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Degradation of oil tank sludge using long-chain alkane-degrading bacteria

Degradation of oil tank sludge using long-chain alkane-degrading bacteria Ann Microbiol (2014) 64:391–395 DOI 10.1007/s13213-013-0643-8 SHORT COMMUNICATION Degradation of oil tank sludge using long-chain alkane-degrading bacteria Toru Matsui & Takahiro Yamamoto & Naoya Shinzato & Tsukasa Mitsuta & Kazuma Nakano & Tomoyuki Namihira Received: 25 September 2012 /Accepted: 2 April 2013 /Published online: 21 April 2013 Springer-Verlag Berlin Heidelberg and the University of Milan 2013 Abstract Bacteria degrading a very long-chain alkane, n- Petroleum is a complex mixture of non-aqueous and hydro- tetracosane, were isolated from enrichment culture of soil in phobic components like n-alkane, aromatics, resins. and Okinawa. Phylogenetic analysis of their16S rRNA sequences asphaltenes. Bioavailability might be the limiting factor revealed that they belong to classes Gammaproteobacteria and controlling the biodegradation of such compounds. Among Actinomycetes. Three isolates belonging to the genera the petroleum fractions, oil tank bottom sludge (OTBS) has Acinetobacter sp., Pseudomonas sp., and Gordonia sp. been regarded as a hazardous and recalcitrant petroleum- showed a stable growth on n-tetracosane and had a wide range derived substance. In addition, the oily wastes in crude storage of assimilation of aliphatic hydrocarbons from C to C , tank bottoms are periodically removed, posing difficulties for 12 30 while not on alkanes shorter than C .Of the isolates, disposal (Ferrari et al. 1996). In the past, these wastes were Gordonia sp. degraded oil tank sludge hydrocarbons efficiently disposed of after mixing them with soil and partially stabiliz- by solving the sludge in a hydrophobic solvent, while ing them with additives such as magnesite; in contrast, now- Acinetobacter sp. showed little degradation, possibly due to adays, they are generally detoxified using expensive physical– the difference in the mechanism of hydrophobic substrate chemical processes. Therefore, biotreatments should be con- incorporation between proteobacteria and actinobacteria. The sidered as an alternative, not only to decrease the volume of data suggested that non-heme di-iron monooxygenases of the waste but also to reduce their toxicity. Bioreactors, bioslurry AlkB-type, not bacterial CYP153 type cytochrome P450 al- techniques, landfarming treatments, or composting comprise kane hydroxylase, was involved in the alkane degradation. bioremediation alternatives for OTBS and for hydrocarbon wastes in general (Marin et al. 2006). Microbial degradation . . Keywords n-alkane Oil tank bottom sludge of aliphatic hydrocarbons have been investigated with the aim . . Biodegradation Actinobacteria Gordonia sp. of bioremediation for petroleum-polluted areas. For example, n-alkane in the petroleum sludge degradation by a bacterial consortium was reported to be enhanced by nutrients and biosurfactant addition (Rahman et al. 2003). Pure cultures, including proteobacteria (Acinetobacter sp., Pseudomonas : : : : T. Matsui (*) T. Yamamoto N. Shinzato K. Nakano sp., and Stenotrophomonas sp.) and firmicutes (Bacillus sp.), T. Namihira have also been reported to degrade petroleum oily sludge Tropical Biosphere Research Center, University of the Ryukyus, (Cerqueira et al. 2011; Verma et al. 2006), while little infor- 1 Sembaru, Nishihara-cho, mation is available on sludge degradation with actinobacteria, Okinawa 903-0213, Japan e-mail: tmatsui@comb.u-ryukyu.ac.jp such as Gordonia sp. and Rhodococcus sp., well known as hydrocarbon degraders (Arenskoetter et al. 2004), especially T. Mitsuta for the degradation of alkanes with carbon number higher than Japan Oil, Gas and Metals National Corporation (JOGMEC), 22. Gallego et al. (2007) examined oil tank bottom sludge Ministry of Economy, Trade and Industry (METI), Japan, Toranomon, Minato-ku, Tokyo 105-0001, Japan degradation using microbial consortia, showing that a consor- tium containing Acinetobacter spp., Nocardioides sp., Present Address: Stenotrophomonas sp., Pseudomonas sp., Aeromonas sp., T. Mitsuta and Rhodotorula spp. resulted in a good cometabolic effects, JX Nippon Oil & Energy Co., Chita, Aichi 478-0046, Japan 392 Ann Microbiol (2014) 64:391–395 emulsification properties, colonization of oil components, and Growth of bacterial cells was estimated by measuring degradative capabilities. In