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Enhancement of phenol biodegradation by Ochrobactrum sp. isolated from industrial wastewaters
International Biodeterioration & Biodegradation 63 (2009) 778–781
Contents lists available at ScienceDirect
International Biodeterioration & Biodegradation journal homepage: www.elsevier.com/locate/ibiod
Enhancement of phenol biodegradation by Ochrobactrum sp. isolated from industrial wastewaters Nur Koçberber Kılıç* Department of Biology, Science Faculty, University of Ankara, 06100 Bes¸ evler, Ankara, Turkey
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 May 2009 Received in revised form 12 June 2009 Accepted 15 June 2009 Available online 5 July 2009
Ochrobactrum sp., was tested with regard to its phenol degradation capacity at different pH levels, and with different carbon sources (mineral salt medium with glucose (MSG) and the same medium with 0.5%, 1%, and 2% (v/v) molasses (MSM)) and phenol concentrations. The highest degradation was in mineral salt medium with 1% (v/v) molasses (45.9%), while degradation was 21.1% in mineral salt medium with 5 g l1 glucose. These data show that the addition of molasses to mineral salt medium enhanced phenol degradation by Ochrobactrum sp. The bacterium can be used effectively to treat wastewaters containing phenol. Ó 2009 Elsevier Ltd. All rights reserved.
Keywords: Degradation Phenol Ochrobactrum sp. Molasses
1. Introduction Phenol is used in many industries such as in the production of polycarbonate resins, paints, explosives, inks, perfumes, textiles, and antibacterial agents. All industries producing or using phenol discharge this pollutant into the environment. Phenolic compounds are toxic and carcinogenic by ingestion, contact, or inhalation, and they have high stability (Van Schie and Young, 2000; Yang and Lee, 2007; Saravanan et al., 2008). Because of the aromatic structure of phenol, it is resistant to natural biodegradation. However, several microorganisms can tolerate phenol and use it as a carbon and energy source (Van Schie and Young, 2000). Aerobic biodegradation of phenol by microorganisms is now well established. During the first step of aerobic degradation, molecular oxygen is used by phenol hydroxylase to form catechol. Catechol can then be degraded via two pathways (ortho- or meta- pathways). In the ortho- pathway, the aromatic ring is cleaved between the catechol hydroxyls by a catechol 1,2-dioxygenase forming 2-hydroxymuconic semialdehyde. The resulting product is metabolized further to Krebs cycle intermediates. In the meta- pathway, ring fission occurs adjacent to the two hydroxyl groups of catechol. The enzyme catechol 2,3-dioxygenase transforms catechol to 2-hydroxymuconic semialdehyde, which is metabolized further to intermediates of the Krebs cycle (Van Schie and Young, 2000).
* Tel.: þ90 312 212 67 20; fax: þ90 312 223 23 95. E-mail address: [email protected] 0964-8305/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibiod.2009.06.006
Degradation of phenol and phenolic compounds has been studied with mainly Pseudomonas species (Powlowski and Shingler, 1994; Gonza´les et al., 2001; Kumar et al., 2005; Kulkarni and Chaudhari, 2006; Afzal et al., 2007; Yang and Lee, 2007; Kotresha and Vidyasagar, 2008; Saravanan et al., 2008). Some other microorganisms like Phanerochaete chrysosporium (Pe´rez et al., 1997), Rhodococcus spp. (Margesin et al., 2005), Candida tropicalis (Yan et al., 2006), Candida sp. (Hu et al., 2006), Bacillus brevis (Arutchelvan et al., 2006), Penicillium chrysogenum (Leita˜o et al., 2007), and Alcaligenes sp. (Nair et al., 2007) also degraded phenol and phenolic compounds. It was reported the isolation and characterization of the bacterium Ochrobactrum sp. (Kılıç et al., 2007). It has been reported that Ochrobactrum species metabolize phenol through catechol, followed by ortho- or meta- pathways (Lechner et al., 1995; El-Sayed et al., 2003), but little information is available about phenol degradation by Ochrobactrum sp. There is no report investigating phenol degradation capacity by Ochrobactrum sp. and the conditions affecting phenol degradation of Ochrobactrum sp. Furthermore, some other phenolic compounds like 4-nitrocatechol (Zhong et al., 2007), p-nitrophenol (Qiu et al., 2007), and 2,4,6tribromophenol (Yamada et al., 2008) were metabolized by Ochrobactrum species. The aim of the study was to examine the biodegradation capacity of Ochrobactrum sp. Different pH levels, carbon sources, and initial phenol concentrations were used to find the highest phenol degradation by Ochrobactrum sp. To improve the phenol degradation capacity of the bacterium, molasses was chosen as an additional carbon source besides phenol due to its low
N.K. Kılıç / International Biodeterioration & Biodegradation 63 (2009) 778–781
