Phenol, Alkylphenols, and Polycyclic Aromatic Hydrocarbons (PAHs

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aromatic hydrocarbons (PAHs) using the bacteria Hydrogenophaga palleronii. ..... Q., Li, R., Li, T., 2013, Biodegradation of phenol by using free and immobilized.

Phenol, Alkylphenols, and Polycyclic Aromatic Hydrocarbons (PAHs) Degradation Using the Bacteria Hydrogenophaga palleronii M. Venkateswar Reddy, Young-Cheol Chang*, Shintaro Kikuchi Department of Applied Sciences, College of Environmental Technology, Muroran Institute of Technology, 27-1 Mizumoto, Muroran, 050-8585, Japan. E-mail: [email protected]

Abstract The present study is the first report of the degradation of phenols, alkylphenols, and polycyclic aromatic hydrocarbons (PAHs) using the bacteria Hydrogenophaga palleronii. H. palleronii showed good growth with various toxic organic compounds. Degradation pattern of all the organic compounds at 100 mg/l concentration were analyzed using High pressure liquid chromatography (HPLC). H. palleronii showed complete removal of phenol, and 4-tert-butylphenol within 96 h, and the bacteria showed 60% and 36% removal of 4-sec-butylphenol and 4-butylphenol within 240 h respectively. Bacteria showed negligible amount of degradation of PAH like naphthalene and phenanthrene. These results make the strain H. palleronii is good candidates for removal of phenols, and some of the alkylphenols in wastewaters. Keywords: Hydrogenophaga palleronii; phenol; alkylphenols; HPLC; acetate. 1. Introduction Industrial development has increased the release of hazardous pollutants such as phenolic compounds into the environment [1]. Huge amount of phenol and phenolics compounds are discharged through effluents from a variety of industries including leather processing, textiles, pharmaceutical, and oil plants [2]. Phenol pollution is of great alarm since this chemical is toxic, mutagenic, and carcinogenic. Because of its several toxic effects, removal of phenol from industrial wastewaters before their release is considered to be obligatory [3]. On the other side Alkylphenols are found in the environment coming from the breakdown of alkylphenol polyethoxylates that are widely used in non-ionic surfactants and detergents. Alkylphenols are toxic to aquatic life and are consider as endocrine disrupters that can cause various harmful effects, including reproductive effects by mimicking the typical female sex hormone, estrogen in aquatic life and in humans. Because of their risk, the US Environmental Protection Agency set a standard for the concentration of alkylphenols in fresh and salt water. Therefore, the industrial effluents containing alkylphenols should be properly treated to remove them. However, alkylphenols are constant in the environment because of their resistance to natural degradation. Using the carbon number of the alkyl chain, alkylphenols are divided into three major groups, short chain (1 to 2), medium chain (3 to 7) [4] and long chain (8 to12) alkylphenols. Wastewaters with high concentration of these organic compounds can be treated mainly by physicochemical processes such as ozonation, Fenton’s reagent, UV, and hydrogen peroxide, but these methods are costly and in-appropriate for large wastewaters volumes [5, 6]. Biological degradation has been utilized as an competent alternative, since it has low associated costs and is more effective in degradation of organic compounds [7]. Hydrogenophaga palleronii (formerly known as Pseudomonas palleronii) is a bacterium from the phylum Proteobacteria, class beta-Proteobacteria, genus of Hydrogenophaga and the family of Comamonadaceae which has the ability to degrade different organic pollutants. Many studies reported the degradation of 4-aminobenzenesulfonic acid, 2,4-dinitrotoluene, and naphthalene by 1

