World Journal of Microbiology & Biotechnology 10, 334-337
Pentachlorophenol degradation by Pseudomonas aeruginosa A. Premalatha and G. Suseela Rajakumar* Five P s e u d o m o n a s species were tested for ability to degrade pentachlorophenol (PCP). P s e u d o m o n a s aeruginosa completely degraded PCP up to 800 mg/1 in 6 days with glucose as co-substrate. With 1000 mg PCP/1, 53% was degraded. NH4 ÷ salts were better at enhancing degradation than organic nitrogen sources and shake-cultures promoted PCP degradation compared with surface cultures. Degradation was maximal at pH 7.6 to 8.0 and at 30 to 37°C. Only PCP induced enzymes that degraded PCP and chloramphenicol inhibited this process. The PCP was degraded to CO 2, with release of Cl-. Key words: Degradation, pentachlorophenol, Pseudomonas aeruginosa.
Pentachlorophenol (PCP) is a recalcitrant biocide used primarily for wood preservation (Chu & Kirsch 1972). Its industrial and agricultural applications have included use as a bactericide, insecticide, herbicide, algicide and fungicide (Crosby 1981). In the leather industry, PCP is used with common salt as a bactericide to preserve raw hide and skin, preventing bacterial damage to skin collagen (Sivaparvathy & Nandy 1973; Nandy & Rao 1977). It is also used as a fungicide during the different tanning operations, to preserve the chrome blue and other tanned leathers and leather articles (Suseela et al. 1988). The United States' Environmental Protection Agency considers PCP a priority toxic pollutant (Sittig 1981) and bioremediation and bioaugmentation techniques have been advocated as the environmentally-friendlyways to detoxify it (Valo et al. 1985; Mikesell & Boyd 1986). Microbial degradations of multi-chlorinated phenols have been reported by several groups (Amy et al. 1985; Apajalahti et al. 1986). The success of bioremediation depends on a good understanding of the biochemical, physiological and ecological principles which govern microbial growth, activity and biological recalcitrance at introduction sites (Fewson 1988). Pseudomonas aeruginosa, isolated by an enrichment culture technique (Suseela et al. 1991) from the soil around the tannery effluent treatment sites of the Central Leather Research Institute, has been used for biodegradation studies of PCP along with other Pseudomonas species. The present study is of some of the various factors that influence PCP degradation by
Materials and Methods Chemicals
PCP was purchased from Aldrich Chemical Company and [U~4C]PCP from Sigma. The purity of the PCP was determined by HPLC. Culture Maintenance All strains of Pseudomonas (see Table 1) were maintained on nu-
trient agar slants and subcultured every 30 days. Media and Cultivation Conditions
The medium used for inoculum preparation and the biodegradation study was a mineral salt medium containing (g/l): K2HPO4, 1.73; KH2PO4, 0.68; NH4NO3, 1.0; MgSO4. 7H20 , 0.1; CaC12. 2H20, 0.02; MnSO4.H20, 0.03; FeSO4.7H20), 0.03; and glucose, 5.0. The medium was adjusted to pH 7.6. The carbon sources were filter-sterilized and added to the medium separately. Pentachlorophenol was prepared as a filter-sterilized stock solution in 0.2 MNaOH. Biodegradation experiments were conducted in 250-ml Erlenmayer flasks, each containing 50 ml medium and inoculated with 2 ml of a late-growth-phase culture. The flasks were incubated at 37°C in an incubator shaker at 300 rev/min. Unless otherwise stated, all the experiments for degradation of PCP were conducted in the mineral salt medium with glucose and ammonium nitrate as the carbon and nitrogen sources, respectively. Cell growth was monitored by measuring optical density at 600 nm. All the experiments were repeated twice in duplicate.
P. aeruginosa.
Mineralization Studies
The authors are with the Bacteriology Laboratory, Central Leather Research Institute, Madras-600 020, India. *Corresponding author.
Mineralization of [U-14C]PCP was studied in 10 ml mineral salt medium using, per flask, either 105 d.p.m. [U-14C]PCP (sp.act. 12.3 mCi/mmol) or 10 mg [U-1K:]PCP (sp.act. 4.1 mCi/mmol).