cost, ready availability, and ease of storage. The major objective of this study was to investigate the potential of using Ochrobactrum sp. to treat industrial wastewaters containing phenol. 2. Materials and methods Phenol was chromatographic grade purchased from Riedel-de Hae¨n (Germany); glucose and inorganic salts from Merck were used in preparing the microbial growth media. The bacterium used in this study was previously isolated and identified by Kılıç et al. (2007). The culture was cultivated at 100 rpm and 30 C for 144 h in a 250 ml flask containing 100 ml of mineral salt (MS) medium in a rotary shaker. The MS medium is composed of (in g l1) KH2PO4, 1.7; (NH4)2SO4, 2.69; MgSO4, 0.2; and CaCI2, 0.03, at pH 7 (Afzal et al., 2007). In the first series of the experiments MS medium with 5 g l1 glucose (MSG) was used, and in the second series MS medium with increasing concentrations of molasses (MSM). Beet molasses solution was approximately equivalent to 10 g l1 sucrose. Control flasks containing phenol without inoculation of culture were also prepared to observe any reactions between media and phenol. To determine the optimum pH for the highest phenol degradation, the pH of the media including approximately 150 mg l1 phenol was adjusted to 6, 7, 8, and 9 with 0.1 M NaOH and 0.1 M HCI. Autoclaved phenol solution and glucose were added directly to the autoclaved MS media to obtain the desired phenol concentration. To determine the effect of carbon sources on phenol degradation, 4 different MS media at predetermined pH (pH 8) and including 50 mg l1 phenol were prepared. In the first MS medium, glucose (5 g l1) was used as a carbon source in addition to phenol. In the other media, in addition to phenol, increasing molasses concentrations (0.5%, 1%, and 2% v/v) were added. The effect of initial phenol concentration on phenol degradation by Ochrobactrum sp. was determined in another series of the experiments. MSG (pH 8; 5 g l1 glucose) with 50–400 mg l1 phenol and MSM (pH 8; 1% (v/v) molasses) including 50–400 mg l1 phenol were used in this series of experiments. The flasks were prepared in three replicates. A 4-ml sample was taken during the incubation period from each flask. Samples were centrifuged at 3421 g for 10 min to remove suspended biomass and the concentration of phenol in the supernatant was determined by high performance liquid chromatography (Shimadzu, Japan), using a C-18 column (250 mm 4.6 mm inner diameter: 5 mm particle size). The mobile phase was acetonitrile:water (60:40 v/v) pumped at 1 ml/min, and the detection was performed with a UV detector set at 275 nm. For the measurement of microbial growth, the biomass concentration was determined by measuring the turbidity of the diluted sample at 540 nm and using a standard curve of absorbance against dry cell mass. Absorbance measurements and centrifugation were performed using a Shimadzu UV 2001 model spectrophotometer (Japan) and Hettich EBA12 model centrifuge (Germany). The experiments were set up in a completely randomized design with three replicates. The data were subjected to analysis of variance using Minitab 14 and significant differences among treatment means were compared by descriptive statistics (S.E.).
Degradation % ¼
h
. i Co 100 Co Cf
779
(1)
Phenol degradation capacity is the concentration of phenol in the biomass and can be calculated based on the mass balance principle from equation (2):
qm ¼
Co Cf
. Xm
(2)
In these two equations, qm (the maximum specific phenol degradation) represents the maximum amount of phenol per unit dry weight of microbial cells (mg g1), Xm maximum dried cell mass (g l1), and C0 and Cf the initial and final concentrations (mg l1), respectively. The effect of the pH value of the media on phenol degradation was determined in samples including approximately 150 mg l1 initial phenol concentration at the end of 144 h of incubation (Fig. 1). Four different pH values (6, 7, 8, and 9) were initially tested to find an appropriate pH for the highest phenol degradation by Ochrobactrum sp. The highest phenol degradation by Ochrobactrum sp. increased with an increase in alkalinity. At pH 6, the bacterium had a yield of phenol degradation of 8.3%, while it was 10.9% at pH 7. The bacterium had the highest yield at pH 8 (18.8%). Previous studies performed with several phenol degrading microorganisms also reported that phenol degradation was achieved more efficiently at alkaline pH levels (Kulkarni and Chaudhari, 2006; Kotresha and Vidyasagar, 2008). Kulkarni and Chaudhari (2006) showed that Pseudomonas putida tolerated 300 mg l1 p-nitrophenol at pH 7.5–9.5. Kotresha and Vidyasagar (2008) observed that the optimum pH was 7 for Pseudomonas aeruginosa MTCC 4996 to degrade 500 mg l1 phenol. Another study performed with p-nitrophenol degrading Ochrobactrum sp. showed that at alkaline pH levels (pH 10) bacteria depleted applied p-nitrophenol after incubation for 8 h (Qiu et al., 2007). At the end of this series of experiments, the maximum phenol degradation by the microorganism occurred at pH 8, whereas the lowest removal of the pollutant was at pH 9 (3.9%). Ochrobactrum sp. was affected negatively in media with a pH level of 9 and grew less at that pH than at other levels. To investigate the effect of different carbon sources on phenol degradation by Ochrobactrum sp., bacteria were grown on 4 different MS media (pH 8) with 50 mg l1 phenol for 144 h (Fig. 2). MSG (with 5 g l1 glucose) with 50 mg l1 phenol and MSM prepared with 0.5%, 1%, and 2% (v/v) molasses and 50 mg l1 phenols were used. Ochrobactrum sp. caused the highest phenol degradation in MSM. In addition, after adding molasses to MS medium, there was
3. Results and discussion Phenol degradation by Ochrobactrum sp. was investigated as a function of initial pH, different carbon sources, and initial phenol concentrations. The percentage degradation of phenol was calculated from equation (1):
Fig. 1. Effect of initial pH on phenol degradation by Ochrobactrum sp. in MSG with 150 mg l1 phenol (T: 30 C; incubation period: 144 h).
