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Introduction

化学物质是现代生活的一部分。他们是爸爸rt of all spheres of human life and used as pharmaceuticals, pesticides, detergents, fertilizers, dyes, preservatives, food additives etc. Pharmaceutical wastes produced on a daily basis may be land filled provided that they are dispersed in large quantities of general waste. Cytotoxic and narcotic drugs, however, should never be land filled, even in small quantities. Mild liquid or semi-liquid pharmaceuticals, such as solutions containing vitamins, cough syrups, intravenous solutions, eye drops, etc. (but not antibiotics or cytotoxic drugs), may be diluted in a large bow of water and discharged into municipal sewers. Petroleum hydrocarbons can be divided into four classes namely saturates, aromatics, the asphaltenes (phenol, fatty acids, ketones, esters and porphyrines), and the resins (pyridines, quinolines, carbazoles, sulfoxides and amides).

Phenol, C₆H₅OH or hydroxybenzene, is an aromatic molecule containing hydroxyl group attached to the benzene ring structure. Phenol commonly known as carbolic acid has a molecular weight of 94.11 gm/M¹. It has a melting point of 43°C and forms white to colorless crystals, colourless to pink solid or thick liquid. Phenol is a constituent of coal tar, and is formed during decomposition of organic materials. Ingestion of even small amounts may cause vomiting, circulatory problems, paralysis, convulsions, and coma. Phenol affects the nervous system and key organs, i.e. spleen, pancreas and kidneys.

Naphthalene PAHs including naphthalene (2 rings), fluorene (3 rings), benzo (a) anthracene, chrysene, fluoranthene, pyrene (4 rings), and benzo (a) pyrene (5 rings). Exposed to naphthalene vapours causes Nausea, vomiting, abdominal pain, and anemia Increased serum bilirubin, met hemoglobin, Heinz bodies, and fragmented red blood cells hematuria, anemia, restlessness, and liver enlargement².

Microbial degradation In aerobic respiration, oxygen acts as the electron acceptor. Molecular oxygen is a reactant for oxygenase enzymes and is incorporated into the final products. In anaerobic respiration, different inorganic electron acceptors are released such as NO₃, SO₄, SO, CO₂and Fe₃during microbial degradation. Following microorganism is responsible for the degradation of phenol and naphthalene.Acinetobactersp.,Arthrobactersp.,³Bacillussp.⁴Micrococcussp.,Nocardiasp.Pseudomonassp.⁵P. pictorum- NICM- 2077,Rhodococcussp, DCB-p0610Fusarium- F. FE11PenicillimmAF2, AF4,⁶Coprinussp.Candidatropicalis. Considering the versatile nature of microorganisms as a good source of degrading the wastes, especially the pharmaceutical residues an attempt has been made to understand the utility of microorganisms for the degradation of pharmaceutical waste.

Material and Methods

Collection of Sample

The soil sample was collected from SaiMeera pharma industry at Chennai. Sample wascollected in polythene bag by using pre-sterilizedspatula.

Screening for Phenol and Naphthalene Degrading Organism

One gm of soil sample was serially dilutedup to 106 dilution, and spreaded on minimal agarmedium containing 10 mg/ 100 ml phenol andnaphthalene which was added after sterilization, andthe plates were kept for incubation at 28°C for 5days⁷.

Enrichment of the Isolated Strains

The 18 hrs selected cultures were prepared in nutrient broth. One ml of this turbid broth was taken and added into 49 ml of liquid mineral medium supplemented with 10 mg of phenol and naphthalene in a sterile 250 ml conical flask and kept in a rotary shaker at 120 rpm for 5 days⁸.

Selection of Potential Phenol and Naphthalene Degrading Strains (Plate Method)

Minimal agar medium was prepared withincreasing concentration of selected phenol andnaphthalene as follows 10 mg/100 ml, 20 mg/100ml, 30 mg/100 ml, 40 mg/100 ml, and 50 mg/100ml of enriched cultures were inoculated on minimalagar media. Plates were incubated at roomtemperature for 48-72 hrs.⁸

Degradation by Free Cells

The selected potential isolates wereinoculated in 10 ml nutrient broth, after 18 hrs ofincubation 5 ml of this nutrient broth was added to95 ml of minimal media containing differentconcentrations of phenol and naphthaleneindividually30mg/100ml, 40mg/100 ml, 50mg /100ml. 3 ml of sample were removed every onehour and microbial growth was measured usingcolorimeter at 600 nm. The aliquot samples werecentrifuged at 1000 rpm for 5 min and supernatantwas obtained for the study of degradation ability ofphenol and naphthalene using 4-amino antipyrenemethod⁴.