© 1994 Rapid Communications of Oxford Ltd
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WorldJournalof Microbiology& Biotechnology,Vo110, 1994
PCP degradation by P. aeruginosa The 14CO2 produced was trapped in ethanolamine-containing scintillation fluid according to the method described by Kirk et al. (1977) and assayed by liquid scintillation spectrometry. The extent of PCP degradation was followed spectrophotometrically~ An equal volume of 0.2 M NaOH was added to the culture medium immediately after the sampling, centrifuged at 7700 x g for 15 min and the absorbance of the clear supernatant produced was then read at 320 nm (8320=2512) to calculate the PCP content. In experiments in which chloramphenicol was added, correction for the chloramphenicol absorption (g320=9772) was made when calculating PCP content. PCP in the culture filtrate was also determined by extracting with ethyl acetate followed by HPLC on a C-18 reverse-phase column. The solvent system used for the mobile phase was methanol/water (80 : 20, v/v). PCP content was determined by integration of the peak height measurements and comparison with an internal standard. The amounts of PCP degraded during the biodegradation studies are expressed as a percentage of PCP in un-inoculated control flasks.
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Chloride in culture filtrates was determined turbidometrically (Frank & Fouch 1992).
I
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Chloride Estimation
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Results and D i s c u s s i o n
20
All the species of Pseudomonas tested (see Table 1) grew well on nutrient broth, peptone, yeast extract and malt extract containing up to 1500 mg PCP/1 but they did not degrade the PCP on these substrates. Only when these Pseudomonas species were grown on glucose could PCP be utilized (Table 1).
Effect of Glucose Concentration
o
J
r
i
f
2
3
4
5
i
I
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Time (days) Figure 1. Influence of glucose concentration on growth (A) and P C P degradation (B). Glucose was added at 0.25 (r-I), 0.5 (e), 1.0 (O) or 2 . 0 % (m). An optical density of 1.0 corresponds to 2.73 g dry cell wt/l.
Glucose was the only carbon source able to support both growth and degradation. Glucose at 0.5% supported complete degradation of 200 mg PCP/1 in 5 days (Figure 1). Above 0.5%, glucose repressed growth. Degradation of PCP by a Flavobacterium sp. was also reported to be repressed by glucose and glutamate (Topp et al. 1988).
it was toxic to P. aeruginosa. Degradation of PCP by a Flavobacterium sp. was decreased considerably below pH 6.9
Effect of pH
Effect of Temperature and Oxygen
The maximal amount of PCP was degraded at pH 7.6 to 8.0. Degradation decreased slowly above pH 8.0 and below pH 7.0
Degradation of PCP was optimal at 30 to 37°C. Degradation was drastically reduced outside of this range. PCP degradation and C1- production was more pronounced in shake cultures than in surface cultures. Similarly, a mixed bacterial population was reported to degrade PCP more effectively in shake culture (Valo et al. 1985).
Table 1. PCP degradation by various Pseudomonas species* Species
Growth after 72 h (optical density)**
PCP degradation after 72 h (%)
Pseudomonas aeruginosa
1.4
Pseudomonas oleovorans
1.8
56
Pseudomonas testosterone
1.7
44
42
Pseudomonas cepacia
1.9
42
Pseudomonas aeruginosa
2.6
69
*PCP was added at 200 mg/I. *'1.0 corresponds to 2.73 g dry cell wtJl.
(Gonzalez & Hei 1991) and degradation by a mixed bacterial population was decreased above pH 8.0 (Valo et aL 1985).
Effect of Nitrogen Source Nitrogen source considerably influenced the degradation of PCP. Organic nitrogen sources were unable to support PCP degradation but gave luxurious growth up to 1500 mg PCP/1. Ammonium salts supported both growth and the degradation whereas nitrate salts failed to support growth of P. aeruginosa. Ammonium chloride and ammonium nitrate were the best nitrogen sources for PCP degradation (Table 2), giving
World Journal of Microbiology & Biotechnology, Vol 1O, 1994
335
A. Premalatha and G. Suseela Rajakumar Table 2. Effect of nitrogen source on PCP degradation by P.
aeruginosa." Nitrogen source
Growth after 72 h (optical density)**
Degradation of PCP after 72 h (%)
2.6 2,7 2.8 3.1
ND ND ND ND
1.7 2.3 2,1 2.0 t .8
76 79 57 41 69
Organic (I g/I) Peptone Malt extract Yeast extract Nutrient broth Ammonium (0.175 g NH4+/I) Ammonium nitrate Ammonium chloride Ammonium sulphate Ammonium acetate Ammonium tar{rate
i
0
i
B 100
V
80
* PCP was added at 200 mg/I. *'1.0 corresponds to 2.73 g dry cell wt/I, ND--No degradation.