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N.K. Kılıç / International Biodeterioration & Biodegradation 63 (2009) 778–781
Fig. 2. Effect of different carbon sources on phenol degradation by Ochrobactrum sp. (phenol concentration: 50 mg l1; T: 30 C; incubation period: 144 h). Fig. 3. Phenol degradation capacity of Ochrobactrum sp. in MSM with 1% (v/v) molasses during the incubation period (T: 30 C).
a significant increase in the degradation of phenol by the bacterium compared to degradation in MSG. In MSG, the microorganism was capable of degrading phenol with a yield of 21.1%. However, with the addition of 0.5% (v/v) molasses instead of glucose to MS medium, degradation of the pollutant increased 1.5 times and was 31.6%. In MS media with 1% (v/v) molasses and 2% (v/v) molasses there was no distinctive difference between the phenol degradation yields of the bacterium. In media with 1% (v/v) molasses the bacterium degraded phenol with a yield of 45.9%, while degradation was 44.7% in media with 2% (v/v) molasses. Therefore, other trials were performed with media including 1% (v/v) molasses. After these experiments, trials were performed in MSM (1% (v/v) molasses) in which Ochrobactrum sp. had the highest phenol degradation capacity. To compare the pollutant degradation yield by Ochrobactrum sp., experiments were also done with MSG. Loh and Wang (1998) reported that additional carbon sources (i.e. glucose) in media support growth and may stimulate the viability of cells and enhance phenol degradation. On the other hand, according to Kulkarni and Chaudhari (2006), at low concentrations of p-nitrophenol, in the presence of glucose 20 and 50 mg l1 p-nitrophenol was rapidly degraded. In the present study, the microorganism did not grow in MS medium with only phenol, but by adding glucose to the medium Ochrobactrum sp. could grow in MS medium including the pollutant and metabolized it. Moreover, when molasses was added to MS medium, the bacterium tolerated much more phenol compared to MSG. The phenol degradation capacity of Ochrobactrum sp. at different phenol concentrations (50, 100, 200, 300, and 400 mg l1) in 2 different media (MSG and MSM) during the incubation period is shown in Table 1 and Fig. 3. The degradation yield decreased with an increase in phenol concentration in MSG. Under these conditions, the maximum yield was 21.1% at 64.0 mg l1 phenol concentration at the end of the incubation period. Ochrobactrum sp. could tolerate phenol up to 304.9 mg l1. Above this concentration the bacterium was affected negatively by the toxic pollutant and could not grow in media with 400.0 mg l1 phenol (Table 1).
In MSM, the maximum phenol degradation was 45.9% at 62.2 mg l1 phenol concentration after incubation for 144 h (Fig. 3). In this medium, the degradation of phenol was rapid at the end of 48 h and the yield was 34.2%. After incubation for 96 h the highest degradation yield was 45.2% by Ochrobactrum sp. At 102.4 mg l1 phenol concentration, the degradation decreased to 23.1% compared to medium including 62.2 mg l1 phenol. However, the phenol removal capacity of the microorganism increased after incubation for 144 h and was 42.5%. In MSM with 219.4 mg l1 phenol, degradation of the pollutant decreased to 8.5% at the end of 48 h of incubation. The microorganism degraded the pollutant with the highest yield, 20.9%, at the end of the incubation period. In MSM including 316.2 mg l1 phenol concentration, Ochrobactrum sp. degraded 20.1% of the applied phenol at the end of the incubation period. The bacterium metabolized 8.0% of the applied phenol after incubation for 48 h and 15.0% after incubation for 96 h in this medium. In contrast to MSG, in MSM with the highest concentration of phenol (405.0 mg l1), Ochrobactrum sp. could tolerate the pollutant and grow. Similar to the other concentrations of phenol, the bacterium had the lowest removal yield after incubation for 48 h, i.e. 5.6%. The highest removal (14.6%) was observed at the end of the incubation period. Comparison of the maximum specific phenol degradation by Ochrobactrum sp. in MSG and MSM with increasing concentration
Table 1 Phenol degradation capacity by Ochrobactrum sp. in MSG (Glucose concentration: 5 g l1; T: 30 C, Incubation period: 144 h). Phenol concentration (mg l1)
Phenol degradation (%)
64.0 111.9 202.9 304.9 400.0
21.1 19.0 17.8 9.6 –
0.8 3.0 0.3 1.4
Fig. 4. Comparison of maximum specific phenol degradation by Ochrobactrum sp. in MSG and MSM (T: 30 C; incubation period: 144 h).
