the percolating fluid of both percolators was examined and found to contain organisms that repeatedly removed added. TeCG (10 p.M). The mixed cultures from ...
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 1988,
p. 3043-3052
Vol. 54, No. 12
0099-2240/88/123043-10$02.00/0 Copyright C 1988, American Society for Microbiology
Degradation and 0-Methylation of Chlorinated Phenolic Compounds by Rhodococcus and Mycobacterium Strains MAX M. HAGGBLOM,t* LIISA J. NOHYNEK, AND MIRJA S. SALKINOJA-SALONEN
Department of General Microbiology, University of Helsinki, Mannerheimintie 172, SF-00280 Helsinki, Finland Received 18 July 1988/Accepted 20 September 1988
Three polychlorophenol-degrading Rhodococcus and Mycobacterium strains were isolated independently from soil contaminated with chlorophenol wood preservative and from sludge of a wastewater treatment facility of a kraft pulp bleaching plant. Rhodococcus sp. strain CG-1 and Mycobacterium sp. strain CG-2, isolated from tetrachloroguaiacol enrichment, and Rhodococcus sp. strain CP-2, isolated from pentachlorophenol enrichment, mineralized pentachlorophenol and degraded several other polychlorinated phenols, guaiacols (2methoxyphenols), and syringols (2,6-dimethoxyphenols) at micromolar concentrations and were sensitive to the toxic effects of pentachlorophenol. All three strains initiated degradation of the chlorophenols by parahydroxylation, producing chlorinated para-hydroquinones, which were then further degraded. Parallel to degradation, strains CG-1, CG-2, and CP-2 also 0-methylated nearly all chlorinated phenols, guaiacols, syringols, and hydroquinones. 0-methylation of chlorophenols was a slow reaction compared with degradation. The preferred substrates of the 0-methylating enzyme(s) were those with the hydroxyl group flanked by two chlorine substituents. 0-methylation was constitutively expressed, whereas degradation of chlorinated phenolic compounds was inducible.
polychlorinated phenolic compounds have been described (3, 5, 10, 11, 14, 16, 17, 29-31, 33, 35, 41), the initial degradation pathways are known in only a few cases (6, 7, 17, 29, 32). To study the mechanisms of chlorophenol degradation, we enriched for and isolated bacteria that degrade polychlorinated phenolic compounds. We used either tetrachloroguaiacol or pentachlorophenol as the enrichment substrate. In this paper we show how three such independently isolated Rhodococcus and Mycobacterium strains mineralized pentachlorophenol and degraded several other chlorinated phenols, guaiacols, and syringols.
Chlorinated phenolic compounds may be removed from the environment by complete or partial biodegradation or by biotransformation. Two different mechanisms have so far been described for aerobic biodegradation of chlorinated phenols. Degradation of mono- and dichlorinated phenols has been shown to proceed by oxygenation into chlorocatechols, with dechlorination only after ring cleavage of the chlorocatechol (9, 20, 21). A different mechanism involving hydrolytic and reductive dechlorinations has been described for degradation of polychlorinated phenols (6, 7, 17, 32). Rhodococcus chlorophenolicus PCP-I and a Flavobacterium sp. begin degradation of polychlorinated phenols by a hydrolytic para-hydroxylation into chlorinated para-hydroquinones (6, 32). In R. chlorophenolicus PCP-I, tetrachlorohydroquinone was further dechlorinated by subsequent hydrolytic and reductive reactions, removing all chlorine substituents before ring cleavage (7). The mechanism of anaerobic degradation of chlorophenols is less well known, and no pure bacterial culture has yet been described. In anaerobic sewage sludge and soil, pentachlorophenol was shown to be reductively dechlorinated (19, 23, 24). Mineralization of the phenol ring was also shown (24). Biotransformation reactions, where the carbon skeleton of the substrate remains unaltered, are an alternative to degradation. Several species of bacteria and fungi have been shown to 0-methylate chlorinated phenols, guaiacols (2methoxyphenols), syringols (2,6-dimethoxyphenols), and hydroquinones (1, 2, 13, 15, 18, 25, 26, 37, 42). The resulting chlorinated methoxybenzenes may resist aerobic microbial attack (1, 2) and have a high potential for bioaccumulation (26, 28). Chlorinated anisoles and veratroles have been found in fish exposed to pulp bleaching effluents, even though these compounds were not detected in the discharged wastewater (28). Even though several pure bacterial cultures degrading
