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Jun 18, 2009 - Synergistic biodegradation of pentachlorophenol by Bacillus cereus (DQ002384), Serratia marcescens (AY927692) and Serratia marcescens ...

World J Microbiol Biotechnol (2009) 25:1821–1828 DOI 10.1007/s11274-009-0083-6

ORIGINAL PAPER

Synergistic biodegradation of pentachlorophenol by Bacillus cereus (DQ002384), Serratia marcescens (AY927692) and Serratia marcescens (DQ002385) Shail Singh Æ B. B. Singh Æ R. Chandra Æ D. K. Patel Æ V. Rai

Received: 24 December 2008 / Accepted: 4 June 2009 / Published online: 18 June 2009 Ó Springer Science+Business Media B.V. 2009

Abstract The consortium of Bacillus cereus (DQ002384), Serratia marcescens (AY927692) and Serratia marcescens (DQ002385) were used for pentachlorophenol (PCP) degradation. The consortia showed better overall removal efficiencies than single strains by utilization of PCP as a carbon and energy source confirmed by pH dependent dye indicator bromocresol purple (BCP) in mineral salt media (MSM). Mixed culture was found to degrade up to 93% of PCP (300 mg/l) as compared to single strains (62.75–90.33%), at optimized conditions (30 ± 1°C, pH 7 ± 0.2, 120 rpm) at 168 h incubation. PCP degradation was also recorded at 20°C (62.75%) and 37°C (83.33%); pH 6 (70%) and pH 9 (75.16%); 50 rpm (73.33%) and 200 rpm (91.63%). The simultaneous release of chloride ion up to 90.8 mg/l emphasized the bacterial dechlorination in the medium. GC–MS analysis revealed the formation of low molecular weight compound, i.e., 6-chlorohydroxyquinol, 2,3,4,6-tetrachlorophenol and tetrachlorohydroquinone, from degraded sample as compared to control.

S. Singh  B. B. Singh  R. Chandra Environmental Microbiology Section, Indian Institute of Toxicology Research, Post Box No. 80, Mahatma Gandhi Marg, Lucknow 226001, India D. K. Patel Analytical Chemistry Section, Indian Institute of Toxicology Research, Post Box No. 80, Mahatma Gandhi Marg, Lucknow 226001, India V. Rai (&) School of Life Sciences, Pandit Ravi Shankar Shukla University, Raipur 492010, India e-mail: [email protected]

Keywords Pentachlorophenol  Degradation  Dechlorination  GC–MS

Introduction With the development of modern agriculture and industry, large quantities of man-made pesticides have been introduced into the environment at concentrations that would cause ecologically undesirable effects. Many of those compounds, such as pentachlorophenol (PCP), are highly resistant to biotic or abiotic degradation, led to substantial environmental contamination. So, it is regulated as one of the priority pollutants by the Environmental Protection Agency EPA (1987). PCP is a chlorinated phenol of widespread use, mainly as herbicides and fungicides (Salmero´n-Alcocer et al. 2007), and in wood protection, tanneries, distilleries, paints manufacturing and pulp and paper mills (Chandra et al. 2006). The effluents discharged from these industries are main source of PCP contamination. In addition, owing to its molecular stability, sorption properties and intensive use, PCP has progressively become a widespread contaminant in the environment (Ma¨nnisto et al. 2001). Microbial degradation is a useful strategy to eliminate these compounds and detoxify wastewaters and polluted environments (Morgan and Watkinson 1989; Puhakka et al. 1995). Several bacterial strains belonging to a variety of genera degrade chlorophenolic compounds (Shimp and Pfaender 1987; Gurujeyalakshmi and Oriel 1989; Chitra et al. 1995; Ha¨ggblom and Valo 1995; Chandra et al. 2006; Singh et al. 2007, 2008). Although, PCP degradation was well studied in pure cultures, the results of such studies are not necessarily relevant to the field because microorganisms in nature grow mostly in mixed cultures. Most studies