the scientific literature on microbial numbers of viable colonies after serial dilution of culture alkane degradation, n-alkanes (linear alkanes) have frequently broths with sterilized saline solution, expressed as colony been referred to as short-chain (C to C ), medium-chain (C to forming units (CFU) per unit of volume. Substrate con- 1 4 5 C ), long-chain (C to C ), and very long-chain n-alkanes sumption was analyzed by gas chromatography (GC) after 9 10 17 (more than C ), according to the length of their linear carbon extracting the reaction solution with n-hexane using n- chain. In addition to the extensive research in microbial degra- tetradecane as the internal standard. The gas chromatograph, dation of n-alkanes with carbon number less than 18, several (Shimadzu GC-14A; Shimadzu, Kyoto, Japan) was fitted reports on very long-chain n-alkanes degradation can be found with a DB-5 capillary column (length 30 m; Agilent tech- (Wentzel et al. 2007). For many n-alkane-degrading bacteria, nologies, CA, USA). Run conditions were as follows: start multiple alkane hydroxylases have been reported exhibiting temperature 100 °C 10 °C /min ramp rate to a final temper- overlapping substrate ranges, including integral membrane ature at 250 °C for 5 min. For OTBS analysis, the extraction non-heme di-iron monooxygenases of the AlkB-type (Smits was with an equal volume of n-hexane, followed by the GC et al. 1999) and cytochrome P450 alkane hydroxylases of the analysis at starting temperature of 80 °C for 4 min, 5 °C/min CYP153 family (van Beilen et al. 2006). ramp rate to a final temperature at 300 °C for 52 min. Here, we describe the isolation of bacteria capable of Biosurfactant production by the isolates were evaluated as degrading recalcitrant, long-chain aliphatic hydrocarbons, the emulsification activity of their culture broth as described such as n-tetracosane, followed by their application for by Saeki et al. (2009). In order to measure the emulsification laboratory scale OTBS remediation. activity of the biosurfactant, 50 μl soybean oil and 1 ml Bacterial strains used in this study were isolated by enrichment culture containing n-tetracosane (CH -(CH ) - 3 2 22 (A) CH ) as the sole carbon and energy source from 500 soils from Okinawa main islands. For the enrichment and alkanes 1 degradation test, basal mineral salts medium No. 11 was used, 0.8 as described previously (Matsui et al. 2009). Enrichment culture was perfomed aerobically at 30 °C, 150 spm with 0.6 reciprocal shaker (Taitec, Tokyo, Japan), for 2 weeks, followed by the repeat of transferring 5 % into the fresh 0.4 medium for 3 times. The obtained enrichment culture was further spread on nutrient agar (Oxoid, Hampshire, UK) 0.2 supplemented with glucose for colony isolation. General DNA manipulations were performed as de- scribedbySambrookand Russell (2001). Chromosomal 0 50 100 150 200 250 DNA from the isolated strains were extracted as described Time (h ) previously (Matsui et al. 2009) and used for polymerase (B) chain reaction (PCR). The 16S rRNA gene, alkB, and cyto- chrome P450 hydroxylase of the CYP153 family homo- logue gene were amplified by PCR using EX Taq DNA polymerase (Takara Bio, Shiga, Japan) and a thermal cycler (type PC-818; Astec, Fukuoka, Japan), as described previ- ously (Matsui et al. 2009; Smits et al. 1999; van Beilen et al. 2006). The PCR-amplified 16S rRNA gene (approximately 1.5 kb long), alkB gene (0.5 kb), and cytochrome P450 hydroxylase gene (0.3 kb) fragments were purified by aga- rose gel electrophoresis, ligated into the pT7 blue vector (Novagen Merck Milllipore, Darmstadt, Germany), and 0.1 used for the transformation of Escherichia coli JM109 0 50 100 150 200 250 (Takara Bio). The nucleotide sequences of both strands of Time (h ) the cloned genes were determined by using the M13-47 and Fig. 1 Time course of n-tetracosane degradation by the isolates. a RV-M primers (Novagen Merck Millipore) and the ABI Residual n-tetracosane (g/l), b cellular growth (CFU/ml). Triangles model 3100 and BigDye terminator kit, v.1.1 (Applied Acinetobacter sp. 49A, squares Pseudomonas sp. 11A, diamonds Biosystems, CA, USA) according to the manufacturer’s Gordonia sp. 30A. Dotted lines results in the absence of cells (a), and in the absence of substrate (b) instructions. Colon y Formin g Unit(X10 /ml) Residual n-tetracosan e (g/l) Ann Microbiol (2014) 64:391–395 393 Table 1 Characterization of n-tetracosane (n-C ) degradation by the