MATERIALS AND METHODS
Abbreviations. The following abbreviations are used: PCP, pentachlorophenol; TeCP, tetrachlorophenol; TCP, trichlorophenol; DCP, dichlorophenol; PCA, pentachloroanisole; TeCA, tetrachloroanisole; TCA, trichloroanisole; DCA, dichloroanisole; TeCG, tetrachloroguaiacol (tetrachloro-2methoxyphenol); TCG, trichloroguaiacol; DCG, dichloroguaiacol; TeCV, tetrachloroveratrole (tetrachloro-1,2-dimethoxybenzene); TCV, trichloroveratrole; DCV, dichloroveratrole; TCS, trichlorosyringol (trichloro-2,6-dimethoxyphenol); DCS, dichlorosyringol; TCTMB, 1,2,3-trichloro-4,5,6-trimethoxybenzene;DCTMB,dichloro-4,5,6-trimethoxybenzene; TeCC, tetrachlorocatechol; TCC, trichlorocatechol; TeCH, tetrachlorohydroquinone (tetrachloro 1,4-dihydroxybenzene); TCH, trichlorohydroquinone; DCH, dichlorohydroquinone; MCH, monochlorohydroquinone; TeCMP, tetrachloro-4-methoxyphenol; TCMP, trichloro-4methoxyphenol; TeCDMB, tetrachloro-1,4-dimethoxybenzene; TCDMB, trichloro-1,4-dimethoxybenzene; GLC, gasliquid chromatography. Enrichment and isolation of chlorophenol-degrading bacteria. Three samples of soil and sludge contaminated with chlorinated phenolic compounds were used as the inoculum to enrich chlorophenol-degrading bacteria. Inoculum A was sludge from an aerated lagoon treating pulp and paper mill effluents; B was chlorophenol-contaminated soil from a
Corresponding author. t Present address: Department of Microbiology, New York University Medical Center, 550 First Ave., New York, NY 10016. *
3043
3044
HAGGBLOM ET AL.
sawmill timber-treating facility; C was a sample of contaminated soil from a location similar to B, after bioremediation by composting (38). Two 500-ml columns with softwood bark chips as a biofilm support were inoculated with 10 to 15 g each of materials A and B and percolated with 200 ml of a mineral salts medium (34). TeCG (>90% purity by GLC) was added weekly to a concentration of 10 to 50 FiM. The concentration of TeCG in the percolating fluid diminished to less than 1 p.M in 1 day because of absorption onto bark chips (4). After 3 months, the percolating fluid of both percolators was examined and found to contain organisms that repeatedly removed added TeCG (10 p.M). The mixed cultures from percolators A and B were further enriched by subculturing in mineral salts medium and feeding with 10 to 20 ,uM TeCG added at 2- to 3-day intervals and finally streaked onto DSM-65 (4.0 g of glucose, 4.0 g of yeast extract, 4.0 g of malt extract, 1,000 ml of H20; pH 7.2) (12) agar with 10 ,uM TeCG. A total of 130 colonies from each mixed culture were tested for TeCG degradation by measuring the removal of TeCG in liquid culture. One colony that degraded TeCG was purified from each culture (A and B) and designated CG-1 and CG-2,
respectively. A third chlorophenol-degrading mixed culture (C) was obtained from a soil dilution of a bioremediation site (38). The culture in minerals salts medium was enriched for a period of 8 months by repeated dilutions and feeding with PCP (5 mg liter-'). The culture was plated on yeast extract agar (3) containing PCP (20 mg liter-1), and one colony that degraded PCP was chosen for further study and designated CP-2. Characterization of isolates. The isolates had the characteristics of the nocardioform actinomycetes and were assigned to the genera of Rhodococcus (CG-1 and CP-2) and Mycobacterium (CG-2). Strains CG-1 and CP-2 exhibited cyclic changes in morphology of cocci to rods to cocci during growth and formed yellowish orange, mucoid colonies on DSM-65 agar. The bacteria contained menaquinones with nine isoprenoid units and one hydrogenated double bond, mycolic acids with 32 to 36 carbon atoms, and tuberculostearic acid (10-methyloctadecanoic acid). Strain CG-2 also exhibited a coccus to rod to coccus cycle of growth, but formed white, wrinkled colonies on DSM-65 agar. This strain contained menaquinones with nine isoprene units and one hydrogenated double bond, mycolic acids with more than 60 carbon atoms, and tuberculostearic acid. A detailed taxonomical description of the strains will be reported elsewhere (M. Haggblom, L. Nohynek, K. Kronqvist, and M.
