Biodegradation of phenol by oxygenase producing thermophilic microorganisms Neetika Mathur1*, Vinod Kumar Nigam2, Ashwani Kumar1 and Purnendu ghosh2 1 Department of Botany, University of Rajasthan, Rajasthan- 302 004 2 Department of Biotechnology, Birla Institute of Scientific Research, Jaipur, Rajasthan-302 001 Abstract: Screening and characterization of phenol degrading bacterial isolates from the soils and sewage samples of desert regions of Rajasthan was performed under batch cultivation. The degradation studies were monitored by the consumption of NADH and it was found that maximum degradation was achieved at 500C. Out of nine phenol adapted oxygenase producing cultures, four cultures BISR 00T1, BISR 00T3, BISR 00T5 and BISR 00T8 showed maximum enzymatic activity. Various biochemical tests were also performed for their characterization. Key words: biodegradation, oxygenases, thermophilic, phenol, pollutants. Introduction: Rapid industrial and economic development, human exploitation of fossil fuels and production of many synthetic compounds have introduced many compounds that are not naturally present in the environment. Many of these xenobiotics substances are toxic to the living system and their presence in the aquatic and terrestrial habitats often have serious ecological consequences. Bioremediation is emerging as most ideal technology for removing pollutants from the environment by the action of microbes or other _______________________________________ * Corresponding author: Neetika Mathur, Department of Botany, University of Rajasthan, Jaipur 302 004; Phone No: 0141-2546381; Email: biological systems, restoring contaminated sites and preventing further pollution (Caplan, 1993; Dua et al., 2002). Phenols and chlorophenols are introduced in the environment in the waste stream of several industrial operations, through its use as biocides or as by-product of other industrial operations, such as pulp bleaching with chlorine, water disinfection or even waste incineration and as degradation product of other chlorinated xenobiotics (Bollag et al., 1986). Other sources of phenol can be oil refineries, chemical plants, explosives manufacturing, resin manufacturing, rubber reclamation, textile mills and plants etc. Phenols have a relatively high oxygen demand (2.4 mg of O2/mg of phenol) for degradation. These are toxic to fish at a level of above 2 mg/L and as little as 0.005mg/L of phenol can impart objectionable tastes and odour to the drinking water (S. N. Jogdand). Because of their toxic effects, phenols and chlorophenols tend to accumulate and in some cases, the contamination of soil and water is of concern (Keither and Tellard 1979, Moos et al., 1983; Borthwick and Schimmel 1978). Several decontamination techniques are available for the removal of contaminants from water such as adsorption, ion exchange, incineration etc., but all are unable to destroy the contaminants. Biodegradation is a technique which could potentially degrade these contaminants to innocuous products (Murialdo et al., 2003). Microbial degradation of phenol and chlorophenols has been reported by several groups (Baker et al., 1980; Pignattelo et al., 1983; Saber and Crawford, 1985; Rozich and Colvin, 1986; Apajalahti and Salkinoja-Salonen, 1986; Radehaus and Schmidt, 1992; Ramos et al., 1995; Lee et al., 1998; Reddy and Gold, 2000; Cortes et al., 2002; Kirchner et al., 2003). Biological methods can reduce phenol down to 0.5-1 mg/L level. Phenol hydroxylase (also known as monooxygenase) catalyses the efficient ortho-hydroxylation of phenol and ultimately its degradation (Kirchner et al., 2003). Phenols are first converted into more reactive dihydroxylated intermediates and then subjected to ring cleavage by molecular oxygen in presence of NAD (P) H dependent flavoprotein monooxygenases (Moonen et al., 2003). The ability of oxygenase to incorporate oxygen into organic compounds is important because many of the hydrophobic pollutants such as polycyclic aromatic hydrocarbons are high in carbon and hydrogen content but low in oxygen content. It is reported that