isolates n-C degradation (%) b c No solvent HMN Pristane Squalane BS (g/l) Pseudomonas sp. strain 11A 71.5 44.1 0 87.5 0.18 Gordonia sp. strain 30A 88.3 100 67.3 100 n.d. Acinetobacter sp. strain 49A 91.2 37.4 36.5 0 0.19 n-C24 degradation in the presence of 10 % (v/v) solvent 2,2,4,4,6,8,8-Heptamethylnonane Biosurfactant production when grown with n-C24 as the sole carbon source Not detected biosurfactant sample were added to 9 ml of 50 mM phos- OTBS was kindly provided by Akita Oil Storage, Akita, phate buffer (pH 7.0) taken in an φ18-mm×125-mm test Japan Various alkanes were supplied by Tokyo Kasei Kogyo, tube. The test tube was vigorously shaken at 345 rpm for Tokyo, Japan. All other materials were of the highest purity 10 min at 30 °C. Thereafter, the optical density was mea- commercially available and were used without further sured at 620 nm using the UV–photometer mini photo 518R purification. (Taitec). The emulsion turbidities of different concentrations Sequence of fragments of 16S rRNA and alkB gene of (0–20 mg/l) of the biosurfactant sample were directly pro- the isolates have been assigned DDBJ/EMBL/GenBank ac- portional to the concentrations of the sample. To estimate cession nos. AB638843–AB638848 and AB638866– the total concentration of the biosurfactant in the culture AB638868, respectively. supernatant, the emulsification activity of the culture super- By the enrichment culture with 3 times transfer on n- natant was measured and then calculated with respect to tetracosane as the sole carbon and energy source, 40 soil soybean oil on the basis of the standard curve that was samples out of 500 exhibited a stable bacterial growth. obtained by using the data of the standard biosurfactant. Eighteen bacterial strains were selected among the isolates The composition of OTBS, analyzed by the TLC-FID meth- from the enrichment culture broths, followed by assimilat- od, modified as described by Cebolla et al. (1995), revealed ing tests of the substrate in liquid cultures. Analysis of the that it consisted of 37.7±1.1 % of saturates, 40.2±2.1 % of 16S rRNA sequences for the isolates revealed that 10 aromatics, 18.5±0.8 % of resins, and 3.5±0.3 % of belonged to Acinetobacter sp., 3 to Pseudomonas sp., and asphaltenes. 5to Gordonia sp. Of the isolates, 3 strains (namely 11A, Fig. 2 Gas chromatograms of the HMN layer after cultivation with OTBS for 7 days in the presence of pristane. a No cells inoculated, b Pseudomonas sp. 11A, c Gordonia sp. 30A, and d Acinetobacter sp. 49A. Peaks at around 10 min were derived from the added HMN and its impurities. Arrows indicates peaks for linear alkanes carbon number 394 Ann Microbiol (2014) 64:391–395 30A, and 49A, belonging to Pseudomonas sp., Gordonia observed only in the case of growing cultures of Gordonia sp., and Acinetobacter sp., respectively) were selected from sp. in the presence of HMN, while no degradation was the each genera, as showing stable growth on n-tetracosane obtained with Pseudomonas sp., and less degradation with as the carbon source, and subjected for further studies. Acinetobacter sp. Microbial incorporation/degradation of Figure 1 shows the typical time courses of n-tetracosane petroleum-derived hydrocarbons were started by two pos- degradation by the selected isolates. Acinetobacter sp. and sible mechanisms, emulsification of the oily droplets or Pseudomonas sp. degraded more than 90 % of n-tetracosane growth in the droplets due to the hydrophobic cell sur- in 120 h of incubation. Gordonia sp. degraded nearly 50 % face (Monticello 2000). Based on these facts, efficient at 60 h, while showing comparable cellular growth to that of degradation of OTBS by Gordonia sp. in the presence of the two other strains. They assimilated n-alkanes ranging HMN could be explained by the latter mechanism, since from C to C , although n-alkanes shorter than C did not the strain degraded efficiently in the presence of the 14 30 8 support the growth of all the strains (data not shown). solvent although no biosurfactant production was In many bacterial species, the oxidation of alkanes is cat- observed. alyzed by non-heme di-iron monooxygenase Alk systems In this study, efficient OTBS degradation was shown (van Beilen and Funhoff 2005), although different pathways using a new isolated Gordonia sp., in the presence of a have been identified (van Beilen et al. 2006). From the ge- hydrophobic solvent. This character might have a potential nome of all the 3 strains, using