Salkinoja-Salonen, manuscript in preparation). Culture conditions. Cells were grown in mineral salts medium (34) supplemented with vitamins (34) and trace elements (8), with 0.1% glucose (CG-2 and CP-2) or 0.1% mannose (CG-1) as a source of carbon, or in DSM-65 medium (12). For some experiments S to 10 ,uM PCP was added to the medium at 24-h intervals. The test substrates were separately added to 2- or 3-day-old cultures to a concentration of 10 ,uM. Cultures were incubated in a gyratory shaker (150 rpm) at 28°C in the dark. The bacterial density in degradation experiments was 108 to 109 ml-1. The toxicity of PCP and PCA to bacteria was determined as described previously for R. chlorophenolicus PCP-I (16). The bacteria were grown in DSM-65 medium in the presence of PCP or PCA at concentrations of 5, 10, 20, 50, and 100 p.M. The culture density at 600 nm was measured after 2 days of incubation and compared with that of a control
APPL. ENVIRON. MICROBIOL.
culture. The concentration causing a 50% inhibition of growth was interpolated from these results. Substrates and reference compounds. Di-, tri-, and tetrachlorophenols used in this study were produced by Fluka AG, Buchs, Switzerland, and Ega-Chemie, Steinheim, Federal Republic of Germany. The preparation of 2346-TeCP contained 20% (wt/wt) PCP. PCP was from E. Merck AG, Darmstadt, Federal Republic of Germany. [U-14C]PCP (3.9 x 10" Bq mmol-1) was from Pathfinder Laboratories Inc., St. Louis, Mo. TeCH and 25-DCH were from Eastman Kodak Co., Rochester, N.Y. All other compounds were synthesized by J. Knuutinen, Department of Chemistry, University of Jyvaskyla, Jyvaskyla, Finland. Chlorinated quaiacols and catechols were synthesized as described by J. Knuutinen (Ph.D. thesis, University of Jyvaskyla, Jyvaskyla,Finland, 1984). One TCG preparation was a 1:1 (wt/wt) mixture of 346-TCG and 356-TCG, designated 346/356-TCG. Another preparation of 346-TCG contained 10% (wt/wt) TeCG. The preparation of 46-DCG contained 30% (wt/wt) 35-DCG. Chlorinated syringols were synthesized by chlorination of syringol with Cl2 in CS2. TCH, 23-DCH, 26-DCH, and MCH were synthesized from chlorinated 4-hydroxybenzaldehydes (22) as described for chlorocatechols by Knuutinen (Ph.D. thesis). Chlorinated 4-methoxyphenols were prepared from chlorinated hydroquinones by limited methylation (J. Knuutinen, P. Autio, P. Klein, S. Kivela, L. Virkki, and M. Lahtipera, Chemosphere, in press). Chloroanisoles, chloroveratroles, and chlorinated 1,4-dimethoxybenzenes were prepared from the corresponding phenols, catechols, and hydroquinones by methylation with diazomethane (40). 