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Biodegradation of phenanthrene with biosurfactant production by a new strain of Brevibacillus sp.
Bioresource Technology 101 (2010) 7980–7983
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Short Communication
Biodegradation of phenanthrene with biosurfactant production by a new strain of Brevibacillus sp. M. Srikanth Reddy, B. Naresh, T. Leela, M. Prashanthi, N.Ch. Madhusudhan, G. Dhanasri, Prathibha Devi * Biotechnology Laboratory, Department of Botany, Osmania University, Hyderabad 500007, India
a r t i c l e
i n f o
Article history: Received 10 December 2009 Received in revised form 3 April 2010 Accepted 7 April 2010 Available online 2 June 2010 Keywords: Biodegradation Phenanthrene Biosurfactant Brevibacillus sp.
a b s t r a c t In this work, a phenanthrene-degrading bacterial strain was isolated by enrichment method from hydrocarbon contaminated sludge samples and identified as Brevibacillus sp. PDM-3 based on morphological, biochemical, chemotaxonomic (FAMEs analysis) and molecular (16S rDNA sequencing) analysis. Growth parameters for efficient degradation of phenanthrene such as nutrient medium, pH, temperature, rpm and inoculum size were standardized and 93% of phenanthrene was degraded in 6 days as analysed by HPLC. The bacterial strain PDM-3 also has the ability to produce biosurfactant during phenanthrene degradation as detected by the surface tension measurements of the culture supernatant and the emulsification index (EI24). The biosurfactant was identified by its functional groups through FT-IR spectroscopy. Phenanthrene degradation and biosurfactant production are associated with each other and can be used in environmental biotechnology. Further, the strain has the ability to degrade other PAHs such as anthracene and fluorene by utilizing them as sole carbon and energy source. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Polyaromatic hydrocarbons (PAHs) are ubiquitous in the environment and comprise over 100 organic compounds containing two or more fused benzene and/or pentacyclic ring structures in linear, angular, or cluster arrangements (Guo et al., 2005). Bioremediation of PAHs depends on their bioavailability, as microorganisms can degrade only water soluble portion of the PAHs (Guo et al., 2005). Surfactants are amphiphilic compounds that can help release hydrocarbons sorbed to soil organic matter by solubilization and emulsification and increase the aqueous concentration of hydrophobic compounds, resulting in high mass transfer ratio (Gottfried et al., 2010). This study was undertaken to isolate and characterize potential PAHs degrading bacteria from contaminated sites through molecular methods for potential use in bioremediation. 2. Methods 2.1. Sampling Sludge samples were collected from three different sites of Hindustan Petrochemical Limited refinery (HPCL) located in Visakhapatnam, Andhra Pradesh, India in sterile plastic containers in April, 2007 and refrigerated for use to isolate bacterial strains. * Corresponding author. Tel.: +91 40 27423874; fax: +91 40 27090020. E-mail address: [email protected] (P. Devi). 0960-8524/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2010.04.054
2.2. Enrichment culture for isolation and screening of phenanthrenedegrading bacterial strains The petrochemical sludge samples were cultured on PAH enriched culture medium according to Kiyohara et al. (1982). Phenanthrene (250 mg/l), a three ring PAH with aqueous solubility of 1.29 mg/l was used as the sole carbon and energy source to enrich the mineral salts medium (MSM) (composition: KH2PO4 1.0 g, Na2HPO42H2O 1.25 g, (NH4)2SO4 1.0 g, MgSO47H2O 0.5 g, CaCl2 0.05 g and FeSO4 0.05 g per litre with the trace element solution comprising FeSO4 40 lg, MnSO4 40 lg, ZnSO4 20 lg, CuSO4 5 lg, CoCl2 4 lg, Na2MoO4 5 lg, CaCl2 0.5 lg, KH2PO4 136 lg and NaCl 1 mg) (1 gm of sludge sample in 50 ml of sterilized culture medium taken in a conical flask) and incubated at 30 °C on orbital shaker at 150 rpm for 7 days. A 2 ml of aliquot was transferred every week to fresh sterile medium and incubated under above conditions. One millilitre of the culture (after six transfers) was diluted with saline solution (0.9% NaCl) and plated on MSM agar plates over which, an ethereal solution of phenanthrene (250 mg/l) was uniformly sprayed wherein, the ether vaporizes leaving a thin-white layer of phenanthrene. The plates were incubated at 30 °C and after 3– 7 days, colonies of candidate phenanthrene-degrading strains were picked up on the basis of clear zones around them and further purified by repetitive streaking on fresh plates. The pure cultures of the final bacterial isolates were preserved under refrigeration on nutrient agar slants or as glycerol stocks at 20 °C.