degenerated primers, PCR for efficient sludge degradation by combination with a con- products of the expected size were obtained for the alkB gene. ventional physico-chemical tank sludge treatment process No amplification was observed for the cytochrome P450, such as crude oil washing (Mitchel 1994), which is widely suggesting that only non-heme di-iron alkane hydroxylase used for OTBS removal from the tank. could be involved in the degradation. Sequence analysis of Acknowledgments This work was supported by Japan Oil, Gas and the alkB gene homologue obtained from the 3 strains’ genome Metals National Corporation (JOGMEC), Ministry of Economy, Trade revealed each one belongs to the same families as those and Industry (METI), Japan. OTBS analysis by Idemitsu Techno-research classified from 16S rRNA sequences. Homology in the primary Center in Idemitsu Kosan, Co., Chiba, Japan, is acknowledged. sequences from 11A, 30A, and 49A strains with the closest proteins were 99.4 (Pseudomonas aeruginosa PAO1, reference No. 2618382EFW), 100 (Gordonia amarae DSM43392, D1MBU3) and 97.3 % (Acinetobacter calcoaceticus PHEA- References 2, F0KMZ3), respectively. Roles of these genes in hydrocar- bons degradation should further be examined. Arenskoetter M, Broker D, Steinbuechel A (2004) Biology of the To facilitate the fluidity of OTBS to enhance its degra- metabolically diverse genus Gordonia. Appl Environ Microbiol dation, since no degradation was observed when cultured 70:3195–3204 Cebolla VL, Vela J, Membrado L, Ferrando AC (1995) TLC-FID in with the oil tank sludge as the sole source of carbon (data quantitative hydrocarbon group type analysis (HGTA) of not shown), water/solvent two-phase cultivation was further asphaltenes and other heavy fossil fuels. J Chromatogr Sci examined with the 3 strains studied (Table 1). To examine 33:417–425 the effect of solvents for enhancement of the substrate Cerqueira VS, Hollenbach EB, Maboni F, Vainstein MH, Camargo FA, do Carmo RPM, Bento FM (2011) Biodegradation potential of solubility, we used heptamethyl-nonane (HMN), pristane, oily sludge by pure and mixed bacterial cultures. Bioresour and squalane as solvents for the enhancement of solubility Technol 102:11003–11010 of OTBS, because they are known as biocompatible and Ferrari MD, Neirotti E, Albornoz C, Mostazo MR, Cozzo M (1996) resistant to microbial attack (Hori et al. 2002; Muñoz et al. Biotreatment of hydrocarbons from petroleum tank bottom sludges in soil slurries. Biotechnol Lett 18:1241–1246 2008). As showninTable 1, P. aeruginosa and Gallego JL, Garcia-Martinez MJ, Llamas JF, Belloch C, Pelaez AI, Acinetobacter sp. showed no degradation in the presence Sanchez J (2007) Biodegradation of oil tank bottom sludge using of pristane and squalane, respectively, while significant microbial consortia. Biodegradation 18:269–281 degradations were observed in the presence of all the sol- Hori K, Matsuzaki Y, Tanji Y, Unno H (2002) Effect of dispers- ing oil phase on the biodegradability of a solid alkane vents tested when using Gordonia sp., resulting in n- dissolved in non-biodegradable oil. Appl Microbiol Biotechnol tetracosane degradation of 100, 67.3, and 100 % when using 59:574–579 HMN, pristane, and squalane, respectively. Production of Marin JA, Moreno JL, Hernandez T, Garcia C (2006) Bioremediation surface-active compounds (bioemulsifiers or biosurfactant) by composting of heavy oil refinery sludge in semiarid condi- tions. Biodegradation 17:251–261 was detected only in Pseudomonas sp. strain 11A and Matsui T, Kato K, Namihira T, Shinzato N, Semba H (2009) Stereo- Acinetobacter sp. 49A, but not in Gordonia sp. 30A specific degradation of phenylsuccinate by actinomycetes. (Table 1). The effect of HMN addition on hydrocarbon Chemosphere 76:1278–1282 degradation was further examined for OTBS degradation Mitchel RB (1994) Regime design matters: intentional oil pollution as shown in Fig. 2. Significant alkanes degradation was and treaty compliance. Int Organ 48:425–458 Ann Microbiol (2014) 64:391–395 395 Monticello DJ (2000) Biodesulfurization and the upgrading of petro- Smits TH, Rothlisberger M, Witholt B, van Beilen JB (1999) Molec- leum distillates. Curr Opin Biotechnol 11:540–546 ular screening for alkane hydroxylase genes in gram-negative and Muñoz R, Chambaud M, Bordel S, Villaverde S (2008) A systematic gram-positive strains. Environ Microbiol 1:307–317 selection of the non-aqueous phase in a bacterial two liquid phase van