You are on page 1of 6

American-Eurasian J. Agric. & Environ. Sci.

, 16 (7): 1341-1346, 2016

ISSN 1818-6769
© IDOSI Publications, 2016
DOI: 10.5829/idosi.aejaes.2016.16.7.12956

Anionic Surfactant Degradation by Pseudomonas in Hospital Wastewater.

Case Study: Shahid Beheshti Hospital in Abadan City, Iran
Ghodous Fathi, 2Farzaneh Janmohammadi,
Daem Roshani and 4Kayvan Farahmandi

Health Center, Kurdistan University of Medical Sciences, Sanandaj, Iran
Health Center, Kurdistan University of Medical Sciences, Sanandaj, Iran
Social Determinants of Health Research Center,
Kurdistan University of Medical Sciences, Sanandaj, Iran
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


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.

Am-Euras. J. Agric. & Environ. Sci., 16 (7): 1341-1346, 2016

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 K2HPO4, 1.5g KH2PO4, 0.5g NaCl, 0.5g NH4Cl, 0.14g
Na2SO4, 0.15g MgCl2.6H2O compounds. After preparing RESULTS
the culture medium, its pH was adjusted to 40 gL 1sodium
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

Am-Euras. J. Agric. & Environ. Sci., 16 (7): 1341-1346, 2016

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 E1 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.

Am-Euras. J. Agric. & Environ. Sci., 16 (7): 1341-1346, 2016

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

Am-Euras. J. Agric. & Environ. Sci., 16 (7): 1341-1346, 2016

Impacts of Temperature and pH: The studied bacteria Ambily & Jisha [16] showed that the bacteria had the
showed maximum efficacy in eliminating alkylbenzene maximum efficiency in eliminating anionic surfactants at
sulfonate at 30°C and a pH of seven (Figs 4 & 5). The 30°C. Bared Razika proved that Pseudomonas bacteria had
bacterial activity reduced at decreased temperatures, the maximum impact on eliminating phenol and acid
reaching a minimum at 20°C. This might be due to the benzoic acid; however, according to the study by
climatic conditions in Abadan and the consequent Behrooz et al. [17] the maximum efficiency in eliminating
adaptation of the bacteria to such weather conditions. naphthalene occurred at a pH of 8. Syed Mohd et al.
According to the previous studies, the highest efficacies [18]studied anionic surfactants removal by P. aeruginosa
of different Pseudomonas bacterial species in eliminating bacterium and proved that the highest removal occurs in
pollutants occurred at different temperatures and pHs. C/N = 1.8 ratio. However, this bacterium showed its
highest efficacy in C/N = 1.14 ratio in the study of Ainon
Nitrogen and Carbon Source: Bacterial behavior was et al. [19]. It is recommended that the research conditions
assessed for six days in a culture environment containing in the treatment plant be maintained for maximum activity
NH4Cl as a source of nitrogen at three levels (0.25, 0.5 and of the bacteria. For generalizing the results of the present
1mg/l) as well as alkylbenzene sulfonate as a source of study to the real world, we also recommend that the
carbon also at three levels ( 5, 10 and 15 mg/l). The elimination efficiency of the studied bacteria be evaluated
obtained results showed that the studied bacteria in an actual treatment plant.
exhibited their maximum efficiency in eliminating
alkylbenzene sulfonate composition at the nitrogen and ACKNOWLEDGMENTS
carbon contents of 0.25mg/l and 5 mg/l, respectively.
Bacterial activity decreased considerably upon decreasing We are very grateful to vice chancellor for research
the nitrogen and carbon contents. This might be due to
& technology, Kurdistan University of Medical Sciences
the increased toxicity of these compounds for the bacteria
and the respected health center staff.
(Figs 6 & 7).
1. Amouei, A., A. Hosseinali, F. Hourieh, F. Hossein,
In this study, Pseudomonas bacteria was isolated and
B. Reyhaneh and N. Dariush, 2012. Investigation of
purified for the purpose of isolating and identifying
hospital wastewater treatment plant efficiency in
indigenous bacteria in the effluent produced by Shahid
north of Iran during 2010-2011. International Journal
Beheshti Hospital, Abadan and examining the potential
of Physical Sciences, 7(31): 5213-5217.
biological decomposition of the organic matter
2. Abd El-Gawad H.S., 2014. Aquatic environmental
alkylbenzene sulfonate in the presence of this bacterial
monitoring and removal efficiency of detergents.
species. The bacteria were identified using biochemical
culture method. The results showed that they could Water Science, 28(1): 51-64.
effectively eliminate 73% of the alkylbenzene sulfonate 3. Ebrahimi, A., M. Hassan Ehrampoosh, M. Reza
composition within 144 hours. Chaturvedi & Kumar [14] samaei, E. Shahsavani, H. Hosseini, H. Hashemi,
isolated Pseudomonas alcaligenes and P. mendocina P. Talebi, S.V. Ghelmani, M. Dehghan and
bacteria from municipal wastewater, which were able to M. Honardoost, 2015. Survey on removal efficiency
remove respectively 99% and 98% of anionic surfactants. of linear alkylbenzenesulfonate in Yazd stabilization
Amirmozafari et al. [9] showed that Pseudomonas beteli pond. Int. J Env. Health. Eng., 4(10):…..???.
was capable of eliminating 96.4% of alkylbenzene 4. Ghodous, F., J.Y. Reza, K. Esmaeil and J. Farzaneh,
sulfonate within ten days. According to the obtained 2015. Bioremediation of anionic surfactants in
results, the optimal conditions for Pseudomonas bacteria hospital wastewater. Case study: Shahid Beheshti
to eliminate 73% of the alkylbenzene sulfonate Hospital in Abadan City, Iran. AES Bioflux.,
composition with a probability of 95% were: nitrogen 8(1): 50-58.
content = 0.25 mg/l, pH = 7, temperature = 30 °C and 5. Akpor, O.B., 2010. Muchie M.Bioremediation of
carbon content = 5 mg/l. Peressutti et al. [15] showed polluted wastewater influent: Phosphorus and
that maximum efficiency of Pseudomonas bacteria nitrogen. Scientific Research and Essays.
in eliminating anionic surfactants occured 37°C. 5(21): 3222-3230.

