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Phenol biodegradation by Rhodococcus coprophilus isolated from semi arid soil samples of pali, Rajasthan.

Introduction

Phenol and its derivatives are hazardous environmental pollutants, which enter in to the environment through waste water discharges from a variety of industries like leather, textiles, dyes, pharmaceuticals etc. These materials have adverse effects on environment and the health of human beings. Due to their toxicity, United States environmental protection agency has included them on the list of priority pollutants [1] Though different Physico-chemical treatments are effective for the degradation of phenolic compounds but they frequently produce hazardous compounds which are toxic for the living organisms. The microbiological dissimilation of phenolic derivatives has been extensively studied and the major catabolic pathways of these compounds to common cellular metabolites have been clarified [2,3,4,]. Bioremediation is generally preferred method for environmental clean up of phenol due to lower costs and the complete mineralisation. Several bacterial strains like Pseudomonas, Ochrabactrum, Serratia, Bacilli, and Acinetobacter etc. were reported for phenol degradation [5,6,7]. Thus isolation and characterization of microorganisms that are capable of degrading high phenol concentrations, is of considerable interest.

In the present case studies, we report the degradation of phenol by Rhodococcus strain belonging Nocardiaceae family, the main objectives of the investigation are to isolate and characterize a bacterial strain capable of degrading phenol, to study the degradation ability of the isolate at initial high phenol concentrations, and to determine the phenol degradative pathway of the organism.

Materials and Methods

Isolation and identification of bacterial cultures

Bacterial culture used in this investigation was isolated from the soil samples collected near to the contaminated sites of dye and textiles industries located in pali district of Rajasthan (India). A 5gm soil sample was suspended in 100m1 M9 medium[8]containing 800mg/1 of phenol as sole source of carbon and incubated in 250m1 flasks at 37[degrees]C on an orbital shaker at 170 rpm for a period of 10 days. After incubation period, the soil particles were allowed to settle and 5m1 of particulate free suspension was used to inoculate a 100m1 M9 media containing 80mg phenol, four such transfers were made and every time the enriched population was plated on M9 medium plates containing phenol as a sole carbon source After fourth transfer, isolated colonies were restreaked. Further the individual colonies were transferred on M9 agar medium containing phenol and checked for a prominent colony type The isolate was purified after several repeated transfers on M9 agar plates This bacterium was identified as Rhodococcus coprophilus, based on the morphological and biochemical properties[9]. Bacterium was further confirmed through FAME (Fatty Acid Methyl Ester) analysis by MTCC, IMTECH, Chandigarh, India.

Phenol degradation studies

Phenol degradation ability of Rhodococcus coprophilus was studied by growing culture in M9 medium with varying initial concentrations (600mg, 800mg, 1000mg, 1200 mg per litre) The flasks were incubated on orbital shaker for a period of 5 days at 37[degrees]C For each experiment freshly prepared inoculums (O.D values between 0.4- 0.7) was used. The samples were analysed for cell biomass concentration for every 6 hours period of interval.

Catechol utilisation studies

Bacterium was also tested for its ability to utilise catechol when supplemented as the substrate in the M9 medium .For this the bacterial strain was grown with 10mM to 50mM catechol concentration in M9 medium at 37[degrees]C for 4 days . Further samples were analysed for cell biomass concentration for every 4 hours spectrophotometrically at 600nm.

Enzyme assay

Enzyme activities of the isolate was determined in the cell free extracts at 37[degrees]C To attain required cell biomass was cultured aerobically to mid -log phase and cells were harvested by centrifugation at 10,000g for 10 minutes Cell pellet was washed twice with saline and resuspendedlOmM Tris Hcl buffer(pH-8). Cell disruption was carried out by sonication (Vibracell, U.S) for 20min with 20 kHz and 10 cycles, all the operations were done at 4[degrees]C. Cell debris was removed by centrifugation 40,000g for 30 minutes and the supernatant was use for enzyme assays. Activity of catechol 1,2 dioxygenase and catechol 2,3 dioxygenase were done by detecting the formation of key intermediates of degradation pathway such as cis,cis muconic acid and 2-hydroxy muconic semialdehyde at 260 and 375 nm at 25[degrees]C. The reaction mixture contained 2.8m1 of 50mM sodium phosphate buffer (PH-7.2), 20[micro]l of 0.1mm catechol and 80[micro]l of cell free extract. The specific activity was measured in micromoles of product formed per min per mg of protein. Protein concentration was determined using Bradford method [10].

