Anionic Surfactant Degradation by Pseudomonas in Hospital Wastewater. Case Study: Shahid Beheshti Hospital in Abadan City, Iran

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1 American-Eurasian J. Agric. & Environ. Sci., 16 (7): , 2016 ISSN IDOSI Publications, 2016 DOI: /idosi.aejaes Anionic Surfactant Degradation by Pseudomonas in Hospital Wastewater. Case Study: Shahid Beheshti Hospital in Abadan City, Iran 1 2 Ghodous Fathi, Farzaneh Janmohammadi, 3 4 Daem Roshani and Kayvan Farahmandi 1 Health Center, Kurdistan University of Medical Sciences, Sanandaj, Iran 2 Health Center, Kurdistan University of Medical Sciences, Sanandaj, Iran 3 Social Determinants of Health Research Center, Kurdistan University of Medical Sciences, Sanandaj, Iran 4 MD-TUMS (Tehran university of medical sciences), Iran Abstract: The high volume of detergents consumed daily for hospital waste treatment poses a serious threat to both human health and the environment. Through isolating and purifyingthe indigenous bacteria in the waste produced at Shahid Beheshti Hospital, Abadan, this study attempted to assess their impacts on eliminating anionic surfactants. Decomposition rate was assessed through the methylene blue method using a Dr5000 spectrophotometer at a wavelength of 650nm. In this way. Pseudomonas bacteria were isolated. Within 144 hours, this bacterial species was able to eliminate 73% of alkylbenzenesulfonate under the following ambient conditions: ph = 7, temperature = 30 C, nitrogen content = 0.25 mg/l and carbon content = 5mg/l. Research conditions in the treatment plant be maintained for maximum activity of the bacteria. For generalizing the results of the present study to the real world, we also recommend that the elimination efficiency of the studied bacteria be evaluated in an actual treatment plant. Key words: Anionic surfactant Hospital wastewater Alkylbenzenesulfonate Mineral culture medium INTRODUCTION marine life and cause excessive growth of algae in acceptor water resources (rivers). In addition, detergents Hospital waste contains large quantities of disrupt the effective enzymatic activity in respiration pathogens and hazardous compounds, which seriously of bacteria [3]. Today, detergents are widely used at threaten human health as well as the environment [1]. In health centers throughout Iran [4]. Disadvantages of the general, hospital/health centers effluent is, qualitatively, chemical methods currently used for eliminating almost similar to that produced by urban sewage; contaminants include increased toxicity for aquatic however, they might contain potentially toxic and organisms, increased sludge production, reduced infectious compounds, which can endanger not only sludge stabilization, increased filamentous growth, the environment, but also the health of Health Sector decreased sludge dewatering characteristics and staff and the society as a whole [2]. Therefore, it is increased costs [5]. Using microorganisms is a useful essential to supervise and manage hospital waste technology for treating sewage of all kind and it appropriately to prevent environmental pollution [1, 2]. can be considered as a useful tool for filtering Surfactants are substances with very high solubility, different types of contaminants [6]. This method can be which reduce oxygen transfer through reducing an appropriate alternative for chemical filtration of surface tension of water. They are extremely toxic for pollutants [7]. The process of bioremediation is defined Corresponding Author: Ghodous Fathi, Health Center, Kurdistan University of Medical Sciences, Sanandaj, Iran. 1341

