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Isolation and characterization of marine diesel oil-degrading Acinetobacter sp. strain Y2

Isolation and characterization of marine diesel oil-degrading Acinetobacter sp. strain Y2 Ann Microbiol (2013) 63:633–640 DOI 10.1007/s13213-012-0513-9 ORIGINAL ARTICLE Isolation and characterization of marine diesel oil-degrading Acinetobacter sp. strain Y2 Qun Luo & Jian-Guo Zhang & Xian-Rong Shen & Zheng-Qiu Fan & Ying He & Deng-Yong Hou Received: 10 December 2011 /Accepted: 16 July 2012 /Published online: 16 September 2012 Springer-Verlag and the University of Milan 2012 Abstract The marine diesel oil-degrading bacterium Acineto- production, transportation, and disposal. There have been bacter sp. strain Y2 was isolated from oil-polluted seawater many incidents of large-scale spills all over the world, the sampled from Dinghai port, Zhoushan City, Zhejiang Province, majority with serious consequences to the environment. The China. The isolated bacterium was identified as Acinetobacter recent oil spill accident in the Gulf of Mexico and the oil sp. based on its 16S rDNA gene sequence as well as various pipeline explosion accident in the city of Dalian, China had morphological and physiological characteristics. The degrada- devastating impacts on the marine environment. It is esti- tion characteristics of strain Y2 were studied and its parameters mated that hundreds of millions of liters of petroleum enter for oil degradation optimized. These optimal conditions were into the marine environment each year. Oil pollution not determined to be an initial pH of 7.5, an incubation temperature only causes serious damage to fisheries, but it also has of 30 °C, an initial diesel oil concentration of 2 % (v/v), and an adverse effects on the natural environment and ecosystem initial inoculating bacteria concentration of 3×10 cells/mL. (Yamamoto et al. 2003; Jeong and Cho 2007). In areas The results from the gas chromatography–mass spectrometry affected by petroleum pollution, local populations of inver- analysis showed that strain Y2 could almost completely de- tebrates, birds, and mammals may be greatly reduced and grade all components of diesel oil, with a degradation ratio of vegetation may die. In general, those organisms worst af- up to 80 % after 10 days of incubation at the optimal conditions. fected are the ones which inhabit shallow water and the littoral zone and those, such as marine mammals, which . . . Keywords Biodegradation Isolation Characterization are obliged to cross the air–sea interface. Acinetobacter sp. The traditional methods to cope with petroleum pollu- tion are confined to physical and chemical containment. These are usually applied to clean up high concentrations of polluting oil, such as those due to an oil spill, but their Introduction effects on low concentrations of oil pollution are not very good; moreover, they are generally expensive and may be a Petroleum pollution has become a serious environmental source of secondary pollution (Yang et al. 2004). The problem, especially in marine environments, due to oil seriousness of the petroleum pollution problem has moti- vated researchers to explore the possibilities of microbio- Q. Luo logical degradation (i.e., bioremediation) of petroleum. Department of Nuclear Science and Engineering, Naval University Biological methods represent a popular option for the of Engineering, Wuhan 430033, China clean-up of environments polluted with hydrocarbons, and they may represent an efficient, economic, and versa- : : : : Q. Luo J.-G. Zhang X.-R. Shen (*) Y. He D.-Y. Hou tile alternative to physicochemical treatments (Venosa and The Naval Medical Research Institute, Zhu 2003). The first major large-scale use of bioremedia- Shanghai 200433, China tion methods to clean up an oil spill was during the Exxon e-mail: xianrong_sh@yahoo.com Valdez accident in 1989. Since then, the development of Z.-Q. Fan bioremediation methods for marine oil pollution has Department of Environmental Science & Engineering, attracted worldwide interest and become an active research Fudan University, area (Liu et al. 2010). Shanghai 200433, China 634 Ann Microbiol (2013) 63:633–640 Table 1 Environmental conditions of the seawater in Dinghai port Materials and methods pH Temperature Salinity Dissolved Total Oil Media (°C) (‰) oxygen dissolved concentration (mg/L) solids (‰) (mg/L) Basal salts medium (BSM; 25 g NaCl, 0.7 g KCl, 0.7 g 8.12 16.8 32.15 5.60 24.3 1.031 MgSO ·7H O, 1 g NH NO ,2gKH PO ,3g 4 2 4 3 2 4 Na HPO ·2H O) was sterilized for 20 min at 121 °C, 2 4 2 supplemented with 2 % microelements (sterilized by Diverse petroleum-degrading bacteria inhabit marine filtration through a 0.22-μm membrane), and the pH environments. Over the past years, diverse bacteria capable adjusted to 7.5. The medium was supplemented with diesel oil as the sole carbon source. The microelement of degrading petroleum hydrocarbons have been isolated and characterized from coastal and oceanic environments, solution contained (per liter) 4 g MgSO ·7H O, 1 g 4 2 including the genera Pseudomonas (El-Naas et al. 2009), CuSO ·5H O, 1 g MnSO ·H O, 1gFeSO ·7H O, and 1 g 4 2 4 2 4 2 Vibrio, Flavobacterium, Marinomonas (Gauthier et al. CaCl .Luria–Bertani (LB) medium contained (per liter) 1992), Halomonas, Alcanivorax (Olivera et al. 2009), 10.0 g NaCl, 10.0 g peptone, 5.0 g yeast extract, and 1 L Cycloclasticus (Kasai et al. 2002), Marinobacter, Neptuno- distilled water), and the solution pH was adjusted to 7.4. Solid monas (Li and Chen 2009), Oleiphilus, Mycobacterium LB medium was prepared by adding 20.0 g agar to 1 L LB (Pagnout et al. 2007), among others. The enormity of the medium. HLB medium contained (per liter) 30.0 g NaCl, environmental problem calls for bioremediation as a poten- 10.0 g peptone, 5.0 g yeast extract, and 1 L distilled water. tially useful tool, based mainly on the use of indigenous All chemicals used were analytical grade. All organic bacteria, in the cleaning and treatment of petroleum- solvents used were high-performance liquid chromatographic contaminated seawater (Swannell et al. 1996). Recently, dif- (HPLC) grade and purchased from Tedia Company (Fairfield, ferent aspects of microbial biodegradation, such as the OH). petroleum-metabolizing ability of bacteria, petroleum degra- dation mechanisms, factors affecting microbial degradation Sampling (Ilori et al. 2005), assimilation of the degradation-related enzyme into bacterial cellular activities, and discovery of the Seawater samples were obtained in December 2009 from degradation-related gene, have been researched (Tomohisa et Dinghai port. Environmental conditions which affect the al. 2010;Seksan et al. 2009). degradation of diesel oil, such as temperature, salinity, oil In this study, a number of highly efficient indigenous concentration, and dissolved oxygen content, were moni- diesel oil-degrading bacterial strains were isolated from tored according to the People’s Republic of China national