2,4,6-Tribromophenol, used as an internal standard in analysis, was from Fluka AG. Analysis. Mineralization of [14C]PCP was monitored for both by trapping evolved 14CO2 and by following the concentration of PCP in the culture by GLC. Experiments were performed in 100-ml flasks with 20 ml of medium, closed with a rubber stopper with a 2-ml cup containing 0.5 ml of 1 M NaOH to absorb CO2. The NaOH solution was collected at specified times for the measurement of radioactivity and replaced by fresh NaOH solution. A 5-ml volume of scintillation fluid (Pico-Fluor 30, Packard Instrument Co., Inc., Rockville, Md.) was added to the 0.5 ml of the NaOH solution, and the radioactivity was measured with a scintillation counter (Rac Beta, LKB Instruments, Inc., Rockville, Md.) for 10 min at 150C. For GLC analysis the chlorinated phenolic compounds, with 2,4,6-tribromophenol added as an internal standard, were acetylated in buffer solution as described previously (16). The acetylated derivatives, together with anisoles, veratroles, and trimethoxybenzenes, were extracted into heptane and analyzed by GLC, using a gas chromatograph (Fractovap 2300, Carlo Erba Strumentazione) equipped with a capillary column (CP Sil 5, Chrompack, Middelburg, The Netherlands) and an electron capture 63Ni detector. The repeatability of analysis was ±+10%. For analysis of degradation intermediates, 1 ml of 10% Na2CO3 and 1 ml of acetic anhydride was added to 20 ml of culture, the acetyl derivatives were extracted with 5 ml of pentane, and the extract was concentrated in a stream of N2 to 40 p.l. Metabolites were identified by GLC-mass spectrometry, using a gas chromatograph (HP 5880, Hewlett-Packard Co., Palo Alto, Calif.) equipped with an Ultra 2 (Hewlett-Packard) or an HP-1 (Hewlett-Packard) capillary column and a mass selective detector (HP 5970 A, Hewlett-Packard). The retention times of the authentic compounds and ions used for selective ion monitoring are listed in Table 1.
DEGRADATION AND 0-METHYLATION OF CHLOROPHENOLS
VOL. 54, 1988
TABLE 1. Mobility of reference compoundsa in GLC with two different columnsb Compound
23-DCP 24-DCP 25-DCP 26-DCP 34-DCP 35-DCP 234-TCP 235-TCP 236-TCP 245-TCP 246-TCP 345-TCP 2345-TeCP 2346-TeCP 2356-TeCP PCP 23-DCA 24-DCA 25-DCA 26-DCA 34-DCA 35-DCA 234-TCA 235-TCA 236-TCA 245-TCA 246-TCA 345-TCA 2345-TeCA 2346-TeCA
2356-TeCA PCA 345-TCC 346-TCC TeCC 34-DCG 35-DCG 36-DCG 45-DCG
46-DCG 56-DCG 345-TCG 346-TCG 356-TCG 456-TCG TeCG 34-DCV 35-DCV 36-DCV 45-DCV 345-TCV 346-TCV TeCV 35-DCS TCS 13-DCTMB TCTMB TCH TeCH 235-TCMP 236-TCMP TeCMP TCDMB TeCDMB
2,4,6-Tribromophenol
Ion(s) monitored (mle)
161.8, 163.8 161.8, 163.8 161.8, 163.8 161.8, 163.8 161.8, 163.8 161.8, 163.8 195.8, 197.8 195.8, 197.8 195.8, 197.8 195.8, 197.8 195.8, 197.8 195.8, 197.8 229.8, 231.8 229.8, 231.8 229.8, 231.8 265.8, 267.8 175.9, 177.9 175.9, 177.9 175.9, 177.9 175.9, 177.9 175.9, 177.9 175.9, 177.9 209.8, 211.8 209.8, 211.8 209.8, 211.8 209.8, 211.8 209.8, 211.8 209.8, 211.8 243.8, 245.8 243.8, 245.8 243.8, 245.8 277.8, 279.8 211.9, 213.9 211.9, 213.9 245.8, 247.8 191.9, 193.9 191.9, 193.9 191.9, 193.9 191.9, 193.9 191.9, 193.9 191.9, 193.9 225.9, 227.9 225.9, 227.9 225.9, 227.9 225.9, 227.9 259.8, 261.8 205.9, 207.9 205.9, 207.9 205.9, 207.9 205.9, 207.9 239.9, 241.9 239.9, 241.9 273.9, 275.9 221.9, 223.9 255.9, 257.9 235.9, 237.9 269.9, 271.9 211.9, 213.9 245.8, 247.8 225.9, 227.9 225.9, 227.9 259.8, 261.8 239.9, 241.9 273.9, 275.9 329.7