Results

Isolation of phenol degrading bacterium

A number of about eight to ten individual bacterial isolates were obtained, which showed phenol tolerance. On further screening by enrichment culture method only one bacterium exhibited, high degradation ability. This bacterium was identified as Rhodococcus coprophilus on the basis of morphological and biochemical properties (Table. 1). It is a gram positive, coccoid rod shaped bacterium capable of utilising phenol as a sole source of carbon and energy.

Growth curve using phenol as sole source of carbon

Rhodococcus coprophilus has effectively utilized phenol as sole source of carbon. The Rhodococcus strain was grown at different concentrations of phenol ranging from 600mg to 1000mg/1. However 800mg/1 of phenol was found to be the effective concentration for the growth of Rhodococcus coprophilus where it has reached to a maximum O.D. of 0.45 at [A.sub.600]. A typical growth curve was presented in (Figure. 1). It is clear from the growth curve that there was initial lag phase of 24 hours and maximum growth occurred at 64 hours (Figure 1).

[FIGURE 1 OMITTED]

Growth on catechol as a substrate

In order to establish the formation of catechol from phenol, preliminary studies were conducted to assess the ability of Rhodococcu coprophilus to grow on catechol as sole source of carbon. As expected catechol also served as source of carbon where Rhodococcus coprophilus was effectively grown in M9 medium containing various concentrations of catechol. Catechol at the concentration of 0. 1 mm was found to be the optimum concentration for the growth of Rhodococcus coprophilus. At this concentration it has attained maximum growth of 0.046 O D. at 16 hours.

The growth of bacterial culture was well coincided with the disappearance of catechol from the culture medium (Figure 2).

[FIGURE 2 OMITTED]

Enzyme activity

In order to further elucidate the phenol degradation pathway and to know the role of catechol in phenol degradation by, Rhodococcus coprophilus key enzymes that are responsible for the ring cleavage reactions have been assayed. The activity levels of catechol 1, 2-dioxygenase and Catechol 2, 3 -dioxygenase serve as evidence to assess the pathway being followed while phenol is being degraded by Rhodococcus coprophilus. Therefore, the enzyme assays were performed with the cell free extracts prepared from the Rhodococcus coprophilus grown in phenol. In the cell free extracts of phenol grown cells, catechol 2, 3 -dioxygenase activity was not noticed and hence hydroxymuconic semialdehyde is not found to be an intermediate in degradation of phenol degradation pathway. Considerable amount of catechol 1, 2 dioxygenase activity was noticed in the cell free extracts of phenol grown cells. Further the increase in the absorbance at 260 nm due to the formation of cis,cis- muconic acid was noticed. The specific activity of this enzyme was found to be 15.266 [mu] moles of cis, cis muconic acid formed per min per mg of protein. The results are presented in Table 2.

Discussion

Phenol degrading aerobic bacteria are able to convert phenol into nontoxic intermediates of the tricarboxylic acid cycle via an ortho or meta pathway [11]. The aromatic ring is initially monohydroxylated by a monooxygenase at a position ortho to the pre-existing hydroxyl group [12]. Aromatic monooxygenases are divided into two groups; activated-ring monooxygenases (monocomponent) and nonactivated-ring enzymes (multicomponent). Multicomponent aromatic monooxygenases contain at least two components[ 11,13] one responsible for hydroxylation (the oxygenase that binds substrate and oxygen), the other responsible for electron transfer from NAD(P)H to the oxygenase (the reductase that binds NAD(P)H). The former is generally an Oligomeric protein, the later a monomeric iron-sulfur flavoproein[11].