enzyme catechol 2, 3-dioxygenase catalyzes the phenol degradation by meta-cleavage pathway in aerobic, sporulating, motile, rod shaped thermophilic bacteria characterized as Bacillus species with growth temperature optima of 500-600C (Ali Saiqa et al., 1998). These thermophilic bacteria can be used in treatment of phenolic waste water. The biotechnological significance and the benefits of the use of thermophilic bacteria in enhancing hydrocarbon removal under different environmental conditions were investigated. Mesophilic sources of phenol have been investigated but due to thermal instability of the enzymes the attention is being made to search for thermostable enzymes (Moiseeva et al., 2002). Many of the organic compounds are first degraded to catechol or protocatechuate by oxygenases (both dioxygenases and monooxgenases). The intermediates are metabolized by a ring cleavage type of dioxgenases to either beta-ketoadipate or 2-keto-4-hydroxyvalerate and finally metabolized by TCA cycle. In the present study, the organisms isolated from the hot desert region of Rajasthan are selected for evaluating their phenol degrading capabilities. The enzymes (whole cells) have also been characterized at various physico chemical parameters. MATERIALS AND METHODS: Isolation and growth conditions: Mineral Base Medium was used for isolation of phenol degrading oxygenase producing thermophilic microorganisms. The Mineral Base Medium includes; yeast extract (2g/L) and 5 X mineral base {NaCl 5g/L, KH2PO4 1.35g/L, K2HPO4 0.87g/L, MgSO4.7H2O 1g/L, CaCl2 0.05g/L, FeCl3 1.25g/L}, 1 ml/ L of trace element solution SL6 (CoCl2 0.2g/L, H3BO3 0.3g/L, ZnSO4.7H2O 0.1g/L, MnCl2.4H2O 0.03g/L, Na2MoO4.H2O 0.03g/L, NiCl2.6H2O 0.02g/L and CuCl2.2H2O 0.01g/L), and filter sterilized thiamine HCl 3µg/l. The phenol (1g/L) was added in the medium as a carbon source. The pH of the medium was adjusted to 7.0 before sterilization. The phenol was added into the medium after sterilization. A number of soil and sewage samples were collected from different regions of Rajasthan. 1g of soil/ sewage sample was added to 100 ml of Mineral Base Medium (MBM) and kept on orbital shaker at 500C and 200 rpm for enrichment. The samples were withdrawn aseptically at different time intervals (24 h, 48 h, 72 h, etc.) and spreaded on agar plates and incubated at 500C for 24 h. Pure colonies were obtained after several streaking. The isolates were grown in the liquid medium (100 mL) for 24 h and centrifuged at 10,000 rpm for 10 min at 40C. The cell pellet after washing was dissolved in 2 mL of buffer and stored at 40C till further use. Finally, the pure colonies were observed for oxygenase activity. Cell growth was measured turbidimetrically at 600nm. Residual phenol estimation: The consumption of phenol in culture broth was measured with respect to the standard plot of phenol by Folin’s ciocalteau method (Sadasivam and Manickam, 1992). Oxygenase assay: The oxygenase assay was performed by measuring the consumption of NADH at 340 nm using whole cells of different isolates (Kirchner et al. 2003). The 3 mL reaction mixture consisted of 1.9 mL of 0.1M phosphate buffer (pH 7.2), 0.5 mL substrate phenol (final concentration 116.7mM), 0.5 mL NADH (final concentration 166M), 0.1mL of whole cells and incubated at 500C for 2 h. The reaction mixture was centrifuged at 10,000 rpm for 5 min and absorbance of supernatant was monitored at 340 nm against control. Enzyme activity is expressed in terms of I.U. which is defined as the amount of NADH (M) consumed per mL of cells per minute. Biochemical tests for the cultures: A number of biochemical tests (Carbohydrates hydrolysis, nitrate reduction, catalase, methyl red test, gelatin and starch hydrolysis etc) were performed for the initial characterization of cultures. RESULTS AND DISCUSSION: Nine phenol adapted cultures were isolated from soil and sewage samples, which were grown in liquid medium for different time intervals and residual phenol concentration along with enzyme activity was measured. The results are given in Figure1. Out of nine cultures, seven