M.S. Reddy et al. / Bioresource Technology 101 (2010) 7980–7983
2.3. Identification, morphological and biochemical characterization of the isolates The bacteria were identified by Bright field and Electron microscopy and the KB003 Hi25™ – Himedia bacterial identification kit was used for biochemical characterization through colorimetric identification. Antibiotic susceptibility was studied by nine types of antibiotic discs (Himedia) according to manufacturer’s instructions. For chemotaxonomic characterization, FAMEs analysis was performed by Gas–Liquid-Chromatography with the Sherlock MIS (Microbial Identification System, MIDI, Inc., Newark, Del) software. 2.4. Molecular characterization of the bacterial isolate Genomic DNA of the selected bacterial isolate was amplified by using the primers:16S1 (50 -GAGTTTGATCCTGGCTCA-30 ) and 16S2 (50 -CGGCTACCTTGTTACGACTT-30 ), which are complementary to the conserved regions at the 50 and 30 ends of the 16S rDNA corresponding to positions 9–27 and 1477–1498 of the Escherichia coli 16S rRNA gene. Amplified DNA fragments were separated by 1% (w/v) agarose gel electrophoresis. The separated bands were visualized under UV transillumination and then digitalized by Electrophoresis Documentation and Analysis System (Kodak DS, USA). The DNA fragment identified as 16S rDNA was eluted and purified using the clean Genei kit (Bangalore Genei) and sequenced with Gene Amp PCR machine (9600, Perkin–Elmer). The purified sequencing reaction mixtures were electrophoresed using ABI Prism Model 377 version 2.1.1 automatic DNA sequencer. The results were subjected to Bioinformatics analysis for identification of the isolates. 2.5. Biodegradation assay Biodegradation experiments were performed by inoculating exponential phase culture of the bacterial strain in MSM medium containing 250 mg/l phenanthrene and incubated under standard-
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ized conditions of temperature 35 °C, pH 7.0, rpm 175 and inoculum size of 5 vol. The amount of phenanthrene degraded was estimated every 12 h for 6 days with High Performance Liquid Chromatography (HPLC) (Story et al., 2004). The reverse phase HPLC of Shimadzu model LC-10AT chromatograph equipped with SPD-M20A prominence Photo Diode Array UV-vis detector set at 254 nm and a CBM-20A model prominence Communication Bus Module (CBM) provided with Class-VP software was used for degradation studies with an injection volume of 20 ll. Separation was achieved on Octyldecyl Silane Hypersil column (250 by 4.6 mm and 5-lm particle size) with a linear gradient of acetonitrile in 10 mM KH2PO4 buffer (pH 3.5) (0 min, 50:50, 0–30 min, ramp to 0:100 and 30–50 min isocratic at 0:100). The experiment was repeated twice. Peak area values obtained by the samples were compared with those of calibration curve with R2 value of 0.9907 and the rate of degradation calculated by using Class-VP software provided with the HPLC system. 2.6. Detection and identification of biosurfactant produced during biodegradation The biosurfactant produced by the bacterial isolate during biodegradation of phenanthrene was detected by measuring the surface tension (by K100 ring surface tensiometer of Kruss, Hamburg, Germany). Emulsification index of the culture supernatant which estimates the ability of biosurfactant to emulsify liquid hydrocarbons, such as kerosene or jojoba oil was determined by vortexing 6 ml of hydrocarbon and 4 ml of aqueous supernatant at high speed for 2 min. and incubated at 25 °C for 24 h. Emulsification index value (E24) was calculated as: E24 = (Height of emulsion layer/ Height of total mixture) 100. To purify the biosurfactant, the culture medium was separated from the bacterial cells by centrifugation and subjected thrice to solvent extraction (Rahman et al., 2003). The purified product was analysed by FT-IR spectroscopy for identification of functional groups by neat KBr (Potassium bromide) pellet method by placing
Fig. 1. Phylogenetic position of the strain PDM-3 among related bacteria.