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deal with pure cultures growing on a single carbon and energy source (Ha¨ggblom 1992; McAllister et al. 1996; Wittmann et al. 1998; Tuomel et al. 1999), whereas the degradation of PCP in mixed cultures has been investigated in lesser extent (Klecka and Maier 1988; De Los CobosVasconcelos et al. 2006). Some authors emphasize that, when mixed cultures are used, substrate competition and crossed inhibition could severely affect the microbial growth and biodegradation rates (Silver and Mateles 1969). In our previous work, we have isolated, optimized and characterized three bacterial strains, i.e., Bacillus cereus (DQ002384), Serratia marcescens (AY927692) and Serratia marcescens (DQ002385) for PCP degradation (Chandra et al. 2006; Singh et al. 2007). The maximum degradation of PCP was shown by B. cereus (DQ002384), Ser. marcescens (AY927692) and Ser. marcescens (DQ002385) up to 62.75, 90.33 and 86.6%, respectively. In our present study, we describe the PCP degradation at different pH, temperature and aeration rate by mixed culture of three previously proven strains of our previous study.

Materials and methods Chemicals All reagents used were of analytical grade. Synthetic PCP (FW 266.30), 2-chlorophenol (MW 128), 2,4-dichlorophenol (FW 162), 2,4,5-trichlorophenol (FW 197.4), 6-chlorohydroxyquinol (MW 179.61) and tetrachlorohydroquinone (MW 247.89) were purchased from Sigma Chemicals (USA). All solutions were prepared in Milli-Q water (Elix, Millipore Purification System, France). Mixed culture preparation The mixed culture, used in this study, was biochemically identified as B. cereus (DQ002384), Ser. mercescens (AY927692) and Ser. mercescens (DQ002385) and further confirmed by 16S rRNA gene sequencing (Chandra et al. 2006; Singh et al. 2007). One percent of inoculum of B. cereus (6.0 9 105 cfu/ml), Ser. mercescens (2.5 9 103 cfu/ml) and Ser. mercescens (2.87 9 106 cfu/ml) was taken from each bacterial strains grown in 250 ml Erlenmeyer flask containing mineral salt media (MSM) of following composition (mg/l): K2HPO4, 85; KH2PO4, 17; MgSO4, 30; FeSO47H2O, 30; CaSO4, 30; MnSO4H2O, 30; (NH4)2 SO4, 17; trace element solution (1 ml/l), containing PCP (300 mg/l), amended with 1% glucose (w/v) at pH 7.0 ± 0.2, and incubated at 30 ± 1°C at 120 rpm (Innova 4230, USA). Each culture was capable of growth in MSM containing PCP as sole carbon source.

