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ISSN: 2155-6199
Journal of Bioremediation & Biodegradation
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Biodegradation of Phenol by Native Bacteria Isolated From Dioxin Contaminated Soils

Han Ba Bui1, Lan Thi Nguyen2 and Long Duc Dang2*
1Department of Biotechnology, Faculty of Chemical Engineering, Da Nang University of Technology, The University of Da Nang, Da Nang, Vietnam and Faculty of Biotechnology and Environmental Technology, Duc Tri College, Da Nang, Vietnam
2Department of Biotechnology, Faculty of Chemical Engineering, Da Nang University of Technology, The University of Da Nang, Da Nang, Vietnam
Corresponding Author : Long Duc Dang
Department of Biotechnology
Faculty of Chemical Engineering
Da Nang University of Technology
The University of Da Nang
Da Nang, Vietnam
Tel: (+84) 01643 733 070
E-mail: [email protected]
Received September 14, 2012; Accepted September 28, 2012; Published September 30, 2012
Citation: Bui HB, Nguyen LT, Dang LD (2012) Biodegradation of Phenol by Native Bacteria Isolated From Dioxin Contaminated Soils. J Bioremed Biodeg 3:168. doi: 10.4172/2155-6199.1000168
Copyright: © 2012 Bui HB, et al. This is an open-a ccess 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

In this investigation, aerobic bacteria in soil contaminated with dioxin (taken from Da Nang airport’s area in Vietnam) were isolated and selected for their ability to degrade phenol using enrichment technique containing phenol as sole source of carbon and energy (100 mg/L phenol in a mineral salt medium). Four strains (designated D1.1, D1.3, D1.4, and D1.6) were obtained and characterized. The results showed that these bacteria were highly effective for the removal of phenol. After 120 hours of culture, strain D1.4 degraded 54.84% and 44.19% phenol from the initial concentrations of 100 mg/L and 1000 mg/L, respectively; strain D1.6 degraded 66.45% of phenol from the initial concentration of 1500 mg/L. The combination of those bacteria in the same medium had a positive effect on the phenol degradation activity. The outcome of the study can contribute new useful resources for treatments of wastewater and soils contaminated with phenolic wastes.

Keywords
Dioxin-contaminated soil; Phenol degradation; Aerobic bacteria; Isolation
Introduction
In the development of the world today, human health and the environment have become the most pressing issues. Due to those concerns, biodegradation of aromatic compounds have received a great attention because of their toxicity and persistence in the environment. Among all those compounds, phenol and their derivatives are ones among the most prevalent forms of chemical pollutants since they are commonly used to produce many resins, dyes, paints, varnishes, detergents, herbicides and pharmaceutical drugs. They are also byproducts of many big industries such as petroleum processing, coke conversion, steel manufacturing [1-3]. Phenol can occur naturally in some agricultural products, animal wastes and decomposition of organic materials [4]. However, it is documented to have harmful effects on human health and the environment. Phenol is a water-soluble and highly mobile neurotoxin and can cause damage to the human body and other living organisms through ingestion, inhalation or contact [1,5]. As a potent air pollutant, phenol can contribute to damage to structures and ozone layer and may reduce visibility and heat balance of the atmosphere [5]. Therefore, phenol has been declared to be a hazardous substance and a hazardous air pollutant by the United States Environmental Protection Agency [6].
A variety of physical, chemical and biological methods have been used for the safe removal of such a chemical from the environment. One of the cheapest and safest solutions for this is the bioremediation using microorganisms [7]. In general, it is difficult to degrade phenol by biological methods when its concentration is above 200 mg/L and the microorganisms are completely deactivated at concentrations larger than 3000 mg/L [8]. Nevertheless, microbial degradation of phenol with different initial concentrations ranging from 50-2000 mg/L have been carried out in various reactor systems (e.g., shake flask, fluidized-bed reactor, continuous stirred tank bioreactor, etc.) using a variety of fungi and bacteria such as Candida tropicalis, Acinetobacter calcoaceticus, Alcaligenes eutrophus, Pseudomonas putida, Burkholderia cepacia G4, Bacillus stearothermophilus, etc. [9,10]. Those microorganisms usually have been isolated from environmental samples with high concentrations of pollutants. In Vietnam, due to historical reasons, there are many sites contaminated heavily with chemical reagents, especially dioxin. In those sites there are serious environmental problems, but also exist certain microorganism strains with special ability to metabolize toxic chemicals. In this study we tried to exploit that situation by isolating naturally occurring bacterial strains present in those contaminated sites and characterizing their efficiency of phenol degradation.