M.S. Reddy et al. / Bioresource Technology 101 (2010) 7980–7983
In addition to phenanthrene, the bacterial strain was tested (by colorimetric measurement of turbidity at 600 nm) for growth on MSM medium containing other PAHs viz. anthracene and fluorene at a concentration of 250 mg/l.
3. Results and discussion 3.1. Isolation, identification and characterization of phenanthrenedegrading bacteria Eight bacterial strains were isolated by enrichment culture of sludge samples of which six were bacilli (75%) and two were cocci, out of which, one highly degrading bacillus strain was selected and characterized. Microscopic studies showed that the selected bacterial strain was Gram positive, motile, spore forming, aerobic and the cells are straight rods, occurring singly and in pairs. The strain showed positive oxidase activity, negative reactions for ONPG hydrolysis, methyl red and Voges Proskauer’s test and positive reduction of nitrate and utilization of citrate. Whereas, rhamnose, cellobiose and saccharose were utilized with acid production, it was resistant to Oxytetracycline, and Penicillin-G, and susceptible to Novobiocin, Nitrofurazone and Nalidixic acid. Further, the dominant fatty acids found in the bacterial strain were 15:0 iso (50.83%) and 15:0 anteiso (22.16%) (FAMEs analysis). The bacteria was identified as a species of Brevibacillus and placed in a systematic position according to Bergey’s manual of systematic bacteriology.
3.2. Molecular characterization of the bacterial strain and phylogenetic analysis Partial sequencing of the 1508 nucleotides length of 16S rDNA of the bacterial strain and phylogenetic analysis showed that it was closely related to Brevibacillus parabrevis with bootstrap value of 93 with a close relationship with different strains of their respective species groups with bootstrap values of more than 50 (Fig. 1). Distance matrix calculated for the strain with some of the most related strains indicates that the strain has 99% sequence similarity with B. parabrevis, which was earlier classified as Bacillus brevis based on 16S rRNA gene sequence and phylogenetic study (Takagi et al., 1993; Shida et al., 1996). Hence, the strain was identified as B. parabrevis and given the strain name of PDM-3. The bacterial strain PDM-3 was deposited in China Type Culture Collection Centre (CTCCC) with deposit number CCTCC AB 209087T and its 16S rDNA sequence was deposited in the GenBank database with the accession number FN185993.
3.3. Biodegradation assay The HPLC analysis showed that the rate of phenanthrene degradation was very low during the first 48 h but increased steadily and by 144 h (6 days), 93.92% of the phenanthrene was degraded (Fig. 2A). There was a perfect positive correlation between percentage of degradation and time, significant at the 0.01 level (2-tailed) according to the Pearson correlation of 0.985 (=1). The present rate of phenanthrene degradation is similar if not higher than earlier reports (Zhao et al., 2008, 2009; Guo et al., 2005; Wang et al., 2007, 2008).
As an indication of production of surfactant by B. parabrevis PDM-3, a feeble initial decrease in surface tension was observed up to 60 h followed by a drastic decrease (47.4 mN/m in 132 h). Later, the surface tension stabilized at 47.1 mN/m, with a decrease of 23 mN/m in 144 h (6 days) and agrees with the fact that a good biosurfactant producing bacterium decreases the surface tension of the culture supernatant more than 20 mN/m (Nayak et al., 2009). Emulsification index of the biosurfactant produced by PDM-3 for 24 h showed the maximum value of 57 which was stable for 72 h, similar to Nayak et al. (2009). The phenanthrene degradation increased after 60 h of incubation and was completed by 144 h with the production of the biosurfactant which started from 60 h increased up to 144 h with a negative correlation (Pearson correlation) of 0.981 (=1) between surface tension and percentage of degradation, significant at the 0.01 level (2-tailed) (Fig. 2A), proving that the Brevibacillus strain initially utilized the aqueous soluble phenanthrene and excreted the biosurfactant into the medium which in turn enhanced the aqueous solubility of remaining phenanthrene and led to increased bioavailability of the phenanthrene to the bacteria indicat-
ST vs % of phenanthrene degradation 100
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2.7. Utilization of other PAHs as growth substrates by the bacteria
3.4. Detection and identification of the biosurfactant produced during biodegradation
Surface Tension (mN/m)
a drop of the chloroform dissolved crude biosurfactant on the KBr pellet and measuring the same.