6o
40
100% degradation by day 5. PCP degradation by a mixed bacterial population was also enhanced by ammonium salts
(J 20
(Valo et al. 1985). 0
i ~
m
4
Effect of PCP Concentration Degradation of PCP at various concentrations was studied in mineral salt medium with 0.5% glucose and 0.175 g ammonium nitrogen (as ammonium chloride)/1. PCP was completely degraded at up to 600 mg/1 in 5 days. After adapting the cells to PCP, the resistance and the degradation efficiency of P. aeruginosa increased up to 1000 mg PCP/I. Adapted cells degraded PCP at 800 mg/1 completely in 6 days (Figure 2) but at 1000 rag/1 only 53% degradation was observed. The growth of the bacterium started after a lag of about 3 days at 800 or 1000 mg PCP/1. There was a linear relationship between the length of the lag phase and PCP concentration, as was also observed for an Arthrobacter strain (Stanlake & Finn 1982); the lag was only 8 h in cells not exposed to PCP, or exposed to PCP at up to 600 mg/l. Pseudomonas aeruginosa can degrade chlorophenols other than PCP (Suseela et al. 1991).
Effect of Chloramphenicol In one set of flasks chloramphenicol at 150 to 300 rag/1 was added to non-proliferating cells of P. aeruginosa (6.4 g dry cell weight/1 and incubated for 4 h before the addition of 200 mg/l PCP. Another set of flasks received PCP and chloramphenicol together. Control flasks received only PCP. PCP degradation was inhibited in all the flasks containing chloramphenicol. Thus enzyme synthesis for the biodegradation of PCP is inhibited by chloramphenicol and is inducible in nature and not constitutively expressed. Degradation of trichlorophenol (TCP) was inhibited when chloramphenicol was added to cultures of strain GPI of an Azotobacter sp. before the induction of TCP-degrading enzyme (Li et al.
336
wo~Jd Journal of Microbiology & Biotechnology,"Vol 16, 1994
6
Time (days) Figure 2. Influence of PCP concentration on growth and
degradation. PCP was added at 0 ( A ) , 200 (O), 400 (o), 600 (I-I), 800 (11) or 1000 mg/I (A). An optical density of 1.O corresponds to 2.73 g dry cell wt/I.
1991). Pentachlorophenol degradation by Rhodococcus chloramphenolicus (Apajalahti et al. 1986) and by a Flavobacterium sp. (Steiert et al. 1987) was also reported to be induced by polychlorinated substrates and inhibited by chloramphenicol at 500 mg/l.
Degradation of [UJ4C]PCP Biodegradation of [U-14C]PCPat 10s d.p.m, per flask was studied. The results shows that most of the [U-14C]PCP was degraded to 14CO2.About 74% of the label was found as 14CO2 after 7 days of incubation (Figure 3), 17% in the culture filtrate, and 5% in the cell mass. The results show that P. aeruginosa utilizes PCP as an energy source along with glucose for its metabolism. To conclude, P. aeruginosa, which is widely distributed in soil and sewage, is highly resistant to PCP and degrades it to CO2 and C1-, may effectively be utilized to decontaminate PCP-polluted environments. PCI'-contaminatedsoil and natural waters have already been decontaminated by inoculating large numbers of Flavobacterium sp. (Crawford & Mohn 1985; Martinson et al. 1986). Similarly Edgehill & Finn (1983) also reported that they could rapidly decontaminate PCP-contami-
PCP degradation by P. aeruginosa 18000.
14000'
12000'
E~ 10000.
8000,
6000-
4OO0
Time (days)
Figure 3. Mineralization of [UJ4C]PCP and the cumulative production of 14C02. About lOSd.p.m. [UJ4C]PCP was added to each flask either at sp. act. 12.3 mCi/mmol (0) or at sp.act. 4.1 mCi/mmol (o).
nated soil by inoculating Arthrobacter sp. The ability of P. aeruginosa to mineralize relatively high concentrations of PCP might be attributed to its production of extracellular surfaceactive detergents (Devander et al. 1992) which act as an emulsifying agent to solubilize and disperse hydrocarbons. Studies on the isolation and identification of metabolites produced during PCP degradation will be published elsewhere.
Acknowledgement AP is very grateful to the Council of Scientific and Industrial Research (CSIR), India, for awarding her a Junior Research Fellowship.
