCBs and related compounds. Neurofoxicology 13, 27-36.
(Received in revisedform 23 May 1993; accepted 27 May 1993)