10 0 144
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B
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1.0 0.8 0.6 0.4 MSM-1 + Anthracene MSM-1 + Fluorene MSM-1 + Phenanthrene MSM-1 + Glucose
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Time (hrs) Fig. 2. Phenanthrene degradation by the strain PDM-3. (A) Relationship of phenanthrene degradation and biosurfactant production. (B) Comparison of the growth of the strain on phenanthrene and other PAHs.
M.S. Reddy et al. / Bioresource Technology 101 (2010) 7980–7983
ing an association between biodegradation of hydrocarbons and production of biosurfactants, an ability widely used in environmental biotechnology (Nayak et al., 2009). The present results support the previous reports on production of biosurfactant (Zhenyong and Jonathan, 2009; Kolomytsevaa et al., 2009). The functional groups observed through FT-IR spectroscopy were a strong C@O stretching at 1736 cm1, OH group at 3435 cm1, C–H stretching at 2924 cm1, C–H stretching in CH3 at 2853 cm1, CH bending at 1461 and 1367 cm1 and C–O stretching in esters and their presence is a characteristic of a biosurfactant of glycolipid nature (Heyd et al., 2008). 3.5. Utilization of other PAHs as growth substrates by PDM-3 The ability of Brevibacillus sp. PDM-3 to degrade other three ring PAHs viz. anthracene and fluorene was confirmed by the growth of the organism on MSM-1 (containing 250 mg/l of anthracene or fluorene) (Fig. 2B). The growth in presence of fluorene was higher as compared to that of anthracene or phenanthrene. Zhao et al. (2008, 2009) demonstrated that the phenanthrene-degrading isolates can utilize other PAHs. 4. Conclusions The bacterial strain Brevibacillus sp. PDM-3 has been identified and characterized presently using morphological, biochemical, chemotaxonomic (FAMEs analysis), molecular (16S rDNA sequencing) and phylogenetic analysis. The bacterial strain could carry out efficient degradation with production of biosurfactants. Further, the strain also has the ability to degrade other PAHs such as anthracene and fluorene. Although there are several reports of biodegradation by Brevibacillus sp., this is the first report on Brevibacillus sp. regarding its activity of degrading phenanthrene as sole carbon and energy source, and its ability to produce biosurfactants during the degradation. Acknowledgements We wish to thank Dr. R.B.N. Prasad, Head, Lipids and Fats Division, Indian Institute of Chemical Technology (CSIR), Hyderabad, India for providing the K100 Ring Surface Tensiometer facility for surface tension measurement.
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References Gottfried, A., Singhal, N., Elliot, R., Swift, S., 2010. The role of salicylate and biosurfactant in inducing phenanthrene degradation in batch soil slurries. Applied Microbiology Biotechnology (Published online: 10 February 2010). Guo, C.L., Zhou, H.W., Wong, Y.S., Tam, N.F.Y., 2005. Isolation of PAH-degrading bacteria from mangrove sediments and their biodegradation potential. Marine Pollution Bulletin 51, 1054–1061. Heyd, M., Kohnert, A., Tan, T.H., Nusser, M., Kirschhofer, F., Brenner-Weiss, G., Franzreb, M., Berensmeier, R., 2008. Development and trends of biosurfactant analysis and purification using rhamnolipid as an example. Annals of Bioanalytical Chemistry 391, 1579–1590. Kiyohara, H., Nagao, K., Yana, K., 1982. Rapid screen for bacterial degrading, waterinsoluble, solid hydrocarbons on agar plates. Applied and Environmental Microbiology 43, 454–457. Kolomytsevaa, M., Randazzob, D., Baskunova, B.P., Scozzafavab, A., Brigantib, F., Golovlevaa, L.A., 2009. Role of surfactants in optimizing fluorene assimilation and intermediate formation by Rhodococcus rhodochrous VKM B-2469. Bioresource Technology 100 (2), 839–844. Nayak, A.S., Vijaykumar, M.H., Karegoudar, T.B., 2009. Characterization of biosurfactant produced by Pseudomonas sp. PNK-04 and its application in bioremediation. International Biodeterioration and Biodegradation 63, 73–79. Rahman, K.S.M., Rahman, T.J., Kourkoutas, Y., Petsas, I., Marchant, R., Banat, I.M., 2003. Enhanced bioremediation of n-alkane in petroleum sludge using bacterial consortium amended with rhamnolipid and micronutrients. Bioresource Technology 90, 159–168. Shida, O., Takagi, H., Kadowaki, K., Komagata, K., 1996. Proposal for two new genera, Brevibacillus gen. nov. and Aneurinibacillus