References Amy, P.S., Schulke, J.W., Frazier, L.M. & Seidler, R.J. 1985 Characterization of aquatic bacteria and cloning of genes specifying partial degradation of 2,4dichlorophenoxyacetic acid. Applied and Environmental Microbiology 49, 1237-1245. Apajalhti, J.H.A., Karpanoja, P. & Salkinoja-Salonen, M.S. 1986 Rhodococcus chlorophenolicus sp. nov., a chlorophenol mineralizing actinomycete. International Journal of Systematic Bacteriology 36, 246--251. Chu, J.P. & Kirsch, E.J. 1972 Metabolism of pentachlorophenol by an axenic bacterial culture. Applied Microbiology 23, 10331035. Crawford, R.L. & Mohn, W.W. 1985 Microbiological removal of pentachlorophenol from soil using a Flavobacterium sp. Enzyme and Microbial Technology 7, 617-620. Crosby, D.G. 1 9 8 1 Environmental chemistry of pentachlorophenol. Applied and Environmental Microbiology 53, 1051-1080.
Devander, K.J., Hung, L. & Trevors, J.T. 1992 Effects of addition of P. aeruginosa UG2 or biosurfactant on biodegradation of selected hydrocarbons in soil. Journal of Industrial Microbiology 10, 87-93. Edgehill, R.V. & Finn, R.K. 1983 Microbial treatment of soil to remove pentachlorophenol. Applied and Environmental Microbiology 45, 1122-1125. Fewson, C.A. 1988 Biodegradation of xenobiotics and other persistence compounds: the case of recalcitrance. Trends in Biotechnology 6, 148-153. Frank, K.H. & Fouch, D.D. 1992 Utilization of 3-chloro-2methyl benzoic acid by Pseudomonas cepaciae MB2 through meta fission pathway. Applied and Environmental Microbiology 57, 1920-1928. Gonzalez, J.R. & Hei, W.-S. 1991 Effect of glutamate on the biodegradation of pentachlorophenol. Applied Microbiology and Biotechnology 35, 100-104. Kirk, T.K., Schultz, E., Connors, W.J., Lorenz, L.F. & Zeikus, J.G. 1977 Influence of culture parameters on lignin metabolism by Phanerochaete chrysoporium. Archives of Microbiology 117, 277-285. Li, D.-Y., Eberspacher, J., Kuntzer, B.W.J. & Lingens, F. 1991 Degradation of 2,4,6-trichlorophenol by Azotobacter sp. strain GPI. Applied and Environmental Microbiology 57, 19201928. Martinson, M.M., Steiert, J.G., Saber, D.L., Mohan, W.W. & Crawford, D.L. 1986 Microbiological decontamination of pentachlorophenol contaminated water. In Biodeterioration 6, eds Llewellyn, G.C. & O'Rear, C.E. pp. 529-534. Slough, UK: CAB International. Mikesell, M.D. & Boyd, S.A. 1986 Complete reductive dechlorination and mineralization of pentachlorophenol by anaerobic microorganisms. Applied and Environmental Microbiology 52, 861-865. Nandy, S.C. & Rao, R.B. 1977 Preservation of raw hides and skin. Leather Science 18, 285-293. Sittig, M. 1981 Handbook of Toxic Hazardous Chemicals, Park Ridge, NJ: Noyes. Sivaparvathy, M. & Nandy, S.C. 1973 Evaluation of preservatives for skin preservation. Journal of the American Leather Chemists" Association 68, 349-353. Stanlake, G.J. & Finn, R.K. 1982 Isolation and characterization of a pentachlorophenol-degrading bacterium. Applied and Environmental Microbiology 44, 1421-1427. Steiert, J.G., Pignatello, J.J. & Crawford, R.L. 1987 Degradation of chlorinated phenols by pentachlorinated phenol degrading bacterium. Applied and Environmental Microbiology 53, 907-910. Suseela, R.G., Basu, S.K. & Nandy, S.C. 1991 Degradation of pentachlorophenol by P. aeruginosa. Indian Journal of Environmental Health 33, 425--432. Suseela R.G., Ramesh, R. & Nandy, S.C. 1988 Fungal growth on leathers as influenced by tanning. Das Leder 40, 24-30. Topp, E., Crawford, R.L. & Hanson, R.S. 1988 Influence of readily metabolizable carbon on pentachlorophenol metabolism by a pentachlorophenol-degrading Flavobacterium sp. Applied and Environmental Microbiology 54, 2452-2459. Valo, R., Apajalahti, J. & Salkinoja-Salonen,M. 1985 Studies on the physiology of degradation of pentachlorophenol. Applied Microbiology and Biotechnology 21, 313-319.