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The stability of mixed culture was maintained in same media. Degradation of PCP by mixed culture The degradation studies were performed by inoculating 1% inoculum size of 6.2 9 105 CFU/ml mixed culture cells to 250 ml Erlenmeyer flask containing 99 ml MSM, PCP (300 mg/l) and 1% glucose (w/v) at pH 7.0 ± 0.2, incubated at 30 ± 1°C at 120 rpm in refrigerated incubator shaker up to 168 h. The growth of the bacterial cells was determined by measuring 5 ml of sample volume for optical density (OD) at 620 nm and PCP concentration was determined by UV–Vis spectrophotometer (GBC Cintra-40, Australia) at 320 nm at every 24 h interval up to 168 h incubation period. The stability of mixed culture for PCP degradation were optimized at different environmental conditions, i.e., temperature (20, 30 and 37°C), pH (6.0, 7.0 and 9.0) and aeration rate (50, 120 and 200 rpm). MSM containing PCP without inoculum was used as control for the PCP degradation. The confirmatory test for the tolerance and accumulation of PCP by these mixed culture were performed using bromocresol purple (BCP-16 mg/l) containing MSM (Martins et al. 1997). Residual PCP was detected in the culture fluid using HPLC reported by Singh et al. (2007). Simultaneously, PCP degradation was also determined by estimation of the chloride ion released in aqueous media at every 24 h interval up to 168 h using method of Bergmann and Sanik (1957). This was also quantified by Orion ion analyzer model 960 (Boston, USA) using calibrated selective chloride ion electrode. The pH of the medium was measured with the selective pH electrode (9172 BN) of Thermo Orion (Model 960). Dissolved oxygen (DO) was measured as partial oxygen pressure using a Clark-type polarographic DO probe model-835A, USA of (083010F) electrode, Thermo Orion (detection level, 0.1 mg/l). All experiments were carried out in triplicates; simultaneously a control was also performed in triplicates. The values were presented as mean ± SD (n = 3) and statistically analyzed by an overall one-way analysis of variance (ANOVA) and when differences observed were significant, the mean were compared by Tukey–Kramer Multiple Comparison Test. GC–MS analysis The same extracted sample given in above section were used for GC–MS analysis for qualitative estimation of PCP and its metabolites at 168 h incubation period along with experimental control as method reported earlier (Singh et al. 2008).

Determination of ring cleavage

Results and discussion Degradation of PCP by mixed culture Bacterial growth, measured by absorbance at 620 nm showed the stationary phase after a 120 h incubation period during PCP degradation (Fig. 1). The maximum degradation of PCP (93%) was shown by mixed culture within a 168 h incubation period under optimized condition (30 ± 1°C, pH 7.0 ± 0.2 and 120 rpm). Mixture showed that it supported the growth of three bacterial strains and simultaneously degraded PCP fast as compared to them alone. They grew equally well in the medium and were compatible with each other, the proportion of each strains were estimated at the end of treatment (data not shown). Further, HPLC analysis confirmed the degradation of PCP by disappearance of the peak of PCP as compared to control as shown in Fig. 2. Bacteria initially utilized glucose for its growth and subsequently utilized PCP as a co-metabolites which was shown by change in colour of media of BCP from blue to yellow due to the acidic pH resulting from release of Clion as well as H? from PCP aromatic ring during dechlorination in the medium (Martins et al. 1997). This phenomenon indicated that PCP degradation was occurring. Our result obtained is in agreement with the findings of Premalatha and Rajkumar (1994).

Absorbance (620 nm)

For ring cleavage activity, 10 ml of degraded sample was centrifuged at 5,000g for 20 min to remove the bacterial biomass. The supernatant and pellet portion were collected for extracellular and intracellular activity, respectively. Bacterial cells were lysed by adding 2 mg/ml of lysozyme in TES buffer (1 ml) [10 mM Tris–HCl (pH 7.5), 1 mM EDTA and 100 mM NaCl] in pellet portion, kept it at 1 h for proper lysis of bacterial cell. The mixture was treated with toluene (1 ml) and 0.01 M catechol (1 M). The development of yellow colour was observed for meta cleavage. In absence of colour, the mixture was shaken for 1 h at 170 rpm for formation of b-ketoadipic acid (Rothera reaction), which indicated the presence of ortho fission (Holding and Collee 1971). In this procedure, the above mixture was acidified with 2 ml HCl followed by addition of 1 ml NaNO3 (1%), concentrated ammonia 15 ml and 1 ml ferrous sulphate solution (10%). In supernatant, same process was followed for meta and ortho cleavage. The development of a reddish brown colour indicated a typical Rothera reaction and the presence of ortho cleavage. MSM containing PCP having no bacterial culture was used as control for the ring cleavage assay.