gen. nov. International Journal of Systematic Bacteriology 46 (4), 939–946. Story, S.P., Kline, E.L., Hughes, T.A., Riley, M.B., Hayasaka, S.S., 2004. Degradation of aromatic hydrocarbons by Sphingomonas paucimobilis strain EPA505. Archives of Environmental Contamination and Toxicology 47, 168–176. Takagi, H., Shida, O., Kadowaki, K., Komagata, K., Udaka, S., 1993. Characterization of Bacillus brevis with descriptions of B. migulans sp. nov., B. choshinensis sp. nov., B. parabrebrevis sp. nov., and B. galactophilus sp. nov. International Journal of Systematic Bacteriology 43 (2), 221–231. Wang, J., Xu, H.K., Guo, S.H., 2007. Isolation and characteristics of a microbial consortium for effectively degrading phenanthrene. Petroleum Science 4, 68– 75. Wang, J., Xu, H., An, M., Yan, G., 2008. Kinetics and characteristics of phenanthrene degradation by microbial consortium. Petroleum Science 5, 73–78. Zhao, H.P., Wang, L., Ren, J.R., Li, Z., LI, M., Gao, H.W., 2008. Isolation and characterization of phenanthrene degrading strains Sphingomonas sp. ZP1 and Tistrella sp. ZP5. Journal of Hazardous Materials 152, 1293–1300. Zhao, H.P., Wu, Q.S., Wang, L., Zhao, X.T., Gao, H.W., 2009. Degradation of phenanthrene by bacterial strain isolated from soil in oil refinery fields in Shanghai, China. Journal of Hazardous Materials 164 (2–3), 863–869. Zhenyong, Z., Jonathan, W., 2009. Biosurfactants from Acinetobactor calcoaceticus BU03 enhance the solubility and biodegradation of phenanthrene. Environmental Technology 30 (3), 291–299.
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Short Communication
Biodegradation of phenanthrene with biosurfactant production by a new strain of Brevibacillus sp. M. Srikanth Reddy, B. Naresh, T. Leela, M. Prashanthi, N.Ch. Madhusudhan, G. Dhanasri, Prathibha Devi * Biotechnology Laboratory, Department of Botany, Osmania University, Hyderabad 500007, India
a r t i c l e
i n f o
Article history: Received 10 December 2009 Received in revised form 3 April 2010 Accepted 7 April 2010 Available online 2 June 2010 Keywords: Biodegradation Phenanthrene Biosurfactant Brevibacillus sp.
a b s t r a c t In this work, a phenanthrene-degrading bacterial strain was isolated by enrichment method from hydrocarbon contaminated sludge samples and identified as Brevibacillus sp. PDM-3 based on morphological, biochemical, chemotaxonomic (FAMEs analysis) and molecular (16S rDNA sequencing) analysis. Growth parameters for efficient degradation of phenanthrene such as nutrient medium, pH, temperature, rpm and inoculum size were standardized and 93% of phenanthrene was degraded in 6 days as analysed by HPLC. The bacterial strain PDM-3 also has the ability to produce biosurfactant during phenanthrene degradation as detected by the surface tension measurements of the culture supernatant and the emulsification index (EI24). The biosurfactant was identified by its functional groups through FT-IR spectroscopy. Phenanthrene degradation and biosurfactant production are associated with each other and can be used in environmental biotechnology. Further, the strain has the ability to degrade other PAHs such as anthracene and fluorene by utilizing them as sole carbon and energy source. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Polyaromatic hydrocarbons (PAHs) are ubiquitous in the environment and comprise over 100 organic compounds containing two or more fused benzene and/or pentacyclic ring structures in linear, angular, or cluster arrangements (Guo et al., 2005). Bioremediation of PAHs depends on their bioavailability, as microorganisms can degrade only water soluble portion of the PAHs (Guo et al., 2005). Surfactants are amphiphilic compounds that can help release hydrocarbons sorbed to soil organic matter by solubilization and emulsification and increase the aqueous concentration of hydrophobic compounds, resulting in high mass transfer ratio (Gottfried et al., 2010). This study was undertaken to isolate and characterize potential PAHs degrading bacteria from contaminated sites through molecular methods for potential use in bioremediation. 2. Methods 2.1. Sampling Sludge samples were collected from three different sites of Hindustan Petrochemical Limited refinery (HPCL) located in Visakhapatnam, Andhra Pradesh, India in sterile plastic containers in April, 2007 and refrigerated for use to isolate bacterial strains. * Corresponding author. Tel.: +91 40 27423874; fax: +91 40 27090020. E-mail address: [email protected] (P. Devi). 0960-8524/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2010.04.054