Materials and Methods
Soil samples and chemicals
Soil samples were collected in Da Nang’s airport area in Vietnam. This soil is heavily contaminated with dioxin in Agent Orange being used in Vietnam War (1964-1973). At present, the dioxin concentration in soil there is up to approximately 200 pg TEQ/g soil [11]. Phenol was obtained from Sigma-Aldrich Co., UK. The mineral salt medium (MM) used in this study for bacteria enrichment and isolation was modified from the one used by Fortnagel and coworkers [12] and comprised (per liter): 3.5 g of Na2HPO4.2H2O, 1.0 g of KH2PO4, 0.5 g of (NH4)2SO4; 0.1 g of MgCl2.6H2O, 50 mg of Ca(NO3)2.4H2O, 1 ml of vitamin B12, and 1 ml of trace salt solution. The final pH of the medium was 7.2. The trace salt solution contained 0.01 g MoO3, 0.07 g ZnSO4.5H2O, 0.005 g CuSO4.5H2O, 0.01 g H3BO3, 0.01 g MnSO4.5H2O, 0.01 g CoCl2.6H2O and 0.01 g NiSO4.7H2O in 100 ml water. The MM solid medium contained 10 g of agar (Meck Co., USA) per liter.
Isolation procedure
Soil samples were passed through a sieve (1.7 mm mesh) to remove large pieces of debris and vegetation. A 0.5% agar medium (agar in distilled water) was autoclaved and cooled down (45°C). Then, a 100 ml ml of agar medium was mixed well with 1 g of the sieved soil samples. Then 5 ml of the resulted suspension was spread evenly onto Petri dishes containing the sterile solid MM medium, which was supplemented with phenol (100 mg/L). Those dishes were cultured in an aerobic condition at 30°C for 120 hours. After this incubation, individual colonies were transferred onto new MM agar plates with and without phenol (also 100 mg/L) as the carbon source. And after incubating at 30°C for 120 hours, we selected the colonies that grew on the medium with phenol but were unable to grow on the medium without phenol. Well grown colonies were maintained on nutrient agar slant and stored at 4°C ± 1°C until used for further experiments. Basic biological characteristics of the isolated bacterial strains were carried out by standard laboratory procedures [13].
Growth of the bacterial strains in phenol and aromatic compounds
The isolated cultures were used to inoculate MM media containing phenol at different concentrations and let grow at 28°C on an orbital shaker at 150 rpm. Similar experiments for MM media containing benzene, aniline and dioxin-contaminated soil as the carbon source had also been conducted. Control samples of MM media without phenol or the other organic substrates have been prepared for reference. All the cultures were limited the exposure to light. Samples were aseptically removed at regular intervals and analyzed for growth, substrate removal and pH. The growth was measured by the cell numbers, total protein concentration, and biomass. To estimate the cell number, 5 ml of the culture medium was centrifuged at 4,000 rpm for 20 minutes at 4°C ± 1°C, then the obtained pellet was resuspended in 4 ml of phosphate buffer (0.05M, pH 7.2) and worked up by the method of Bedard et al. [14]. For analysis of the total protein concentration (mg/ml medium), the samples after the centrifugation were washed few times with fresh (phenol-free) MM to remove the substrate. After cell lysis in the presence of NaOH (0.15 M) for 5 min at 95°C [14], the total protein concentration was determined by calibration with Bovine Serum Albumin (BSA) standards according to Biure [15]. The biomass (g/L of medium) is estimated by a dried weight method [10]. Specifically, 10 ml of the culture broth was centrifuged as mentioned above, then the pellet was washed twice and finally transferred from the tube into a pre-weighed 1.2 μm pore filter paper (Whatman GF/C). The filter paper were dried in an oven at 105°C for between 72 hours, cooled in a desiccator at room temperature and reweighed to estimate the dry weight of the biomass. The phenol concentration was using a 4-aminoantipyrine colorimetric approach [16]. The supernatant of the centrifuged culture medium was reacted with 4-aminoantipyrine at pH 7.9 ± 0.1 forming a brownish-orange compound. Subsequently sample absorbance was measured at 500 nm. The phenol concentration was calculated by referring to the standard curve.