N.K. Kılıç / International Biodeterioration & Biodegradation 63 (2009) 778–781
of phenol is summarized in Fig. 4. When the phenol concentration was increased in MSG, the maximum specific phenol degradation also increased, up to 300 mg l1 phenol concentration. In this medium, above 200 mg l1 phenol concentration, the maximum specific phenol degradation decreased according to the lower microbial growth compared to the other concentrations of the pollutant. The maximum specific phenol degradation was 30.1 mg g1 for nearly 200 mg l1 phenol in MSG. In that medium, the maximum specific phenol degradation values were 9.6 mg g1, 16.4 mg g1, and 28.3 mg g1 for approximately 50, 100, and 300 mg l1 phenol concentrations, respectively. Similar to MSG, in MSM, the maximum specific phenol degradation increased with the level of phenol and then decreased. When the phenol concentration was increased in this media, the maximum specific phenol degradation also increased, up to 400 mg l1 phenol concentration. The maximum specific phenol degradation was 50.4 mg g1 for approximately 300 mg l1 phenol concentration. Above 300 mg l1 phenol, the maximum specific phenol degradation was 44.5 mg g1 at 405.0 mg l1 phenol. On the other hand, at the lowest phenol concentration (50 mg l1) the maximum specific phenol degradation was 20.4 mg g1, whereas in MSG it was 9.6 mg g1 at the same phenol concentration. In MSM with 100 mg l1 phenol the maximum specific phenol degradation was 36.3 mg g1, while it was 38.2 mg g1 in the same medium with 200 mg l1 phenol concentration. In trials performed with MS medium including 2 different carbon sources and increasing phenol concentrations, the MSM was more efficient than MSG in the biodegradation process according to the higher maximum specific phenol degradation by Ochrobactrum sp. El-Sayed et al. (2003) previously reported that Ochrobactrum sp. has phenol-degrading activity in MS medium. In that study, the microorganism metabolized all the applied phenol (100 mg l1) after incubation for 30 h. In the present report, degradation capacity was 21.5% in the same medium at the same phenol concentration. However, by adding molasses (1% v/v) to MS medium, the biodegradation capacity of the bacteria was enhanced and was 42.5% with the maximum specific degradation of 36.3 mg g1 at 100 mg l1 phenol concentration at the end of the incubation period. Moreover, the highest maximum specific degradation by Ochrobactrum sp. was 50.4 mg phenol per gram of the microbial biomass at 316 mg l1 phenol after incubation for 144 h. 4. Conclusions For the highest phenol degradation by Ochrobactrum sp. the optimum pH level was 8. In control flasks (media without inoculation, but with phenol) there was no reaction between phenol and media. This result proved that, phenol was degraded only by the bacterium tested in the study. On the other hand, phenol degradation of the microorganism was greatly affected by the addition of different carbon sources. The bacterium had the highest phenol degradation yield in media with 1% (v/v) molasses, while the lowest yield was found in media with 5 g l1 glucose at 50 mg l1 phenol concentration. Ochrobactrum sp. tolerated high concentrations of phenol and had the maximum specific phenol degradation (50.4 mg g1) in media with 1% (v/v) molasses at 316.2 mg l1 phenol. These data indicated that Ochrobactrum sp. has the potential to be used to remove phenol by biodegradation from wastewaters at alkaline pH levels in a safe, economic, and effective way.
781
Acknowledgements Financial support was provided by the Ankara University Research Found.
References Afzal, M., Iqbal, S., Rauf, S., Khalid, Z.M., 2007. Characteristics of phenol biodegradation in saline solutions by monocultures of Pseudomonas aeruginosa and Pseudomonas pseudomallei. Journal of Hazardous Materials 149, 60–66. Arutchelvan, V., Kanakasabai, V., Elangovan, R., Nagarajan, S., Muralikrishnan, V., 2006. Kinetics of high strength phenol degradation using Bacillus brevis. Journal of Hazardous Materials B129, 216–222. El-Sayed, W., Ibrahim, M.K., Abu-Shady, M., El-Beih, F., Ohmura, N., Saiki, H., Ando, A., 2003. Isolation and identification of a novel strain of the genus Ochrobactrum with phenol-degrading activity. Journal of Bioscience Bioengineering 96 (3), 310–312. ˜ a, M., 2001. Biodegradation of phenolic Gonza´les, G., Herrera, G., Garcı´a, MaT., Pen industrial wastewater in a fluidized bed bioreactor with immobilized cells of Pseudomonas putida. Bioresource Technology 80, 137–142. Hu, Z., Wu, Y.R., Lin, B.K., Maskaoui, K., Zhuang, D.H., Zheng, T.L., 2006. Isolation and characterization of two phenol-degrading yeast strains from marine sediment. Bulletin of Environmental Contamination and Toxicology 76, 899–906. Kılıç, N.K., Nielsen, J.L., Yu¨ce, M., Do¨nmez, G., 2007. Characterization of a simple bacterial consortium for effective treatment of wastewaters with reactive dyes and Cr(VI). Chemosphere 67, 826–831. Kotresha, D., Vidyasagar, G.M., 2008. Isolation and characterisation of phenol degrading Pseudomonas aeruginosa MTCC 4996. World Journal of Microbiology and Biotechnology 24, 541–547. Kulkarni, M., Chaudhari, A., 2006. Biodegradation of p-nitrophenol by P. putida. Bioresource Technology 97, 982–988. Kumar, A., Kumar, S., Kumar, S., 2005. Biodegradation kinetics of phenol and catechol using Pseudomonas putida MTCC 1194. Biochemical Engineering Journal 22, 151–159. Lechner, U., Baumbach, R., Becker, D., Kitunen, V., Aulin, G., Salkinoja-Salonen, M., 1995. Degradation of 4-chloro-2-methylphenol by an activated sludge isolate and its taxonomic description. Biodegradation 6, 83–92. Leita˜o, A.L., Duarte, M.P., Santos Oliveira, J., 2007. Degradation of phenol by a halotolerant strain of Penicillium chrysogenum. International Biodeterioration and Biodegradation 59, 220–225. Loh, K., Wang, S., 1998. Enhancement of biodegradation of phenol and a nongrowth substrate 4-chlorophenol by medium augmentation with conventional carbon sources. Biodegradation 8, 329–338. Margesin, R., Fonteyne, P.