2.2. Enrichment culture for isolation and screening of phenanthrenedegrading bacterial strains The petrochemical sludge samples were cultured on PAH enriched culture medium according to Kiyohara et al. (1982). Phenanthrene (250 mg/l), a three ring PAH with aqueous solubility of 1.29 mg/l was used as the sole carbon and energy source to enrich the mineral salts medium (MSM) (composition: KH2PO4 1.0 g, Na2HPO42H2O 1.25 g, (NH4)2SO4 1.0 g, MgSO47H2O 0.5 g, CaCl2 0.05 g and FeSO4 0.05 g per litre with the trace element solution comprising FeSO4 40 lg, MnSO4 40 lg, ZnSO4 20 lg, CuSO4 5 lg, CoCl2 4 lg, Na2MoO4 5 lg, CaCl2 0.5 lg, KH2PO4 136 lg and NaCl 1 mg) (1 gm of sludge sample in 50 ml of sterilized culture medium taken in a conical flask) and incubated at 30 °C on orbital shaker at 150 rpm for 7 days. A 2 ml of aliquot was transferred every week to fresh sterile medium and incubated under above conditions. One millilitre of the culture (after six transfers) was diluted with saline solution (0.9% NaCl) and plated on MSM agar plates over which, an ethereal solution of phenanthrene (250 mg/l) was uniformly sprayed wherein, the ether vaporizes leaving a thin-white layer of phenanthrene. The plates were incubated at 30 °C and after 3– 7 days, colonies of candidate phenanthrene-degrading strains were picked up on the basis of clear zones around them and further purified by repetitive streaking on fresh plates. The pure cultures of the final bacterial isolates were preserved under refrigeration on nutrient agar slants or as glycerol stocks at 20 °C.
M.S. Reddy et al. / Bioresource Technology 101 (2010) 7980–7983
2.3. Identification, morphological and biochemical characterization of the isolates The bacteria were identified by Bright field and Electron microscopy and the KB003 Hi25™ – Himedia bacterial identification kit was used for biochemical characterization through colorimetric identification. Antibiotic susceptibility was studied by nine types of antibiotic discs (Himedia) according to manufacturer’s instructions. For chemotaxonomic characterization, FAMEs analysis was performed by Gas–Liquid-Chromatography with the Sherlock MIS (Microbial Identification System, MIDI, Inc., Newark, Del) software. 2.4. Molecular characterization of the bacterial isolate Genomic DNA of the selected bacterial isolate was amplified by using the primers:16S1 (50 -GAGTTTGATCCTGGCTCA-30 ) and 16S2 (50 -CGGCTACCTTGTTACGACTT-30 ), which are complementary to the conserved regions at the 50 and 30 ends of the 16S rDNA corresponding to positions 9–27 and 1477–1498 of the Escherichia coli 16S rRNA gene. Amplified DNA fragments were separated by 1% (w/v) agarose gel electrophoresis. The separated bands were visualized under UV transillumination and then digitalized by Electrophoresis Documentation and Analysis System (Kodak DS, USA). The DNA fragment identified as 16S rDNA was eluted and purified using the clean Genei kit (Bangalore Genei) and sequenced with Gene Amp PCR machine (9600, Perkin–Elmer). The purified sequencing reaction mixtures were electrophoresed using ABI Prism Model 377 version 2.1.1 automatic DNA sequencer. The results were subjected to Bioinformatics analysis for identification of the isolates. 2.5. Biodegradation assay Biodegradation experiments were performed by inoculating exponential phase culture of the bacterial strain in MSM medium containing 250 mg/l phenanthrene and incubated under standard-
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ized conditions of temperature 35 °C, pH 7.0, rpm 175 and inoculum size of 5 vol. The amount of phenanthrene degraded was estimated every 12 h for 6 days with High Performance Liquid Chromatography (HPLC) (Story et al., 2004). The reverse phase HPLC of Shimadzu model LC-10AT chromatograph equipped with SPD-M20A prominence Photo Diode Array UV-vis detector set at 254 nm and a CBM-20A model prominence Communication Bus Module (CBM) provided with Class-VP software was used for degradation studies with an injection volume of 20 ll. Separation was achieved on Octyldecyl Silane Hypersil column (250 by 4.6 mm and 5-lm particle size) with a linear gradient of acetonitrile in 10 mM KH2PO4 buffer (pH 3.5) (0 min, 50:50, 0–30 min, ramp to 0:100 and 30–50 min isocratic at 0:100). The experiment was repeated twice. Peak area values obtained by the samples were compared with those of calibration curve with R2 value of 0.9907 and the rate of degradation calculated by using Class-VP software provided with the HPLC system. 