showed maximum utilization of phenol for their growth at 40 h of incubation at 500C. In case of cultures BISR 00T1 and BISR 00T2, the utilization of phenol was minimum. By comparing the enzyme activities of nine phenol adapted oxygenase producing isolates (Table 1) four cultures BISR 00T1, BISR 00T3, BISR 00T5 and BISR 00T8 were observed with maximum hydroxylation activity (oxygenase) and selected for further studies. Growth profile: In order to achieve the maximum synthesis of oxygenase enzyme, the growth pattern of the cultures was investigated under optimal conditions (pH 7.0, temperature 500C and rpm 200). The best isolates (BISR 00T1, BISR 00T3, BISR 00T5 and BISR 00T8) were inoculated in to mineral base medium and 2 mL of samples were drawn aseptically at different time interval for analyzing cell biomass. The supernatants were used for estimation of residual phenol (Figure 1). The results are presented in Table 2. It was observed that as the time of incubation increased, the cell biomass also increased. But this increase in cell biomass is limited upto 24 h of incubation, after that it decreased slightly and becomes almost constant. In another experiments the cultures were inoculated in the liquid broth as mentioned above and the whole cells obtained at different time (24h, 48 and 72 h respectively) were evaluated for the oxygenase activity. The results are given in Table 3. It was observed from the table that isolates grown for 48 h of incubation showed maximum enzyme activity. Later aged cells have been found with decrease in oxygenase activity. This might be because of the maturation of cells that did not favour oxygenase production. After optimizing the growth characteristics of all four isolates in the liquid broth, the enzyme oxygenase was characterized in terms of various physiological conditions such as pH, temperature, substrate concentration and substrate specificity etc. Effect of pH on the activity of oxygenase: The effect of pH on the oxygenase activity was investigated by varying the pH of buffer in the range of 6.8-8.0 keeping all other parameters constant (temperature 500C, phenol concentration 117 mM and incubation 1 h). The results are presented in Figure 2. The figure reveals that with increase in pH, the enzyme activity increases upto 7.6 and then decreased for culture BISR 00T1, BISR 00T5 and BISR 00T8 while in case of BISR 00T3 the oxygenase activity continuously decreased. Effect of temperature on activity of oxygenase: The effect of temperature on activity of oxygenase was performed by varying the temperatures of incubation in range of 300C to 600C at optimal conditions and the results are shown in Figure 3. It is clear from the figure that with the increase in temperature, the rate of phenol degradation was maximum at 500C and then decreased for the isolates BISR 00T1, BISR 00T5 and BISR 00T8 respectively. However, for isolate BISR 00T3, the oxygenase activity was maximum at 600C. Effect of substrate concentration on oxygenase activity: The effect of substrate (phenol) concentration on activity of oxygenase was investigated by varying the concentration of phenol in the range of 40 mM to 240 mM keeping all other parameters constant. The result of this experiment is shown in Figure 4. The observation of figure showed that the oxygenase activity for BISR 00T1 started increasing from 40 mM to 80 mM of phenol concentration and decreased at higher concentration. For the isolate BISR 00T3, the oxygenase activity was highest at 40 mM of phenol concentration. In case of BISR 00T5, the enzyme activity increased from 40 mM, then became constant at 120 mM of phenol concentration and then decreased. The utilization of phenol increased from 40 mM to 120 mM by isolate BISR 00T8, and then continuously decreased. This shows that the biodegradation of phenol varies with the different isolates. The isolate BISR 00T3 showed maximum utilization of phenol. Effect of substrate specificity on oxygenase activity: Different substrates (naphthalene and toluene) were used for evaluating the substrate specificity of oxygenase