350

2

300 1.6

Growth rate PCP degradation Chloride ion release

1.2

250 200 150

0.8

100 0.4

50

0 0

24

48

72

96

120

144

0 168

(mg/L)

1823 PCP degradation and chloride ion

World J Microbiol Biotechnol (2009) 25:1821–1828

Time (hrs)

Fig. 1 Growth curve, PCP degradation and chloride ion release in MSM by mixed culture at optimized condition (30 ± 1°C, pH 7.0 and 120 rpm)

The effect of temperature, pH and aeration rate (in rpm) on PCP degradation by mixed culture At low (20°C) as well as high (37°C) temperatures, metabolic activities of bacterial cells were affected adversely, hence a sharp decrease in degradation of PCP was noticed (Table 1). After 168 h incubation, 62.75, 93, and 83.33% of PCP was degraded at 20, 30 and 37°C, respectively. An optimum temperature of 33°C was previously reported for Sphingomonas chlorophenolica RA2, with no growth observed above 36°C (Wittmann et al. 1998). After 168 h incubation, the maximum PCP degradation was observed (93%) at pH 7.0, on other hand, a sharp decline (70 and 75.16%) was recorded at pH 6.0 and 9.0, respectively (Table 1). Wittmann et al. (1998) reported that no growth was observed at pH 4.0. Our results suggested that a neutral to slight alkaline pH was a best suited range for maximum PCP degradation and our result obtained is in agreement with the findings of Wittmann et al. (1998). PCP degradation was recorded at different aeration rate (in rpm) as shown in Table 1. As data indicated, only 73.33% of PCP was degraded at 50 rpm, as compared to 120 and 200 rpm, where 93 and 91.63% degradation of PCP was observed, respectively. High biomass concentration was observed at high aeration rates (MacLeod and Daugulis 2005). Moreover, high aeration rates enhanced fluid-to-particles mass transfer (Arnaud et al. 1992). But it was also observed that very high aeration rate affected adversely on biodegradation of PCP, it may be, due to bacterial cell rupturing (Singh et al. 2007). Effect of temperature, pH and aeration rate on PCP degradation was also optimized for individual strains (Fig. 3). In the course of experiment, a significant decrease in pH (from 7.0 to 4.0 pH) was observed at 96 h, followed by an increase in pH (up to 6.5) at 168 h. Concomitantly, dissolved oxygen (DO) of the media was also changed from

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Fig. 2 Comparative HPLC chromatogram of PCP degradation by B. cereus (DQ002384), Ser. marcescens (AY927692), Ser. marcescens (DQ002385) and mixed culture compared with control after 168 h incubation period at optimized condition (30 ± 1°C, pH 7.0 and 120 rpm)

0.130 0.120 0.110

Control 0.100 0.090

Degraded by B. cereus (DQ002384)

AU

0.080 0.070

Degraded by Ser. mercescens (DQ002385)

0.060 0.050

Degraded by Ser. mercescens (AY927692)

0.040 0.030

Degraded by mixed culture

0.020 0.010 0.000 0.60 0.70 0.80

0.90 1.00 1.10 1.20 1.30 1.40 1.50

1.60 1.70 1.80 1.90 2.00 2.10 2.20

2.30 2.40 2.50

Minutes

Table 1 PCP degradation by mixed culture in 1% glucose containing MSM, in the presence of 300 mg/l of PCP at optimized condition (Temperature 30 ± 1°C, pH 7, 120 rpm) and temperature, pH, aeration rate effect on PCP degradation Time in hours