-A., Redl, B., 2005. Low-temperature biodegradation of high amounts of phenol by Rhodococcus spp. and basidiomycetous yeasts. Research in Microbiology 156, 68–75. Nair, I.C., Jayachandran, K., Shashidhar, S., 2007. Treatment of paper factory effluent using a phenol degrading Alcaligenes sp. under free and immobilized conditions. Bioresource Technology 98, 714–716. ´ Perez, R.R., Benito, G.G., Miranda, P., 1997. Chlorophenol degradation by Phanerochaete chrysosporium. Bioresource Technology 60, 207–213. Powlowski, J., Shingler, V., 1994. Genetics and biochemistry of phenol degradation by Pseudomonas sp. CF600. Biodegradation 5, 219–236. Qiu, X., Zhong, Q., Li, M., Bai, W., Li, B., 2007. Biodegradation of p-nitrophenol by methyl parathion-degrading Ochrobactrum sp. B2. International Biodeterioration and Biodegredation 59, 297–330. Saravanan, P., Pakshirajan, K., Saha, P., 2008. Biodegradation of phenol and m-cresol in a batch and fed batch operated internal loop airlift bioreactor by indigenous mixed microbial culture predominantly Pseudomonas sp. Bioresource Technology 99, 8553–8558. Van Schie, P.M., Young, L.Y., 2000. Biodegradation of phenol: mechanisms and applications. Bioremediation Journal 4 (1), 1–18. Yamada, T., Takahama, Y., Yamada, Y., 2008. Biodegradation of 2,4,6-tribromophenol by Ochrobactrum sp. strain TB01. Bioscience Biotechnology and Biochemistry 72 (5), 1264–1271. Yan, J., Jianping, W., Jing, B., Daoquan, W., Zongding, H., 2006. Phenol biodegradation by the yeast Candida tropicalis in the presence of m-cresol. Biochemical Engineering Journal 29, 227–234. Yang, C., Lee, C., 2007. Enrichment, isolation, and characterization of phenol degrading Pseudomonas resinovorans strain P-1 and Brevibacillus sp. strain P-6. International Biodeterioration and Biodegradation 59, 206–210. Zhong, Q., Zhang, H., Bai, W., Li, M., Li, B., Qiu, X., 2007. Degradation of aromatic compounds and degradative pathway of 4-nitrocatechol by Ochrobactrum sp. B2. Journal of Environmental Science and Health Part A 42, 2111–2116.
Contents lists available at ScienceDirect
International Biodeterioration & Biodegradation journal homepage: www.elsevier.com/locate/ibiod
Enhancement of phenol biodegradation by Ochrobactrum sp. isolated from industrial wastewaters Nur Koçberber Kılıç* Department of Biology, Science Faculty, University of Ankara, 06100 Bes¸ evler, Ankara, Turkey
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 May 2009 Received in revised form 12 June 2009 Accepted 15 June 2009 Available online 5 July 2009
Ochrobactrum sp., was tested with regard to its phenol degradation capacity at different pH levels, and with different carbon sources (mineral salt medium with glucose (MSG) and the same medium with 0.5%, 1%, and 2% (v/v) molasses (MSM)) and phenol concentrations. The highest degradation was in mineral salt medium with 1% (v/v) molasses (45.9%), while degradation was 21.1% in mineral salt medium with 5 g l1 glucose. These data show that the addition of molasses to mineral salt medium enhanced phenol degradation by Ochrobactrum sp. The bacterium can be used effectively to treat wastewaters containing phenol. Ó 2009 Elsevier Ltd. All rights reserved.
Keywords: Degradation Phenol Ochrobactrum sp. Molasses
1. Introduction Phenol is used in many industries such as in the production of polycarbonate resins, paints, explosives, inks, perfumes, textiles, and antibacterial agents. All industries producing or using phenol discharge this pollutant into the environment. Phenolic compounds are toxic and carcinogenic by ingestion, contact, or inhalation, and they have high stability (Van Schie and Young, 2000; Yang and Lee, 2007; Saravanan et al., 2008). Because of the aromatic structure of phenol, it is resistant to natural biodegradation. However, several microorganisms can tolerate phenol and use it as a carbon and energy source (Van Schie and Young, 2000). Aerobic biodegradation of phenol by microorganisms is now well established. During the first step of aerobic degradation, molecular oxygen is used by phenol hydroxylase to form catechol. Catechol can then be degraded via two pathways (ortho- or meta- pathways). In the ortho- pathway, the aromatic ring is cleaved between the catechol hydroxyls by a catechol 1,2-dioxygenase forming 2-hydroxymuconic semialdehyde. The resulting product is metabolized further to Krebs cycle intermediates. In the meta- pathway, ring fission occurs adjacent to the two hydroxyl groups of catechol. The enzyme catechol 2,3-dioxygenase transforms catechol to 2-hydroxymuconic semialdehyde, which is metabolized further to intermediates of the Krebs cycle (Van Schie and Young, 2000).
