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Degradation of phenols by thermophilic and halophilic bacteria isolated from a marine brine sample
JOURNAL OF FERMENTATION AND BIOENGINEERING Vol. 74, No. 5, 297-300. 1992
Degradation of Phenols by Thermophilic and Halophilic Bacteria Isolated from a Marine Brine Sample HIDESHI YANASE, l KAZUYOSHI ZUZAN, I KEIKO KITA, I SATOSHI SOGABE, 2 AND NOBUO KATO ]*
Department of Biotechnology, Tottori University, Tottori 680,t and Nippon Halogens Chemical Co., Ltd., Tokyo 103,2 Japan Received 11 June 1992/Accepted 6 August 1992
Two thermophilic bacteria that degrade phenol, strains 401 and 501, were isolated from a brine sample from a submarine gas field. Strain 401 was identified as being Bacillus stearothermophUus. Strain 501 was tentatively identified as a previously unknown species in the family of Rhizobiaceae. Strains 401 and 501 completely degraded phenol at the concentrations of 1,000 and 700 rag~l, respectively, while growing on a nutrient broth at 50°C. Bacterial cells grown on a medium containing phenol degraded a variety of phenolic compounds. Strain 401 tolerated up to 10% NaCI, and strain 501 absolutely required seawater for growth and phenol degradation. Such thermophilic bacteria that tolerate salt and phenol might be useful for the treatment of industrial wastes containing phenols.
Phenols are hazardous pollutants produced in oil refineries, petrochemical plants, and other industrial settings. Various kinds of phenol-degrading bacteria (I-3), yeasts (4, 5), and an immobilized mixed culture of a bacterium and a yeast (6) have been evaluated for their usefulness in controlling phenol pollution. Brine from a submarine gas field near Niigata Prefecture, Japan, is used for commercial iodine production. The brine contains a variety of phenols (total concentration, about 10mg//), so waste treatment is necessary before discharge of the effluent into the environment after iodine extraction. The concentration of phenols in the brine was significantly decreased when it was aerated at 50°C, and we isolated two bacterial strains from a fresh brine sample. The isolates were thermophilic, required seawater or were salt-tolerant, and highly tolerant of phenol. In recent petroleum processes improved in terms of energy reduction, the temperatures of effluents are becoming higher. High salt concentrations of some effluents are also a problem in microbial waste treatment. During weathering of spilled oil in the sea, chemical and biological oxidation occurs in the initial stage (7), during which a variety of phenolic compounds probably accumulate in the seawater. Our isolates may be useful for the treatment of such effluents and of seawater polluted with oil. In this report, we describe the properties of the degradation of phenols by these bacteria.
solution instead of the seawater. The composition of the metal salt solution was as follows (mg/l of medium): MgSO4.7H20, 20; CaC12.2H20, 4; H3BO3, 5; CuSO45H20, 0.4; KI, l; FeSO4.7H20, 2; MnSO,.4--7H20, 4; and ZnSO4.7H20, 4. After the medium was autoclaved, an appropriate amount of phenol was added. Culture was done in a 300-ml Erlenmeyer flask containing 50ml of medium, on a reciprocal shaker (100 strokes/min). Agar (15 g//) was added to the seawater nutrient broth for slope or plate cultures. Isolation of phenol-degrading bacteria Phenoldegrading bacteria were isolated from brine that had just emerged from a natural gas well under the sea near Niigata Prefecture, Japan. The salt composition of the brine was almost the same as that of seawater. The temperature of the brine flowing into an aqueduct, where samples for bacterial isolation were collected, was 50°C. Peptone (0.2%), NH4C1 (0.1%), K2HPO4 (0.01%), and phenol (200 mg//) were added to 50mi of the brine sample in a 300-ml Erlenmeyer flask, and the mixture was shaken at 50°C for 3 d. The phenol disappeared during this treatment. Then 5 ml of the culture was transferred into 50mi of seawater nutrient broth containing 200 rag~! phenol, and the broth was incubated at 50°C for 3 d. All cultures were transferred to fresh medium every 3 d. Pure bacteriai cultures were obtained by dilution of the final cultures and inoculation of agar plates containing 200 mg/l phenol. Single colonies were isolated, transferred onto slope agar with no phenol, grown at 50°C for 2 d, and stored at 5°C. Bacterial cells suspended in sterile artificial seawater containing 10% (vol/vol) glycerol were stored frozen at - 8 0 ° C in plastic vials. Under these conditions, the isolates were viable and did not lose phenol degradation activity for at least 2 years. Organisms Two phenol-degrading bacteria, strains 401 and 501, were isolated as mentioned above. Their identification was carried out by the National Collection of Industrial and Marine Bacteria, Ltd., Scotland. Strain 401 was identified as being Bacillus stearothermophilus subgroup 2 (8). The principal bacteriological characteristics of strain 501 were Gram-variable, short rods (1 x 1.5 pm), no spores, motile, did not grow on nutrient agar without sea
MATERIALS AND METHODS Media and cultivation Seawater nutrient broth (pH 7.0) consisted of 10 g of tryptone, 5 g of yeast extract, and 1 l of artificial seawater. Glucose medium (pH 7.0) consisted of 5 g of glucose, 2 g of NaNOj, 2 g of (NH4)2SO4, 2g of K2HPO4, 1 g of KH2PO4, 0.2g of yeast extract, and 1 1 of artificial seawater. The composition of the artificial seawater was as follows (g/0: NaC1, 23.5; KCI, 0.66; CaC12.H20, 1.1; Na2SO4, 3.91; KBr, 0.10; SrC126H20, 0.024; NaHCO3, 0.19; MgCI2-6H20, 4.98; and HaBOj, 0.03. Fresh water medium had a metal salt *Corresponding author.
