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ISSN: 2155-6199
Journal of Bioremediation & Biodegradation

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Degradation of Phenol by Mixed Culture of Locally Isolated Pseudomonas Species

Sadia Afrin Jame1, A.K.M. Rashidul Alam1, A.N.M. Fakhruddin1* and Md. Khorshed Alam2
1Department of Environmental Sciences, Jahangirnagar University, Savar, Dhaka 1342, Bangladesh
2Institute of Food & Radiation Biology (IFRB), Atomic Energy Research Establishment (AERE), Ganakbari, Savar, Dhaka 1344, Bangladesh
Corresponding Author : Dr. A. N. M. Fakhruddin
Associate Professor
Department of Environmental Sciences
Jahangirnagar University
Savar, Dhaka-1342
Tel: +8801718217979
E-mail: [email protected]
Received: August 04, 2010; Accepted: September 14, 2010; Published: September 17, 2010
Citation: Jame SA, Rashidul Alam AKM, Fakhruddin ANM, Alam MK (2010) Degradation of Phenol by Mixed Culture of Locally Isolated Pseudomonas Species. J Bioremed Biodegrad 1:102. doi:10.4172/2155-6199.1000102
Copyright: © 2010 Jame SA, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Abstract

Textile, pharmaceuticals and automobile waste most often contain phenolic wastes. An attempt was made to degrade phenol using locally isolated bacteria. Moreover, as in nature pure culture is rarely found rather than mixed culture, therefore, attempt was also made to improve the degradation using mixed culture of Pseudomonas species. All isolates could completely degrade phenol up to 600 ppm. Isolate Pseudomonas FA degraded 800 ppm phenol completely in 72 hours, but the isolates Pseudomonas SA, TK and KA degraded only 39.33, 43.83 and 33.16% of 800 ppm phenol respectively in 96 hours. Complete removal time was also shorter for the isolate Pseudomonas FA compares to the other isolates. Patterns of growth were similar for all of the isolates, but maximum growth was found with the isolate FA on 600 ppm phenol. Complete degradation time was decreased with mixed culture and removal rate of phenol was 25 ppm/h in mixed culture of all combinations and was higher than that of the single culture of the isolates. In mixed culture study, the growth of bacteria was also increased.