Retention time b (min) in column:
1
2
12.60 12.17 12.12 11.82 13.02 12.42 14.95 14.32 14.14 14.39 13.58 15.18 16.82 16.07 16.02 18.30 11.80 11.16 11.17 10.03 11.41 10.80 14.41 13.75 12.65 13.67 12.07 13.92 16.41 14.85 14.87 17.31 18.22 17.45 19.56 15.02 14.48 14.20 15.53 14.82 15.44 17.04 16.27 16.25 17.37 18.57 13.88 13.33 12.22 14.38 16.12 14.59 17.16 16.31 18.70 14.40 17.06 17.98 19.35 16.75 17.40 18.29 16.07 17.20 17.21
12.96 12.48 12.46 12.19 13.32 12.66 15.30 14.61 14.51 14.68 13.88 15.47 17.13 16.39 16.31 18.61 12.14 11.60 11.51 10.23 11.65 11.00 14.78 14.08 12.92 13.99 12.26 14.17 16.79 15.08 15.05 17.59 18.60 17.85 19.96 15.36 14.75 14.53 15.94 15.24 15.89 17.42 16.58 16.55 17.80 18.91 14.23 13.60 12.47
14.75 16.45 14.82 17.44 16.64 19.08 14.66 17.40 18.34 19.68 17.08 17.86 18.64 16.46 17.53 17.63
3045
RESULTS Degradation and 0-methylation of chlorinated phenols, guaiacols, and syringols. We tested the ability of TeCGdegrading Rhodococcus sp. strain CG-1 and Mycobacterium sp. strain CG-2 and PCP-degrading Rhodococcus sp. strain CP-2 to degrade 29 different chlorinated phenolic compounds. The chlorophenols, chloroguaiacols, or chlorosyringols were added to 2-day-old cultures to a concentration of 10 ,uM. As a test for the inducibility of degradation, chloramphenicol (60 pug ml-') was added to parallel cultures to inhibit protein synthesis. The degradation and transformation of the chlorinated substrates by strains CG-1, CG-2, and CP-2 are shown in Fig. 1. The results show that the three strains, whether enriched on TeCG or PCP, degraded many different chlorinated phenols, guaiacols, and syringols. The strains also 0-methylated most of these compounds into the corresponding chlorinated anisoles, veratroles, and trimethoxybenzenes, respectively. The three strains differed from each other in their degradation and transformation specificities. In sterile medium the chlorinated phenols, guaiacols, and syringols were stable, with a recovery of over 90% after 2 days. The metabolites produced by the bacteria were identified by mass spectra and retention times in two different GLC columns, using authentic compounds as a reference (Table 1). The mass spectra of authentic PCA, TeCV, and TCTMB and of metabolites produced from PCP, TeCG, and TCS by strain CG-1 are compared in Fig. 2. Rhodococcus sp. strain CG-1, isolated from a TeCG enrichment culture, degraded several chlorophenols, chloroguaiacols, and chlorosyringols in 48 h, namely, PCP, 2346and 2356-TeCP, 235-, 236-, and 246-TCP, 24- and 34-DCP, TeCG, 346- and 346/356-TCG, and TCS, without accumulation of metabolites detectable by GLC (Fig. 1A). Noninduced cells did not significantly degrade any of the chlorinated phenolic compounds in the presence of chloramphenicol. However, the cells did 0-methylate all chlorophenols, chloroguaiacols, and chlorosyringols (Fig. 1B). 