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water, grew at 30 to 55°C, was catalase- and oxidase-positive, and was not fermentative in glucose OF. Strain 501 was similar to Agrobacterium in being urease- and aesculin hydrolysis-positive, but it did not utilize malate as a carbon source. The strain may belong to the family Rhizobiaceae (9). Analyses Growth was monitored by measurement of the optical density at 610 nm. Protein was measured with a protein assay kit (Japan Bio-Rad Laboratories, Tokyo) with bovine serum albumin as the standard. Phenol and its related compounds were assayed by gas chromatography with a glass column (0.26 x 200cm) filled with Unisole F-200 (30/60 mesh, Gasukuro Kogyo, Inc., Tokyo) at 180°C, with nitrogen carrier gas (60ml/min) and an FID detector. The supernatant of the culture broth or reaction mixture was directly injected. Phenols were also determined by high performance liquid chromatography (HPLC) with a Waters pBondasphere Ct8 (3.9 × 150 ram) column with detection at 275 nm and the mobile phase was methanol : water (40 : 60) at a flow rate of 0.8 ml/min. Enzyme assay Bacterial cells grown in seawater nutrient broth containing 200 mg/l phenol at 50°C for 24 h were suspended in 50 mM Tris-HCl (pH 7.5) containing 1 mM dithiothreitol and disrupted with a sonic oscillator (19kHz) under N2 gas for 15 rain at below 5°C. The cell-free extract was obtained by centrifugation at 12,000g for 15 min. Phenol 2-monooxygenase (EC 1.14. 13.7) was assayed by three methods, those of Powlowski and Shingler (I0), Neujahr and Gaal (4), and Gurujeyalakshmi and Oriel (3). The activities of catechol 1,2-dioxygenase (EC 1.13.11.1) and catechol 2,3-dioxygenase ('EC 1.13.11.2) were determined by the methods of Nakazawa and Nakazawa (11) and Nozaki (12), respectively. Materials Tryptone and yeast extract were obtained from Difco Laboratories (Detroit, Mich., USA). All other chemicals were obtained from commercial sources and were of reagent grade. RESULTS AND DISCUSSION
Bacteria isolated Of the two strains of phenoldegrading bacteria, 401 and 501, that were isolated from a brine sample of a submarine gasfield, strain 401 was identiffed as being B. stearothermophilus subgroup 2, members of which are salt-tolerant, and have been isolated from various natural sources, including seawater (8). B. stearothermophilgs strain BR219, which was isolated from a sample of river sediment, grows on phenol (3). Our strain 401 and strain B219 were different with respect to NaCI tolerance; strain 401 grew in 10% NaCI and stain BR219 grows in 3% but not in 5% NaC1. Strain 501 had an absolute requirement for seawater for growth. Strain 501 did not seem to be any type of marine Pseudomonas, Vibrio, Deleya, Marinomonas, or Alteromonas sp., and was identified as a previously unknown member of the family Rhizobiaceae. An unnamed species of Rhizobiaceae was isolated from the Baltic Sea (13), but such bacteria isolated from marine samples have not otherwise been reported. Further bacterial studies are needed for the precise taxonomy of such unusual bacteria which inhabit the marine environment. Neither isolate used phenol as a sole carbon source, and could degrade phenol only when growing on nutrient broth or a glucose medium. Neither isolate grew on the petrochemicals tested, such as benzene, toluene, xylene,
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FIG. I. Effect of temperature on growth (broken lines) and phenol degradation (solid lines) by strains 401 (A) and 501 (B). Seawater nutrient broth containing 200 mg/I phenol was used. Cultures were shaken at 37 (e), 50 (,,), 55 (,t), and 65°C (O). n-paraffins, and so on. Enzymes related to phenol degradation In the assay of phenol 2-monooxygenase, no reproducible activity was detected in cell-free extracts of either strain grown on a medium containing phenol. On the other hand, pyrocatechol was detected by HPLC in the reaction mixtures with the resting cells of strains 401 and 501 at the rates of 0.29 and 0.14nmol.mg cells-~.min, respectively. This could be explained by the instability of the enzyme or the decomposition of complexes formed by the phenol 2-mono0xygenase system (I0) when the cells are disrupted. In the case of strain 401, the activity of catechol 2,3-dioxygenase (specific activity, 0.015 pmol/min-mg) was higher than that of catechol 1,2-dioxygenase (0.003 ttmol/min-
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Time (It) FIG. 2.