seawater sampled from Dinghai port, Zhoushan city, standard method (2007). The environmental conditions of Zhejiang Province, China, and a Acinetobacter sp. strain seawater in Dinghai port are shown in Table 1. The average Y2 was identified based on its 16S rDNA sequence as oil concentration reached 1.031 mg/L, which is twofold well as various morphological and physiological charac- higher than the fourth class quality standard according to teristics. The phylogenesis and biodegradation character- the sea water quality standard of the People’s Republic of istics of strain Y2 were studied and its parameters for oil China. Samples were collected in triplicate using 4-L steril- degradation optimized. This information can help in ized brown bottles, kept on ice during transportation to the defining the optimum conditions for the development laboratory, stored at room temperature, and treated within of an effective bioremediation technology for marine 24 h. environmental clean-up operations. Fig. 1 Biodegradation ratio of diesel oil by the isolated bacterial strains after a 10-day incubation. The incubation conditions were pH 7.5, 30 °C, an initial diesel oil concentra- tion of 2 % (v/v) and an initial concentration of inoculated bacteria of 4×10 cells/mL Ann Microbiol (2013) 63:633–640 635 Table 2 Physiological and biochemical characteristics of strain Y2 diesel oil was then added at a concentration of 0.5 % (v/v). The flask was incubated aerobically at 30 °C on an orbital Physiological and biochemical reactions Characteristic shaker at 200 rpm, and 2-mL aliquots were transferred Catalase + weekly to fresh BSM medium and incubated under the same Oxidase - conditions. The concentration of diesel oil in the BSM medium was increased weekly by 0.5 % up to 2.5 % (v/v). Anaerobic growth - Growth factors required - All enrichment experiments were performed in triplicate. Isolation and purification procedures were carried out on Utilization of carbon source LB agar plates by conventional spread plate techniques. Glucose - Pure bacterial strains obtained were kept on LB slant culture D-Fructose - at 4 °C for further analysis and stored as liquid cultures L-Arabinose - containing 15% glycerol at −80 °C. D-Ribose - Na-Citrate + Morphological and physiological characteristics Sodium acetate + Utilization of nitrogen source After the bacteria had grown on agar plates, the morphological Ammonium sulfate + properties of the cells were examined by light microscopy and Potassium nitrate + transmission electron microscopy (TEM). The presence or +, Positive; −, negative absence of flagella was examined by TEM using cells from exponentially growing cultures. Physiological and biochemi- cal characteristics were determined according to the “Manual Enrichment and isolation of diesel oil-degrading bacteria of determinative bacteriology” (Xiu and Miao 2001). We used diesel oil as the sole carbon and energy source to enrich petroleum-degrading bacteria because diesel oil was Analysis of 16S rDNA sequence and phylogenetic analysis the main oil pollution compound of seawater in the port. About 5-mL samples of seawater were transferred into 250- One isolated strain, denoted Y2, grown on agar plates was mL conical flasks containing 100 mL BSM liquid medium; used for PCR amplification of 16S rDNA using the primer Acinetobacter johnsonii DSM 6963 (X81663) Acinetobacter bouvetii 4B02 (AF509827) Acinetobacter schindleri LUH5832 (AJ278311) Acinetobacter gyllenbergii 1271 (AJ293694) Acinetobacter haemolyticus DSM 6962 (X81662) 44 Acinetobacter beijerinckii 58a (AJ626712) Acinetobacter tandoii 4N13 (AF509830) 19 T Acinetobacter parvus LUH4616 (AJ293691) 18 Acinetobacter tjernbergiae 7N16 (AF509825) Acinetobacter berezinae ATCC 17924 (Z93443) 98 Acinetobacter guillouiae ATCC 11171 (X81659) Acinetobacter ursingii LUH 3792 (AJ275038) Acinetobacter lwoffii DSM 2403 (X81665) Acinetobacter calcoaceticus DSM 30006 (X81661) Acinetobacter junii LMG 998 (AM410704) Acinetobacter baumannii DSM 30007 (X81660) Acinetobacter gerneri 9A01 (AF509829) Acinetobacter radioresistens DSM 6976 (X81666) Y2 Acinetobacter venetianus RAG-1 (AJ295007) Acinetobacter towneri AB1110 (AF509823) Acinetobacter baylyi B2 (AF509820) 82 Acinetobacter soli B1 (EU290155) Alkanindiges illinoisensis MVAB Hex1 (AF513979) Perlucidibaca piscinae IMCC1704 (DQ664237) Psychrobacter aestuarii SC35 (EU939718) Enhydrobacter aerosaccus LMG 21877 (AJ550856) Moraxella cuniculi CCUG 2154 (AF005188) Moraxella catarrhalis ATCC 25238 (U10876) Moraxella caviae CCUG 355 (AF005187) Moraxella bovoculi 237 (DQ153089) Moraxella lacunata ATCC 17967 (AF005160) 99 Moraxella equi 327/72 (AF005184) 0.01 Fig. 2 Phylogenetic tree of Acinetobacter sp. strain Y2 and related species constructed on the basis of 16S rDNA sequences using the neighbor- joining method.Bar: 0.01 knuc unit 636 Ann Microbiol (2013) 63:633–640 Table 3 Effect of pH on the biodegradation ratio of diesel oil by 200 rpm for about 12 h. When the optical density (OD )of strain Y2 the bacterial suspension was 1.0 (approx. 10 cells/mL), the bacterial suspension was centrifuged for 10 min at pH 3,000 rpm. After the supernatant was discarded and the 6.0 7.0 7.5 8.0 8.5 wet bacteria washed with sterilized BSM liquid medium, the cell suspensions were inoculated into 250-mL conical Biodegradation ratio 44.8 % 57.4 % 75.6 % 65.8 % 45.6 % flasks containing sterilized liquid culture (per flask: 100 mL BSM with a specified amount of diesel oil) and incubated in The incubation conditions were pH 7.5, 30 °C, an initial diesel oil concentration of 2 % (v/v), and an initial inoculated bacteria concen- darkness on an orbital shaker. The degradation ratio was tration of 4×10 cells/mL. All data were obtained after 7 days of detected by infrared spectrometry (Sun et al. 2002). incubation The biodegradation characteristics of strain Y2 were studied and its parameters for oil degradation optimized. set 27 F (5′-AGA GTT TGA TYM TGG CTC AG-3′) and The relationship between the degradation ratio and sev- 1492R (5′-GGH TAC CTT GTT ACG ACT T-3′) (Lane eral parameters were investigated, including incubation 1991). The amplification cycling conditions consisted of time, different initial diesel oil concentrations (v/v: 1, an initial denaturation at 94 °C for 10 min, followed by 30 1.5, 2, 2.5, 3 %), inoculated bacteria concentrations 7 7 7 7 7 cycles of denaturation at 94 °C for 1 min, annealing at 53 °C (1×10 ,2×10 ,3×10 ,4×10 ,5×10 cells/mL), and for 90 s, and elongation at 72 °C for 90 s, with a final various pH (6.0, 7.0, 7.5, 8.0, 8.5) and temperatures elongation at 72 °C for 10 min. The AccuPrep PCR Purifi- (20, 25, 30, 37 °C). The degradation ratio was detected cation kit (Bioneer Corp, Kawasaki, Japan,) was used for after 7 days of incubation. PCR product purification. Sequencing was performed on All biodegradation experiments were performed in triplicate. Sterilized cultures without inoculation were used as negative both strands by the Shanghai Major Bio Technology Co. (Shanghai, China). DNA sequencing using the primers controls. 