0methylation was also observed in the cultures without chloramphenicol. Another strain from a TeCG enrichment, Mycobacterium sp. strain CG-2, degraded several chlorophenols, chloroguaiacols, and chlorosyringols without accumulation of products. PCP, 2345-, 2346-, and 2356-TeCP, 234-, 235-, 236-, and 246-TCP, 24- and 26-DCP, TeCG, 345-, 346-, 346/ 356-, and 456-TCG, 34-, 35-, and 36-DCG, TCS, and 35-DCS were completely removed in the absence of chloramphenicol (Fig. 1C). Also, in the presence of chloramphenicol, uninduced cells of strain CG-2 degraded PCP, 2356-TeCP, 235and 246-TCP, TeCG, 346- and 346/356-TeCG, 36-DCG, and TCS (Fig. 1D). Small amounts of anisoles were also produced from chlorophenols. Rhodococcus sp. strain CP-2, isolated from a PCP enrichment culture, degraded most of the tested chlorinated phenolic compounds, namely, PCP, 2345-, 2346-, and 2356TeCP, 234-, 235-, 236-, and 245-TCP, 25-DCP, TeCG, 345-, 346-, 346/356-, and 456-TCG, 34-, 35-, and 36-DCG, TCS, and 35-DCS (Fig. 1E). In the presence of chloramphenicol, uninduced cells of strain CP-2 0-methylated nearly all of these compounds (Fig. 1F). a Phenols, catechols, guaiacols, hydroquinones, 4-methoxyphenols, and syringols were analyzed as the acetyl derivatives.
b The temperature program was 1 min at 50°C, increasing by 10°C min-' to 260°C. Column 1 was HP-1, and column 2 was Ultra 2 (Hewlett-Packard).
i
E
LiJ 50
I I.
CL C(LcL CLcL cL L(L cLcL
21,
rm
I
0
1As
4IC14
w
iA Metabolite from PCP
11
IIII1i
I
Lnw Lo o en cL cL M gNooX ;J; n*J;Xe*tu) X~ ~ ~ ~ C'
C4 04 04 c04
OI NNNN
ci~~~~~U
I
Pentachloroanisole
IB
263 278 \I \
OCH3
I
y so
ICCl
!
235
ClI
1
!-. LL
IL
1001
I..
L. IL mWe
2744
Metabolite from TeCG
259
;-/ c
-: 50-
196
216
\ Cl
-.A. .L-i
IL.,L
6
LL
100-
231
II. . h...
-1
200
100
.1
\
300
m/e
Tetrachloroveratrole
274 259
OCH3
0o ' 50-
50-I
OCH3
Cl
196
LI
+
216
I
11
.
k. IIL L
1-Li. I Ii 100
L 200
300
m/e
100
%E 100-
Metabolite from TCS
270 255
E
.-
212
S
227
50
4-
L. L N CM4 ,>en
U.
.~NNN
N
C NCON N c~
-
h.,J.,- L ft 1,
l.I
o) s.tXXXwwU
IDIl
IL ..
200
100
m m w
M/e
M
300
E
50
16o
FIG. 1. Degradation and 0-methylation of chlorinated phenols, guaiacols, and syringols by Rhodococcus sp. strain CG-1 (A and B), Mycobacterium sp. strain CG-2 (C and D), and Rhodococcus sp strain CP-2 (Eand F). Bars indicate the amount (percentage of initial substrate concentration) of substrate (-) and 0-methylated product (LI) found in the cultures after 48 h of incubation in the presence (+Cm, 60 Vig ml-') or absence (-Cm) of chloramphenicol.