At optimized condition

Temperature 20°C

pH 37°C

Aeration rate

6

9

50 rpm

200 rpm

Degradation of PCP (%) 0

0

0

0

0

0

0

0

24

11.30 ± .01

06.66 ± .07

10.00 ± .02

10.08 ± .01

13.03 ± .01

08.86 ± .02

10.03 ± .06

48

26.80 ± .01

11.60 ± .06

25.00 ± .09

16.65 ± .03

18.66 ± .02

17.71 ± .02

19.65 ± .05

72

42.60 ± .02

21.66 ± .05

43.31 ± .08

25.00 ± .04

21.00 ± .04

22.00 ± .02

23.00 ± .04

96

57.83 ± .01

31.60 ± .04

53.32 ± .07

25.62 ± .03

27.63 ± .04

24.66 ± .04

26.61 ± .03

120

73.30 ± .01

41.62 ± .03

73.33 ± .06

56.61 ± .06

52.65 ± .06

53.56 ± .05

55.66 ± .04

144

92.93 ± .02

62.60 ± .02

83.12 ± .05

69.85 ± .04

74.95 ± .06

73.16 ± .05

91.45 ± .07

168

93.01** ± .01

62.75** ± .01

83.33* ± .07

70.00** ± .06

75.16** ± .10

73.33** ± .07

91.63* ± .10

All the values are mean ± SD (n = 3) and compared vertically ** significant, ANOVA P \ 0.01; * less significant, ANOVA P \ 0.05; rest are all highly significant, ANOVA P \ 0.001

4.5 to 2 mg/l at 168 h as compared to control 4.8 mg/l. The decline in pH and subsequent change in DO were indicative of the depletion of PCP from media and further increase in pH was confirmed the PCP biotransformation (Singh et al. 2008). In the standard degradation experiment, the incubated flasks were in cotton-plugged shake flasks. Under these conditions, atmospheric oxygen is admitted to the system. As degradation was not apparently affected by low O2

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concentration, inhibition is more likely to be a metabolism dependent event. The decrease of DO may be attributed to glucose fermentation, contribute to decrease its concentration in the medium. However, the degradation at low oxygen concentrations can be considered to indicate the facultative microaerophilic nature of the bacteria. The removal of PCP from media and simultaneous release of chloride ion also gave strong evidence for the bacterial degradation of PCP. At 20 and 37°C temperature,

World J Microbiol Biotechnol (2009) 25:1821–1828 B cereus

100

Ser. marcescens

Ser. marcescens

90 80

% degradation of PCP

Fig. 3 Effect of temperature, pH and aeration rate on PCP degradation (%) for individual strains—B. cereus (DQ002384), Ser. marcescens (AY927692) and Ser. marcescens (DQ002385)

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70 60 50 40 30 20 10 0 20 °C

80.5 and 86.2 mg/l of chloride were released, respectively. Chloride release was also affected by pH and aeration rate, i.e., 74 mg/l at pH 6.0, 80.3 mg/l at pH 9.0, 77 mg/l at 50 rpm and 90 mg/l at 200 rpm. But best result (90.8 mg/l) was observed at optimized conditions (Fig. 1). No growth and PCP degradation was observed in control sample during experiment. GC–MS analysis Result obtained for degradation of PCP through GC–MS analysis of the ethyl acetate extractable products show formation of 6-chlorohydroquinol (RT = 10.72 min), tetrachloro-p-hydroquinone (RT = 13.89 min) and 2,3,4,6tetrachlorophenol (RT = 10.87 min) in the extract of degraded samples by mixed culture at 168 h whereas, in control sample, only PCP (RT = 13.34 min) is identified using the NIST mass database (Fig. 4a, e). At 168 h about 93% of the PCP had been degrade showed small peak and only very less PCP remained in media, indicating the formation of large peak of 6-chlorohydroxyquinol, 2,3,4,6tetrachlorophenol and tetrachlorohydroquinone. It assures from the results that the PCP degradation is at its end of destination. In addition to this, many intermediates products were formed during PCP degradation such as 2(1H)-quinolinone (RT 8.885), 2H-1-benzopyran-2-one 3-chlo (RT 9.197), 6chlorohydroxyquinol (RT 10.535), benzo (F) quinoline (RT 11.251), tetrachlorohydroquinone (RT 14.00) by B. cereus (DQ002384), propanamide 2-hydroxy (RT 3.72), aloxiprin (RT 3.71), 4(3H)-pyrimidinone (RT 6.61), 4Hpyran-4-one 2,3 dihydro-3,5 (RT 6.94), 4-hexen-3-one 4,5dimethyl (RT 7.77), 6-chlorohydroxyquinol (RT 10.61), 2,3,4,6-tetrachlorophenol (RT 10.61), furan, 2-(2,4dichlorophenyl) (RT 10.77), tetrachlorohydroquinone (RT 13.51), phthalates (RT 20.12) by Ser. marcescens (AY927692) and 2(1H)-quinoline (RT 8.89), 2,4,6-