produced from the different thermophilic bacterial isolates. Different concentrations of naphthalene (2.13 mM to 5.3 mM) and toluene (2.25 mM to 9.0 mM) were incubated with the whole cells at optimal conditions and the oxygenase activity was monitored. The results are given in Table 4 and 5 respectively. The observation of Table 4 shows that oxygenase activity expressed by the isolates BISR 00T1 and BISR 00T5 increased from 2.13 mM to 5.3 mM of naphthalene concentration respectively. Same observation were also be noticed for isolates BISR 00T3 and BISR 00T8 with a little variation. The observation of Table 5 in case of toluene as substrate showed that the oxygenase activity for the isolates BISR 00T1 increased from 2.25 mM to 9.0 mM. For isolates BISR 00T3 and BISR 00T8 the oxygenase activity increased from 2.25 mM to 4.5 mM and then decreased. The oxygenase activity was highest at 2.25 mM of toluene concentration in case of the isolate BISR 00T5 and then decreased. Biochemical Test: All the cultures have been characterized by different biochemical tests and the results are shown in Table 6. The table revealed that all isolates are able to degrade carbohydrates (glucose and sucrose) except lactose. All the isolates showed positive results for catalase and nitrate reduction test but negative for starch hydrolysis. Conclusions: Out of nine phenol adapted oxygenase producing isolates from soil and sewage samples, four isolates showed maximum oxygenase activity at 500C. The isolate BISR 00T3 showed maximum utilization of phenol during the enrichment. The growth profile showed that the enzyme production was maximum at 48 h of incubation. For isolates BISR 00T1, BISR 00T5 and BISR 00T8, the optimum pH was 7.6 and optimum temperature was 500C for oxygenase activity while for the isolate BISR 00T3, the optimum pH was 6.8 and optimum temperature was 600C respectively. REFERENCES 1. Alis S, Lafuente R.F. and Cowan D.A. 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Isolates Abs at 340 NADH Residual NADH Consumption Enzyme Activity % Amt (µM) % Amt (µM) BISR 00T1 0.1755 31.22 51.82 68.45 113.62 0.3156 BISR 00T2 0.8107 - - - - - BISR 00T3 0.0585 10.40 17.27 89.6 148.73 0.4131 BISR 00T4 0.0748 13.3 22.09 86.7 143.92 0.3997 BISR 00T5 0.0552 9.82 16.30 90.18 149.69 0.4158 BISR 00T6 0.2410 42.87 71.17 57.13 94.83 0.2634 BISR 00T7 0.0769 13.68 22.71 86.32 143.29 0.3980 BISR 00T8 0.0763 13.57 22.53 86.43 143.47 0.3985 BISR 00T9 0.0784 13.94 23.15 86.06 142.85 0.3968 Control 0.5621 100 166 -- -- -- Table 2: Growth profile of four isolates with respect to time. Time Period (h) OD at 600nm and Dry cell biomass (g/L) BISR 00T1 BISR 00T3 BISR 00T5 BISR 00T8 18 0.589 (0.896) 0.612 ( 0.93) 0.601 (0.63) 0.562 (1.05) 24 0.680 (1.05) 0.732 (1.10) 0.630 (0.70) 0.784 (1.25) 40 0.446 (1.00) 0.540 (0.95) 0.449 (0.80) 0.688 (1.05) 72 0.402 (0.80) 0.706 (0.90) 0.448 (0.65) 0.586 (0.95) Table 3: Oxygenase activity of isolates at different ages of their growth. Time of growth ( h ) Oxygenase activity (IU) BISR 00T1 BISR 00T3 BISR 00T5 BISR 00T8 24 0.8270 0.8342 0.7956 0.4377 48 0.8440 0.8426 0.8155 0.4314 72 0.3640 0.5493 0.3135 0.7297 96 0.2468 0.5813 0.0903 0.1359 Table 4: Effect of naphthalene concentration on oxygenase activity. Naphthalene Concentration (mM) Oxygenase Activity (IU) BISR 00T1 BISR 00T3 BISR 00T5 BISR 00T8 2.13 0.8965 0.8857 0.8112 0.8702 3.2 0.9120 0.8890 0.7382 0.8746 5.3 0.9148 0.8346 0.8651 0.8667 Table 5: Effect of toluene concentration on oxygenase activity. Toluene Concentration (mM) Oxygenase Activity (IU) BISR 00T1 BISR 00T3 BISR 00T5 BISR 00T8 2.25 0.8997 0.00 0.8445 0.7935 4.5 0.9131 0.9089 0.8380 0.8011 9.0 0.9159 0.8754 0.8077 0.6033 Table 6: Results of Biochemical Tests Phenol Adapted Oxygenase Producing Cultures Carbohydrates Test Nitrate Catalase Methyl Red Gelatin Starch Dextrose Sucrose Lactose BISR 00T1 + + - + + + + - BISR 00T2 + + - + + - - - BISR 00T3 + + - + + - - - BISR 00T4 + + - + + + + - BISR 00T5 + - - + + - - - BISR 00T6 + + - + + + + - BISR 00T7 + + - + + + + - BISR 00T8 + + - + + + + - BISR 00T9 + + - + + + + -