338 F (5′-ACT CCT ACG GGW GGC WGC AG-3′), 536R (5′-ATT ACC GCG GCT GCT GG-3′) and 907 F Gas chromatography–mass spectrometry analysis (5′-AAA CTC AAA KGA ATT GAC GG-3′) (Quan et al. 2005) were determined directly from the purified PCR The degradation effect of diesel oil was detected by gas product. The DNA sequence of the cloned 16S rDNA frag- chromatography–mass spectrometry (GC-MS; Focus DSQ ments was compared using EzTaxon Server ver. 2.1 (http:// GC-MS; Thermo Fisher Scientific, Waltham, MA). The 147.47.212.35:8080), and sequences were analyzed phylo- diesel oil remaining in the liquid culture was extracted with genetically using the software Clustal_X ver. 1.83 (Thomp- dichloromethane three times. The organic phase was then son et al. 1997). The phylogenetic tree was constructed dehydrated with anhydrous sodium sulfate, and 1 μL of the using the neighbor joining method in the software program organic phase was analyzed by GC–MS. The gas chromato- Mega 4 (Tamura et al. 2007). graph was equipped with a split–splitless injector (split ratios of 50:1) and an HP-5 MS column (30 m×0.25 mm× 0.25 μm; Biodegradation assays Agilent Technologies, Santa Clara, CA). The oven temperature was initially held at 60 °C for 2 min and then programmed to −1 Assays on the degradation of diesel oil were performed in 300 °C at a rate of 20 °C min where it was held for 5 min. The liquid culture using washed cell suspensions. Bacteria iso- temperatures of the injector, transfer line, and ionization source lated from agar plates were inoculated into HLB medium were all 250 °C. The electron impact ionization was tuned to 70 and incubated aerobically at 30 °C on an orbital shaker at eV, and helium was used as carrier gas at an average linear flow Fig. 3 Effect of inoculating bacteria concentration on the biodegradation ratio of diesel oil. The incubation conditions were pH 7.5, 30 °C, an initial diesel oil concentration of 2 % (v/v). All data were obtained at 7 days of incubation Ann Microbiol (2013) 63:633–640 637 Fig. 4 Effect of initial diesel oil concentration on the growth of strain Y2 and the biodegradation ratio of diesel oil. The incubation conditions were pH 7.5, 30 °C, and an initial concentration of inoculated bacteria of 3×10 cells/mL. All data were obtained at 7 days of incubation -1 of 1.0 mL min . The mass spectra were recorded within 41– days were round or nearly round, 1–2mmindiameter, 400 amu to collect the total ion current (TIC) chromatograms. bamboo-yellow in color, smooth, moist, and low convex. The edge of colonies was even on the LB solid plates. The cells were Gram-negative, strictly aerobic, oxidase negative, Calculation method andcatalasepositive.In a youngculture, theyappeared as The degradation ratio was defined as follows: short rods, 0.9–1.2 μm in diameter and 1.9–2.2 μm long, and in the stationary phase of growth, they appeared as coccid cells, η ¼ðÞ C  C =C  100%; 0 1 0 either singly, in pairs, or occasionally as short chains or irreg- where C is the residual diesel oil in the control sample; ular clumps. Spore formation was not observed. Strain Y2 was unable to use glucose, D-fructose, L-arabinose, or D-ribose as a C is the residual diesel oil in the test sample. The value of the degradation ratio is the average of three samples. The carbon source but could use sodium citrate and sodium acetate as the sole source of carbon and potassium nitrate and ammo- mean and standard deviation (SD) of triplicate measure- ments were calculated by Excel for Windows (Microsoft, nium sulfate as the sole nitrogen source, without additional growth factors. The physiological and biochemical character- Redwood, USA). istics of strainY2are showninTable 2. Results and discussion 16S rDNA sequence and phylogenetic analysis The almost complete 16S rDNA gene sequences of strain Isolation of diesel oil-degrading bacteria Y2 was a continuous stretch of 1,465 kb. Subsequent 16S rDNA-based phylogenetic analysis demonstrated that the Eight strains (denoted Y1, Y2, Y3, Y4, Y5, Y6, Y7, and Y8, respectively) of diesel oil-degrading bacteria were isolated. strain belonged to the genus Acinetobacter sp. Fig. 2 shows The degradation radto of diesel oil by these isolated strains the relationship between the isolated strain and the nearest after a 10-day incubation is shown in Fig. 1. The biodegra- phylogenetic relatives. Similarity calculations after neighbor- dation ratio of diesel oil by strain Y2 was up to 80 %. Strain joining analysis indicate that the closest relative of strain Y2 is Y2 was therefore identified in more detail and selected for Acinetobacter venetianus RAG-1 (100 %). further study of its oil-degrading characteristics. Effect of environmental conditions on the degradation Morphological and biochemical characteristics of diesel oil Strain Y2 was subjected to morphological and biochemical A series of degradation tests were carried out at various studies. Strain Y2 colonies grown on nutrient agar plates for 3 pH (6.0–8.5), incubation times (1–10 days), temperatures Fig. 5 Effect of temperature on the biodegradation ratio of diesel oil by strain Y2. The incubation conditions were pH 7.0, an initial diesel oil concentration of 2% (v/v), and an initial concentration of inoculated bacteria of 3×10 cells/mL. All data were obtained at 7 days of incubation 638 Ann Microbiol (2013) 63:633–640 Fig. 6 Changes in the growth of strain Y2 and the diesel oil biodegradation ratio with incubation time. The incubation conditions were pH 7.5, 30 °C, an initial diesel oil concentration of 2 % (v/v), and an initial concentration of inoculated bacteria of 3×10 cells/mL (20–37 °C), initial diesel oil concentrations (v/v: 1, 1.5, ratio was higher at initial oil concentrations (1, 1.5, and 2 %) 2, 2.5, 3 %), and inoculated bacteria concentrations (1× than at high initial oil concentrations (2.5 and 3.0 %). The 7 7 7 7 7 10 ,2×10 ,3×10 ,4×10 ,5×10 cells/mL). highest degradation ratio was achieved at an initial oil pH is an important environmental factor which impacts concentration of 2 % (v/v), possibly because when the initial oil biodegradation. As shown in Table 3, the degradation concentration of diesel oil is too high, the oil would cover ratio was the highest at pH approximately 7.5–8.0, and the surface of the water and prevent oxygen from dissolving Acinetobacter sp. strain Y2 was able to degrade oil when into the liquid culture. Aerobic conditions are generally the pH ranged from 6 to 8.5. Previous studies have shown considered to necessary for extensive degradation of oil that the degradation of oil increases with increasing pH and hydrocarbons in the environment since major degradation that optimum degradation occurs under slightly alkaline pathways for both saturates and aromatics involve oxy- conditions (Dibble and Bartha 1979; Foght and Westlake genases (Cerniglia 1992). The study of Camilli et al. 1987). The pH is an important environmental factor which (2010) reported that the dissolved