Dihydroxyl-substituted aromatic compound, one hydroxyl in ortho (catechol) position relative to the other hydroxyl group, can be cleaved by ring-cleavage dioxygenases. All these enzymes have Fe, which participates in catalysis, in the active site. These enzymes do not have a cofactor requirement, in contrast to the ring-cleavage dioxygenases. Cleavage of catechol can occur in two ways, each with a distinct catalytic mechanism. Cleavage can occur either between two hydroxyl groups (ortho, or intradiol) or proximal to one of the two hydroxyl groups (meta or extradiol) [14].

Nevertheless only a few gram positive genera like Corynebacterium, Streptomyces and Rhodococcus are able to mineralize a wide array of aromatic compounds [15,16,17,18,191. Where as metabolism of phenolic compounds in gram negative bacteria are well elucidated up to the molecular and genetic levels. In our studies we have made an attempt to elucidate metabolic pathway of Rhodococcus sp. a gram positive phenol degrader, which represents a versatile property of aromatic compound degradation. In Rhodococcus coprophilus we have observed formation of catechol and cis cis muconic acid during the process of phenol degradation. These observations were further strengthened by detecting the activities of relevant enzymes in the cell free extracts prepared from the phenol grown culture.

Therefore we propose that in, Rhodococcus coprophilus phenol is degraded by initial hydroxylation to convert it into catechol. Once catechol is formed it appears that it is going to be cleaved by following the ortho cleavage pathway to yield cis,cis muconic acid which will subsequently gets converted in to TCA (Tricarboxylic acid cycle) intermediate to generate energy and other vital bio molecules necessary for the growth and survival of the organism. Therefore, the present investigation results suggest that elucidation of metabolic pathways of phenol degrading strains can be used as a effective tools in enhancing biodegradation properties of phenol degrading bacterium.

References

[1] Neumann, G., Teras, R., Monson, L., Kivisar, M., Schauer, F. and Heipieper, J. H., 2004, "Simultaneous degradation of Atrazine and phenol by Pseudomonas sp." Appl.Environ. Microbiol., 70, pp. 1907-1912.

[2] Hinteregger, C. R., Leinter, M., Loidl, A., and Streichshier, F., 1992," Degradation of phenol and phenolic compounds by Pseudomonas EKII". Appl.Microbiol Biotechnol., 37, pp. 252-295.

[3] Dua, M., Singh, A., Sethunathan, N., and Johri, A.K., 2002, "Biotechnology and bioremediation: Successes and limitations". Appl. Microbiol. Biot., 59,pp. 143-152.

[4] Lovely, D. R., 2003, "Cleaning up with genomics: applying molecular biology of self-bioremediation". Nat. Rev. Microbiol., 1, pp. 35-44.

[5] Balasankar, T., and Nagarajan, S., 2000, "Biodegradation of phenol by Bacillus brevi". Asian. J. Microbiol. Biotech Env. Sci., 2, pp. 155-158

[6] Grit, N., Riho, T., Liis, T., Maia, K., Frieder, S., and Hermann, J. H.,2004, "Simultaneous degradation of atrazine and phenol by Pseudomonas sp. Strain ADP: effects of toxicity and adaptation". Appl. Environ. Microbiol., 70,pp. 1907-1912

[7] Pradhan, N., and Ingle, A.O., 2007, "Mineralization of Phenol by a Serratia plymuthica Strain GC isolated from sludge sample". Int. Biodeterior. Biodegrad., 60, pp. 103-108.

[8] Sambrook, J., Fritsch, E. F., and Maniatis, T., 2001, Molecular cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold spring, Harbor, N.Y

[9] Cappuccino, J.G. and N. Sherman., 2002, In: Microbiol. A laboratory manual Pearson Education, San Francisco, CA., 6th, edn

[10] Bradford, M., 1976, "A rapid sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding". Anal.biochem., 72,pp. 248-254.

[11] Powlowski, J., and Shinger, V., 1990' "In vitro analysis and polypeptide requirements of multicomponent phenol hydroxylase from Pseudomonas sp. strain CF600". J. Bacteriol., 172, pp. 6834-6840.