(Received in revised form 8 December 1993; accepted 9 December 1993)
World Journal of Microbiology & Biotechnology, Vol 10, 1994
337
Pentachlorophenol degradation by Pseudomonas aeruginosa A. Premalatha and G. Suseela Rajakumar* Five P s e u d o m o n a s species were tested for ability to degrade pentachlorophenol (PCP). P s e u d o m o n a s aeruginosa completely degraded PCP up to 800 mg/1 in 6 days with glucose as co-substrate. With 1000 mg PCP/1, 53% was degraded. NH4 ÷ salts were better at enhancing degradation than organic nitrogen sources and shake-cultures promoted PCP degradation compared with surface cultures. Degradation was maximal at pH 7.6 to 8.0 and at 30 to 37°C. Only PCP induced enzymes that degraded PCP and chloramphenicol inhibited this process. The PCP was degraded to CO 2, with release of Cl-. Key words: Degradation, pentachlorophenol, Pseudomonas aeruginosa.
Pentachlorophenol (PCP) is a recalcitrant biocide used primarily for wood preservation (Chu & Kirsch 1972). Its industrial and agricultural applications have included use as a bactericide, insecticide, herbicide, algicide and fungicide (Crosby 1981). In the leather industry, PCP is used with common salt as a bactericide to preserve raw hide and skin, preventing bacterial damage to skin collagen (Sivaparvathy & Nandy 1973; Nandy & Rao 1977). It is also used as a fungicide during the different tanning operations, to preserve the chrome blue and other tanned leathers and leather articles (Suseela et al. 1988). The United States' Environmental Protection Agency considers PCP a priority toxic pollutant (Sittig 1981) and bioremediation and bioaugmentation techniques have been advocated as the environmentally-friendlyways to detoxify it (Valo et al. 1985; Mikesell & Boyd 1986). Microbial degradations of multi-chlorinated phenols have been reported by several groups (Amy et al. 1985; Apajalahti et al. 1986). The success of bioremediation depends on a good understanding of the biochemical, physiological and ecological principles which govern microbial growth, activity and biological recalcitrance at introduction sites (Fewson 1988). Pseudomonas aeruginosa, isolated by an enrichment culture technique (Suseela et al. 1991) from the soil around the tannery effluent treatment sites of the Central Leather Research Institute, has been used for biodegradation studies of PCP along with other Pseudomonas species. The present study is of some of the various factors that influence PCP degradation by
Materials and Methods Chemicals
PCP was purchased from Aldrich Chemical Company and [U~4C]PCP from Sigma. The purity of the PCP was determined by HPLC. Culture Maintenance All strains of Pseudomonas (see Table 1) were maintained on nu-
trient agar slants and subcultured every 30 days. Media and Cultivation Conditions
The medium used for inoculum preparation and the biodegradation study was a mineral salt medium containing (g/l): K2HPO4, 1.73; KH2PO4, 0.68; NH4NO3, 1.0; MgSO4. 7H20 , 0.1; CaC12. 2H20, 0.02; MnSO4.H20, 0.03; FeSO4.7H20), 0.03; and glucose, 5.0. The medium was adjusted to pH 7.6. The carbon sources were filter-sterilized and added to the medium separately. Pentachlorophenol was prepared as a filter-sterilized stock solution in 0.2 MNaOH. Biodegradation experiments were conducted in 250-ml Erlenmayer flasks, each containing 50 ml medium and inoculated with 2 ml of a late-growth-phase culture. The flasks were incubated at 37°C in an incubator shaker at 300 rev/min. Unless otherwise stated, all the experiments for degradation of PCP were conducted in the mineral salt medium with glucose and ammonium nitrate as the carbon and nitrogen sources, respectively. Cell growth was monitored by measuring optical density at 600 nm. All the experiments were repeated twice in duplicate.
P. aeruginosa.
Mineralization Studies
The authors are with the Bacteriology Laboratory, Central Leather Research Institute, Madras-600 020, India. *Corresponding author.
Mineralization of [U-14C]PCP was studied in 10 ml mineral salt medium using, per flask, either 105 d.p.m. [U-14C]PCP (sp.act. 12.3 mCi/mmol) or 10 mg [U-1K:]PCP (sp.act. 4.1 mCi/mmol).