oxygen concentrations impacts microbial growth through a number of mechanisms. in the plume of the Gulf of Mexico oil spill suggested that First, the charge of proteins, nucleic acids, and other bio- microbial respiration rates within the plume were not appre- logical macromolecules changes with pH; thus, pH affects ciably more than 1 micromolar oxygen per day. Many microbial biological activity. Second, the environmental pH studies have shown that oxygen depletion leads to sharply changes the electric charge in the cell membrane, which in reduced biodegradation activities (Bossert and Bartha 1984) turn affects the ability of the microbial cell to absorb and subsequently to a decreased biodegradation ratio. nutrients. Third, pH modifies nutrient availability and the The effect of environmental temperature on oil degradation toxicity of hazardous substances. Since the pH of our sea- was also investigated because of the significant role of tem- water samples was about 8.0, our results demonstrate that perature. As shown in Fig. 5, four different temperatures (20, the investigated bacteria could effectively degrade the diesel 25, 30, and 37 °C) were evaluated for their effect on diesel oil oil in this environment. degradation. The results showed that the biodegradation ratio The effect of the inoculated bacteria concentration on the was only about 15 % at the lowest temperature tested (20 °C) biodegradation ratio of diesel oil and the cell density at the end but that it increased up to approximately 60–70 % at high of the experiments are shown in Fig. 3. The results indicate that temperatures (30, 37 °C). Temperature not only controls the the degradation ratio of Acinetobacter sp. strain Y2 decreased nature and extent of microbial hydrocarbon metabolism, but it when the initial concentration of inoculating bacteria was too also directly affects the physicochemical behavior of oil hydro- 7 7 low (1×10 cells/mL) or too high (5×10 cells/mL). carbons, such as viscosity, diffusion, and volatilization, which The biodegradation ratio of diesel oil at different initial changes the oil composition and bioavailability of the water- oil concentrations are shown in Fig. 4. The biodegradation soluble components. At low temperatures, the viscosity of the Fig. 7 The chromatogram of diesel oil remaining in Acinetobacter sp. diesel oil concentration of 2 % (v/v), and an initial concentration of strain Y2 culture (b) and abiotic control (a) media after 10 days of inoculated bacteria was 3×10 cells/mL incubation. The incubation conditions were pH 7.5, 30 °C, an initial Ann Microbiol (2013) 63:633–640 639 oil increases, while the volatility of toxic low-molecular- environment, a study of the effect of environmental conditions weight hydrocarbons decreases (Atlas and Bartha 1972), there- on the degradation of petroleum was required. Therefore, we by explaining why the ratio of degradation generally decreases studied such environmental factors as salinity, dissolved oxy- with decreasing temperature. gen, nutrient salts, and the mechanisms of degradation of The relationship between bacterial growth and biodegra- strain Y2 in the marine environment in order to determine dation ratio is shown in Fig. 6. The degradation ratio and the effect of these factors on the degradation of petroleum. The cell density (OD ) increased sharply up to 42 % and 2.0, degradation capabilities of diesel oil by Acinetobacter sp. respectively, on the first day of culture. The ratio of degra- strain Y2 suggest that this strain can be exploited further for dation subsequently increased with increasing cell density the development of effective bioremediation technology for on the first 4 days. Cell density started to decline from the the remediation of hydrocarbon pollution in the marine fifth day onwards, and the ratio of degradation increased environment. slowly, probably because the nutrient condition was rich at the beginning, so bacteria grew massively. Beginning on the Acknowledgments This work was supported by a grant from Shanghai fifth day, the degradation ratio increased slowly and the Natural Science Foundation of China (No. 08ZR1401300) bacteria growth volume began to decrease due to limitations imposed by metabolic wastes and available nutrients. Based on our experimental results, we determined the References optimal culture conditions for strain Y2 to be an initial pH ranging from 7.5 to 8.0, an incubation temperature of 30 °C, Atlas RM, Bartha R (1972) Biodegradation of petroleum in seawater at an initial diesel oil concentration of 2 % (v/v), and an initial low temperatures. Can J Microbiol 18:1851–1855 inoculating bacteria concentration of 3×10 cells/mL. The Bossert I, Bartha R (1984) The fate of petroleum in soil ecosystems. In: Atlas RM (ed) Petroleum microbiology. Macmillan , New York, degradation ratio of diesel oil reached as high as 80 % under pp 435–476 these optimal conditions after 10 days of incubation. Camilli R, Reddy CM, Yoerger DR, Van Mooy BA, Jakuba MV, Kinsey JC, McIntyre CP, Sylva SP, Maloney JV (2010) Tracking Gas chromatography–mass spectrometry analysis hydrocarbon plume transport and biodegradation at Deepwater Horizon. Science 330(6001):201–204 Cerniglia CE (1992) Biodegradation of polycyclic aromatic hydrocarbons. Diesel oil in the culture medium of Acinetobacter sp. strain Biodegradation 3:351–368 Y2 was analyzed by GC–MS after 10 days of culture Dibble JT, Bartha R (1979) Effect of environmental parameters on (Fig. 7). The components of diesel oil identified on the biodegradation of oil sludge. Appl Environ Microbiol 37:729–739 El-Naas MH, Al-Muhtaseb SA, Makhlouf S (2009) Biodegradation of chromatogram were mixed hydrocarbons (C11–C21), and phenol by Pseudomonas putida immobilized in polyvinyl alcohol all components of the diesel oil had been degraded by (PVA) gel. J Hazard Mater 164:720–725 Acinetobacter sp. strain Y2. Acinetobacter is one of the Foght JM, Westlake DWS (1987) Biodegradation of hydrocarbons in representative genera capable of growing on n-alkanes (Tani freshwater. In: Vandermeulen JH, Hrudey SE (eds) Oil in fresh- water: chemistry, biology, countermeasure technology. Pergamon et al. 2002). They are therefore frequently detected in waste Press, New York, pp 217–230 water treatment processes, seawater, and sewage sludge Gauthier MJ, Lafay B, Christen R, Fernandez L, Acquaviva M, Bonin (Gottsching and Schmidt. 