FIG. and
TCS
TeCV,
3046
2.
by Rhodococcus
and TCTMB.
360
260
Mass spectra of metabolites sp.
strain
produced from PCP, TeCG, CG-1
and
authentic
PCA,
DEGRADATION AND 0-METHYLATION OF CHLOROPHENOLS
VOL. 54, 1988
100 .1
100 ,uM), whereas strain CG-2 was sensitive to PCA (50% inhibition of growth, 10 ,uM) (data not shown). DISCUSSION Three bacterial strains were independently isolated from sludge from a pulp and paper mill wastewater treatment plant and from chlorophenol-contaminated soil. Rhodococcus sp. strain CG-1 and Mycobacterium sp. strain CG-2, A
2346-TeCP
2345-TeCP
KP
'cY S. o
0
--V 0
24
24
48
B
2356-TeCP
231*6-TeCP
2345-TeCP
.*/K-
/o/
-a c c
24
48
2A
48
time (h) FIG. 7. 0-methylation of PCP, 2345-TeCP, 2346-TeCP, and 2356-TeCP (-) into the respective anisoles (O) by Rhodococcus sp. strain CG-lM (A) and Mycobacterium sp. strain CG-2M (B).
3050
HAGGBLOM ET AL.
APPL. ENVIRON. MICROBIOL.
TABLE 2. 0-methylation of trichlorophenols by Rhodococcus sp. strain CG-1M and Mycobacterium sp. strain CG-2M % of initial substrate concn after 48 h Substrate added (10 ,uM)
234-TCP 235-TCP 236-TCP 245-TCP 246-TCP 345-TCP
CG-2M
CG-1M
Phenol remaining
Anisole produced
Phenol remaining
Anisole produced
58 59 10 55 37 97
35 40 90 34 63 3
47 91 52 67 40 93
33 5 48 31 60 1
enriched on TeCG, and Rhodococcus sp. strain CP-2, enriched on PCP, readily mineralized PCP and removed several other polychlorinated phenols, 2-methoxyphenols (guaiacols), and 2,6-dimethoxyphenols (syringols). The three strains degraded chlorophenols, chloroguaiacols, and chlorosyringols at micromolar concentrations and were sensitive to the toxic effects of PCP. This is similar to what was shown for R. chlorophenolicus PCP-I (5, 16, 17). Chlorophenol-mineralizing bacteria were thus obtained independently of the selective agent, PCP or TeCG. This indicates that the ability to utilize these chemicals may be metabolically related. In fact, all three strains initiated the degradation of polychlorinated phenols by para hydroxylation into chlorinated hydroquinones. Such has previously also been shown to occur in R. chlorophenolicus PCP-I (6), A
10
A
C
0
~~~~~~~0
0
LeziG
i 24
time (h)
48
A~~~~
a Flavobacterium sp. (32), and a PCP-degrading bacterium, KC-3 (29). In R. chlorophenolicus PCP-I (6) and in the Flavobacterium sp. (32) the hydroxyl group originated from water. R. chlorophenolicus PCP-I also degraded chlorinated guaiacols and syringols via similar hydroxylation (17). Demethylation of TeCG and TeCC was not observed. The same degradation pathway was thus used by the bacteria from enrichment cultures on PCP and on TeCG, which already contains two oxygen atoms on the aromatic ring. R. chlorophenolicus PCP-I degrades TeCH further through hydrolytic and reductive dechlorinations, removing all chlorines before ring cleavage (7). These results show that chlorinated parahydroquinones are central metabolites in the aerobic degradation of polychlorinated phenolic compounds, in contrast to mono- and dichlorophenols, which are generally channeled through chlorocatechols (9, 20, 21). The ability of Rhodococcus sp. strain CG-1 and Mycobacterium sp. strain CG-2 to degrade chlorinated phenolic compounds was easily lost after repeated transfers without a polychlorinated substrate. Rhodococcus sp. strain CP-2 did not lose its ability to degrade chlorinated phenolic compounds and neither