Determination of enzyme activities in cell extracts
Manganese Peroxidase (MnP) is an enzyme catalyzing the Mn(II) and H2O2 dependent oxidation of lignin and a variety of phenols. We measured the MnP activities of the isolated bacterial using an assay based on the oxidative coupling of 3-methyl-2-benzothiazolinone hydrazone (MBTH) and 3-(dimethylamino) benzoic acid (DMAB) [17]. The reaction of MBTH and DMAB in the presence of H2O2, Mn(II), and MnP gives a deep purple-blue color with the absorption peak at 590 nm. Catalase and oxidase activities were measured by traditionally biochemical methods [18].
Results
Enrichment, isolation and characterization of phenol-degrading bacteria
Starting with two different dioxin-contaminated soil samples at Da Nang airport (D1 and D2 location), we have been able to isolate four bacteria strains (D1.1, D1.3, D1.4, and D1.6) from the D1 sample (Figure 1). The isolates were all Gram-positive, formed yellowish and slimy colonies, and grew under strictly aerobic conditions. The strains reacted negatively to acid resistance test, produced no H2S and NH3, and showed catalase but no oxidase activity (table 1). From the second location (D2), we failed to isolate any aerobic bacteria.
The growth of the bacteria in media containing phenol or other organics
The growth of four bacterial strains on MM containing phenol as the sole source carbon and energy were shown in Figure 2. The bacterial strains D1.3, D1.4, and D1.6 were very quickly to reach the log phase (several hours), while the strain D1.1 took longer and had lower number of cells and protein concentration. It should be noted that the amounts of protein produced by the bacteria were not tightly linked to the cell numbers, especially in the case of the strain D1.3. It may be due to the differences in contents of those cells in different development stages.
After 120 hrs of incubation, the biomass of the strain D1.6 was highest (0.8 g/L), followed by D1.4 (0.73 g/L), and the lowest value was belonged to the strain D1.1 (0.24 g/L). Those growth profiles indicated that the strain D1.1 grew more poorly in the phenol medium. Also after the incubation, the phenol amounts that were degraded by the four strains- D1.1, D1.3, D1.4, and D1.6- were 31.98%, 51.64%, 54.84% and 47.07%, respectively. Clearly, the phenol-degradation capabilities of the strains were not proportionally related to their growth. The reason is that the phenol degrading activity is dependent on amounts and characteristics of metabolic enzyme systems in those strains, not on their cell numbers.
In order to know more metabolic characterization of the bacteria strain, we grew them in various liquid media containing other organic carbon sources, from simple sugars to toxic aromatic compounds. The strains all developed very well on the media containing glucose, lactose and mannitol, respectively. They also showed the ability to grow in the media containing benzene and aqueous extract of the dioxin-contaminated soil, but not in aniline medium (the quantitative data were not shown here).
Enzyme activities in crude cell extracts
After 120 hrs of culture, the biomass of each strain was obtained and the cells were lysed. The activities of MnP of the four cell extracts shown that, the strain D1.4 had the highest enzyme activity, followed by the strain D1.3, then the strain D1.6, and the strain D1.1 had the lowest activity (Table 2). This pattern was similar to the variation of the phenol-degrading capability of those strains.