2007; Phrommanicha et al. P, Bertrand JC (1992) Marinobacter hydrocarbonoclasticus gen. 2009). Microorganisms are known to attack and degrade a nov., sp. nov., a new, extremely halotolerant, hydrocarbon-degrading marine bacterium. Int J Syst Bacteriol 42(4):568–576 specific component as compared with other components of Gottsching A, Schmidt S (2007) Productive degradation of the biocide petroleum. Obayori et al. (2009) observed that the same benzyl benzoate by Acinetobacter sp. strain AG1 isolated from compounds in different crude oil samples were degraded the River Elbe. Res Microbiol 158:251–257 to different extents by the same organisms. Acinetobacter Ilori MO, Amobi CJ, Odocha AC (2005) Factors affecting biosurfactant production by oil degrading Aeromonas spp. isolated from a tropical sp. strain Y2 has the unusual characteristic of being able to environment. Chemosphere 61:985–992 grow on a broad range of n-alkanes, which suggests that Jeong WG, Cho SM (2007) Long-term effect of polycyclic aromatic strain Y2 could potentially be useful for the remediation of hydrocarbon on physiological metabolisms of the Pacific oyster, hydrocarbon pollution in the marine environment. Crassostrea gigas. Aquaculture 265:343–350 Kasai Y, Kishira H, Harayama S (2002) Bacteria belonging to the genus Cycloclasticus play a primary role in the degradation of aromatic hydrocarbons released in a marine environment. Appl Conclusion Environ Microbiol 68:5625–5633 Lane DJ (1991) 16S/23S rRNA sequencing. In: Goodfellow M, Stack- ebrandt E (eds) Nucleic acid techniques in bacterial systematics. Acinetobacter sp strain Y2 has a high ability to degrade Wiley, Chichester, pp 115–175 diesel oil in a wide pH, temperature and initial diesel oil Li JL, Chen BH (2009) Effect of nonionic surfactants on biodegrada- concentration range. Given the complexity of oil products tion of phenanthrene by a marine bacteria of Neptunomonas and environmental conditions in a contaminated natural naphthovorans. J Hazard Mater 162:66–73 640 Ann Microbiol (2013) 63:633–640 Liu YC, Li LZ, Wu Y et al (2010) Isolation of an alkane-degrading contaminants, 4th edn. China Environmental Science Press, Bei- Alcanivorax sp. strain 2B5 and cloning of the alkB gene. Bioresour jing, pp 491–495 Technol 101:310–316 Swannell RPJ, Lee K, McDonagh M (1996) Field evaluations of Obayori OS, Adebusoye SA, Adewale AO, Oyetibo GO, Oluyemi OO, marine oil spill bioremediation. Microbiol Rev 60:342–365 Amokun RA, Ilori MO (2009) Differential degradation of crude Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: Molecular oil (Bonny Light) by four Pseudomonas strains. J Environ Sci Evolutionary Genetics Analysis (MEGA) software version 4.0. 21:243–248 Mol Biol Evol 24:1596–1599 Olivera NL, Nievas ML, Lozada M (2009) Isolation and characterization Tani A, Ishige T, Sakai Y, Kato N (2002) Two acyl-CoA dehydro- of biosurfactant-producing Alcanivorax strains: hydrocarbon acces- genases of Acinetobacter sp. strain M-1 that uses very long-chain sion strategies and alkanehydroxylase gene analysis. Res Microbiol n-alkanes. J Biosci Bioeng 94(4):326–329 160:19–26 Tomohisa K, Miyanaga A, Kanaya S, Morikawa M (2010) Gene Pagnout C, Frache G, Poupin P, Maunit B, Muller JF, Ferard JF (2007) cloning and characterization of an aldehyde dehydrogenase from Isolation and characterization of a gene cluster involved in PAH long-chain alkane-degrading Geobacillus thermoleovorans B23. degradation in Mycobacterium sp. strain SNP11: Expression in Extremophiles 14:33–39 Mycobacterium smegmatis mc2155. Res Microbiol 158:175– Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG 186 (1997) The CLUSTAL_X windows interface: flexible strategies Phrommanicha S, Suanjitb S, Upathamc S, Gramsd SV, Kruatrachued for multiple sequence alignment aided by quality analysis tools. M, Pokethitiyookd P, Korgee G, Hofmanne A (2009) Quantitative Nucleic Acids Res 25:4876–4882 detection of the oil-degrading bacterium Acinetobacter sp. strain Venosa AD, Zhu XQ (2003) Biodegradation of crude oil contaminating MUB1 by hybridization probe based real-time PCR. Microbiol marine shorelines and freshwater wetlands. Spill Sci Technol B 8 Res 164:486–492 (2):163–178 Quan ZX, Bae HS, Baek JH, Chen WF, Im WT, Lee ST (2005) Xiu ZD, Miao YC (2001) Manual of determinative bacteriology. Science Rhizobium daejeonense sp. nov. isolated from a cyanide treatment Press, Beijing bioreactor. Int J Syst Evol Microbiol 55:2543–2549 Yamamoto T, Nakaoka M, Komatsu T, Kawai H, Ohwada K, Marine Seksan P, Sudarat S, Suchart U, Suksiri VG, Maleeya K, Prayad P, Gunter Life Research Group of Takeno (2003) Impacts by heavy-oil spill K, Annemarie H (2009) Quantitative detection of the oil-degrading from the Russian tanker Nakhodka on intertidal ecosystems: re- bacterium Acinetobacter sp. strain MUB1 by hybridization probe covery of animal community. Mar Pollut Bull 47:91–98 base dreal-time PCR. Microbiol Res 164:486–492 Yang X, Sun Y, Qian S (2004) Biodegradation of seven polychlorinated Sun ZG, Huang YR et al (2002) Determination methods for examina- biphenyls by a newly isolated aerobic bacterium (Rhodococcus sp. tion of water and wastewater. In: Wei FS (ed) Organic R04). J Ind Microbiol. Biotechnol 31:415–420 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Annals of Microbiology Springer Journals

Isolation and characterization of marine diesel oil-degrading Acinetobacter sp. strain Y2

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Springer Journals
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Copyright © 2012 by Springer-Verlag 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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1590-4261
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1869-2044
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10.1007/s13213-012-0513-9
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Abstract

Ann Microbiol (2013) 63:633–640 DOI 10.1007/s13213-012-0513-9 ORIGINAL ARTICLE Isolation and characterization of marine diesel oil-degrading Acinetobacter sp. strain Y2 Qun Luo & Jian-Guo Zhang & Xian-Rong Shen & Zheng-Qiu Fan & Ying He & Deng-Yong Hou Received: 10 December 2011 /Accepted: 16 July 2012 /Published online: 16 September 2012 Springer-Verlag and the University of Milan 2012 Abstract The marine diesel oil-degrading bacterium Acineto- production, transportation, and disposal. There have been bacter sp. strain Y2 was isolated from oil-polluted seawater many incidents of large-scale spills all over the world, the sampled from Dinghai port, Zhoushan City, Zhejiang Province, majority with serious consequences to the environment. The China. The isolated bacterium was identified as Acinetobacter recent oil spill accident in the Gulf of Mexico and the oil sp. based on its 16S rDNA gene sequence as well as various pipeline explosion accident in the city of Dalian, China had morphological and physiological characteristics. The degrada- devastating impacts on the marine environment. It is esti- tion characteristics of strain Y2 were studied and its parameters mated that hundreds of millions of liters of petroleum enter for oil degradation optimized. These optimal conditions were into the marine environment each year. Oil pollution not determined to be an initial pH of 7.5, an incubation temperature only causes serious damage to fisheries, but it also has of 30 °C, an initial diesel oil concentration of 2 % (v/v), and an adverse effects on the natural environment and ecosystem initial inoculating bacteria concentration of 3×10 cells/mL. (Yamamoto et al. 2003; Jeong and Cho 2007). In areas The results from