Beilen JB, Funhoff EG (2005) Expanding the alkane oxygenase bioreactor treating alpha-pinene. Appl Microbiol Biotechnol toolbox: new enzymes and applications. Curr Opin Biotechnol 79:33–41 16:308–314 Rahman KS, Rahman TJ, Kourkoutas Y, Petsas I, Marchant R, Banat van Beilen JB, Funhoff EG, van Loon A, Just A, Kaysser L, Bouza M, IM (2003) Enhanced bioremediation of n-alkane in petroleum Holtackers R, Rothlisberger M, Li Z, Witholt B (2006) Cyto- sludge using bacterial consortium amended with rhamnolipid chrome P450 alkane hydroxylases of the CYP153 family are and micronutrients. Bioresour Technol 90:159–168 common in alkane-degrading eubacteria lacking integral mem- Saeki H, Sasaki M, Komatsu K, Miura A, Matsuda H (2009) Oil spill brane alkane hydroxylases. Appl Environ Microbiol 72:59–65 remediation by using the remediation agent JE1058BS that con- Verma S, Bhargava R, Pruthi V (2006) Oily sludge degradation by tains a biosurfactant produced by Gordonia sp. Strain JE-1058. bacteria from ankleshwar, India. Int Biodet Biodeg 57:207–213 Bioresour Technol 100:572–577 Wentzel A, Ellingsen TE, Kotlar HK, Zotchev SB, Throne-Holst M Sambrook J, Russell DW (2001) Molecular cloning: a laboratory (2007) Bacterial metabolism of long-chain n-alkanes. Appl manual, 3rd edn. Cold Spring Harbor Laboratory, New York Microbiol Biotechnol 76:1209–1221 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Annals of Microbiology Springer Journals

Degradation of oil tank sludge using long-chain alkane-degrading bacteria

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Springer Journals
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Copyright © 2013 by Springer-Verlag Berlin Heidelberg and the University of Milan
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Life Sciences; Microbiology; Microbial Genetics and Genomics; Microbial Ecology; Fungus Genetics; Medical Microbiology; Applied Microbiology
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Abstract

Ann Microbiol (2014) 64:391–395 DOI 10.1007/s13213-013-0643-8 SHORT COMMUNICATION Degradation of oil tank sludge using long-chain alkane-degrading bacteria Toru Matsui & Takahiro Yamamoto & Naoya Shinzato & Tsukasa Mitsuta & Kazuma Nakano & Tomoyuki Namihira Received: 25 September 2012 /Accepted: 2 April 2013 /Published online: 21 April 2013 Springer-Verlag Berlin Heidelberg and the University of Milan 2013 Abstract Bacteria degrading a very long-chain alkane, n- Petroleum is a complex mixture of non-aqueous and hydro- tetracosane, were isolated from enrichment culture of soil in phobic components like n-alkane, aromatics, resins. and Okinawa. Phylogenetic analysis of their16S rRNA sequences asphaltenes. Bioavailability might be the limiting factor revealed that they belong to classes Gammaproteobacteria and controlling the biodegradation of such compounds. Among Actinomycetes. Three isolates belonging to the genera the petroleum fractions, oil tank bottom sludge (OTBS) has Acinetobacter sp., Pseudomonas sp., and Gordonia sp. been regarded as a hazardous and recalcitrant petroleum- showed a stable growth on n-tetracosane and had a wide range derived substance. In addition, the oily wastes in crude storage of assimilation of aliphatic hydrocarbons from C to C , tank bottoms are periodically removed, posing difficulties for 12 30 while not on alkanes shorter than C .Of the isolates, disposal (Ferrari et al. 1996). In the past, these wastes were Gordonia sp. degraded oil tank sludge hydrocarbons efficiently disposed of after mixing them with soil and partially stabiliz- by solving the sludge in a hydrophobic solvent, while ing them with additives such as magnesite; in contrast, now- Acinetobacter sp. showed little degradation, possibly due to adays, they are generally detoxified using expensive physical– the difference in the mechanism of hydrophobic substrate chemical processes. Therefore, biotreatments should be con- incorporation between proteobacteria and actinobacteria. The sidered as an alternative, not only to decrease the volume of data suggested that non-heme di-iron monooxygenases of the waste but also to reduce their toxicity. Bioreactors, bioslurry AlkB-type, not bacterial CYP153 type cytochrome P450 al- techniques, landfarming treatments, or composting comprise kane hydroxylase, was involved in the alkane degradation. bioremediation alternatives for OTBS and for hydrocarbon wastes in general (Marin et al. 2006). Microbial degradation . . Keywords n-alkane