* Tel.: þ90 312 212 67 20; fax: þ90 312 223 23 95. E-mail address: [email protected] 0964-8305/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibiod.2009.06.006
Degradation of phenol and phenolic compounds has been studied with mainly Pseudomonas species (Powlowski and Shingler, 1994; Gonza´les et al., 2001; Kumar et al., 2005; Kulkarni and Chaudhari, 2006; Afzal et al., 2007; Yang and Lee, 2007; Kotresha and Vidyasagar, 2008; Saravanan et al., 2008). Some other microorganisms like Phanerochaete chrysosporium (Pe´rez et al., 1997), Rhodococcus spp. (Margesin et al., 2005), Candida tropicalis (Yan et al., 2006), Candida sp. (Hu et al., 2006), Bacillus brevis (Arutchelvan et al., 2006), Penicillium chrysogenum (Leita˜o et al., 2007), and Alcaligenes sp. (Nair et al., 2007) also degraded phenol and phenolic compounds. It was reported the isolation and characterization of the bacterium Ochrobactrum sp. (Kılıç et al., 2007). It has been reported that Ochrobactrum species metabolize phenol through catechol, followed by ortho- or meta- pathways (Lechner et al., 1995; El-Sayed et al., 2003), but little information is available about phenol degradation by Ochrobactrum sp. There is no report investigating phenol degradation capacity by Ochrobactrum sp. and the conditions affecting phenol degradation of Ochrobactrum sp. Furthermore, some other phenolic compounds like 4-nitrocatechol (Zhong et al., 2007), p-nitrophenol (Qiu et al., 2007), and 2,4,6tribromophenol (Yamada et al., 2008) were metabolized by Ochrobactrum species. The aim of the study was to examine the biodegradation capacity of Ochrobactrum sp. Different pH levels, carbon sources, and initial phenol concentrations were used to find the highest phenol degradation by Ochrobactrum sp. To improve the phenol degradation capacity of the bacterium, molasses was chosen as an additional carbon source besides phenol due to its low
N.K. Kılıç / International Biodeterioration & Biodegradation 63 (2009) 778–781
cost, ready availability, and ease of storage. The major objective of this study was to investigate the potential of using Ochrobactrum sp. to treat industrial wastewaters containing phenol. 2. Materials and methods Phenol was chromatographic grade purchased from Riedel-de Hae¨n (Germany); glucose and inorganic salts from Merck were used in preparing the microbial growth media. The bacterium used in this study was previously isolated and identified by Kılıç et al. (2007). The culture was cultivated at 100 rpm and 30 C for 144 h in a 250 ml flask containing 100 ml of mineral salt (MS) medium in a rotary shaker. The MS medium is composed of (in g l1) KH2PO4, 1.7; (NH4)2SO4, 2.69; MgSO4, 0.2; and CaCI2, 0.03, at pH 7 (Afzal et al., 2007). In the first series of the experiments MS medium with 5 g l1 glucose (MSG) was used, and in the second series MS medium with increasing concentrations of molasses (MSM). Beet molasses solution was approximately equivalent to 10 g l1 sucrose. Control flasks containing phenol without inoculation of culture were also prepared to observe any reactions between media and phenol. To determine the optimum pH for the highest phenol degradation, the pH of the media including approximately 150 mg l1 phenol was adjusted to 6, 7, 8, and 9 with 0.1 M NaOH and 0.1 M HCI. Autoclaved phenol solution and glucose were added directly to the autoclaved MS media to obtain the desired phenol concentration. To determine the effect of carbon sources on phenol degradation, 4 different MS media at predetermined pH (pH 8) and including 50 mg l1 phenol were prepared. In the first MS medium, glucose (5 g l1) was used as a carbon source in addition to phenol. In the other media, in addition to phenol, increasing molasses concentrations (0.5%, 1%, and 2% v/v) were added. The effect of initial phenol concentration on phenol degradation by Ochrobactrum sp. was determined in another series of the experiments. MSG (pH 8; 5 g l1 glucose) with 50–400 mg l1 phenol and MSM (pH 8; 1% (v/v) molasses) including 50–400 mg l1 phenol were used in this series of experiments. The flasks were prepared in three replicates. A 4-ml sample was taken during the incubation period from each flask. Samples were centrifuged at 3421 g for 10 min to remove suspended biomass and the concentration of phenol in the supernatant was determined by high performance liquid chromatography (Shimadzu, Japan), using a C-18 column (250 mm 4.6 mm inner diameter: 5 mm particle size). The mobile phase was acetonitrile:water (60:40 v/v) pumped at 1 ml/min, and the detection was performed with a UV detector set at 275 nm. For the measurement of microbial growth, the biomass concentration was determined by measuring the turbidity of the diluted sample at 540 nm and using a standard curve of absorbance against dry cell mass. Absorbance measurements and centrifugation were performed using a Shimadzu UV 2001 model spectrophotometer (Japan) and Hettich EBA12 model centrifuge (Germany). The experiments were set up in a completely randomized design with three replicates. The data were subjected to analysis of variance using Minitab 14 and significant differences among treatment means were compared by descriptive statistics (S.E.).