2.6. Detection and identification of biosurfactant produced during biodegradation The biosurfactant produced by the bacterial isolate during biodegradation of phenanthrene was detected by measuring the surface tension (by K100 ring surface tensiometer of Kruss, Hamburg, Germany). Emulsification index of the culture supernatant which estimates the ability of biosurfactant to emulsify liquid hydrocarbons, such as kerosene or jojoba oil was determined by vortexing 6 ml of hydrocarbon and 4 ml of aqueous supernatant at high speed for 2 min. and incubated at 25 °C for 24 h. Emulsification index value (E24) was calculated as: E24 = (Height of emulsion layer/ Height of total mixture) 100. To purify the biosurfactant, the culture medium was separated from the bacterial cells by centrifugation and subjected thrice to solvent extraction (Rahman et al., 2003). The purified product was analysed by FT-IR spectroscopy for identification of functional groups by neat KBr (Potassium bromide) pellet method by placing
Fig. 1. Phylogenetic position of the strain PDM-3 among related bacteria.
M.S. Reddy et al. / Bioresource Technology 101 (2010) 7980–7983
In addition to phenanthrene, the bacterial strain was tested (by colorimetric measurement of turbidity at 600 nm) for growth on MSM medium containing other PAHs viz. anthracene and fluorene at a concentration of 250 mg/l.
3. Results and discussion 3.1. Isolation, identification and characterization of phenanthrenedegrading bacteria Eight bacterial strains were isolated by enrichment culture of sludge samples of which six were bacilli (75%) and two were cocci, out of which, one highly degrading bacillus strain was selected and characterized. Microscopic studies showed that the selected bacterial strain was Gram positive, motile, spore forming, aerobic and the cells are straight rods, occurring singly and in pairs. The strain showed positive oxidase activity, negative reactions for ONPG hydrolysis, methyl red and Voges Proskauer’s test and positive reduction of nitrate and utilization of citrate. Whereas, rhamnose, cellobiose and saccharose were utilized with acid production, it was resistant to Oxytetracycline, and Penicillin-G, and susceptible to Novobiocin, Nitrofurazone and Nalidixic acid. Further, the dominant fatty acids found in the bacterial strain were 15:0 iso (50.83%) and 15:0 anteiso (22.16%) (FAMEs analysis). The bacteria was identified as a species of Brevibacillus and placed in a systematic position according to Bergey’s manual of systematic bacteriology.
3.2. Molecular characterization of the bacterial strain and phylogenetic analysis Partial sequencing of the 1508 nucleotides length of 16S rDNA of the bacterial strain and phylogenetic analysis showed that it was closely related to Brevibacillus parabrevis with bootstrap value of 93 with a close relationship with different strains of their respective species groups with bootstrap values of more than 50 (Fig. 1). Distance matrix calculated for the strain with some of the most related strains indicates that the strain has 99% sequence similarity with B. parabrevis, which was earlier classified as Bacillus brevis based on 16S rRNA gene sequence and phylogenetic study (Takagi et al., 1993; Shida et al., 1996). Hence, the strain was identified as B. parabrevis and given the strain name of PDM-3. The bacterial strain PDM-3 was deposited in China Type Culture Collection Centre (CTCCC) with deposit number CCTCC AB 209087T and its 16S rDNA sequence was deposited in the GenBank database with the accession number FN185993.
3.3. Biodegradation assay The HPLC analysis showed that the rate of phenanthrene degradation was very low during the first 48 h but increased steadily and by 144 h (6 days), 93.92% of the phenanthrene was degraded (Fig. 2A). There was a perfect positive correlation between percentage of degradation and time, significant at the 0.01 level (2-tailed) according to the Pearson correlation of 0.985 (=1). The present rate of phenanthrene degradation is similar if not higher than earlier reports (Zhao et al., 2008, 2009; Guo et al., 2005; Wang et al., 2007, 2008).