© 1994 Rapid Communications of Oxford Ltd
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WorldJournalof Microbiology& Biotechnology,Vo110, 1994
PCP degradation by P. aeruginosa The 14CO2 produced was trapped in ethanolamine-containing scintillation fluid according to the method described by Kirk et al. (1977) and assayed by liquid scintillation spectrometry. The extent of PCP degradation was followed spectrophotometrically~ An equal volume of 0.2 M NaOH was added to the culture medium immediately after the sampling, centrifuged at 7700 x g for 15 min and the absorbance of the clear supernatant produced was then read at 320 nm (8320=2512) to calculate the PCP content. In experiments in which chloramphenicol was added, correction for the chloramphenicol absorption (g320=9772) was made when calculating PCP content. PCP in the culture filtrate was also determined by extracting with ethyl acetate followed by HPLC on a C-18 reverse-phase column. The solvent system used for the mobile phase was methanol/water (80 : 20, v/v). PCP content was determined by integration of the peak height measurements and comparison with an internal standard. The amounts of PCP degraded during the biodegradation studies are expressed as a percentage of PCP in un-inoculated control flasks.
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Chloride in culture filtrates was determined turbidometrically (Frank & Fouch 1992).
I
I
i
I
I
I
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Chloride Estimation
I
80
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1D 03 o~ -o Q_
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40
L~
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Results and D i s c u s s i o n
20
All the species of Pseudomonas tested (see Table 1) grew well on nutrient broth, peptone, yeast extract and malt extract containing up to 1500 mg PCP/1 but they did not degrade the PCP on these substrates. Only when these Pseudomonas species were grown on glucose could PCP be utilized (Table 1).
Effect of Glucose Concentration
o
J
r
i
f
2
3
4
5
i
I
6
7
Time (days) Figure 1. Influence of glucose concentration on growth (A) and P C P degradation (B). Glucose was added at 0.25 (r-I), 0.5 (e), 1.0 (O) or 2 . 0 % (m). An optical density of 1.0 corresponds to 2.73 g dry cell wt/l.
Glucose was the only carbon source able to support both growth and degradation. Glucose at 0.5% supported complete degradation of 200 mg PCP/1 in 5 days (Figure 1). Above 0.5%, glucose repressed growth. Degradation of PCP by a Flavobacterium sp. was also reported to be repressed by glucose and glutamate (Topp et al. 1988).
it was toxic to P. aeruginosa. Degradation of PCP by a Flavobacterium sp. was decreased considerably below pH 6.9
Effect of pH
Effect of Temperature and Oxygen
The maximal amount of PCP was degraded at pH 7.6 to 8.0. Degradation decreased slowly above pH 8.0 and below pH 7.0
Degradation of PCP was optimal at 30 to 37°C. Degradation was drastically reduced outside of this range. PCP degradation and C1- production was more pronounced in shake cultures than in surface cultures. Similarly, a mixed bacterial population was reported to degrade PCP more effectively in shake culture (Valo et al. 1985).
Table 1. PCP degradation by various Pseudomonas species* Species
Growth after 72 h (optical density)**
PCP degradation after 72 h (%)
Pseudomonas aeruginosa
1.4
Pseudomonas oleovorans
1.8
56
Pseudomonas testosterone
1.7
44
42
Pseudomonas cepacia
1.9
42
Pseudomonas aeruginosa
2.6
69
*PCP was added at 200 mg/I. *'1.0 corresponds to 2.73 g dry cell wtJl.
(Gonzalez & Hei 1991) and degradation by a mixed bacterial population was decreased above pH 8.0 (Valo et aL 1985).
Effect of Nitrogen Source Nitrogen source considerably influenced the degradation of PCP. Organic nitrogen sources were unable to support PCP degradation but gave luxurious growth up to 1500 mg PCP/1. Ammonium salts supported both growth and the degradation whereas nitrate salts failed to support growth of P. aeruginosa. Ammonium chloride and ammonium nitrate were the best nitrogen sources for PCP degradation (Table 2), giving
World Journal of Microbiology & Biotechnology, Vol 1O, 1994
335
A. Premalatha and G. Suseela Rajakumar Table 2. Effect of nitrogen source on PCP degradation by P.
aeruginosa." Nitrogen source
Growth after 72 h (optical density)**
Degradation of PCP after 72 h (%)
2.6 2,7 2.8 3.1
ND ND ND ND
1.7 2.3 2,1 2.0 t .8
76 79 57 41 69
Organic (I g/I) Peptone Malt extract Yeast extract Nutrient broth Ammonium (0.175 g NH4+/I) Ammonium nitrate Ammonium chloride Ammonium sulphate Ammonium acetate Ammonium tar{rate
i
0
i
B 100
V
80
* PCP was added at 200 mg/I. *'1.0 corresponds to 2.73 g dry cell wt/I, ND--No degradation.