[12] Katayama, K., Tobita, K., and Harayama, K., 1992, Detection of R-ketoadipic acid in the phenol degradation using whole cells of Rhodotorula rubra. J. Gen. Appl. Microbiol., 36, pp.497-499

[13] Ehrt, S., Schimer, F., and Hillen, W., 1995, "Genetic organization, nucleotide sequence and regulation of expression of expression of genes encoding phenol hydroxylase and catechol 1, 2 dioxygenase in Acinetobacter calcaceticus NCIB.8250". Mol. Microbiol., 18, pp.13-20.

[14] Harayama, S., and Timmis, K.N., 1992, "Aerobic biodegradation of aromatic hydrocarbons by bacteria". In: Sigel H., Sigel A (eds) Metal ions in biological systems. Mareel Dekker, New York, 28,pp. 99-156

[15] Apajalahti, J. H. Kdrpdnoja, P., and Salkinoja-Salonen, M. S., 1986, "Rhodococcus chlorophenolicus sp. nov., a chlorophenol-mineralizing actinomycete". Int.J.Syst Bacteriol 36, pp.246-251.

[16] Eulberg, D., Golovleva, L.A., and Schlomann, M.,1997, "Characterization of catechol catabolic genes from Rhodococcus erythropolis 1CP." J. Bacteriol. 179, pp. 370-381.

[17] Golovleva, L.A., Zaborina, O., Pertsova, R., Baskunov, B., Schurukhin, Y., Kuzmin, S., 1991, "Degradation of polychlorinated phenols by Streptomyces rochei 303". Biodegradation. 2,pp.201-208.

[18] Ishiyama, D., Vujaklija, D., and Davies, J., 2004, "Novel Pathway of Salicylate Degradation by Streptomyces sp. Strain WA46". Appl. Environ Microbio1.70, pp. 1297-1306

[19] Itoh, N., Yoshida, K., and Okada, K.,1996," Isolation and identification of styrene-degrading Corynebacterium strains, and their styrene metabolism". Biosci. Biotechnol. Biochem. 60, pp. 1826-1830

(1) Adimulam Nagamani and (2) Madan Lowry

Lecturer in Biotechnology, Mahatma Institute of Applied Sciences, Jaipur Engineering College and Research centre (JECRC) Foundation, Shri Ram Ki nangal, Via-vatika, Tonk Road, Sitapura, Jaipur-303905, India Email: nagamani.adimulam@gmail.com Assistant Professor, Department of Zoology, University of Rajasthan, Jaipur-302017, India Email: drmlowry3@rediffmail.com
Table 1: Morphological and biochemical characteristics of
Rhocococcus coprophilus.

Characteristics                  Rhodococcus coprophilus

Gram Staining                    Positive
Cell Shape                       Coccoid rod
Motility                         Negative
Oxygen Requirement               Aerobic
Colony                           Dense, light red
Catalase test                    Positive
Nitrate Reduction test           Negative
Hydrogen Sulphide Production     Negative
Indole Production test           Negative
Fermentation Test
  Dextrose                       Positive
  Lactose                        Positive
  Sucrose                        Positive
Gelatin Liquefaction test        Negative
Starch Hydrolysis                Negative
Oxidase test                     Positive
Citrate Utilization test         Positive
Growth Temperature               37[degrees]C

Table 2: Rate of Enzyme formation in Rhodococcus coprophilus.

S.no    Enzyme                   Enzyme activity
                                 (Absorbance)

1       Catechol 1,2             0.033 [+ or -] 0.001
        dioxygenase

2       Catechol 2,3             --
        dioxygenase

S.no    Protein                  Specific activity
        concentration            ([micro]M/mg/min)
        (mg/ml)

1       0.002                    15.266 [+ or -] 0.3

2       --                       --

Mean [+ or -] SD values of triplicate readings were done.
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Title Annotation:Rajasthan, India
Author:Nagamani, Adimulam; Lowry, Madan
Publication:International Journal of Applied Environmental Sciences
Article Type:Report
Geographic Code:9INDI
Date:Sep 1, 2009
Words:2371
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