Bioremediation of pharmaceutical industrial effluent by organisms screened from effluent discharged soil

Transcription

1 World Journal of Pharmaceutical Sciences ISSN (Print): ; ISSN (Online): Published by Atom and Cell Publishers All Rights Reserved Available online at: Original Article Bioremediation of pharmaceutical industrial effluent by organisms screened from effluent discharged soil Prince L, Marudhupandiyar College, Thanjavur. Tamilnadu, India ABSTRACT Received: / Revised: / Accepted: / Published: Two bacterial strains were isolated from pharmaceutical effluents discharged soil. The samples were identified as Klebsiella pneumonia and Pseudomonas aeruginosa the biological degradation was carried on two bacterial isolates separately added to pharmaceutical effluent water. After proper interval were analysed the physic chemical parameters such as ph, Turbidity, Chemical oxygen demand (COD), Biochemical oxygen demand (BOD), Total solids (mg/l), Total dissolved solid (TDS), Chlorides (mg/l), Alkalinity, Hardness (mg/l) and Aromatic organic compound of phenol (Folin Ciocalteu method). The maximum degradation was observed at 48 hours in Pseudomonas aeruginosa (72%) and Klebsiella pneumoniae maximum degradation was observed at 48 hours (70%). On comparative study of the biodegradation ability of Bacillus subtilis and Alcaligenes eutrophus maximum biodegradation ability was observed in Bacillus subtilis. Finally, it was concluded that Pseudomonas aeruginosa can able to highly degrade phenolic content in the pharmaceutical effluent. It can be improved and used industrially for the large scale treatment of pharmaceutical waste effluent water and improvement of its quality for its reuse. Pseudomonas aeruginosa can be recommended for controlling the pollution caused by pharmaceutical effluent. Keywords: Klebsiella pneumoniae, Pseudomonas aeruginosa, Pharmaceutical effluents and phenol. INTRODUCTION In the developing countries, rapid urbanization and industrialization is making the satisfactory collection, treatment and disposal of liquid effluents a formidable problem with serious implications for public health (1). Many Industrial products which has aromatic compounds contaminates the ground water and gets accumulated in plant/animals leading to serious toxicological problems (2). The pharmaceutical waste effluents are typically, toxic coloured, inorganic, organic, and turbid with high suspended solids. Many organic compounds are recalcitrant in nature; it may be carcinogenic, mutagenic and tetratogenic, cyclophosphamade and itosfomide were not degradable in effluent (3).Phenol, one of the most common environmental pollutants is a simple organic compound possessing a hydroxyl group attached to benzene ring or to another more complex aromatic ring system (4). Biological wastewater treatment is the most widely accepted process due to its ease of handling and economic feasibility (5). Basically reduces the pollutents concentration through microbial coagulation and removal on nonsettlable organic colloidal solids (6). In this study the isolation of bacteria capable of bioremediation of pharmaceutical industrial effluent from effluent discharged soil through repetitive enrichment culture method. MATERIALS AND METHODS Collection of sample: The soil sample were collected from madras pharmaceuticals effluents discharged area at Chennai and stored at 4 C until further analysis. Collection of pharmaceutical effluent: The effluent sample were collected from aeropae of effluent treatment plant at Madras pharmaceuticals and stored at 4 C until further analysis. Isolation and purification of bacteria: The nutrient agar medium was used to isolate the bacterial strains from the samples. The cell counting techniques in agar plates was followed to determine the population density for each sample. A series of dilutions were made to reduce the cells in the samples. 0.1ml of diluted sample was spreaded onto the surface of Nutrient agar medium in the Petri dishes and incubated at 37 C and *Corresponding Author Address: Prince L, Marudhupandiyar College, Thanjavur. Tamilnadu, India; prince @gmail.com