6o
40
100% degradation by day 5. PCP degradation by a mixed bacterial population was also enhanced by ammonium salts
(J 20
(Valo et al. 1985). 0
i ~
m
4
Effect of PCP Concentration Degradation of PCP at various concentrations was studied in mineral salt medium with 0.5% glucose and 0.175 g ammonium nitrogen (as ammonium chloride)/1. PCP was completely degraded at up to 600 mg/1 in 5 days. After adapting the cells to PCP, the resistance and the degradation efficiency of P. aeruginosa increased up to 1000 mg PCP/I. Adapted cells degraded PCP at 800 mg/1 completely in 6 days (Figure 2) but at 1000 rag/1 only 53% degradation was observed. The growth of the bacterium started after a lag of about 3 days at 800 or 1000 mg PCP/1. There was a linear relationship between the length of the lag phase and PCP concentration, as was also observed for an Arthrobacter strain (Stanlake & Finn 1982); the lag was only 8 h in cells not exposed to PCP, or exposed to PCP at up to 600 mg/l. Pseudomonas aeruginosa can degrade chlorophenols other than PCP (Suseela et al. 1991).
Effect of Chloramphenicol In one set of flasks chloramphenicol at 150 to 300 rag/1 was added to non-proliferating cells of P. aeruginosa (6.4 g dry cell weight/1 and incubated for 4 h before the addition of 200 mg/l PCP. Another set of flasks received PCP and chloramphenicol together. Control flasks received only PCP. PCP degradation was inhibited in all the flasks containing chloramphenicol. Thus enzyme synthesis for the biodegradation of PCP is inhibited by chloramphenicol and is inducible in nature and not constitutively expressed. Degradation of trichlorophenol (TCP) was inhibited when chloramphenicol was added to cultures of strain GPI of an Azotobacter sp. before the induction of TCP-degrading enzyme (Li et al.
336
wo~Jd Journal of Microbiology & Biotechnology,"Vol 16, 1994
6
Time (days) Figure 2. Influence of PCP concentration on growth and
degradation. PCP was added at 0 ( A ) , 200 (O), 400 (o), 600 (I-I), 800 (11) or 1000 mg/I (A). An optical density of 1.O corresponds to 2.73 g dry cell wt/I.
1991). Pentachlorophenol degradation by Rhodococcus chloramphenolicus (Apajalahti et al. 1986) and by a Flavobacterium sp. (Steiert et al. 1987) was also reported to be induced by polychlorinated substrates and inhibited by chloramphenicol at 500 mg/l.
Degradation of [UJ4C]PCP Biodegradation of [U-14C]PCPat 10s d.p.m, per flask was studied. The results shows that most of the [U-14C]PCP was degraded to 14CO2.About 74% of the label was found as 14CO2 after 7 days of incubation (Figure 3), 17% in the culture filtrate, and 5% in the cell mass. The results show that P. aeruginosa utilizes PCP as an energy source along with glucose for its metabolism. To conclude, P. aeruginosa, which is widely distributed in soil and sewage, is highly resistant to PCP and degrades it to CO2 and C1-, may effectively be utilized to decontaminate PCP-polluted environments. PCI'-contaminatedsoil and natural waters have already been decontaminated by inoculating large numbers of Flavobacterium sp. (Crawford & Mohn 1985; Martinson et al. 1986). Similarly Edgehill & Finn (1983) also reported that they could rapidly decontaminate PCP-contami-
PCP degradation by P. aeruginosa 18000.
14000'
12000'
E~ 10000.
8000,
6000-
4OO0
Time (days)
Figure 3. Mineralization of [UJ4C]PCP and the cumulative production of 14C02. About lOSd.p.m. [UJ4C]PCP was added to each flask either at sp. act. 12.3 mCi/mmol (0) or at sp.act. 4.1 mCi/mmol (o).
nated soil by inoculating Arthrobacter sp. The ability of P. aeruginosa to mineralize relatively high concentrations of PCP might be attributed to its production of extracellular surfaceactive detergents (Devander et al. 1992) which act as an emulsifying agent to solubilize and disperse hydrocarbons. Studies on the isolation and identification of metabolites produced during PCP degradation will be published elsewhere.
Acknowledgement AP is very grateful to the Council of Scientific and Industrial Research (CSIR), India, for awarding her a Junior Research Fellowship.