Effect of seawater concentrations on growth (broken
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VoL. 74, 1992
DEGRADATION OF PHENOLS BY BACTERIA
rag). The significant activities of phenol hydroxylation and catechol 2,3-dioxygenase were found only in the cells grown on the medium containing phenol. These results suggest that a meta phenol degradation pathway may be present in strain 401. The enzyme activities related to phenol degradation in strain 501 were too low (0.001 y m o l / m i n - m g ) to assume a degradation pathway. Growth temperature and pH The temperature range for both growth and phenol degradation by strain 401 was 37 to 65°C and that for strain 501 was 30 to 55°C. The optimum temperature for phenol degradation by both strains was found to be 50°C (Fig. 1). Strain 401 and 501 grew and degraded phenol in the pH range of 6 to 9 (optimal pH of 8.0), in seawater nutrient broth containing 200 mg/I phenol. Effect of seawater on growth and phenol degradation Growth and phenol degradation by strain 501 were affected by the seawater concentration, and the greatest activities were obtained in nutrient broth containing artificial seawater at full strength (Fig. 2). The rates of growth and phenol degradation in the medium containing 10% seawater were lower than in the media containing the higher seawater concentrations. For growth and phenol degradation, the seawater could be replaced by NaC1 or NazSO4 at a concentration of 0.47 M (equivalent to the concentration of NaC1 in the artificial seawater), but not by KC1 or sucrose (Table I). Thus, strain 501 required sodium ion at a fairly high concentration for growth. Such a requirement is one physiological criterion in the identification of marine bacteria. Strain 401 did not absolutely require seawater, although the growth rate was 150% more with 50% seawater than (a)
(A)
TABLE 1. Sodium ion requirement for growth and phenol degradation by strain 501 Addition to basal medium° (0.47 M) NaCI Na2SO4 KCI MgCIz Sucrose (Seawater)b
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FIG. 3. Degradation of phenol by strains 401 (A) and 501 (B) while growing. Cultivation was in seawater nutrient broth containing phenol at the concentration of 1000 ( • ), 700 ( a ), 500 (A), 300 (A), 200 (e), or 100 (O) mg/l.
Growth (OD610,m) 4.18 4.40 0.03 0.02 0.24 5.70
Phenol degradation (%) 100 82.8 2.7 1.1 3. I 100
Cultivation was at 50°C for 24 h. a Fresh water nutrient broth was the basal medium except for bseawater (seawater nutrient broth). growth in a medium with fresh water (data not shown). Phenol degradation by the strain was not affected by the addition of seawater. Degradation of phenol and related compounds Figure 3 shows growth and phenol degradation by strains 401 and 501 in seawater nutrient broth containing several concentrations of phenol. Strain 401 completely degraded 1,000 mg/l phenol in 96 h, although growth was prevented at this concentration. The ability to degrade phenol by strain 501 was less than that of strain 401, and its growth was inhibited by phenol at the concentration of 1,000 mg/l. The maximum phenol concentration that could be completely degraded by strain 501 was 700 mg//, which disappeared completely after 144 h of cultivation. During phenol degradation by strain 501, the medium became TABLE 2. Degradation of phenolic compounds by resting cells of strain 501 Phenols substituted by
t
299
(Phenol) o-Fluoro m-Fluoro p-Fluoro o-Chloro m-Chloro p-Chloro o-Bromo m-Bromo p-Bromo o-Methyl m-Methyl p-Methyl o-Ethyl m-Ethyl p-Ethyl 2,3-Dimethyl 2,4-Dimethyl 2,5-Dimethyl 3,4-Dimethyl 3,5-Dimethyl 2,3,5-Trimethyl 2,3,6-Trimethyl 2,4,6-Trimethyl (Pyrocatechol) (l-Naphthol)
Degradation (%) cells grown with 10% seawater 100% seawater 11.7 100a 6.4 92.0 10.5 72.2 0 17.0 10.6 98.7 0.2 61.9 2.2 14.8 5.8 53.2 0 17.2 0 0 0 71.5 1.4 50.8 2.4 23.8 4.9 57.5 3.4 20.1 2.5 31.7 14.4 37.7 0.2 27.9 10.8 18.3 0 13.7 7.6 53.7 21.4 20.1 2.0 17.2 2.6 0 1.7 10.8 0 0
The reaction mixture contained 100 mM MOPS buffer (pH 7.0), 0.1 mM phenolic compound, and 4.5 mg/mi cells in a total volume of 2.2 ml in a test tube (18 x 180 ram). The reaction occurred at 50°C for 30 rain with reciprocal shaking (230strokes/rnin). a Ninety percent of the phenol was degraded for 20 min under the reaction conditions.