4-Amino Antipyrene Method

The phenol and naphthalene compoundsreacts with 4-amino antipyrene at pH 7.9 ± 0.1 in the presence of potassium ferriccyanide (K₃Fe (CN) 6) and forms a pink colored antipyrene dye. This dye kept in aqueous solution and the absorbance was monitored at 500 nm.⁴

Optimization of Phenol and Naphthalene Degradation by Potential Strain Effect of pH and Temperature

The minimal agar medium was preparedwith different pH (5, 7 and 9) and differenttemperature (28ºC, 37ºC, 45ºC) for 48-72 hrs alsosupplemented with 50mg phenol and naphthaleneseparately.

Preparation of Immobilized Cells

酚和萘(0.01%)作为唯一source of carbon and kept in rotary shaker at120rpm for 72 hrs at 30ºC. 3gm of sodium alginatewas dissolved in 100 ml of distilled water and mixed thoroughly and autoclaved. Then it was stirred with 3 ml of potential broth culture using magnetic stirrer. Then the final mixture was extruded through a needle into 200 ml of 0.05M CaCl₂solution to yield 3 to 4 mm diameter alginate beads. After 20 min, the beads were filtered out and stored in 0.05 % CaCl₂solution in the refrigerator.⁹

降解苯酚的d Naphthalene by Immobilized Cells

15 gm of immobilized beads were placedinto 100 ml of mineral medium containing thedesired amount of phenol (30 mg/100 ml, 40 mg/100 ml, and 50 mg/100 ml). Aliquot samples of thebroth culture were taken at regular interval for thestudy of degradation by 4-amino antipyrenemethods.

Identification of Potential Strains

Phenotypic characteristics such as micromorphology (gram staining, capsule staining endospore staining and motility), cultural characteristics (on basal media, differential media and selective media), and biochemical characteristics (catalase, oxidase, IMViC). Based on the results of studied phenotypic characteristics all the selected bacterial isolates are identified with the help of Bergey's Manual of Determinative Bacteriology¹⁰.

Results and Discussion

Screening for Phenol and Naphthalene Degrading Organism

During the third day of incubation, twocolonies from phenol containing plate and threecolonies from naphthalene containing plate wereobserved and the plates were further incubated.After 5 days, another two colonies from phenolcontaining plate and three colonies fromnaphthalene containing plate morphologicallydifferent colonies were observed. Totally 10 isolateswere selected as phenol and naphthalene degradingbacterial colonies and named as strain P1 to P10.The stock cultures for the selected strains weremaintained. Rigo and Alegre, used phenol as thesole carbon source in the defined mineral saltmedium for the isolation process7, in the present study were used as phenol and naphthalene as thesole carbon source it is the screening method forthe degradation.

Selection of Potential Phenol and Naphthalene Degrading Strains

The enrichment medium which is used forthe enrichment of an isolated strains from mixedcultures which show good variations of species inmixed cultures⁸. In this study, Minimal medium was使用堡he enrichment purpose. Better growth wasobserved after five days. After incubation, all thestrains namely P1 to P10 shows growth in 10 mg of phenol and naphthalene, but P7 and P10 show maximum degradation against all the concentrations of phenol and naphthalene. From these, P7 and P10 were selected as potential strains and used further studies (Table 1). On serial dilution two colonies from phenol plates and three colonies from naphthalene plates were observed within two days, in the next three days two colonies from phenol and three colonies from naphthalene were isolated. Totally ten colonies were observed on the mineral salt medium.