did the previously isolated chlorophenol degrader R. chlorophenolicus PCP-I (J. Apajalahti and M. Haggblom, unpublished results). Rhodococcus sp. strains CG-1 and CP-2 and Mycobacterium sp. CG-2 differ from R. chlorophenolicus PCP-I (3, 5) in that they 0-methylated nearly all chlorophenols, chloroguaiacols, and chlorosyringols, whereas R. chlorophenolicus PCP-I only methylated chlorinated para-hydroquinones (18). R. chlorophenolicus was also shown to 0-methylate polychlorinated para-phenoxyphenols (39). 0-methylation of chlorinated phenolic compounds was constitutively expressed and a slow reaction compared with the inducible
degradation. The 0-methylating enzyme(s) in Rhodococcus sp. strains CG-1 and CP-2 and Mycobacterium sp. strain CG-2 preferred a substrate with the hydroxyl group flanked by two chlorine substituents. R. chlorophenolicus PCP-I 0-methylated chlorinated hydroquinones into 4-methoxyphenols, selectively methylating the hydroxyl group flanked by two chlorine atoms also (18). A similar substrate specificity is also noticeable in other 0-methylating bacteria (2, 27). Even though Rhodococcus sp. strain CG-1M and Mycobacterium sp. strain CG-2M had a preference for ortho-chlorinated substrates, they were able to 0-methylate almost all chlorophenols, chloroguaiacols, and chlorosyringols studied. Published work elsewhere with both gram-positive and gramnegative bacteria (1, 2, 25, 26) and with bacterial cell extracts (27, 36) has shown that the bacterial 0-methylating enzyme(s) has a broad substrate specificity. It has been suggested that 0-methylation might function as a detoxification mechanism. Rhodococcus sp. strains CG-1 and CP-2 were sensitive to the toxic effects of PCP, but the same concentration of the methylation product PCA was
FIG. 8. 0 methylation of PCP (-) into PCA (O), TeCG (-) into TeCV (O), and TCS (A) into TCTMB (A) by Rhodococcus sp. strain CG-1M (A) and Mycobacterium sp. strain CG-2M (B).
nontoxic. Mycobacterium sp. strain CG-2 was, however, sensitive to both PCP and PCA. Chlorinated anisoles and veratroles were less toxic than the corresponding phenols and guaiacols to both gram-negative and gram-positive bacteria (1). 0-methylation of chlorinated phenols into anisoles and guaiacols into veratroles has been reported for several bacteria (1, 2, 25, 26, 37) and fungi (13, 15) and may thus be a significant alternative to degradation. We show in this paper that the enzymes for the degradation or 0-methylation of chlorinated phenolic compounds may be present in the same organism (Fig. 9).
VOL. 54, 1988
DEGRADATION AND 0-METHYLATION OF
OCH3 OCl Cl
OH
ci
ClI
10.
ci
c
11. 12.
OCH3
OH
OH
OH CI
CO1 HC
13.
14.
Cl
OCH3
OCH3
15. 16.
17.
CO2
18.
FIG. 9. Suggested sequence for degradation and 0-methylation of polychlorinated phenols by Rhodococcus and Mycobacterium strains. ACKNOWLEDGMENTS We thank Risto Valo for donating strain CP-2, Jussi Uotila for assistance in tending the enrichment percolators, and Riitta Boeck for her help in the laboratory. We thank Juha Knuutinen and co-workers (University of Jyvaskyla, Jyvaskyla, Finland) for the synthesis of model compounds. This work was financially supported by the Academy of Finland (grant 03/159), by the Maj and Tor Nessling Foundation, and by the University of Helsinki (M. Haggblom). LITERATURE CITED
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