30 °C

37 °C

pH 6

pH 7

pH 9

50 rpm 120 rpm 200 rpm

trichlorophenol (RT 8.89), 6-chlorohydroxyquinol (RT 10.70), 2,3,4,6-tetrachlorophenol (RT 10.70), benzo (F) quinoline (RT 11.21), tetrachlorohydroquinone (RT 13.87), Di-N-octyl-phthalate (RT 17.85) by Ser. marcescens (DQ002385) as shown in (Fig. 4b–d). The identification of tetrachlorohydroquinone would suggest that their products are produced by a reductive dechlorination were intermediates prior to ring cleavage in PCP degradation. Phthalates was found in chromatogram at the end of (RT = 19.90 min) due to contamination in water bottles packing because it used in plastic material as a plasticizer that gives flexibility to rigid plastics. GC–MS analysis revealed the formation of low molecular weight compounds released during PCP degradation was previously reported by Gerlach and Emon (1997). This result showed a considerable qualitative difference in the pattern of compounds obtained by PCP degradation by mixed culture in comparison to that of control sample. Controls did not show the formation of metabolites from PCP. The results show that the PCP dechlorination pathway can be tracked from the GC–MS analysis. Similar to the results of previously reported by Piccininia et al. (1998), Hong et al. (2000), Suegara et al. (2005). Therefore, this mixed culture may be used for bioremediation of PCP containing wastewater from pulp paper mill, pharmaceuticals, pesticides and agro based industries. Metabolites formation mechanisms based on bond cleavage. The C–Cl bonds are more fragile in PCP although the O–H bond is not very much stronger. Suegara et al. (2005) calculated the C–Cl bond dissociation energies in each of the PCP molecule substitutes. The C–Cl bond dissociation energy in position 4 (para) was the lowest. Position 3 and 5 (meta) were second from the lowest. It was, therefore, determined that the C–Cl bond in position 4 is the easiest to dissociate while those in positions 3 and 5 were the second easiest ones to dissociate. The hardest to disassociate appeared to be the bonds in positions 2 and 6 (ortho).

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World J Microbiol Biotechnol (2009) 25:1821–1828 PCP degradation

PCP degradation ControlMLPBasic010307 13.34 100

Strain6MLPacedic010307

Scan EI+ TIC 4.61e7

a

Scan EI+ TIC 1.48e8

11.25

100

b

%

%

14.00 13.73

13.38

9.20

0

11.87

0 4.00

6.00

8.00

10.00

12.00

14.00

16.00

18.00

20.00

22.00

4.00

24.00

6.00

8.00

10.00

12.00

Time

PCP degradation

16.00

18.00

20.00

PCP degradation

Strain7MLPacedic010307 100

14.00

Time

Strain9OLPAcedic020307

Scan EI+ TIC 6.64e7

10.61

c

100

Scan EI+ TIC 1.24e8

11.21

d

10.70

%

%

13.87

7.77

10.77 3.72

4.14

13.51 13.34

6.61 9.20

13.23

6.06

8.89

20.12

0

11.87

16.44

17.85

17.03

20.99 21.98 23.77

0 4.00

6.00

8.00

10.00

12.00

14.00

16.00

18.00

20.00

22.00

24.00

Time

4.00

6.00

8.00

10.00

12.00

14.00

16.00

18.00

20.00

22.00

24.00

Time

PCP degradation Mix MLPBasic010307 100

Scan EI+ TIC 7.87e7

10.72

e

10.87

13.89

%

22.43

9.18 19.90

15.82

22.03

22.78

0 4.00

6.00

8.00

10.00

12.00

14.00

16.00

18.00

20.00

22.00

Time

Fig. 4 a–e GC–MS chromatogram obtained after PCP degradation. a Control sample, b B. cereus (DQ002384), c Ser. marcescens (AY927692), d Ser. marcescens (DQ002385) and e mixed culture after 168 h incubation period at optimized condition (30 ± 1°C, pH 7.0 and 120 rpm)