Keywords
Biodegradation; Phenol; Mixed culture; Pseudomonas
Introduction
Production and uses of synthetic organic chemicals has increased enormously during the last few decades. Phenol is the simplest aromatic compound derived from benzene by adding a hydroxyl group to a carbon to replace hydrogen. It is found in nature in some foods, in human and animal wastes and decomposing organic material [1]. United States Environmental Protection Agency regulates phenol under the Clean Water Act (CWA) and the Clean Air Act (CAA) and has designated it as a hazardous substance and a hazardous air pollutant (HAP) [2]. Phenol is on the list of chemicals appearing in “Toxic Chemicals Subject to Section 313 of the Emergency Planning and Community Right-to-Know Act of 1986” and has been assigned a reportable quantity (RQ) limit of 1,000 pounds [2]. Phenol is also considered to be an extremely hazardous substance as it can remain in the air, soil and water for much longer periods of time if a large amount of it is released at a time, or if it is constantly released to the environment. It is moderately toxic and can be absorbed into the body by ingestion, inhalation and contact. It affects the central nervous system, and so symptoms of exposure can include muscle weakness and tremors, loss of coordination, paralysis, convulsions, coma and respiratory arrest [3]. Phenol is harmful for the non living environment also. Effects on the non-living environment include damage to structures by acidic air pollutants, effects on ozone layer and the earth’s heat balance and reduced visibility [4].
Mixed culture means that several kinds of bacteria are mixed simultaneously [5]. A defined mixed culture is a miniature of complex microorganisms in nature. To treat special waste, adaptation is generally needed. If microorganisms that can degrade these materials are added, the adaptation time might be reduced. In this study, pure culture of few locally isolated Pseudomonas sp. were carried out to see their ability to degrade various concentrations of phenol as Pseudomonas being a most widely prevalent microorganisms in the environment and it generally dominates the biological treatment system. Two known bacteria, Pseudomonas putida CP1, Pseudomonas putida A (a) were also used for the mixed culture study. Studying the interactions of different species in xenobiotics degradation is highly relevant for natural processes and also for bioremediation purposes under non sterile conditions [6]. The study therefore aims to study the rate of degradation of phenol when supplied as a sole source of carbon at various concentrations by the bacterial isolates and to evaluate the degradation capability of bacterial isolates by mixed culture in order to determine their practical applicability.
Materials and Methods
Microorganisms
Pseudomonas putida CP1 and Pseudomonas putida A (a) was obtained from the stock culture of Food Microbiology and Industrial Irradiation Division, Institute of Food and Radiation Biology, Bangladesh Atomic Energy Research Establishment, Savar, Dhaka, Identification of these two isolates were described elsewhere [7]. The remaining four locally isolated bacterial were Pseudomonas SA, TK, KA and FA. Identification of the local isolates was also described [8].
Chemicals and media
Phenol used in this study was obtained from Sigma-Aldrich Co., UK. Bacteria capable of degrading phenol were cultured on minimal medium [9] comprised K2HPO4- 4.36 g/l; Na2HPO4-3.45 g/l; NH4Cl- 1.0 g/l; MgSO4.6H2O – 0.912 g/l; trace salt solution- 1ml/l. The pH of the medium was adjusted to 7.0 with 2M NaOH. The trace salt solution contained 4.77 g-CaCl2. 2H2O; 0.37 g -FeSO4. 7H2O; 0.37 g-CoCl2.6H2O, 0.10 g-MnCl2.4H2O and 0.02 g-NaMoO4. 2H2O in 100 ml water
Inoculums
Cells from the Pseudomonas minimal medium were used to inoculate nutrient broth (15 ml). The harvested cells were centrifuged at 5000 rpm for 10 minutes and washed twice with 0.01M sodium phosphate buffer and final pellet resuspended in the same buffer. Bacterial suspension (≈107/ml) was used to inoculate 95 ml sterile minimal medium containing phenol in 250 ml conical flasks to give the appropriate final concentration of the added substrates. After inoculation, flasks were incubated in an orbital shaker at 120 rpm 30°C. Samples were aseptically removed at regular intervals and analyzed for growth, substrate removal and pH. Growth of the organisms was monitored by measuring optical density at 660 nm. For mixed culture study, equal amounts of different inoculums were taken and the ultimate volume was fixed to 5 ml in 250 ml conical flasks to give the appropriate final concentration of the added inoculums. After inoculation, flasks were incubated in an orbital shaker at 120 rpm at 30°C. Rest of all the procedures were followed as above.
Chemical analyses
Samples were then centrifuged at 5000 rpm for 10 minutes; the supernatants were then analyzed for phenol. Phenol concentration was determined by using the 4-aminoantipyrene colorimetric method [10].
Results
Degradation of various concentrations (400 to 800 ppm) of phenol by the Pseudomonas isolates when grown as single culture
table 1 illustrates removal rate, complete removal time and removal efficiency of the Pseudomonas isolates FA, SA, TK, and KA when grown on various concentrations (400 to 800ppm) of phenol as sole source of carbon and energy. All isolates could completely degrade phenol up to 600 ppm. Isolate Pseudomonas FA degraded 800 ppm phenol completely in 72 hours, but the isolates Pseudomonas SA, TK and KA degraded only 39.33, 43.83 and 33.16% of 800 ppm phenol respectively in 96 hours. Complete removal time was also shorter for the isolate Pseudomonas FA compares to the other isolates.