using Hydrogenophaga palleronii, but there are no reports regarding the degradation of phenol, alkylphenols, and polycyclic aromatic hydrocarbons (PAHs) using this strain. So, objective of the present study is to evaluate the feasibility of phenol, alkylphenols, and PAH compounds degradation using H. palleronii. Here, we report the growth pattern of H. palleronii using different organic compounds as substrate. We further show the capacity of this strain for degradation of those organic compounds using high performance liquid chromatography (HPLC). 2. Materials and methods 2.1. Chemicals In this study we selected 3 different types of toxic organic compounds like phenol, alkyl phenols, and PAH for degradation using the bacteria H. palleronii. In alkyl phenols we selected six different types of compounds i.e., 4-chlorophenol (4-CP), 4-n-butylphenol (4-BP), 4-n-nonylphenol (4-NP), 4-secbutylphenol (4-s-BP), 4-tert-butylphenol (4-t-BP), and p-tert-octylphenol (p-t-OP). In PAH compounds we selected 2 types i.e., naphthalene, and phenanthrene. All chemicals used were of analytical grade and were purchased from Tokyo Chemical Industry (Tokyo, Japan). 2.2 Culture media For the growth of H. palleronii nutrient broth, and mineral salt medium (MSM) was used as the media. The MSM contained 1.0 g (NH4)2SO4, 1.0 g K2HPO4, 0.2 g NaH2PO4, 0.2 g MgSO4 7H2O, 0.05 g NaCl, 0.05 g CaCl2, 8.3 mg FeCl3 6H2O, 1.4 mg MnCl2 4H2O, 1.17 mg Na2MoO4 2H2O, and 1 mg ZnCl2 per one liter of deionized water [8]. The pH of the medium was adjusted to 7 and autoclaved before adding to the flasks. 2.3 Growth of H. palleronii H. palleronii was cultivated in MSM medium at 30oC by supplementing with three different types of toxic organic compounds i.e., phenol, alkyl phenols (4-CP, 4-BP, 4-NP, 4-s-BP, 4-t-BP, and p-t-OP), and PAH (naphthalene and phenanthrene) at 100 mg/l final concentration as the sole carbon and energy source. The growth of H. palleronii with toxic organic compounds was compared with nontoxic compound like acetate at same concentration (100 mg/l). For growth curve study, a loop of H. palleronii strain was initially inoculated into 50 ml of nutrient broth in 100 ml flasks, and 1 ml of the overnight grown culture was then inoculated to different shake flasks containing 100 ml of MSM medium with different aromatic compounds. Samples were collected at different time intervals, and growth was monitored spectrometrically by measuring the absorbance at 600 nm using UVspectrometer (UV-1800, Shimadzu, Japan). 2.4 Degradation experiments by H. palleronii To investigate the degradation of a variety of toxic organic compounds by H. palleronii, culture was grown in MSM medium under aerobic condition. The toxic organic compounds such as phenol, 4-CP, 4-BP, 4-NP, 4-s-BP, 4-t-BP, p-t-OP, naphthalene, and phenanthrene at a final concentration of 100 mg/l in MSM medium were added in to separate conical flasks. A 10 g/l substrate stock solution in methanol was used. Naphthalene and phenanthrene stock solution in n-hexane was used since those compounds were not solubilized in methanol. For all the experiments the flasks were shaken at 180 rpm at 30°C for 240 h under aerobic condition. Samples were collected at different time intervals for HPLC analysis, and were acidified with phosphoric acid (10 %, wv−1) to stop the biological reaction, extracted with an equal volume of 1:1 (v v−1) ethyl acetate, shaken for 3 minutes, and centrifuged at 8,000×g for 10 min. The organic layer was then analyzed directly by HPLC. The degradation patterns of different toxic organic compounds were analyzed on HPLC (Shimadzu) with an SPD-10AV UV/Vis detector at 277 nm and Shim-pack VP-ODS column (4.5×150 mm diameter, particle size 5 μm; Shimadzu, Kyoto, Japan). Filtered and degassed mobile phase mixture composed of acetonitrile/water (4:1 v v−1) was used as mobile phase at a flow rate of 1.0 ml/min. The column was maintained at a temperature of 40°C in a thermostat chamber. Degradation concentrations were calculated from the area of the curve obtained for 1 mM of the standards. The detection limit was 0.03 mg/l. Recovery of samples was 99.5% in percent. All results were presented as average and standard deviation of the data from three independent experiments. 2

3. Results and discussion 3.1 Growth curve H. palleronii was cultivated in MSM medium at 30oC by supplementing with three different types of toxic organic compounds i.e., phenol, alkylphenols (4-CP, 4-BP, 4-NP, 4-s-BP, 4-t-BP, and p-t-OP), and PAH (naphthalene and phenanthrene) at 100 mg/l final concentration as the sole carbon and energy source. Growth study results denoted that different toxic organic compounds showed significant influence and there is a marked difference on the growth of bacteria. Among all the different toxic organic compounds, bacteria showed higher growth with phenol. The growth of H. palleronii was 7.3 times higher with phenol, when growth was compared with non toxic compound like acetate (Figure 1a). Growth results indicating that the strain H. palleronii has potential application for the degradation of phenol contaminated sites. Among six different alkylphenols, H. palleronii showed higher growth with 4-t-BP, and 4-s-BP (Figure 1b). If the growth was compared with acetate, the growth of H. palleronii was 6.9 times higher with 4-t-BP, followed by 6.25 times with 4-s-BP, 3.35 times with 4-NP, 1.74 times with p-t-OP, and 1.33 times with 4-CP when compared with acetate. H. palleronii showed lower growth with 4-BP and it was 1.9 times lower when compared with acetate. In the case of PAH compounds, H. palleronii showed lowest growth with both the PAH compounds i.e., naphthalene and phenanthrene than acetate (Figure 1c).

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Figure 1: Growth curve of H. palleronii using (a) phenol; (b) Alkylphenols; and (c) Poly cyclic aromatic compounds as substrates with respect to time.