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J. FERMENT.BIOEN~.,
dark brown. This might be caused by accumulation of a catechol as a degradation intermediate. Both strains degraded a variety of phenols substituted with halogen, methyl, ethyl, dimethyl, or trimethyl at different positions. Table 2 shows the degradation of phenols by resting cells of strain 501 grown in media conraining 100 or 10% seawater. The phenol degradation activity was greater with seawater. The activity of the cells grown with 10% seawater was not different when the reaction mixtures were prepared with seawater or distilled water. These results suggest that the formation of phenoldegrading enzyme(s), rather than the catalytic activity of the enzyme(s), is regulated by ions in seawater, probably sodium ions. Cells grown in the medium with 100% seawater generally were most active toward o-substituted phenols, less so for m-substituted ones, and the least f o r p substituted compounds. The activity of phenol degradation in strain 501 was induced 3.6-fold by the addition of phenol and 2.5 fold by the addition of o-cresol when either addition was at the concentration of 200 m g / l . Cells grown in medium containing o-cresol had the same pattern of degradation activities for phenolic compounds as shown in Table 2 (data not shown), indicating that the same enzyme system as is present in cells grown with phenol was formed in response to o-cresol. REFERENCES 1. Antai, S . P . and Crawford, D.L.: Degradation of phenol by
Streptomyces setonii. Can. J. Microbiol., 29, 142-143 (1983). 2. Bayly, R.C. and Wigmore, G.L.: Metabolism of phenol and
cresols by mutants of Pseudomonas putida. J. Bacteriol., 113, 1112-1120 (1973).
3. Gurujeyalakshmi, G. and Oriel, P.: Isolation of phenol-degrading Bacillus stearothermophilus and partial characterization of the phenol hydroxylase. Appl. Environ. Microbiol., 55, 500-502 0989). 4. Neujahr, H.Y. and Gaal, A.: Phenol hydroxylase from yeast: purification and properties of the enzyme from Trichosporon cutaneum. Eur. J. Biochem., 35, 386-400 (1973). 5. Neujahr, H.Y., Lindsjo, S., and Varga, J.M.: Oxidation of phenols by cells and cell-free enzyme from Candida tropicalis. Antonie van Leeuwenhoek J. Microbiol. Serol., 40, 209-216 (1974). 6. Morsen, A. and Rehm, H. J.: Degradation of phenol by a defined mixed culture immobilized by adsorption on activated carbon and sintered glass. Appl. Microbiol. Biotechnol., 33, 206-212 (1990). 7. Literathy, P., Haider, S., Samhan, O., and Morel, G.: Experimental studies on biological and chemical oxidation of dispersed oil in sea water. Wat. Sci. Tech., 21,845-856 (1989). 8. Claus, D. and Berkeley, R. C.: Genus Bacillus, p. 1105-1139. In Krieg, N. R. and Holt, J. G. (ed.), Bergey's manual of systematic bacteriology, vol. 2. Williams and Wilkins, Baltimore, London (1986). 9. Jordan, D.C.: Family III Rhizobiaceae, p. 234-254. In Krieg, N. R. and Holt, J. G. (ed.), Bergey's manual of systematic bacteriology, vol. I. Williams and Wilkins, Baltimore, London (1984). 10. Powlowski, J. and Shingler, V.: In vitro analysis of polypeptide requirements of multicomponent phenol hydroxylase from Pseudomonas sp. strain CF600. J. Bacteriol., 172, 6834-6840 (1990). 11. Nakazawa, T. and Nakazawa, A.: Pyrocatechase (Pseudomonas), p. 518-522. In Tabor, H. and Tabor, C. W. (ed.), Methods in enzymology, vol. 17A. Academic Press, New York (1970). 12. Nozaki, M.: Metapyrocatechase (Pseudomonas), p. 522-526. In Tabor, H. and Tabor, C. W. (ed.), Methods in enzymology, vol. 17A. Academic Press, New York (1970). 13. de Smedt, J. and de Ley, J.: Intra- and intergeneric similarities of Agrobacterium ribosomal ribonucleic acid cistrons. Int. J. Syst. Bacteriol., 27, 222-240 (1977).