Degradation % ¼
h
. i Co 100 Co Cf
779
(1)
Phenol degradation capacity is the concentration of phenol in the biomass and can be calculated based on the mass balance principle from equation (2):
qm ¼
Co Cf
. Xm
(2)
In these two equations, qm (the maximum specific phenol degradation) represents the maximum amount of phenol per unit dry weight of microbial cells (mg g1), Xm maximum dried cell mass (g l1), and C0 and Cf the initial and final concentrations (mg l1), respectively. The effect of the pH value of the media on phenol degradation was determined in samples including approximately 150 mg l1 initial phenol concentration at the end of 144 h of incubation (Fig. 1). Four different pH values (6, 7, 8, and 9) were initially tested to find an appropriate pH for the highest phenol degradation by Ochrobactrum sp. The highest phenol degradation by Ochrobactrum sp. increased with an increase in alkalinity. At pH 6, the bacterium had a yield of phenol degradation of 8.3%, while it was 10.9% at pH 7. The bacterium had the highest yield at pH 8 (18.8%). Previous studies performed with several phenol degrading microorganisms also reported that phenol degradation was achieved more efficiently at alkaline pH levels (Kulkarni and Chaudhari, 2006; Kotresha and Vidyasagar, 2008). Kulkarni and Chaudhari (2006) showed that Pseudomonas putida tolerated 300 mg l1 p-nitrophenol at pH 7.5–9.5. Kotresha and Vidyasagar (2008) observed that the optimum pH was 7 for Pseudomonas aeruginosa MTCC 4996 to degrade 500 mg l1 phenol. Another study performed with p-nitrophenol degrading Ochrobactrum sp. showed that at alkaline pH levels (pH 10) bacteria depleted applied p-nitrophenol after incubation for 8 h (Qiu et al., 2007). At the end of this series of experiments, the maximum phenol degradation by the microorganism occurred at pH 8, whereas the lowest removal of the pollutant was at pH 9 (3.9%). Ochrobactrum sp. was affected negatively in media with a pH level of 9 and grew less at that pH than at other levels. To investigate the effect of different carbon sources on phenol degradation by Ochrobactrum sp., bacteria were grown on 4 different MS media (pH 8) with 50 mg l1 phenol for 144 h (Fig. 2). MSG (with 5 g l1 glucose) with 50 mg l1 phenol and MSM prepared with 0.5%, 1%, and 2% (v/v) molasses and 50 mg l1 phenols were used. Ochrobactrum sp. caused the highest phenol degradation in MSM. In addition, after adding molasses to MS medium, there was
3. Results and discussion Phenol degradation by Ochrobactrum sp. was investigated as a function of initial pH, different carbon sources, and initial phenol concentrations. The percentage degradation of phenol was calculated from equation (1):
Fig. 1. Effect of initial pH on phenol degradation by Ochrobactrum sp. in MSG with 150 mg l1 phenol (T: 30 C; incubation period: 144 h).
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Fig. 2. Effect of different carbon sources on phenol degradation by Ochrobactrum sp. (phenol concentration: 50 mg l1; T: 30 C; incubation period: 144 h). Fig. 3. Phenol degradation capacity of Ochrobactrum sp. in MSM with 1% (v/v) molasses during the incubation period (T: 30 C).
a significant increase in the degradation of phenol by the bacterium compared to degradation in MSG. In MSG, the microorganism was capable of degrading phenol with a yield of 21.1%. However, with the addition of 0.5% (v/v) molasses instead of glucose to MS medium, degradation of the pollutant increased 1.5 times and was 31.6%. In MS media with 1% (v/v) molasses and 2% (v/v) molasses there was no distinctive difference between the phenol degradation yields of the bacterium. In media with 1% (v/v) molasses the bacterium degraded phenol with a yield of 45.9%, while degradation was 44.7% in media with 2% (v/v) molasses. Therefore, other trials were performed with media including 1% (v/v) molasses. After these experiments, trials were performed in MSM (1% (v/v) molasses) in which Ochrobactrum sp. had the highest phenol degradation capacity. To compare the pollutant degradation yield by Ochrobactrum sp., experiments were also done with MSG. Loh and Wang (1998) reported that additional carbon sources (i.e. glucose) in media support growth and may stimulate the viability of cells and enhance phenol degradation. On the other hand, according to Kulkarni and Chaudhari (2006), at low concentrations of p-nitrophenol, in the presence of glucose 20 and 50 mg l1 p-nitrophenol was rapidly degraded. In the present study, the microorganism did not grow in MS medium with only phenol, but by adding glucose to the medium Ochrobactrum sp. could grow in MS medium including the pollutant and metabolized it. Moreover, when molasses was added to MS medium, the bacterium tolerated much more phenol compared to MSG. The phenol degradation capacity of Ochrobactrum sp. at different phenol concentrations (50, 100, 200, 300, and 400 mg l1) in 2 different media (MSG and MSM) during the incubation period is shown in Table 1 and Fig. 3. The degradation yield decreased with an increase in phenol concentration in MSG. Under these conditions, the maximum yield was 21.1% at 64.0 mg l1 phenol concentration at the end of the incubation period. Ochrobactrum sp. could tolerate phenol up to 304.9 mg l1. Above this concentration the bacterium was affected negatively by the toxic pollutant and could not grow in media with 400.0 mg l1 phenol (Table 1).