the gas chromatography–mass spectrometry affected by petroleum pollution, local populations of inver- analysis showed that strain Y2 could almost completely de- tebrates, birds, and mammals may be greatly reduced and grade all components of diesel oil, with a degradation ratio of vegetation may die. In general, those organisms worst af- up to 80 % after 10 days of incubation at the optimal conditions. fected are the ones which inhabit shallow water and the littoral zone and those, such as marine mammals, which . . . Keywords Biodegradation Isolation Characterization are obliged to cross the air–sea interface. Acinetobacter sp. The traditional methods to cope with petroleum pollu- tion are confined to physical and chemical containment. These are usually applied to clean up high concentrations of polluting oil, such as those due to an oil spill, but their Introduction effects on low concentrations of oil pollution are not very good; moreover, they are generally expensive and may be a Petroleum pollution has become a serious environmental source of secondary pollution (Yang et al. 2004). The problem, especially in marine environments, due to oil seriousness of the petroleum pollution problem has moti- vated researchers to explore the possibilities of microbio- Q. Luo logical degradation (i.e., bioremediation) of petroleum. Department of Nuclear Science and Engineering, Naval University Biological methods represent a popular option for the of Engineering, Wuhan 430033, China clean-up of environments polluted with hydrocarbons, and they may represent an efficient, economic, and versa- : : : : Q. Luo J.-G. Zhang X.-R. Shen (*) Y. He D.-Y. Hou tile alternative to physicochemical treatments (Venosa and The Naval Medical Research Institute, Zhu 2003). The first major large-scale use of bioremedia- Shanghai 200433, China tion methods to clean up an oil spill was during the Exxon e-mail: xianrong_sh@yahoo.com Valdez accident in 1989. Since then, the development of Z.-Q. Fan bioremediation methods for marine oil pollution has Department of Environmental Science & Engineering, attracted worldwide interest and become an active research Fudan University, area (Liu et al. 2010). Shanghai 200433, China 634 Ann Microbiol (2013) 63:633–640 Table 1 Environmental conditions of the seawater in Dinghai port Materials and methods pH Temperature Salinity Dissolved Total Oil Media (°C) (‰) oxygen dissolved concentration (mg/L) solids (‰) (mg/L) Basal salts medium (BSM; 25 g NaCl, 0.7 g KCl, 0.7 g 8.12 16.8 32.15 5.60 24.3 1.031 MgSO ·7H O, 1 g NH NO ,2gKH PO ,3g 4 2 4 3 2 4 Na HPO ·2H O) was sterilized for 20 min at 121 °C, 2 4 2 supplemented with 2 % microelements (sterilized by Diverse petroleum-degrading bacteria inhabit marine filtration through a 0.22-μm membrane), and the pH environments. Over the past years, diverse bacteria capable adjusted to 7.5. The medium was supplemented with diesel oil as the sole carbon source. The microelement of degrading petroleum hydrocarbons have been isolated and characterized from coastal and oceanic environments, solution contained (per liter) 4 g MgSO ·7H O, 1 g 4 2 including the genera Pseudomonas (El-Naas et al. 2009), CuSO ·5H O, 1 g MnSO ·H O, 1gFeSO ·7H O, and 1 g 4 2 4 2 4 2 Vibrio, Flavobacterium, Marinomonas (Gauthier et al. CaCl .Luria–Bertani (LB) medium contained (per liter) 1992), Halomonas, Alcanivorax (Olivera et al. 2009), 10.0 g NaCl, 10.0 g peptone, 5.0 g yeast extract, and 1 L Cycloclasticus (Kasai et al. 2002), Marinobacter, Neptuno- distilled water), and the solution pH was adjusted to 7.4. Solid monas (Li and Chen 2009), Oleiphilus, Mycobacterium LB medium was prepared by adding 20.0 g agar to 1 L LB (Pagnout et al. 2007), among others. The enormity of the medium. HLB medium contained (per liter) 30.0 g NaCl, environmental problem calls for bioremediation as a poten- 10.0 g peptone, 5.0 g yeast extract, and 1 L distilled water. tially useful tool, based mainly on the use of indigenous All chemicals used were analytical grade. All organic bacteria, in the cleaning and treatment of petroleum- solvents used were high-performance liquid chromatographic contaminated seawater (Swannell et al. 1996). Recently, dif- (HPLC) grade and purchased from Tedia Company (Fairfield, ferent aspects of microbial biodegradation, such as the OH). petroleum-metabolizing ability of bacteria, petroleum degra- dation mechanisms, factors affecting microbial degradation Sampling (Ilori et al. 2005), assimilation of the degradation-related enzyme into bacterial cellular activities, and discovery of the Seawater samples were obtained in December 2009 from degradation-related gene, have been researched (Tomohisa et Dinghai port. Environmental conditions which affect the al. 2010;Seksan et al. 2009). degradation of diesel oil, such as temperature, salinity, oil In this study, a number of highly efficient indigenous concentration, and dissolved oxygen content, were moni- diesel oil-degrading bacterial strains were isolated from tored according to the People’s Republic of China national seawater sampled from Dinghai port, Zhoushan city, standard method (2007). The environmental conditions of Zhejiang Province, China, and a Acinetobacter sp. strain seawater in Dinghai port are shown in Table 1. The average Y2 was identified based on its 16S rDNA sequence as oil concentration reached 1.031 mg/L, which is twofold well as various morphological and physiological charac- higher than the fourth class quality standard according to teristics. The phylogenesis and biodegradation character- the sea water quality standard of the People’s Republic of istics of strain Y2 were studied and its parameters for oil China. Samples were collected in triplicate using 4-L steril- degradation optimized. This information can help in ized brown bottles, kept on ice during transportation to the defining the optimum conditions for the development laboratory, stored at room temperature, and treated within of an effective bioremediation technology for marine 24 h. environmental clean-up operations. Fig. 1 Biodegradation ratio of diesel oil by the isolated bacterial strains after a 10-day incubation. The incubation conditions were pH 7.5, 30 °C, an initial diesel oil concentra- tion of 2 % (v/v) and an initial concentration of inoculated bacteria of 4×10 cells/mL Ann Microbiol (2013) 63:633–640 635 Table 2 Physiological and biochemical characteristics of strain Y2 diesel oil was then added at a concentration of 0.5 % (v/v). The flask was incubated aerobically at 30 °C on an orbital Physiological and biochemical reactions Characteristic shaker at 200 rpm, and 2-mL aliquots were transferred Catalase + weekly to fresh BSM medium and incubated under the same Oxidase - conditions. The concentration of diesel oil in the BSM medium was increased weekly by 0.5 % up to 2.5 % (v/v). Anaerobic growth - Growth factors required - All enrichment experiments were performed in triplicate. Isolation and purification procedures were carried out on Utilization of