2 allowed to grow for 24hrs. Single developed colony was picked on the NA plates and sub cultured to purification. Pure bacterial strains were obtained after successive transfer of individual colony in NA plates and incubated for 24hrs at 37 C temperature. Prince, World J Pharm Sci 2016; 4(3): Standards are also prepared by using various concentration of catechol. A blank containing all the reagents except sample was used to adjust the absorbance to zero. RESULT AND DISCUSSION Identification of bacteria: The two major isolates obtained from the samples were identified by morphological, cultural and biochemical characteristics. The morphological characteristics were identified by gram s staining method and motility test. The cultural characteristics were identified by growing the cultures in nutrient agar medium and Macconkey agar medium. The biochemical characteristics were identified by catalase test. Oxidase test, indole test, methyl red test, vogesproskauer test, Nitrate test, Citrate test, Urease test, trible sugar ion test (TSI) and Carbohydrate fermentation test. Degradation studies: 10ml of two isolates namely Klebsiella pneumonia and Pseudomonas aeruginosa were added to 90 ml of Pharmaceutical effluent water separately and carried out standard flask culture experiments. The flask were checked microscopically for their characteristics and compared with that observed in the initial inoculums. The inoculated effluent samples were incubated for 24 hrs, after incubation analysis the physic chemical parameters. Analytical methods: ph was measured with the help of ph meter (Model No. 101E) of Electronic India, standardized with ph buffer 4, 7 and 9. The turbidity was determined by turbidity meter (HI Microprocessor turbidity meter). Total dissolved solid (TDS) was estimatd by evaporation method at 180 C. Chemical oxygen demand (COD) and Biochemical oxygen demand (BOD) analyses on the clear supernatant (Centrifuged at 6000 g for 30 min) of sample were performed according Standard methods. Sufficient amount of Hg So 4 (Hgso4/cl= 10 was added to the sample to overcome the chloride ion interference in COD measurements. Total solids (mg/l), Chloride (mg/l), Alkalinity and Hardness (mg/l) above all parameters were analyzed by standard procedure mentioned in APHA (1995). Phenol estimation: Aromatic organic compound of phenol was estimated by FolinCiocalteu method. 1ml of sample was pipette out in test tube and 1ml of FolinCiocalteu reagent (20% sodium carbonate) and 2ml of Na 2Co 3 solution was added. The tube was shacked well and kept in boiling water bath for 1min. Cooled under running tap water. The blue solution was diluted to 25ml with water and its absorbance was measured at 650nm. In the present study was aimed to biological pharmaceutical effluent discharged soil. Many different isolates were obtained from the pharmaceutical effluent discharged soil, but only two major colonies were taken and they were identified based on morphological, culture and biochemical characteristics. The results were presented in table1. Two isolates Klebsiella pneumoniae (Isolate I) and Pseudomonas aeruginosa (Isolate II) were obtained. They are gram negative. Morphologically Klebsiella pneumoniae is nonmotile rod shaped bacterium. Pseudomonas aeruginosa is motile, translucent, bluish pigment diffused colonies (7) the previous study was isolated from the effluent water. In order to study the physicochemical characteristics from the pharmaceutical effluents were analyzed. The Klebsiella pneumonia and Pseudomonas aeruginosa was used for effluent treatment. The results presented in the table 2 and table 3. Pollution is a major environmental issue in the world due to its adverse effect on human health. Discharge of urban, industrial and agricultural wastes have increased the quantum of various chemicals that enter the receiving waters which alter considerably their physicochemical characteristics (8). In another study Siddique et al. (10) noted that the release of chloride ions probably led to the formation of HCl which in turn reduced the ph of the medium. Similarly treated pharmaceutical effluents ph was reduced. Phenol concentration was estimated by Folin Ciocalteu method using Pseudomonas aeruginosa and Klebsiella pneumoniae. The maximum degradation was observed at 48 hours in Pseudomonas aeruginosa (72%) and Klebsiella pneumoniae maximum degradation was observed at 48 hours (70%). Kumar et al. (9) has compared the physical chemical and microbial characters of waste effluent and treated effluent outgoing into inland surface. The data indicates that the total solids and total dissolved solids in treated effluents were reduced in the outgoing waste water. CONCLUSION The study carried out was biodegradation of pharmaceutical effluent. The pharmaceutical effluent was collected from the industry PHARM PRODUCTS situated in Thanjavur. The raw effluent contains number of bacterial species. 420