In MSM, the maximum phenol degradation was 45.9% at 62.2 mg l1 phenol concentration after incubation for 144 h (Fig. 3). In this medium, the degradation of phenol was rapid at the end of 48 h and the yield was 34.2%. After incubation for 96 h the highest degradation yield was 45.2% by Ochrobactrum sp. At 102.4 mg l1 phenol concentration, the degradation decreased to 23.1% compared to medium including 62.2 mg l1 phenol. However, the phenol removal capacity of the microorganism increased after incubation for 144 h and was 42.5%. In MSM with 219.4 mg l1 phenol, degradation of the pollutant decreased to 8.5% at the end of 48 h of incubation. The microorganism degraded the pollutant with the highest yield, 20.9%, at the end of the incubation period. In MSM including 316.2 mg l1 phenol concentration, Ochrobactrum sp. degraded 20.1% of the applied phenol at the end of the incubation period. The bacterium metabolized 8.0% of the applied phenol after incubation for 48 h and 15.0% after incubation for 96 h in this medium. In contrast to MSG, in MSM with the highest concentration of phenol (405.0 mg l1), Ochrobactrum sp. could tolerate the pollutant and grow. Similar to the other concentrations of phenol, the bacterium had the lowest removal yield after incubation for 48 h, i.e. 5.6%. The highest removal (14.6%) was observed at the end of the incubation period. Comparison of the maximum specific phenol degradation by Ochrobactrum sp. in MSG and MSM with increasing concentration
Table 1 Phenol degradation capacity by Ochrobactrum sp. in MSG (Glucose concentration: 5 g l1; T: 30 C, Incubation period: 144 h). Phenol concentration (mg l1)
Phenol degradation (%)
64.0 111.9 202.9 304.9 400.0
21.1 19.0 17.8 9.6 –
0.8 3.0 0.3 1.4
Fig. 4. Comparison of maximum specific phenol degradation by Ochrobactrum sp. in MSG and MSM (T: 30 C; incubation period: 144 h).
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of phenol is summarized in Fig. 4. When the phenol concentration was increased in MSG, the maximum specific phenol degradation also increased, up to 300 mg l1 phenol concentration. In this medium, above 200 mg l1 phenol concentration, the maximum specific phenol degradation decreased according to the lower microbial growth compared to the other concentrations of the pollutant. The maximum specific phenol degradation was 30.1 mg g1 for nearly 200 mg l1 phenol in MSG. In that medium, the maximum specific phenol degradation values were 9.6 mg g1, 16.4 mg g1, and 28.3 mg g1 for approximately 50, 100, and 300 mg l1 phenol concentrations, respectively. Similar to MSG, in MSM, the maximum specific phenol degradation increased with the level of phenol and then decreased. When the phenol concentration was increased in this media, the maximum specific phenol degradation also increased, up to 400 mg l1 phenol concentration. The maximum specific phenol degradation was 50.4 mg g1 for approximately 300 mg l1 phenol concentration. Above 300 mg l1 phenol, the maximum specific phenol degradation was 44.5 mg g1 at 405.0 mg l1 phenol. On the other hand, at the lowest phenol concentration (50 mg l1) the maximum specific phenol degradation was 20.4 mg g1, whereas in MSG it was 9.6 mg g1 at the same phenol concentration. In MSM with 100 mg l1 phenol the maximum specific phenol degradation was 36.3 mg g1, while it was 38.2 mg g1 in the same medium with 200 mg l1 phenol concentration. In trials performed with MS medium including 2 different carbon sources and increasing phenol concentrations, the MSM was more efficient than MSG in the biodegradation process according to the higher maximum specific phenol degradation by Ochrobactrum sp. El-Sayed et al. (2003) previously reported that Ochrobactrum sp. has phenol-degrading activity in MS medium. In that study, the microorganism metabolized all the applied phenol (100 mg l1) after incubation for 30 h. In the present report, degradation capacity was 21.5% in the same medium at the same phenol concentration. However, by adding molasses (1% v/v) to MS medium, the biodegradation capacity of the bacteria was enhanced and was 42.5% with the maximum specific degradation of 36.3 mg g1 at 100 mg l1 phenol concentration at the end of the incubation period. Moreover, the highest maximum specific degradation by Ochrobactrum sp. was 50.4 mg phenol per gram of the microbial biomass at 316 mg l1 phenol after incubation for 144 h. 4. Conclusions For the highest phenol degradation by Ochrobactrum sp. the optimum pH level was 8. In control flasks (media without inoculation, but with phenol) there was no reaction between phenol and media. This result proved that, phenol was degraded only by the bacterium tested in the study. On the other hand, phenol degradation of the microorganism was greatly affected by the addition of different carbon sources. The bacterium had the highest phenol degradation yield in media with 1% (v/v) molasses, while the lowest yield was found in media with 5 g l1 glucose at 50 mg l1 phenol concentration. Ochrobactrum sp. tolerated high concentrations of phenol and had the maximum specific phenol degradation (50.4 mg g1) in media with 1% (v/v) molasses at 316.2 mg l1 phenol. These data indicated that Ochrobactrum sp. has the potential to be used to remove phenol by biodegradation from wastewaters at alkaline pH levels in a safe, economic, and effective way.
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Acknowledgements Financial support was provided by the Ankara University Research Found.
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