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3.2 Degradation of toxic organic compounds 3.2.1 Phenol degradation H. palleronii strains were incubated with phenol at 100 mg/l concentration in MSM medium. Samples were collected at different time intervals, i.e., 0 h, 24 h, 48 h, 72 h, 96 h, 120 h, and 144 h, and 240 h. The remaining concentration of phenol in the medium was analyzed by HPLC. Resting cell analysis was done using HPLC to know degradation pattern of phenol. H. palleronii showed complete removal of phenol within 96 h (Figure 2). There are many reports on the degradation of phenol using different types of bacteria, but no reports till date regarding the degradation of phenol using H. palleronii. 100 80 60 40 20 0

Figure 2: Degradation of various toxic organic compounds using the bacteria H. palleronii. 3

3.2.2 Alkylphenols degradation For degradation of alkylphenols, H. palleronii strains were incubated with six different types of alkylphenols (4-CP, 4-BP, 4-NP, 4-s-BP, 4-t-BP, p-t-OP) at 100 mg/l concentration in MSM medium. Samples were collected at different time intervals, and the remaining concentration of alkylphenols in the medium was analyzed by HPLC. H. palleronii showed complete (100%) removal of 4-t-BP within 120 h, 60% removal of 4-s-BP, 36% removal of 4-BP, and 8% removal of p-t-OP within 240 h (Figure 2). H. palleronii can’t degrade the 4-NP and 4-CP, even though it showed growth in growth curve analysis. There are no reports till date regarding degradation of alkylphenols using H. palleronii. 3.2.3 PAH compounds degradation H. palleronii showed negligible amount of degradation of PAH compounds i.e., naphthalene (6%), and phenanthrene (8%) (Figure 2). Lowest degradation of PAH compounds was supported by the lowest growth of H. palleronii. Bacteria showed lower growth and degradation with PAH compounds due to their complex nature and presence of more than one phenyl rings in their structure. Xin et al., (2010) reported that H. palleronii LHJ38 can grow on naphthalene as sole carbon and energy source, and the naphthalene (2 g/L) can be degraded completely with in 132 h [11]. 4. Conclusions The bacterial strains H. palleronii were effective in the degradation of phenol, and some of the alkylphenols. But the bacteria were unable to degrade PAH compounds like naphthalene and phenanthrene. Our results indicate that the bacteria have potential for use in situ bioremediation of phenol and alkylphenols contaminated soils or wastewater treatment plant. However, further research is necessary to understand the metabolites formed during the degradation process, and types of genes involved in the bioremediation process. Acknowledgements This work was supported by a Grant-in-Aid for Scientific Research (No. 26340067) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. References [1] Krastanov, A., Alexieva, Z., Yemendzhiev, H., 2013, Microbial degradation of phenol and phenolic derivatives: Engineering in Life Sciences, 13, 76-87. [2] Jiang, L., Ruan, Q., Li, R., Li, T., 2013, Biodegradation of phenol by using free and immobilized cells of Acinetobacter sp. BS8Y: Journal of Basic Microbiology, 53, 224-230. [3] Lu, D., Zhang, Y., Niu, S., Wang, L., Lin, S., Wang, C., Ye, W., Yan, C., 2012, Study of phenol biodegradation using Bacillus amyloliquefaciens strain WJDB-1 immobilized in alginatechitosan-alginate (ACA) microcapsules by electrochemical method: Biodegradation, 23, 209-219. [4] Takeo, M., Prabu, S.K., Kitamura, C., Hirai, M., Takahashi, H., Kato, D., Negoro, S., 2006, Characterization of alkylphenol degradation gene cluster in Pseudomonas putida MT4 and evidence of oxidation of alkylphenols and alkylcatechols with medium-length alkyl chain: Journal of Bioscience and Bioengineering, 102, 352–361. [5] Elahwany, A.M.D., Mabrouk, M.E.M., 2013, Isolation and characterization of a phenol degrading strain of Alcaligenes sp. AM4: Acta Biologica Hungarica, 64, 106-117. [6] Lobo, C.C., Bertola, N.C., Contreras, E.M., 2013, Stoichiometry and kinetic of the aerobic oxidation of phenolic compounds by activated sludge: Bioresource Technology, 136, 58-65. [7] Park, M.R., Kim, D.J., Choi, J.W., Lim, D.S., 2013, Influence of immobilization of bacterial cells and TiO2 on phenol degradation: Water, Air & Soil Pollution, 224, 1-9. [8] Chang, Y.C., Takada, K., Choi, D.B., Toyama, T., Sawada, K., Kikuchi, S., 2013, Isolation of biphenyl and polychlorinated biphenyl-degrading bacteria and their degradation pathway: Applied Biochemistry and Biotechnology, 170, 381–398.

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