Table 1: Selection of potential phenol and naphthalene degrading strains Click here to view table

Degradation by Free Cells (4-Amino Antipyrene Method)

The phenol and naphthalene in differentconcentration were added to mineral media fordegradation by the potential isolates was studied by the colour reaction of chemical (phenol andnaphthalene) with potassium ferric cyanide at pH7.9. The optical density (O.D) value was measuredfor the change in colour by using colorimeter. Forthe degradation studies, various concentrations ofphenol and toluene in (25 mg / L, 50 mg / L, 75 mg/ L and 100 mg / L) were used. It is beneficial forthe study of biodegradation to quantity their pollutanteffect studied4. For this present study, increasingconcentration of phenol and naphthalene such as30 mg/ 100ml, 40 mg/ 100ml and 50 mg/ 100ml ofphenol and naphthalene were used for thedegradation process. Out of the two strains P7 andP10 were selected as potential strains. (Fig. 1 to 4).

Optimization of Phenol and Naphthalene Degradation by Potential Strain P7 and P10 Effect of pH

After incubation, the plates containing bothphenol and naphthalene in different pH conditionwere observed and the better growth was observedby P7 and P10 at neutral pH in the phenolplate and neutral pH in naphthalene plate. (Table 2& 3).

Table 2: Effect of pH Click here to view table

Table 3: Effect of pH Click here to view table

Effect of Temperature

After incubation, the plates containing bothphenol and naphthalene in different temperatureconditions were observed. The strains P7 andP10 indicate better growth in 28°C and 37°C inphenol plate and 28°C in naphthalene plate. (Table4 & 5).

Table 4: Effect of temperature Click here to view table

Table 5: Effect of temperature Click here to view table

For the optimization process, different pH, temperature and enzymatic activity of Serratia plymuthica isolate from sludge sample reported¹¹which shows the optimization of the degradation at certain pH and temperature optimized the cells by applying different pH and temperature for the microbial degradation of phenol¹². In the same way in the work degradation of phenol by the influence of optimum pH was already reported¹³. In this study P7 and P10 strain show better growth in neutral pH. P7 and P10 strain shown better growth in 28°C and 37°C for phenol degradation. P7 and P10 strain shown better growth in 28°C for naphthalene degradation.

Graph 1: Phenol degradation by free cells of P7 Click here to view graph

Graph 2: Naphthalene degradation by free cells of P7 Click here to view graph

Graph 3: Phenol degradation by free cells of P10 Click here to view graph

Graph 4: Naphthalene degradation by free cells of P10 Click here to view graph

Graph 5: Phenol degradation by immobilized cells of P7 Click here to view graph

Graph 6: Naphthalene degradation by immobilized cells of P7 Click here to view graph

Degradation studies by immobilized cells (4-amino antipyrene method)

The degradation of phenol andnaphthalene in different concentration were addedto mineral media by the potential isolates wasstudied by the colour reaction of chemical (phenoland naphthalene) with potassium ferric cyanide atpH 7.9. The optical density value was measuredfor the change in colour by using colorimeter.Recently, for the better advantage in degradationof various chemicals the free microbial cells wereimmobilized. So that they can be reused for the cycleand compare to free cells these shows on betterresults. (Figs. 5 to 8) Arthrobacter sp is immobilizedfor the phenol degradation studied¹⁴. Free andalginate entrappedPseudomonassp is used for thenaphthalene biodegradation¹⁵.

Identification of Potential Strain

Based on the phenotypic studied, thepotential strains were identified asBacillussp (P7).Brachybacterium sp (P10). In this study,Bacillus(P7) and Brachybacterium (P10) are the potentialstrain for degradation process. In which,Bacillusisalready reported in the phenol and toluenedegradation⁴and Brachybacterium is a new strainto degrade phenol and naphthalene.

Graph 4: Naphthalene degradation by free cells of P10 Click here to view graph

Graph 5: Phenol degradation by immobilized cells of P7 Click here to view graph

Acknowledgments

I wish to sincerely record my deepestgratitude to Dr. K.R. Venkatesan M.A., M.Phil.,Ph.D., Principal, Sri Sankara Arts and ScienceCollege, Kanchipuram for their valuable andenthusiastic encouragement at very state of thiswork. I express my sincere thank to Dr. R.Balagurunathan for his support during this researchendeavor.

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