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The bacterial enzyme PCP-4-monooxygenase from Flavobacterium sp. strain ATCC 39723 catalyzes the oxygenolytic removal of the first chlorine from PCP. PCP-4monooxygenase is a FAD-binding, NADPH-requiring oxygenase with similar functional domains as other bacterial flavoprotein monooxygenase specific for phenolic substrate. PCP-4-monooxygenase converts PCP to 2,3,5,6tetrachloro-p-hydroquinone (TeCH) in the presence of oxygen and NADPH, which is further converted to 2,6dichlorohydroquinone (DiCH) by tetrachloro-p-hydroquinone reductive dehalogenase enzyme. This enzyme not only catalyzes dehalogenation but also removes hydrogen, nitro, amino, and cyano groups from the benzene ring at the para position in relation to the hydroxyl of phenol. The result of the study indicated conversion of PCP into TeCH, which further converted to 2,6-DiCH. The identification of intermediary metabolites is a significant step in complete mineralization of PCP. Extraction methods are critical steps and selection of time points for sampling is another step, which must be optimized for degradation of PCP. Two methods are adopted for extraction of PCP and its intermediary metabolites for the complete mineralization of PCP. Results obtained for degradation of PCP through GC–MS analysis of the DCM extractable products show formation of tetrachloro-p-hydroquinone and 2,6-dichlorohydroquinone in the extracts as reported earlier by Shah and Thakur (2003). Ring fission in PCP The chloride released by microbial action was determined over the period up to 168 h. The chloride release was due to dechlorination of PCP, which indicates its utilization by bacterial strain. The ring fission of PCP was determined. Yellow colour not appeared after mixing catechol in cell suspension, which indicated the absence of meta cleavage by visual inspection. Moreover, a negative Rothera test was observed over the period 0–144 h, which indicated the absence of ortho cleavage. However, a positive Rothera test was also observed after 144 h (data not shown). The exact enzymatic mechanism is not known and we do not have a proper explanation for ring cleavage enzymatic activity at this stage. The simultaneous release of chloride ion also corroborated the previous findings of dechlorination during PCP degradation as described by Mohn and Kennedy (1992). Alteration of optimized pH might be inhibitory to the activity of the enzymes responsible for PCP degradation in bacteria (Miller et al. 2004). Consequently, DO uptake increases in media and pH decline PCP degradation due to release of H? and Cl- ions in medium, which makes medium acidic and increase in DO level due to continuous degradation.

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Conclusion The microbial consortium was able to co-metabolically degrade PCP, using glucose as the primary carbon and energy source with over all removal efficiency up to 93% under optimized condition (30 ± 1°C, pH 7.0 ± 0.2 and 120 rpm) at 168 h incubation period. In all cultures, the microbial consortium showed better overall removal efficiencies than single strains. To the knowledge of the authors, this is the first report on attempt to identify three intermediates as 6-chlorohydroxyquinol, 2,3,4,6-tetrachlorophenol and tetrachlorohydroquinone using mixed culture of B. cereus and two strains of Ser. mercescens. It is our view that the above information would be useful for modeling and designing the units treating pentachlorophenol contaminate wastewaters. Acknowledgments Authors are thankful to Director, ITRC, Lucknow, for his encouragement. The financial support from Council of Science and Technology, U.P. is also highly acknowledged.

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