Degradation of Phenols by Thermophilic and Halophilic Bacteria Isolated from a Marine Brine Sample HIDESHI YANASE, l KAZUYOSHI ZUZAN, I KEIKO KITA, I SATOSHI SOGABE, 2 AND NOBUO KATO ]*
Department of Biotechnology, Tottori University, Tottori 680,t and Nippon Halogens Chemical Co., Ltd., Tokyo 103,2 Japan Received 11 June 1992/Accepted 6 August 1992
Two thermophilic bacteria that degrade phenol, strains 401 and 501, were isolated from a brine sample from a submarine gas field. Strain 401 was identified as being Bacillus stearothermophUus. Strain 501 was tentatively identified as a previously unknown species in the family of Rhizobiaceae. Strains 401 and 501 completely degraded phenol at the concentrations of 1,000 and 700 rag~l, respectively, while growing on a nutrient broth at 50°C. Bacterial cells grown on a medium containing phenol degraded a variety of phenolic compounds. Strain 401 tolerated up to 10% NaCI, and strain 501 absolutely required seawater for growth and phenol degradation. Such thermophilic bacteria that tolerate salt and phenol might be useful for the treatment of industrial wastes containing phenols.
Phenols are hazardous pollutants produced in oil refineries, petrochemical plants, and other industrial settings. Various kinds of phenol-degrading bacteria (I-3), yeasts (4, 5), and an immobilized mixed culture of a bacterium and a yeast (6) have been evaluated for their usefulness in controlling phenol pollution. Brine from a submarine gas field near Niigata Prefecture, Japan, is used for commercial iodine production. The brine contains a variety of phenols (total concentration, about 10mg//), so waste treatment is necessary before discharge of the effluent into the environment after iodine extraction. The concentration of phenols in the brine was significantly decreased when it was aerated at 50°C, and we isolated two bacterial strains from a fresh brine sample. The isolates were thermophilic, required seawater or were salt-tolerant, and highly tolerant of phenol. In recent petroleum processes improved in terms of energy reduction, the temperatures of effluents are becoming higher. High salt concentrations of some effluents are also a problem in microbial waste treatment. During weathering of spilled oil in the sea, chemical and biological oxidation occurs in the initial stage (7), during which a variety of phenolic compounds probably accumulate in the seawater. Our isolates may be useful for the treatment of such effluents and of seawater polluted with oil. In this report, we describe the properties of the degradation of phenols by these bacteria.
solution instead of the seawater. The composition of the metal salt solution was as follows (mg/l of medium): MgSO4.7H20, 20; CaC12.2H20, 4; H3BO3, 5; CuSO45H20, 0.4; KI, l; FeSO4.7H20, 2; MnSO,.4--7H20, 4; and ZnSO4.7H20, 4. After the medium was autoclaved, an appropriate amount of phenol was added. Culture was done in a 300-ml Erlenmeyer flask containing 50ml of medium, on a reciprocal shaker (100 strokes/min). Agar (15 g//) was added to the seawater nutrient broth for slope or plate cultures. Isolation of phenol-degrading bacteria Phenoldegrading bacteria were isolated from brine that had just emerged from a natural gas well under the sea near Niigata Prefecture, Japan. The salt composition of the brine was almost the same as that of seawater. The temperature of the brine flowing into an aqueduct, where samples for bacterial isolation were collected, was 50°C. Peptone (0.2%), NH4C1 (0.1%), K2HPO4 (0.01%), and phenol (200 mg//) were added to 50mi of the brine sample in a 300-ml Erlenmeyer flask, and the mixture was shaken at 50°C for 3 d. The phenol disappeared during this treatment. Then 5 ml of the culture was transferred into 50mi of seawater nutrient broth containing 200 rag~! phenol, and the broth was incubated at 50°C for 3 d. All cultures were transferred to fresh medium every 3 d. Pure bacteriai cultures were obtained by dilution of the final cultures and inoculation of agar plates containing 200 mg/l phenol. Single colonies were isolated, transferred onto slope agar with no phenol, grown at 50°C for 2 d, and stored at 5°C. Bacterial cells suspended in sterile artificial seawater containing 10% (vol/vol) glycerol were stored frozen at - 8 0 ° C in plastic vials. Under these conditions, the isolates were viable and did not lose phenol degradation activity for at least 2 years. Organisms Two phenol-degrading bacteria, strains 401 and 501, were isolated as mentioned above. Their identification was carried out by the National Collection of Industrial and Marine Bacteria, Ltd., Scotland. Strain 401 was identified as being Bacillus stearothermophilus subgroup 2 (8). The principal bacteriological characteristics of strain 501 were Gram-variable, short rods (1 x 1.5 pm), no spores, motile, did not grow on nutrient agar without sea
MATERIALS AND METHODS Media and cultivation Seawater nutrient broth (pH 7.0) consisted of 10 g of tryptone, 5 g of yeast extract, and 1 l of artificial seawater. Glucose medium (pH 7.0) consisted of 5 g of glucose, 2 g of NaNOj, 2 g of (NH4)2SO4, 2g of K2HPO4, 1 g of KH2PO4, 0.2g of yeast extract, and 1 1 of artificial seawater. The composition of the artificial seawater was as follows (g/0: NaC1, 23.5; KCI, 0.66; CaC12.H20, 1.1; Na2SO4, 3.91; KBr, 0.10; SrC126H20, 0.024; NaHCO3, 0.19; MgCI2-6H20, 4.98; and HaBOj, 0.03. Fresh water medium had a metal salt *Corresponding author.