Oil tank bottom sludge of aliphatic hydrocarbons have been investigated with the aim . . Biodegradation Actinobacteria Gordonia sp. of bioremediation for petroleum-polluted areas. For example, n-alkane in the petroleum sludge degradation by a bacterial consortium was reported to be enhanced by nutrients and biosurfactant addition (Rahman et al. 2003). Pure cultures, including proteobacteria (Acinetobacter sp., Pseudomonas : : : : T. Matsui (*) T. Yamamoto N. Shinzato K. Nakano sp., and Stenotrophomonas sp.) and firmicutes (Bacillus sp.), T. Namihira have also been reported to degrade petroleum oily sludge Tropical Biosphere Research Center, University of the Ryukyus, (Cerqueira et al. 2011; Verma et al. 2006), while little infor- 1 Sembaru, Nishihara-cho, mation is available on sludge degradation with actinobacteria, Okinawa 903-0213, Japan e-mail: tmatsui@comb.u-ryukyu.ac.jp such as Gordonia sp. and Rhodococcus sp., well known as hydrocarbon degraders (Arenskoetter et al. 2004), especially T. Mitsuta for the degradation of alkanes with carbon number higher than Japan Oil, Gas and Metals National Corporation (JOGMEC), 22. Gallego et al. (2007) examined oil tank bottom sludge Ministry of Economy, Trade and Industry (METI), Japan, Toranomon, Minato-ku, Tokyo 105-0001, Japan degradation using microbial consortia, showing that a consor- tium containing Acinetobacter spp., Nocardioides sp., Present Address: Stenotrophomonas sp., Pseudomonas sp., Aeromonas sp., T. Mitsuta and Rhodotorula spp. resulted in a good cometabolic effects, JX Nippon Oil & Energy Co., Chita, Aichi 478-0046, Japan 392 Ann Microbiol (2014) 64:391–395 emulsification properties, colonization of oil components, and Growth of bacterial cells was estimated by measuring degradative capabilities. In the scientific literature on microbial numbers of viable colonies after serial dilution of culture alkane degradation, n-alkanes (linear alkanes) have frequently broths with sterilized saline solution, expressed as colony been referred to as short-chain (C to C ), medium-chain (C to forming units (CFU) per unit of volume. Substrate con- 1 4 5 C ), long-chain (C to C ), and very long-chain n-alkanes sumption was analyzed by gas chromatography (GC) after 9 10 17 (more than C ), according to the length of their linear carbon extracting the reaction solution with n-hexane using n- chain. In addition to the extensive research in microbial degra- tetradecane as the internal standard. The gas chromatograph, dation of n-alkanes with carbon number less than 18, several (Shimadzu GC-14A; Shimadzu, Kyoto, Japan) was fitted reports on very long-chain n-alkanes degradation can be found with a DB-5 capillary column (length 30 m; Agilent tech- (Wentzel et al. 2007). For many n-alkane-degrading bacteria, nologies, CA, USA). Run conditions were as follows: start multiple alkane hydroxylases have been reported exhibiting temperature 100 °C 10 °C /min ramp rate to a final temper- overlapping substrate ranges, including integral membrane ature at 250 °C for 5 min. For OTBS analysis, the extraction non-heme di-iron monooxygenases of the AlkB-type (Smits was with an equal volume of n-hexane, followed by the GC et al. 1999) and cytochrome P450 alkane hydroxylases of the analysis at starting temperature of 80 °C for 4 min, 5 °C/min CYP153 family (van Beilen et al. 2006). ramp rate to a final temperature at 300 °C for 52 min. Here, we describe the isolation of bacteria capable of Biosurfactant production by the isolates were evaluated as degrading recalcitrant, long-chain aliphatic hydrocarbons, the emulsification activity of their culture broth as described such as n-tetracosane, followed by their application for by Saeki et al. (2009). In order to measure the emulsification laboratory scale OTBS remediation. activity of the biosurfactant, 50 μl soybean oil and 1 ml Bacterial strains used in this study were isolated by enrichment culture containing n-tetracosane (CH -(CH ) - 3 2 22 (A) CH ) as the sole carbon and energy source from 500 soils from Okinawa main islands. For the enrichment and alkanes 1 degradation test, basal mineral salts medium No. 11 was used, 0.8 as described previously (Matsui et al. 2009). Enrichment culture was perfomed aerobically at 30 °C, 150 spm with 0.6 reciprocal shaker (Taitec, Tokyo, Japan), for 2 weeks, followed by the repeat of transferring 5 % into the fresh 0.4 medium for 3 times. The obtained enrichment culture was further spread on nutrient agar (Oxoid, Hampshire, UK) 0.2 supplemented with glucose for colony isolation. General DNA manipulations were performed as de- scribedbySambrookand Russell (2001). Chromosomal 0 50 100 150 200 250 DNA from the isolated strains were extracted as described Time (h ) previously (Matsui et al. 2009) and used for polymerase (B) chain reaction (PCR). The 16S rRNA gene, alkB, and cyto- chrome P450 hydroxylase of the CYP153 family homo- logue gene were amplified by PCR using EX Taq DNA polymerase (Takara Bio, Shiga, Japan) and a thermal cycler (type PC-818; Astec, Fukuoka, Japan), as described previ- ously (Matsui et al. 2009; Smits et al. 1999; van Beilen et al. 2006). The PCR-amplified 16S rRNA gene (approximately 1.5 kb long), alkB gene (0.5 kb), and cytochrome P450 hydroxylase gene (0.3 kb) fragments were purified by aga- rose gel electrophoresis, ligated into the pT7 blue vector (Novagen Merck Milllipore, Darmstadt, Germany), and 0.1 used for the transformation of Escherichia coli JM109 0 50 100 150 200 250 (Takara Bio). The nucleotide sequences of both strands of Time (h ) the cloned genes were determined by using the M13-47 and Fig. 1 Time course of n-tetracosane degradation by the isolates. a RV-M primers (Novagen Merck Millipore) and the ABI Residual n-tetracosane (g/l), b cellular growth (CFU/ml). Triangles model 3100 and BigDye terminator kit, v.1.1 (Applied Acinetobacter sp. 49A, squares Pseudomonas sp. 11A, diamonds Biosystems, CA, USA) according to the manufacturer’s Gordonia sp. 30A. Dotted lines results in the absence of cells (a), and in the absence of substrate (b) instructions. Colon y Formin g Unit(X10 /ml) Residual n-tetracosan e (g/l) Ann Microbiol (2014) 64:391–395 393 Table 1 Characterization of n-tetracosane (n-C ) degradation by the isolates n-C degradation (%) b c No solvent HMN Pristane Squalane BS (g/l) Pseudomonas sp. strain 11A 71.5 44.1 0 87.5 0.18 Gordonia sp. strain 30A 88.3 100 67.3 100 n.d. Acinetobacter sp. strain 49A 91.2 37.4 36.5 0 0.19 n-C24 degradation in the presence of 10 % (v/v) solvent 2,2,4,4,6,8,8-Heptamethylnonane Biosurfactant production when grown with n-C24 as the sole carbon source Not detected biosurfactant sample were added to 9 ml of 50 mM phos- OTBS was kindly provided by Akita Oil Storage, Akita, phate buffer (pH 7.0) taken in an φ18-mm×125-mm test Japan Various alkanes were supplied by Tokyo Kasei Kogyo, tube. The test tube was vigorously shaken at 345 rpm for Tokyo, Japan. All other materials were of the highest purity 10 min at 30 °C. Thereafter, the optical density was mea- commercially available and were used without further sured at 620 nm using the UV–photometer mini photo 518R purification. (Taitec). The emulsion turbidities of different concentrations Sequence of fragments of 16S rRNA and alkB gene of (0–20 mg/l) of the biosurfactant sample were directly pro- the isolates have been assigned DDBJ/EMBL/GenBank ac- portional to the concentrations of the sample. To estimate cession nos. AB638843–AB638848 and AB638866– the total concentration of the biosurfactant in the culture AB638868, respectively. supernatant, the emulsification activity of the culture super- By the enrichment culture with 3 times transfer on n- natant was measured and then calculated with respect to tetracosane as the sole carbon and energy source, 40 soil soybean oil on the basis of the standard curve that was samples out of 500 exhibited a stable bacterial growth. obtained by using the data of the standard biosurfactant. Eighteen bacterial strains were selected among the isolates The composition of OTBS, analyzed by the TLC-FID meth- from the enrichment culture broths, followed by assimilat- od, modified as described by Cebolla et al. (1995), revealed ing tests of the substrate in liquid cultures. Analysis of the that it consisted of 37.7±1.1 % of saturates, 40.2±2.1 % of 16S rRNA sequences for the isolates revealed that 10 aromatics, 18.5±0.8 % of resins, and 3.5±0.3 % of belonged to Acinetobacter sp., 3 to Pseudomonas sp., and asphaltenes. 