carbon source LB agar plates by conventional spread plate techniques. Glucose - Pure bacterial strains obtained were kept on LB slant culture D-Fructose - at 4 °C for further analysis and stored as liquid cultures L-Arabinose - containing 15% glycerol at −80 °C. D-Ribose - Na-Citrate + Morphological and physiological characteristics Sodium acetate + Utilization of nitrogen source After the bacteria had grown on agar plates, the morphological Ammonium sulfate + properties of the cells were examined by light microscopy and Potassium nitrate + transmission electron microscopy (TEM). The presence or +, Positive; −, negative absence of flagella was examined by TEM using cells from exponentially growing cultures. Physiological and biochemi- cal characteristics were determined according to the “Manual Enrichment and isolation of diesel oil-degrading bacteria of determinative bacteriology” (Xiu and Miao 2001). We used diesel oil as the sole carbon and energy source to enrich petroleum-degrading bacteria because diesel oil was Analysis of 16S rDNA sequence and phylogenetic analysis the main oil pollution compound of seawater in the port. About 5-mL samples of seawater were transferred into 250- One isolated strain, denoted Y2, grown on agar plates was mL conical flasks containing 100 mL BSM liquid medium; used for PCR amplification of 16S rDNA using the primer Acinetobacter johnsonii DSM 6963 (X81663) Acinetobacter bouvetii 4B02 (AF509827) Acinetobacter schindleri LUH5832 (AJ278311) Acinetobacter gyllenbergii 1271 (AJ293694) Acinetobacter haemolyticus DSM 6962 (X81662) 44 Acinetobacter beijerinckii 58a (AJ626712) Acinetobacter tandoii 4N13 (AF509830) 19 T Acinetobacter parvus LUH4616 (AJ293691) 18 Acinetobacter tjernbergiae 7N16 (AF509825) Acinetobacter berezinae ATCC 17924 (Z93443) 98 Acinetobacter guillouiae ATCC 11171 (X81659) Acinetobacter ursingii LUH 3792 (AJ275038) Acinetobacter lwoffii DSM 2403 (X81665) Acinetobacter calcoaceticus DSM 30006 (X81661) Acinetobacter junii LMG 998 (AM410704) Acinetobacter baumannii DSM 30007 (X81660) Acinetobacter gerneri 9A01 (AF509829) Acinetobacter radioresistens DSM 6976 (X81666) Y2 Acinetobacter venetianus RAG-1 (AJ295007) Acinetobacter towneri AB1110 (AF509823) Acinetobacter baylyi B2 (AF509820) 82 Acinetobacter soli B1 (EU290155) Alkanindiges illinoisensis MVAB Hex1 (AF513979) Perlucidibaca piscinae IMCC1704 (DQ664237) Psychrobacter aestuarii SC35 (EU939718) Enhydrobacter aerosaccus LMG 21877 (AJ550856) Moraxella cuniculi CCUG 2154 (AF005188) Moraxella catarrhalis ATCC 25238 (U10876) Moraxella caviae CCUG 355 (AF005187) Moraxella bovoculi 237 (DQ153089) Moraxella lacunata ATCC 17967 (AF005160) 99 Moraxella equi 327/72 (AF005184) 0.01 Fig. 2 Phylogenetic tree of Acinetobacter sp. strain Y2 and related species constructed on the basis of 16S rDNA sequences using the neighbor- joining method.Bar: 0.01 knuc unit 636 Ann Microbiol (2013) 63:633–640 Table 3 Effect of pH on the biodegradation ratio of diesel oil by 200 rpm for about 12 h. When the optical density (OD )of strain Y2 the bacterial suspension was 1.0 (approx. 10 cells/mL), the bacterial suspension was centrifuged for 10 min at pH 3,000 rpm. After the supernatant was discarded and the 6.0 7.0 7.5 8.0 8.5 wet bacteria washed with sterilized BSM liquid medium, the cell suspensions were inoculated into 250-mL conical Biodegradation ratio 44.8 % 57.4 % 75.6 % 65.8 % 45.6 % flasks containing sterilized liquid culture (per flask: 100 mL BSM with a specified amount of diesel oil) and incubated in The incubation conditions were pH 7.5, 30 °C, an initial diesel oil concentration of 2 % (v/v), and an initial inoculated bacteria concen- darkness on an orbital shaker. The degradation ratio was tration of 4×10 cells/mL. All data were obtained after 7 days of detected by infrared spectrometry (Sun et al. 2002). incubation The biodegradation characteristics of strain Y2 were studied and its parameters for oil degradation optimized. set 27 F (5′-AGA GTT TGA TYM TGG CTC AG-3′) and The relationship between the degradation ratio and sev- 1492R (5′-GGH TAC CTT GTT ACG ACT T-3′) (Lane eral parameters were investigated, including incubation 1991). The amplification cycling conditions consisted of time, different initial diesel oil concentrations (v/v: 1, an initial denaturation at 94 °C for 10 min, followed by 30 1.5, 2, 2.5, 3 %), inoculated bacteria concentrations 7 7 7 7 7 cycles of denaturation at 94 °C for 1 min, annealing at 53 °C (1×10 ,2×10 ,3×10 ,4×10 ,5×10 cells/mL), and for 90 s, and elongation at 72 °C for 90 s, with a final various pH (6.0, 7.0, 7.5, 8.0, 8.5) and temperatures elongation at 72 °C for 10 min. The AccuPrep PCR Purifi- (20, 25, 30, 37 °C). The degradation ratio was detected cation kit (Bioneer Corp, Kawasaki, Japan,) was used for after 7 days of incubation. PCR product purification. Sequencing was performed on All biodegradation experiments were performed in triplicate. Sterilized cultures without inoculation were used as negative both strands by the Shanghai Major Bio Technology Co. (Shanghai, China). DNA sequencing using the primers controls. 338 F (5′-ACT CCT ACG GGW GGC WGC AG-3′), 536R (5′-ATT ACC GCG GCT GCT GG-3′) and 907 F Gas chromatography–mass spectrometry analysis (5′-AAA CTC AAA KGA ATT GAC GG-3′) (Quan et al. 2005) were determined directly from the purified PCR The degradation effect of diesel oil was detected by gas product. The DNA sequence of the cloned 16S rDNA frag- chromatography–mass spectrometry (GC-MS; Focus DSQ ments was compared using EzTaxon Server ver. 2.1 (http:// GC-MS; Thermo Fisher Scientific, Waltham, MA). The 147.47.212.35:8080), and sequences were analyzed phylo- diesel oil remaining in the liquid culture was extracted with genetically using the software Clustal_X ver. 1.83 (Thomp- dichloromethane three times. The organic phase was then son et al. 1997). The phylogenetic tree was constructed dehydrated with anhydrous sodium sulfate, and 1 μL of the using the neighbor joining method in the software program organic phase was analyzed by GC–MS. The gas chromato- Mega 4 (Tamura et al. 2007). graph was equipped with a split–splitless injector (split ratios of 50:1) and an HP-5 MS column (30 m×0.25 mm× 0.25 μm; Biodegradation assays Agilent Technologies, Santa Clara, CA). The oven temperature was initially held at 60 °C for 2 min and then programmed to −1 Assays on the degradation of diesel oil were performed in 300 °C at a rate of 20 °C min where it was held for 5 min. The liquid culture using washed cell suspensions. Bacteria iso- temperatures of the injector, transfer line, and ionization source lated from agar plates were inoculated into HLB medium were all 250 °C. The electron impact ionization was tuned to 70 and incubated aerobically at 30 °C on an orbital shaker at eV, and helium was used as carrier gas at an average linear flow Fig. 3 Effect of inoculating bacteria concentration on the biodegradation ratio of diesel oil. The incubation conditions were pH 7.5, 30 °C, an initial diesel oil