297
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YANASEET AL.
water, grew at 30 to 55°C, was catalase- and oxidase-positive, and was not fermentative in glucose OF. Strain 501 was similar to Agrobacterium in being urease- and aesculin hydrolysis-positive, but it did not utilize malate as a carbon source. The strain may belong to the family Rhizobiaceae (9). Analyses Growth was monitored by measurement of the optical density at 610 nm. Protein was measured with a protein assay kit (Japan Bio-Rad Laboratories, Tokyo) with bovine serum albumin as the standard. Phenol and its related compounds were assayed by gas chromatography with a glass column (0.26 x 200cm) filled with Unisole F-200 (30/60 mesh, Gasukuro Kogyo, Inc., Tokyo) at 180°C, with nitrogen carrier gas (60ml/min) and an FID detector. The supernatant of the culture broth or reaction mixture was directly injected. Phenols were also determined by high performance liquid chromatography (HPLC) with a Waters pBondasphere Ct8 (3.9 × 150 ram) column with detection at 275 nm and the mobile phase was methanol : water (40 : 60) at a flow rate of 0.8 ml/min. Enzyme assay Bacterial cells grown in seawater nutrient broth containing 200 mg/l phenol at 50°C for 24 h were suspended in 50 mM Tris-HCl (pH 7.5) containing 1 mM dithiothreitol and disrupted with a sonic oscillator (19kHz) under N2 gas for 15 rain at below 5°C. The cell-free extract was obtained by centrifugation at 12,000g for 15 min. Phenol 2-monooxygenase (EC 1.14. 13.7) was assayed by three methods, those of Powlowski and Shingler (I0), Neujahr and Gaal (4), and Gurujeyalakshmi and Oriel (3). The activities of catechol 1,2-dioxygenase (EC 1.13.11.1) and catechol 2,3-dioxygenase ('EC 1.13.11.2) were determined by the methods of Nakazawa and Nakazawa (11) and Nozaki (12), respectively. Materials Tryptone and yeast extract were obtained from Difco Laboratories (Detroit, Mich., USA). All other chemicals were obtained from commercial sources and were of reagent grade. RESULTS AND DISCUSSION
Bacteria isolated Of the two strains of phenoldegrading bacteria, 401 and 501, that were isolated from a brine sample of a submarine gasfield, strain 401 was identiffed as being B. stearothermophilus subgroup 2, members of which are salt-tolerant, and have been isolated from various natural sources, including seawater (8). B. stearothermophilgs strain BR219, which was isolated from a sample of river sediment, grows on phenol (3). Our strain 401 and strain B219 were different with respect to NaCI tolerance; strain 401 grew in 10% NaCI and stain BR219 grows in 3% but not in 5% NaC1. Strain 501 had an absolute requirement for seawater for growth. Strain 501 did not seem to be any type of marine Pseudomonas, Vibrio, Deleya, Marinomonas, or Alteromonas sp., and was identified as a previously unknown member of the family Rhizobiaceae. An unnamed species of Rhizobiaceae was isolated from the Baltic Sea (13), but such bacteria isolated from marine samples have not otherwise been reported. Further bacterial studies are needed for the precise taxonomy of such unusual bacteria which inhabit the marine environment. Neither isolate used phenol as a sole carbon source, and could degrade phenol only when growing on nutrient broth or a glucose medium. Neither isolate grew on the petrochemicals tested, such as benzene, toluene, xylene,
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FIG. I. Effect of temperature on growth (broken lines) and phenol degradation (solid lines) by strains 401 (A) and 501 (B). Seawater nutrient broth containing 200 mg/I phenol was used. Cultures were shaken at 37 (e), 50 (,,), 55 (,t), and 65°C (O). n-paraffins, and so on. Enzymes related to phenol degradation In the assay of phenol 2-monooxygenase, no reproducible activity was detected in cell-free extracts of either strain grown on a medium containing phenol. On the other hand, pyrocatechol was detected by HPLC in the reaction mixtures with the resting cells of strains 401 and 501 at the rates of 0.29 and 0.14nmol.mg cells-~.min, respectively. This could be explained by the instability of the enzyme or the decomposition of complexes formed by the phenol 2-mono0xygenase system (I0) when the cells are disrupted. In the case of strain 401, the activity of catechol 2,3-dioxygenase (specific activity, 0.015 pmol/min-mg) was higher than that of catechol 1,2-dioxygenase (0.003 ttmol/min-
o( 0
10
20
30
40
50
Time (It) FIG. 2.