Am-Euras. J. Agric. & Environ. Sci., 16 (7): 1341-1346, 2016

6. Moghbeli, M., F. Shakeri and H. Hashemi- 13. Oluwafemi, S.O., B.S. Lateef and S.O. Oluwatoba,
Moghaddam, 2011. Separation of mercury resistant 2014. Biodegradation of Fresh and Used Engine Oils
bacteria from wastewater of milk, detergent and by Pseudomonas aeruginosa LP5. J Bioremed
ceramic industry. Journal of Chemical Health Risks, Biodeg., 5: 1 2-7.
1(1): 19-22. 14. Chaturvedi, V. and A. Kumar, 2010. Isolation of
7. Louvado, A., F.J. Coelho, P. Domingues, A.L. Santos, sodium dodecyl sulfate degrading strains from a
N.C. Gomes, A. Almeida and A. Cunha, 2012. detergent polluted pond situated in Varanasi city,
Isolation of surfactant-resistant Pseudomonads India. Journal of Cell and Molecular Biology,
from the estuarine surface microlayer..Journal of 8(2): 103-111.
Microbiology and Biotechnology, 22(3): 283-291. 15. Peressutti, S.R., N.L. Olivera, P.A. Babay,
8. Nilanjana, D. and Preethy Ch., 2011. Microbial M. Costagliola and HM. Alvarez, 2008.
Degradation of Petroleum Hydrocarbon Degradation of linear alkylbenzenesulfonate by a
Contaminants: An Overview. Biotechnology bacterial consortium isolated from the aquatic
Research International Article. ID 941810, pp: 13. environment of Argentina. Journal of Applied
9. Amirmozafari, N., F. Malekzadeh, F. Hosseini Microbiology, 105(2): 476-484.
and N. Ghaemi, 2007. Isolation and identification 16. Ambily, PS. and M.S. Jisha, 2012. Biodegradation of
of anionic surfactant degrading bacteria from anionic surfactant, sodium dodecyl sulphate by
activated sludge. Iranian Biomedical Journal, Pseudomonas aeruginosa MTCC 10311.J Environ
11(2): 81-86. Biol., 33(4): 717-20.
10. Venkatesh Ch and K. Ashok, 2010. Isolation of 17. Behrooz, K., H. Maryam and E. Mehry, 2015.
sodium dodecyl sulfate degrading strains from a Biodegradation of naphthalene using Pseudomonas
detergent polluted pond situated in Varanasi city, aeruginosa by up flow anoxic–aerobic continuous
India. Journal of Cell and Molecular Biology, flow combined bioreactor. Journal of Environmental
8(2): 102-111. Health Science & Engineering, 13: 26 2-10.
11. Sushma, P., A. Anvita, S. Nishmitha and S. Melwyn, 18. Syed Mohd A., M. Mahamood, M.Y. Shukor and
2012. Degradation of anionic surfactants by Bacillus N.A. Shamaan, 2010. Isolation and characterization
sdbtilis and Bacillus cereus. Journal of Pharmacy and of SDS degrading Pseudomonas aeruginosa sp.
Biological Sciences, 3: 42-45. strain D1. Australian Journal of Basic and Applied
12. Aju K.A., A.F. Pallickalvaliyaveettil and Sciences, 4(10): 5000-5011.
J. ManakulamSh, 2015. Biodegradation of Linear 19. Ainon, H., S. Noramiza and R. Shahidan, 2013.
AlkylbenzeneSulfonate (LAS) by Immobilized Screening and optimization of biosurfactant
Pseudomonas sp. Advances in Chemical Engineering production by the hydrocarbon–degrading bacteria.
and Science, 5: 465-475. Sains Malaysiana, 42(5): 615-623.