Ability to grow in different phenol concentrations of the bacterial strains
All of the four isolates were grew in a series of liquid MM with the phenol concentrations varied from 100 mg/L, to 300, 1000, and 1500 mg/L. Based on the resulted cell numbers, protein concentrations, biomass, and phenol concentrations, we could observe that the bacterial strains D1.1 only grew well in 100 mg/L phenol, but grew poorly in the higher concentrations of phenol. Meanwhile, the strain D1.3 only grew poorly when the concentration of phenol was up to 1500 mg/L, and the strains D1.4 and D1.6 grew well in the whole range of the phenol concentration. The growth of two bacterial strains (D1.4 and D1.6), who seemed to adapt best to phenolic environment, were illustrated in Figure 3 with the case of the phenol concentration of 1,000 mg/L for the strain D1.4 and of 1,500 mg/L for the strain D1.6.
Phenol degradation ability of a mixed culture of the bacterial strains and the native soil microorganism systems
A synergetic action of microorganisms are usually helpful in metabolize and degrade organic chemicals [5,19]. To test this feature of the isolates here, we mixed the four strains together in an equal fashion and grew this mixed culture in a MM containing 100 mg/L. For comparison, the two initially dioxin-contaminated soil samples were also cultured in the same medium with the amounts of soil were calculated so that they carried similar numbers of the bacterial cells. The phenol degradation activities of those cultures during 120 hrs of incubation were shown in Figure 4.
Influence of environmental factors on the phenol-degrading capability of the bacterial strains
From the results mentioned above, we found that the strain D1.4 had the best ability to degrade phenol in MM. Therefore, we investigated the influence of environmental factors on the phenol-degrading activity of this strain. This line of experiments would be helpful to identify optimal conditions of phenol degradation using the isolated bacteria. Figure 5 represented the effects of NaCl, glucose concentrations and pH of the growth medium on the final phenol concentration after 120 hours of incubation of the strain D1.4 in the MM containing 100 mg/L phenol.
Discussions
Many works on biodegradation of phenol using pure or mixed microorganisms isolated from various environmental sources have been reported [2-5,7-10,19-22]. However, studies on phenol degrading ability of microorganisms from dioxin-contaminated soils have not been reported. In this study, from that kind of soils we have isolated successfully four aerobic bacteria strains that all can use phenol as the source of carbon and energy for their growth. In addition, those bacteria also can utilize several toxic aromatic compounds, such as benzene and dioxin derivatives. Due to this kind of metabolic capability, those bacteria strains can degrade phenol and different aromatic contaminants. The phenol degradation capabilities were not the same for those isolates. Through culturing the strains in a wide range of phenol concentration, we selected out two strains, D1.4 and D1.6, which can grow and metabolize a very high concentration of phenol (1,500 mg/L). Our assay suggested that the phenol degrading activity of the bacteria were closely linked to the activity of manganese peroxidase, an important enzyme for degradation of a wide variety of chemicals and polymers. These strains can be the basics of a biological reagent or reactor processing phenol and aromatic chemicals in soil and wastewater. Overall, our study here demonstrates that dioxin-contaminated soils are valuable sources of microorganisms that can be beneficial in environmental protection as well as in other fields.
However, we have not been able to isolate all the phenol-degrading microorganisms existing in the soil samples. Especially, the soil sample D2 showed a strong phenol-degrading activity in its native state, but it did not result in any isolate in our selection procedure. The procedure was just designed for aerobic bacteria. Maybe in the soil sample D2, the dominant phenol-degrading microorganisms are different, such as anaerobic bacteria, or fungi, or yeast. The mixed culture of the strains had better phenol degradation than each individual bacteria, showing that the synergetic actions of the native organisms could be very important in the degrading capability. We have not investigated thoroughly this kind of synergy in this study, but it will be an important topic so that the best biodegradation reagent for phenol can be construct from the soil samples.
Furthermore, identifying the best reagent is required to combine with identifying the optimal condition of the medium. In general, the presence of NaCl in the medium had a positive effect on the capability of phenol degradation. The bacteria grew better in MM with glucose (in the range of 0.25 - 0.75% w/v) than without glucose. pH of the medium could also influence the ability to degrade phenol of the strain D1.4.
With the results in our study, we have establish the fundamental researches and benefits of utilizing heavily dioxin-contaminated soils’ microorganisms for bioremediation of sites and the treatment of industrial wastewater contaminated with phenolic wastes.
 
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