Effect of seawater concentrations on growth (broken
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VoL. 74, 1992
DEGRADATION OF PHENOLS BY BACTERIA
rag). The significant activities of phenol hydroxylation and catechol 2,3-dioxygenase were found only in the cells grown on the medium containing phenol. These results suggest that a meta phenol degradation pathway may be present in strain 401. The enzyme activities related to phenol degradation in strain 501 were too low (0.001 y m o l / m i n - m g ) to assume a degradation pathway. Growth temperature and pH The temperature range for both growth and phenol degradation by strain 401 was 37 to 65°C and that for strain 501 was 30 to 55°C. The optimum temperature for phenol degradation by both strains was found to be 50°C (Fig. 1). Strain 401 and 501 grew and degraded phenol in the pH range of 6 to 9 (optimal pH of 8.0), in seawater nutrient broth containing 200 mg/I phenol. Effect of seawater on growth and phenol degradation Growth and phenol degradation by strain 501 were affected by the seawater concentration, and the greatest activities were obtained in nutrient broth containing artificial seawater at full strength (Fig. 2). The rates of growth and phenol degradation in the medium containing 10% seawater were lower than in the media containing the higher seawater concentrations. For growth and phenol degradation, the seawater could be replaced by NaC1 or NazSO4 at a concentration of 0.47 M (equivalent to the concentration of NaC1 in the artificial seawater), but not by KC1 or sucrose (Table I). Thus, strain 501 required sodium ion at a fairly high concentration for growth. Such a requirement is one physiological criterion in the identification of marine bacteria. Strain 401 did not absolutely require seawater, although the growth rate was 150% more with 50% seawater than (a)
(A)
TABLE 1. Sodium ion requirement for growth and phenol degradation by strain 501 Addition to basal medium° (0.47 M) NaCI Na2SO4 KCI MgCIz Sucrose (Seawater)b
1 o.
L
"O
2001 ( o
_
/X
! IV"
0
I
12
--O
--I1
I
24
T i m e (h)
96
I
0
12
24
96
Time (h)
FIG. 3. Degradation of phenol by strains 401 (A) and 501 (B) while growing. Cultivation was in seawater nutrient broth containing phenol at the concentration of 1000 ( • ), 700 ( a ), 500 (A), 300 (A), 200 (e), or 100 (O) mg/l.
Growth (OD610,m) 4.18 4.40 0.03 0.02 0.24 5.70
Phenol degradation (%) 100 82.8 2.7 1.1 3. I 100
Cultivation was at 50°C for 24 h. a Fresh water nutrient broth was the basal medium except for bseawater (seawater nutrient broth). growth in a medium with fresh water (data not shown). Phenol degradation by the strain was not affected by the addition of seawater. Degradation of phenol and related compounds Figure 3 shows growth and phenol degradation by strains 401 and 501 in seawater nutrient broth containing several concentrations of phenol. Strain 401 completely degraded 1,000 mg/l phenol in 96 h, although growth was prevented at this concentration. The ability to degrade phenol by strain 501 was less than that of strain 401, and its growth was inhibited by phenol at the concentration of 1,000 mg/l. The maximum phenol concentration that could be completely degraded by strain 501 was 700 mg//, which disappeared completely after 144 h of cultivation. During phenol degradation by strain 501, the medium became TABLE 2. Degradation of phenolic compounds by resting cells of strain 501 Phenols substituted by
t
299
(Phenol) o-Fluoro m-Fluoro p-Fluoro o-Chloro m-Chloro p-Chloro o-Bromo m-Bromo p-Bromo o-Methyl m-Methyl p-Methyl o-Ethyl m-Ethyl p-Ethyl 2,3-Dimethyl 2,4-Dimethyl 2,5-Dimethyl 3,4-Dimethyl 3,5-Dimethyl 2,3,5-Trimethyl 2,3,6-Trimethyl 2,4,6-Trimethyl (Pyrocatechol) (l-Naphthol)
Degradation (%) cells grown with 10% seawater 100% seawater 11.7 100a 6.4 92.0 10.5 72.2 0 17.0 10.6 98.7 0.2 61.9 2.2 14.8 5.8 53.2 0 17.2 0 0 0 71.5 1.4 50.8 2.4 23.8 4.9 57.5 3.4 20.1 2.5 31.7 14.4 37.7 0.2 27.9 10.8 18.3 0 13.7 7.6 53.7 21.4 20.1 2.0 17.2 2.6 0 1.7 10.8 0 0
The reaction mixture contained 100 mM MOPS buffer (pH 7.0), 0.1 mM phenolic compound, and 4.5 mg/mi cells in a total volume of 2.2 ml in a test tube (18 x 180 ram). The reaction occurred at 50°C for 30 rain with reciprocal shaking (230strokes/rnin). a Ninety percent of the phenol was degraded for 20 min under the reaction conditions.