References Amy, P.S., Schulke, J.W., Frazier, L.M. & Seidler, R.J. 1985 Characterization of aquatic bacteria and cloning of genes specifying partial degradation of 2,4dichlorophenoxyacetic acid. Applied and Environmental Microbiology 49, 1237-1245. Apajalhti, J.H.A., Karpanoja, P. & Salkinoja-Salonen, M.S. 1986 Rhodococcus chlorophenolicus sp. nov., a chlorophenol mineralizing actinomycete. International Journal of Systematic Bacteriology 36, 246--251. Chu, J.P. & Kirsch, E.J. 1972 Metabolism of pentachlorophenol by an axenic bacterial culture. Applied Microbiology 23, 10331035. Crawford, R.L. & Mohn, W.W. 1985 Microbiological removal of pentachlorophenol from soil using a Flavobacterium sp. Enzyme and Microbial Technology 7, 617-620. Crosby, D.G. 1 9 8 1 Environmental chemistry of pentachlorophenol. Applied and Environmental Microbiology 53, 1051-1080.
Devander, K.J., Hung, L. & Trevors, J.T. 1992 Effects of addition of P. aeruginosa UG2 or biosurfactant on biodegradation of selected hydrocarbons in soil. Journal of Industrial Microbiology 10, 87-93. Edgehill, R.V. & Finn, R.K. 1983 Microbial treatment of soil to remove pentachlorophenol. Applied and Environmental Microbiology 45, 1122-1125. Fewson, C.A. 1988 Biodegradation of xenobiotics and other persistence compounds: the case of recalcitrance. Trends in Biotechnology 6, 148-153. Frank, K.H. & Fouch, D.D. 1992 Utilization of 3-chloro-2methyl benzoic acid by Pseudomonas cepaciae MB2 through meta fission pathway. Applied and Environmental Microbiology 57, 1920-1928. Gonzalez, J.R. & Hei, W.-S. 1991 Effect of glutamate on the biodegradation of pentachlorophenol. Applied Microbiology and Biotechnology 35, 100-104. Kirk, T.K., Schultz, E., Connors, W.J., Lorenz, L.F. & Zeikus, J.G. 1977 Influence of culture parameters on lignin metabolism by Phanerochaete chrysoporium. Archives of Microbiology 117, 277-285. Li, D.-Y., Eberspacher, J., Kuntzer, B.W.J. & Lingens, F. 1991 Degradation of 2,4,6-trichlorophenol by Azotobacter sp. strain GPI. Applied and Environmental Microbiology 57, 19201928. Martinson, M.M., Steiert, J.G., Saber, D.L., Mohan, W.W. & Crawford, D.L. 1986 Microbiological decontamination of pentachlorophenol contaminated water. In Biodeterioration 6, eds Llewellyn, G.C. & O'Rear, C.E. pp. 529-534. Slough, UK: CAB International. Mikesell, M.D. & Boyd, S.A. 1986 Complete reductive dechlorination and mineralization of pentachlorophenol by anaerobic microorganisms. Applied and Environmental Microbiology 52, 861-865. Nandy, S.C. & Rao, R.B. 1977 Preservation of raw hides and skin. Leather Science 18, 285-293. Sittig, M. 1981 Handbook of Toxic Hazardous Chemicals, Park Ridge, NJ: Noyes. Sivaparvathy, M. & Nandy, S.C. 1973 Evaluation of preservatives for skin preservation. Journal of the American Leather Chemists" Association 68, 349-353. Stanlake, G.J. & Finn, R.K. 1982 Isolation and characterization of a pentachlorophenol-degrading bacterium. Applied and Environmental Microbiology 44, 1421-1427. Steiert, J.G., Pignatello, J.J. & Crawford, R.L. 1987 Degradation of chlorinated phenols by pentachlorinated phenol degrading bacterium. Applied and Environmental Microbiology 53, 907-910. Suseela, R.G., Basu, S.K. & Nandy, S.C. 1991 Degradation of pentachlorophenol by P. aeruginosa. Indian Journal of Environmental Health 33, 425--432. Suseela R.G., Ramesh, R. & Nandy, S.C. 1988 Fungal growth on leathers as influenced by tanning. Das Leder 40, 24-30. Topp, E., Crawford, R.L. & Hanson, R.S. 1988 Influence of readily metabolizable carbon on pentachlorophenol metabolism by a pentachlorophenol-degrading Flavobacterium sp. Applied and Environmental Microbiology 54, 2452-2459. Valo, R., Apajalahti, J. & Salkinoja-Salonen,M. 1985 Studies on the physiology of degradation of pentachlorophenol. Applied Microbiology and Biotechnology 21, 313-319.
(Received in revised form 8 December 1993; accepted 9 December 1993)
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