5to Gordonia sp. Of the isolates, 3 strains (namely 11A, Fig. 2 Gas chromatograms of the HMN layer after cultivation with OTBS for 7 days in the presence of pristane. a No cells inoculated, b Pseudomonas sp. 11A, c Gordonia sp. 30A, and d Acinetobacter sp. 49A. Peaks at around 10 min were derived from the added HMN and its impurities. Arrows indicates peaks for linear alkanes carbon number 394 Ann Microbiol (2014) 64:391–395 30A, and 49A, belonging to Pseudomonas sp., Gordonia observed only in the case of growing cultures of Gordonia sp., and Acinetobacter sp., respectively) were selected from sp. in the presence of HMN, while no degradation was the each genera, as showing stable growth on n-tetracosane obtained with Pseudomonas sp., and less degradation with as the carbon source, and subjected for further studies. Acinetobacter sp. Microbial incorporation/degradation of Figure 1 shows the typical time courses of n-tetracosane petroleum-derived hydrocarbons were started by two pos- degradation by the selected isolates. Acinetobacter sp. and sible mechanisms, emulsification of the oily droplets or Pseudomonas sp. degraded more than 90 % of n-tetracosane growth in the droplets due to the hydrophobic cell sur- in 120 h of incubation. Gordonia sp. degraded nearly 50 % face (Monticello 2000). Based on these facts, efficient at 60 h, while showing comparable cellular growth to that of degradation of OTBS by Gordonia sp. in the presence of the two other strains. They assimilated n-alkanes ranging HMN could be explained by the latter mechanism, since from C to C , although n-alkanes shorter than C did not the strain degraded efficiently in the presence of the 14 30 8 support the growth of all the strains (data not shown). solvent although no biosurfactant production was In many bacterial species, the oxidation of alkanes is cat- observed. alyzed by non-heme di-iron monooxygenase Alk systems In this study, efficient OTBS degradation was shown (van Beilen and Funhoff 2005), although different pathways using a new isolated Gordonia sp., in the presence of a have been identified (van Beilen et al. 2006). From the ge- hydrophobic solvent. This character might have a potential nome of all the 3 strains, using degenerated primers, PCR for efficient sludge degradation by combination with a con- products of the expected size were obtained for the alkB gene. ventional physico-chemical tank sludge treatment process No amplification was observed for the cytochrome P450, such as crude oil washing (Mitchel 1994), which is widely suggesting that only non-heme di-iron alkane hydroxylase used for OTBS removal from the tank. could be involved in the degradation. Sequence analysis of Acknowledgments This work was supported by Japan Oil, Gas and the alkB gene homologue obtained from the 3 strains’ genome Metals National Corporation (JOGMEC), Ministry of Economy, Trade revealed each one belongs to the same families as those and Industry (METI), Japan. OTBS analysis by Idemitsu Techno-research classified from 16S rRNA sequences. Homology in the primary Center in Idemitsu Kosan, Co., Chiba, Japan, is acknowledged. sequences from 11A, 30A, and 49A strains with the closest proteins were 99.4 (Pseudomonas aeruginosa PAO1, reference No. 2618382EFW), 100 (Gordonia amarae DSM43392, D1MBU3) and 97.3 % (Acinetobacter calcoaceticus PHEA- References 2, F0KMZ3), respectively. Roles of these genes in hydrocar- bons degradation should further be examined. Arenskoetter M, Broker D, Steinbuechel A (2004) Biology of the To facilitate the fluidity of OTBS to enhance its degra- metabolically diverse genus Gordonia. Appl Environ Microbiol dation, since no degradation was observed when cultured 70:3195–3204 Cebolla VL, Vela J, Membrado L, Ferrando AC (1995) TLC-FID in with the oil tank sludge as the sole source of carbon (data quantitative hydrocarbon group type analysis (HGTA) of not shown), water/solvent two-phase cultivation was further asphaltenes and other heavy fossil fuels. J Chromatogr Sci examined with the 3 strains studied (Table 1). To examine 33:417–425 the effect of solvents for enhancement of the substrate Cerqueira VS, Hollenbach EB, Maboni F, Vainstein MH, Camargo FA, do Carmo RPM, Bento FM (2011) Biodegradation potential of solubility, we used heptamethyl-nonane (HMN), pristane, oily sludge by pure and mixed bacterial cultures. Bioresour and squalane as solvents for the enhancement of solubility Technol 102:11003–11010 of OTBS, because they are known as biocompatible and Ferrari MD, Neirotti E, Albornoz C, Mostazo MR, Cozzo M (1996) resistant to microbial attack (Hori et al. 2002; Muñoz et al. Biotreatment of hydrocarbons from petroleum tank bottom sludges in soil slurries. Biotechnol Lett 18:1241–1246 2008). 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Journal

Annals of Microbiology – Springer Journals

Published: Apr 21, 2013

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