2 as the highly cost-effective and noninvasive use of the whole compound by solving 10 mg of each compound microorganisms for detoxification and elimination of in 10 ml sterile distilled water [6]. After cooling the culture contaminants from the environment [8]. Through isolating medium at the room temperature up to 45 C, one ml of the and purifying the indigenous bacteria which actively sample was taken and added to it; and then it was kept in decompose alkylbenzenesulfonate in the effluent a shaking incubator at 30 C and 150 rpm for six days. At produced by Shahid Beheshti Hospital in Abadan, we the end of this period and observance of the turbidity attempted in this study to examine the feasibility of caused by bacterial growth, 1 ml was taken and added to eliminating these compounds by using indigenous the new culture medium [10]. These steps were repeated bacteria as well as to study different factors at three times. Finally, 0.5 ml of the bacteria-containing different levels to maximize the associated decomposition culture medium was diluted five times in the final step rate brought about by these bacteria. This study aimed of enrichment and dipped into a SMS (Salt mineral at isolating and purifying indigenous bacteria in the medium) culture medium containing 1% agar to purify the wastewater of Shahid Beheshti Hospital treatment plant bacteria. It was then put in a normal incubator at 37 C for in Abadan, Iran which are capable of removing anionic 72 hours. surfactants. This research was carried out in May to September 2015and in seven steps including sampling, Bacteria Identification: Biochemical tests were used for enrichment, isolation and purification of bacteria, identifying the bacteria [11]. The bacteria were reproduced identification using culturing, reproduction, determination in the mineral culture medium containing alkylbenzene of bacteria efficiency in removing organic materials and sulfonate and they were kept in the shaking incubator for determination of optimal conditions in bacteria growth. six days at 30 C and 150 rpm. To prepare bacterial suspension, the bacteria reproduced in the centrifuge at MATERIALS AND METHODS 3000 rpm for 10 minutes were respectively removed from the culture medium, transferred to the SMS culture Sampling: Two 50mL samples were taken from the medium and counted using dilution method. aerated chamber and settling basin of the active sludge system of Shahid Beheshti Hospital in Abadan. Determination of Bacterial Growth Rate and Analysis Totally, three samples were taken at 7:30 AM, 10:00 AM of Alkylbenzene Sulfonate by Bacteria: The methylene and 13:00 PM; because Abadan is located in a hot and blue method an assay for nonionic surfactants in humid region, its temperature fluctuation during the environmental samples was used [12].An optical day and the time the research was carried out was spectrophotometer at a wavelength of 650 nm was between 25 C in the early morning to 50 C in the middle used for determining degradation rate of alkylbenzene of the day and the activity of different parts of the sulfonate during six days. Five factors were studied at hospital during a day, which increases or decreases three levels to determine optimal conditions. An optical mineral load imposed on the power plant was taken into spectrophotometer at a wavelength of 600 nm was used account. for determining bacteria growth [13]. The cells used in this stage were made of compressed plastic with 3mL volume. Bacteria Isolation and Enrichment: The samples were In every measurement, 0.6 ml of the bacterial suspension taken to the Laboratory of Oil College in Abadan city and was diluted by 2.4 ml of the sterile SMS culture medium. they were mixed under laboratory conditions. The culture The optical spectrophotometer was calibrated in an SMS medium used here was a mineral culture medium with culture medium free from bacteria. 0.5g K2HPO 4, 1.5g KH2PO 4, 0.5g NaCl, 0.5g NH4Cl, 0.14g Na2SO 4, 0.15g MgCl 2.6H2O compounds. After preparing RESULTS the culture medium, its ph was adjusted to 40 gl 1 sodium hydroxide (1 M) and it was sterilized in an autoclave at the Table 1 shows the results of identifying bacteria temperature of 121 C and pressure of 15 Psi for 15 through biochemical tests. The results showed that minutes. Alkylbenzene sulfonate, which is an anionic this is a gram-negative, rod-shaped, facultative aerobic surfactant, was used in this culture medium as the only bacterium. It was a Pseudomonas bacterium source of carbon [9]. One ppt stokes were prepared from 1342

3 Table 1: The results of identification of bacteria using biochemical tests Triple Simmons Methyl Voges-/red The bacteria Lysine Gelatin Lactose Oxidase Indole SIM Sugar Iron Citrate Proskauer Urea Bacteria name tested Row ALK/ ALK Pseudomonas E 1 1 Results of Alkylbenzene Sulfonate Disintegration Rate Studying growth rate of the bacterium in a mineral and Bacterial Growth: Calibration curve to restore culture medium containing 10 PPM of alkylbenzene alkylbenzene sulfonate concentrations is shown in Fig. 1. sulfonate compound and its modest growth in a culture Pseudomonas bacteria were purified upon enriching medium free from alkylbenzene sulfonate as compared the indigenous bacteria isolated from the effluent of with the culture medium containing bacteria indicate that Shahid Beheshti Hospital. The bacteria removed 73% of the bacterium uses this compound as a carbon source the alkylbenzene sulfonate present in the effluent within (Fig. 4). Calibration curve to restore alkylbenzene 144 hours (Fig. 2). The remarkable ability of Pseudomonas sulfonate concentrations is shown in Fig. 1. bacteria in eliminating alkylbenzene sulfonate has been pointed out in previous studies. Fig. 1: Drawing of a calibration curve to restore alkylbenzene sulfonate concentrations Fig. 2: Changes of alkylbenzene sulfonate concentrations by Pseudomonas bacteria. Fig. 3: The growth curve of Pseudomonas bacteria at Ppm10 concentration of alkylbenzene sulfonate compound. 1343

4 Fig. 4: Comparison of alkylbenzene sulfonate removal rates at different temperatures using Pseudomonas bacteria. Fig. 5: Comparison of alkylbenzene sulfonate removal rates at different phs using Pseudomonas bacteria. Fig. 6: Comparison of alkylbenzene sulfonate removal in different values of nitrogen by Pseudomonas bacteria. Fig. 7: Comparison of alkylbenzene sulfonate removal in different values of alkylbenzene sulfonate by Pseudomonas bacteria. 1344