300
YANASEET AL.
J. FERMENT.BIOEN~.,
dark brown. This might be caused by accumulation of a catechol as a degradation intermediate. Both strains degraded a variety of phenols substituted with halogen, methyl, ethyl, dimethyl, or trimethyl at different positions. Table 2 shows the degradation of phenols by resting cells of strain 501 grown in media conraining 100 or 10% seawater. The phenol degradation activity was greater with seawater. The activity of the cells grown with 10% seawater was not different when the reaction mixtures were prepared with seawater or distilled water. These results suggest that the formation of phenoldegrading enzyme(s), rather than the catalytic activity of the enzyme(s), is regulated by ions in seawater, probably sodium ions. Cells grown in the medium with 100% seawater generally were most active toward o-substituted phenols, less so for m-substituted ones, and the least f o r p substituted compounds. The activity of phenol degradation in strain 501 was induced 3.6-fold by the addition of phenol and 2.5 fold by the addition of o-cresol when either addition was at the concentration of 200 m g / l . Cells grown in medium containing o-cresol had the same pattern of degradation activities for phenolic compounds as shown in Table 2 (data not shown), indicating that the same enzyme system as is present in cells grown with phenol was formed in response to o-cresol. REFERENCES 1. Antai, S . P . and Crawford, D.L.: Degradation of phenol by
Streptomyces setonii. Can. J. Microbiol., 29, 142-143 (1983). 2. Bayly, R.C. and Wigmore, G.L.: Metabolism of phenol and
cresols by mutants of Pseudomonas putida. J. Bacteriol., 113, 1112-1120 (1973).
3. Gurujeyalakshmi, G. and Oriel, P.: Isolation of phenol-degrading Bacillus stearothermophilus and partial characterization of the phenol hydroxylase. Appl. Environ. Microbiol., 55, 500-502 0989). 4. Neujahr, H.Y. and Gaal, A.: Phenol hydroxylase from yeast: purification and properties of the enzyme from Trichosporon cutaneum. Eur. J. Biochem., 35, 386-400 (1973). 5. Neujahr, H.Y., Lindsjo, S., and Varga, J.M.: Oxidation of phenols by cells and cell-free enzyme from Candida tropicalis. Antonie van Leeuwenhoek J. Microbiol. Serol., 40, 209-216 (1974). 6. Morsen, A. and Rehm, H. J.: Degradation of phenol by a defined mixed culture immobilized by adsorption on activated carbon and sintered glass. Appl. Microbiol. Biotechnol., 33, 206-212 (1990). 7. Literathy, P., Haider, S., Samhan, O., and Morel, G.: Experimental studies on biological and chemical oxidation of dispersed oil in sea water. Wat. Sci. Tech., 21,845-856 (1989). 8. Claus, D. and Berkeley, R. C.: Genus Bacillus, p. 1105-1139. In Krieg, N. R. and Holt, J. G. (ed.), Bergey's manual of systematic bacteriology, vol. 2. Williams and Wilkins, Baltimore, London (1986). 9. Jordan, D.C.: Family III Rhizobiaceae, p. 234-254. In Krieg, N. R. and Holt, J. G. (ed.), Bergey's manual of systematic bacteriology, vol. I. Williams and Wilkins, Baltimore, London (1984). 10. Powlowski, J. and Shingler, V.: In vitro analysis of polypeptide requirements of multicomponent phenol hydroxylase from Pseudomonas sp. strain CF600. J. Bacteriol., 172, 6834-6840 (1990). 11. Nakazawa, T. and Nakazawa, A.: Pyrocatechase (Pseudomonas), p. 518-522. In Tabor, H. and Tabor, C. W. (ed.), Methods in enzymology, vol. 17A. Academic Press, New York (1970). 12. Nozaki, M.: Metapyrocatechase (Pseudomonas), p. 522-526. In Tabor, H. and Tabor, C. W. (ed.), Methods in enzymology, vol. 17A. Academic Press, New York (1970). 13. de Smedt, J. and de Ley, J.: Intra- and intergeneric similarities of Agrobacterium ribosomal ribonucleic acid cistrons. Int. J. Syst. Bacteriol., 27, 222-240 (1977).