As an indication of production of surfactant by B. parabrevis PDM-3, a feeble initial decrease in surface tension was observed up to 60 h followed by a drastic decrease (47.4 mN/m in 132 h). Later, the surface tension stabilized at 47.1 mN/m, with a decrease of 23 mN/m in 144 h (6 days) and agrees with the fact that a good biosurfactant producing bacterium decreases the surface tension of the culture supernatant more than 20 mN/m (Nayak et al., 2009). Emulsification index of the biosurfactant produced by PDM-3 for 24 h showed the maximum value of 57 which was stable for 72 h, similar to Nayak et al. (2009). The phenanthrene degradation increased after 60 h of incubation and was completed by 144 h with the production of the biosurfactant which started from 60 h increased up to 144 h with a negative correlation (Pearson correlation) of 0.981 (=1) between surface tension and percentage of degradation, significant at the 0.01 level (2-tailed) (Fig. 2A), proving that the Brevibacillus strain initially utilized the aqueous soluble phenanthrene and excreted the biosurfactant into the medium which in turn enhanced the aqueous solubility of remaining phenanthrene and led to increased bioavailability of the phenanthrene to the bacteria indicat-
ST vs % of phenanthrene degradation 100
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% of phenanthrene degradation
2.7. Utilization of other PAHs as growth substrates by the bacteria
3.4. Detection and identification of the biosurfactant produced during biodegradation
Surface Tension (mN/m)
a drop of the chloroform dissolved crude biosurfactant on the KBr pellet and measuring the same.
10 0 144
Time (hrs)
B
1.4 1.2
Growth (at 600nm)
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1.0 0.8 0.6 0.4 MSM-1 + Anthracene MSM-1 + Fluorene MSM-1 + Phenanthrene MSM-1 + Glucose
0.2 0.0 0
10
20
30
40
50
Time (hrs) Fig. 2. Phenanthrene degradation by the strain PDM-3. (A) Relationship of phenanthrene degradation and biosurfactant production. (B) Comparison of the growth of the strain on phenanthrene and other PAHs.
M.S. Reddy et al. / Bioresource Technology 101 (2010) 7980–7983
ing an association between biodegradation of hydrocarbons and production of biosurfactants, an ability widely used in environmental biotechnology (Nayak et al., 2009). The present results support the previous reports on production of biosurfactant (Zhenyong and Jonathan, 2009; Kolomytsevaa et al., 2009). The functional groups observed through FT-IR spectroscopy were a strong C@O stretching at 1736 cm1, OH group at 3435 cm1, C–H stretching at 2924 cm1, C–H stretching in CH3 at 2853 cm1, CH bending at 1461 and 1367 cm1 and C–O stretching in esters and their presence is a characteristic of a biosurfactant of glycolipid nature (Heyd et al., 2008). 3.5. Utilization of other PAHs as growth substrates by PDM-3 The ability of Brevibacillus sp. PDM-3 to degrade other three ring PAHs viz. anthracene and fluorene was confirmed by the growth of the organism on MSM-1 (containing 250 mg/l of anthracene or fluorene) (Fig. 2B). The growth in presence of fluorene was higher as compared to that of anthracene or phenanthrene. Zhao et al. (2008, 2009) demonstrated that the phenanthrene-degrading isolates can utilize other PAHs. 4. Conclusions The bacterial strain Brevibacillus sp. PDM-3 has been identified and characterized presently using morphological, biochemical, chemotaxonomic (FAMEs analysis), molecular (16S rDNA sequencing) and phylogenetic analysis. The bacterial strain could carry out efficient degradation with production of biosurfactants. Further, the strain also has the ability to degrade other PAHs such as anthracene and fluorene. Although there are several reports of biodegradation by Brevibacillus sp., this is the first report on Brevibacillus sp. regarding its activity of degrading phenanthrene as sole carbon and energy source, and its ability to produce biosurfactants during the degradation. Acknowledgements We wish to thank Dr. R.B.N. Prasad, Head, Lipids and Fats Division, Indian Institute of Chemical Technology (CSIR), Hyderabad, India for providing the K100 Ring Surface Tensiometer facility for surface tension measurement.
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