5 Impacts of Temperature and ph: The studied bacteria showed maximum efficacy in eliminating alkylbenzene sulfonate at 30 C and a ph of seven (Figs 4 & 5). The bacterial activity reduced at decreased temperatures, reaching a minimum at 20 C. This might be due to the climatic conditions in Abadan and the consequent adaptation of the bacteria to such weather conditions. According to the previous studies, the highest efficacies of different Pseudomonas bacterial species in eliminating pollutants occurred at different temperatures and phs. Nitrogen and Carbon Source: Bacterial behavior was assessed for six days in a culture environment containing NH4Cl as a source of nitrogen at three levels (0.25, 0.5 and 1mg/l) as well as alkylbenzene sulfonate as a source of carbon also at three levels ( 5, 10 and 15 mg/l). The obtained results showed that the studied bacteria exhibited their maximum efficiency in eliminating alkylbenzene sulfonate composition at the nitrogen and carbon contents of 0.25mg/l and 5 mg/l, respectively. Bacterial activity decreased considerably upon decreasing the nitrogen and carbon contents. This might be due to the increased toxicity of these compounds for the bacteria (Figs 6 & 7). DISCUSSIONS In this study, Pseudomonas bacteria was isolated and purified for the purpose of isolating and identifying indigenous bacteria in the effluent produced by Shahid Beheshti Hospital, Abadan and examining the potential biological decomposition of the organic matter alkylbenzene sulfonate in the presence of this bacterial species. The bacteria were identified using biochemical culture method. The results showed that they could effectively eliminate 73% of the alkylbenzene sulfonate composition within 144 hours. Chaturvedi & Kumar [14] isolated Pseudomonas alcaligenes and P. mendocina bacteria from municipal wastewater, which were able to remove respectively 99% and 98% of anionic surfactants. Amirmozafari et al. [9] showed that Pseudomonas beteli was capable of eliminating 96.4% of alkylbenzene sulfonate within ten days. According to the obtained results, the optimal conditions for Pseudomonas bacteria to eliminate 73% of the alkylbenzene sulfonate composition with a probability of 95% were: nitrogen content = 0.25 mg/l, ph = 7, temperature = 30 C and carbon content = 5 mg/l. Peressutti et al. [15] showed that maximum efficiency of Pseudomonas bacteria in eliminating anionic surfactants occured 37 C. Ambily & Jisha [16] showed that the bacteria had the maximum efficiency in eliminating anionic surfactants at 30 C. Bared Razika proved that Pseudomonas bacteria had the maximum impact on eliminating phenol and acid benzoic acid; however, according to the study by Behrooz et al. [17] the maximum efficiency in eliminating naphthalene occurred at a ph of 8. Syed Mohd et al. [18]studied anionic surfactants removal by P. aeruginosa bacterium and proved that the highest removal occurs in C/N = 1.8 ratio. However, this bacterium showed its highest efficacy in C/N = 1.14 ratio in the study of Ainon et al. [19]. It is recommended that the research conditions in the treatment plant be maintained for maximum activity of the bacteria. For generalizing the results of the present study to the real world, we also recommend that the elimination efficiency of the studied bacteria be evaluated in an actual treatment plant. ACKNOWLEDGMENTS We are very grateful to vice chancellor for research & technology, Kurdistan University of Medical Sciences and the respected health center staff. REFERENCES 1. Amouei, A., A. Hosseinali, F. Hourieh, F. Hossein, B. Reyhaneh and N. Dariush, Investigation of hospital wastewater treatment plant efficiency in north of Iran during International Journal of Physical Sciences, 7(31): Abd El-Gawad H.S., Aquatic environmental monitoring and removal efficiency of detergents. Water Science, 28(1): Ebrahimi, A., M. Hassan Ehrampoosh, M. Reza samaei, E. Shahsavani, H. Hosseini, H. Hashemi, P. Talebi, S.V. Ghelmani, M. Dehghan and M. Honardoost, Survey on removal efficiency of linear alkylbenzenesulfonate in Yazd stabilization pond. Int. J Env. Health. Eng., 4(10):..???. 4. Ghodous, F., J.Y. Reza, K. Esmaeil and J. Farzaneh, Bioremediation of anionic surfactants in hospital wastewater. Case study: Shahid Beheshti Hospital in Abadan City, Iran. AES Bioflux., 8(1): Akpor, O.B., Muchie M.Bioremediation of polluted wastewater influent: Phosphorus and nitrogen. Scientific Research and Essays. 5(21):

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