The growth of potential isolates (Pseudomonas SA, FA, TK and KA) when grown on various concentrations (400 to 800 ppm) is shown in Table 2. Maximum growth was found with the isolate FA on 600 ppm phenol. Patterns of growth were similar for all of the isolates. Pseudomonas FA also grew in the presence of 2- and 4-chlorophenol [8].
Degradation of 600 ppm of phenol by the Pseudomonas isolates when grown as mixed culture
Since all these bacteria degraded 600 ppm phenol, this concentration was selected for mix culture study. Different combination of isolates was used for mixed culture study. Figure 1 and Table 3describe the compassion between mixed and pure cultures in terms of the removal rate, complete degradation time and growth.
From mixed cultures of Pseudomonas putida A (a) and Pseudomonas sp. SA, it was found that P. putida A (a) degraded 600 ppm phenol in 24 hour and Pseudomonas sp. SA degraded the same concentration in 72 hour when they were cultured individually. The mix culture of this two Pseudomonas sp. degraded the same concentration in 20 hour (Figure 1). The OD of mixed culture ranged from 0.25-0.856 whereas the OD of Pseudomonas. sp. SA and P. putida A (a) was 0.2525-0.6420 and 0.26-0.772, respectively.
From mixed cultures of Pseudomonas putida A (a) and Pseudomonas sp. KA, it was observed that P. putida A (a) and Pseudomonas sp. KA degraded 600 ppm phenol in 20 hour in a mix culture, 72 hour and 24 hour were needed to complete the degradation by Pseudomonas sp. KA and P. putida A (a) respectively (Figure 1). The growth of the mixed culture ranged from 0.2330- 0.7350 while the growth ranged from 0.2525 to 0.6420 for P. putida A (a) and 0.1320 to 0.70 for Pseudomonas sp. KA.
From mixed cultures of Pseudomonas putida CP1 and Pseudomonas sp. KA, it was observed that Pseudomonas putida CP1 and Pseudomonas sp. KA degraded 600 ppm phenol in 20 hour while individually they completed degradation in 48 hour and 72 hour respectively (Figure 1). The growth (i.e., OD at 660 nm) was 0.23- 0.727 during different period in mixed culture study, was higher than single culture.
The removal rate of phenol was 25 ppm/h in mixed culture of all combinations and was higher than the pure culture of the isolates.
Discussion
The ability of the bacteria particularly those of genus Pseudomonas to utilize aromatic hydrocarbons has been widely documented [3,11]. Four isolates (Pseudomonas spp. SA, TK, and KA) were isolated which completely degraded 600 ppm of phenol within 72 hour but varying in their removal rate. The concentration above 600 ppm of phenol was degraded by Pseudomonas sp. FA. Among the investigated bacteria, Pseudomonas putida CP1 was able to degrade 600, 800 ppm of phenol, 200 ppm of 2-chlorophenol and 300 ppm of 4-chlorophenol in 48, 72, 100 and 72 hours respectively, and Pseudomonas putida A (a) degraded 600 ppm phenol in 24h and 800 ppm phenol in 48 hours [12,13].
The removal rates of the potential isolates were higher for phenol and they are almost similar for 400 ppm. But in case of 600 ppm of phenol the removal rate in varies in bacterial isolates. The removal rate was lower in every case when the phenol concentration was 600 ppm except Pseudomonas sp. SA. The rate was 16.67 ppm/h for 400 ppm and increased to 19.87 ppm/h for 600 ppm. The removal rate of Pseudomonas putida CP1 was 23.25 ppm/h at 600 ppm and increased to 24.58 ppm/h when the phenol concentration was 800 ppm and 23 ppm/h removal rate was recorded for 800 ppm phenol whereas it was 25 ppm/h at 600 ppm in case of Pseudomonas putida A (a) [12].
The difference of structure and toxicity among phenolic compounds requires that various bacteria have specific qualities to degrade each compound and a mixed culture may be applied [5]. Degradation of mono-chlorophenol and phenol by mixed microbial community were reported [7,14,15]. Although four isolates capable of degrading phenol of various concentrations, most of the cases, the degradation rate of a mixed culture was determined as being significantly higher that for any of the individual strains. Individual organisms may metabolize only a limited range of substrates; therefore it is likely that assemblages of different bacterial species with broader enzymatic capabilities have a greater capacity to degrade complex phenolic compounds [16]. For this reason mixed culture was carried out with the individual degrading isolates.
The removal rates of Pseudomonas sp. SA and KA were 19.87, 13.83 ppm/h, respectively and the removal rate of P. putida was 23.30 ppm/h while the phenol concentration was 600 ppm. When SA and KA mixed with P. putida A (a), the removal rate was increased 25 ppm/h in both cases. The degradation time was also decreased rather than the pure culture indicating positive interaction between them. When organisms were mixed with each other, the degradation time was decreased and the removal rate was increased. This observation was also supported by the higher bacterial growth and possibly both of the isolates contributed to degradation.
Conclusion
Pseudomonas sp. SA, Pseudomonas sp. TK and Pseudomonas sp. KA were able to degrade 600 ppm and Pseudomonas sp. FA was able to degrade phenol up to 800 ppm of phenol. In mixed culture study, the growth of bacteria was increased and degradation rate was also increased.
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  1. nadji bouakaz
    Posted on Jan 18 2017 at 4:17 am
    Hello sir/madam Can you authorize me to consult this article including these figures ''Degradation of Phenol by Mixed Culture of Locally Isolated Pseudomonas Species'' cordially

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