3 Among that two dominant bacterial species were isolated and identified based on cultural, morphological, biochemical characteristics. The identified organisms were Klebsiella pneumoniae and Pseudomonas aeruginosa. The isolated organisms Klebsiella pneumoniae and Pseudomonas aeruginosa were inoculated into the pharmaceutical effluent water and incubated for 1 week. Biodegradation ability was estimated based on physicochemical characters of effluent water. Finally, it was concluded that Pseudomonas aeruginosa can able to highly degrade phenolic content in the pharmaceutical effluent. Prince, World J Pharm Sci 2016; 4(3): Pseudomonas aeruginosa can be improved and used industrially for the large scale treatment of pharmaceutical waste effluent water and improvement of its quality for its reuse. Pseudomonas aeruginosa can be recommended for controlling the pollution caused by pharmaceutical effluent. ACKNOWLEDGMENT The author thankfully acknowledges the laboratory and moral support provided by the director, Specialty Lab and Research at Thanjavur. Table 1: Morphological and biochemical characteristics of bacterial isolates S.No. Morphological and biochemical Isolate I Isolate II test 1 Gram staining Rod Rod Motility Indole test Methyl red test Vogesproskauer test Citrate utilization test Triple sugar iron test Urease hydrolysis test Oxidase test Catalase test Carbohydrate ± AG AG Positive, Negative, AG Acid/Gas, ± Variable Table 2: Degradation studies of dairy effluent using Klebsiella pneumoniae S.No. Physico chemical parameter Raw effluent (hrs) effluent treated with Bacillus subtilis Colour ph Odour BOD (mg/l) COD (mg/l) CaCo 3 (mg/ 1) Total solids(mg/ 1) Total dissolved solids(mg/ 1) Chloride (mg/l) Turbidity(mg/ 1) Yellow 7.8 Unpleasant Yellowish white Colourless Unpleasant odourless

4 ph Prince, World J Pharm Sci 2016; 4(3): Table 3: Degradation studies of dairy effluent using Pseudomonas aeruginosa S.No. Physico chemical parameter Raw effluent (hrs) effluent treated with Pseudomonas aeruginosa Colour ph Odour BOD (mg/l) COD (mg/l) CaCo 3(mg/ 1) Total solids(mg/ 1) Total dissolved solids(mg/ 1) Chloride (mg/l) Turbidity(mg/ 1) Yellow 7.8 Unpleasant Colourless Unpleasant odourless Fig1: ph Klebsiella pneumoniae Pseudomonas aeruginosa Raw Various time of incubation (hrs) 422

5 Fig2: BOD Prince, World J Pharm Sci 2016; 4(3): Fig3: COD 423

6 Prince, World J Pharm Sci 2016; 4(3): Fig4: CaCo 3 Fig5: Total solids 424

7 Fig6: Total dissolved solids Prince, World J Pharm Sci 2016; 4(3): Fig7: Chloride 425

8 Prince, World J Pharm Sci 2016; 4(3): Fig8: Turbidity REFERENCES 1. Ajaybabu P et al. Biodegradation of bulk drug industrial effluents by microbial isolates from soil. J. Scientific and Ind Res 1999; 58: RD2 Prasanna NN et al. Biodegradation of Phenol and Toluene by Bacillus Sp. Pseudomonas sp. and Staphylococcus sp. isolated from pharmaceutical industrial effluent. Advanced Biotech 2008; Kummerer K et al. Biodegratability of antineoplastic compounds in screening tests influence of glucosidation and stereochemistry. Chemoshere 2000; 40(7): Ferhan M et al. Estimation and Removal of Phenol in Pharmaceutical Industrial Effluents from Paracetamol and Aspirin Manufacturing Units.OnLine Journal of Biological Science 2002; 2 (9): Atlas R. Bioremediation, Chem. and Eng. News 1995; 3: Flathman PE et al. bioremediation filed experience, lewispoblishere, Boca Raton, F.L Gibson DT et al. Oxidative degradation of aromatic hydrocarbons by microorganisms. II metabolisms of halogenated aromatic hydrocarbon Biochemistry 1968; 7: Rao, V. N. R. and C. P. Valsaraj, J. Mar. Biol. Ass., India, 1984; 26: Kumar, M., Puri, A., Kumar, A. and Goswami, V.B., Efficiency of biological treatment. Asian. J. Water. Env. Polln., 2007; 4: Siddique, T., Okeke, B.C., Frankenberger, W.T. Jr. Enrichment and Isolation of EndosulfanDegrading Microorganisms. Environ. Qual. 2003; 32,