concentration of 2 % (v/v). All data were obtained at 7 days of incubation Ann Microbiol (2013) 63:633–640 637 Fig. 4 Effect of initial diesel oil concentration on the growth of strain Y2 and the biodegradation ratio of diesel oil. The incubation conditions were pH 7.5, 30 °C, and an initial concentration of inoculated bacteria of 3×10 cells/mL. All data were obtained at 7 days of incubation -1 of 1.0 mL min . The mass spectra were recorded within 41– days were round or nearly round, 1–2mmindiameter, 400 amu to collect the total ion current (TIC) chromatograms. bamboo-yellow in color, smooth, moist, and low convex. The edge of colonies was even on the LB solid plates. The cells were Gram-negative, strictly aerobic, oxidase negative, Calculation method andcatalasepositive.In a youngculture, theyappeared as The degradation ratio was defined as follows: short rods, 0.9–1.2 μm in diameter and 1.9–2.2 μm long, and in the stationary phase of growth, they appeared as coccid cells, η ¼ðÞ C  C =C  100%; 0 1 0 either singly, in pairs, or occasionally as short chains or irreg- where C is the residual diesel oil in the control sample; ular clumps. Spore formation was not observed. Strain Y2 was unable to use glucose, D-fructose, L-arabinose, or D-ribose as a C is the residual diesel oil in the test sample. The value of the degradation ratio is the average of three samples. The carbon source but could use sodium citrate and sodium acetate as the sole source of carbon and potassium nitrate and ammo- mean and standard deviation (SD) of triplicate measure- ments were calculated by Excel for Windows (Microsoft, nium sulfate as the sole nitrogen source, without additional growth factors. The physiological and biochemical character- Redwood, USA). istics of strainY2are showninTable 2. Results and discussion 16S rDNA sequence and phylogenetic analysis The almost complete 16S rDNA gene sequences of strain Isolation of diesel oil-degrading bacteria Y2 was a continuous stretch of 1,465 kb. Subsequent 16S rDNA-based phylogenetic analysis demonstrated that the Eight strains (denoted Y1, Y2, Y3, Y4, Y5, Y6, Y7, and Y8, respectively) of diesel oil-degrading bacteria were isolated. strain belonged to the genus Acinetobacter sp. Fig. 2 shows The degradation radto of diesel oil by these isolated strains the relationship between the isolated strain and the nearest after a 10-day incubation is shown in Fig. 1. The biodegra- phylogenetic relatives. Similarity calculations after neighbor- dation ratio of diesel oil by strain Y2 was up to 80 %. Strain joining analysis indicate that the closest relative of strain Y2 is Y2 was therefore identified in more detail and selected for Acinetobacter venetianus RAG-1 (100 %). further study of its oil-degrading characteristics. Effect of environmental conditions on the degradation Morphological and biochemical characteristics of diesel oil Strain Y2 was subjected to morphological and biochemical A series of degradation tests were carried out at various studies. Strain Y2 colonies grown on nutrient agar plates for 3 pH (6.0–8.5), incubation times (1–10 days), temperatures Fig. 5 Effect of temperature on the biodegradation ratio of diesel oil by strain Y2. The incubation conditions were pH 7.0, an initial diesel oil concentration of 2% (v/v), and an initial concentration of inoculated bacteria of 3×10 cells/mL. All data were obtained at 7 days of incubation 638 Ann Microbiol (2013) 63:633–640 Fig. 6 Changes in the growth of strain Y2 and the diesel oil biodegradation ratio with incubation time. The incubation conditions were pH 7.5, 30 °C, an initial diesel oil concentration of 2 % (v/v), and an initial concentration of inoculated bacteria of 3×10 cells/mL (20–37 °C), initial diesel oil concentrations (v/v: 1, 1.5, ratio was higher at initial oil concentrations (1, 1.5, and 2 %) 2, 2.5, 3 %), and inoculated bacteria concentrations (1× than at high initial oil concentrations (2.5 and 3.0 %). The 7 7 7 7 7 10 ,2×10 ,3×10 ,4×10 ,5×10 cells/mL). highest degradation ratio was achieved at an initial oil pH is an important environmental factor which impacts concentration of 2 % (v/v), possibly because when the initial oil biodegradation. As shown in Table 3, the degradation concentration of diesel oil is too high, the oil would cover ratio was the highest at pH approximately 7.5–8.0, and the surface of the water and prevent oxygen from dissolving Acinetobacter sp. strain Y2 was able to degrade oil when into the liquid culture. Aerobic conditions are generally the pH ranged from 6 to 8.5. Previous studies have shown considered to necessary for extensive degradation of oil that the degradation of oil increases with increasing pH and hydrocarbons in the environment since major degradation that optimum degradation occurs under slightly alkaline pathways for both saturates and aromatics involve oxy- conditions (Dibble and Bartha 1979; Foght and Westlake genases (Cerniglia 1992). The study of Camilli et al. 1987). The pH is an important environmental factor which (2010) reported that the dissolved oxygen concentrations impacts microbial growth through a number of mechanisms. in the plume of the Gulf of Mexico oil spill suggested that First, the charge of proteins, nucleic acids, and other bio- microbial respiration rates within the plume were not appre- logical macromolecules changes with pH; thus, pH affects ciably more than 1 micromolar oxygen per day. Many microbial biological activity. Second, the environmental pH studies have shown that oxygen depletion leads to sharply changes the electric charge in the cell membrane, which in reduced biodegradation activities (Bossert and Bartha 1984) turn affects the ability of the microbial cell to absorb and subsequently to a decreased biodegradation ratio. nutrients. Third, pH modifies nutrient availability and the The effect of environmental temperature on oil degradation toxicity of hazardous substances. Since the pH of our sea- was also investigated because of the significant role of tem- water samples was about 8.0, our results demonstrate that perature. As shown in Fig. 5, four different temperatures (20, the investigated bacteria could effectively degrade the diesel 25, 30, and 37 °C) were evaluated for their effect on diesel oil oil in this environment. degradation. The results showed that the biodegradation ratio The effect of the inoculated bacteria concentration on the was only about 15 % at the lowest temperature tested (20 °C) biodegradation ratio of diesel oil and the cell density at the end but that it increased up to approximately 60–70 % at high of the experiments are shown in Fig. 3. The results indicate that temperatures (30, 37 °C). Temperature not only controls the the degradation ratio of Acinetobacter sp. strain Y2 decreased nature and extent of microbial hydrocarbon metabolism, but it when the initial concentration of inoculating bacteria was too also directly affects the physicochemical behavior of oil hydro- 7 7 low (1×10 cells/mL) or too high (5×10 cells/mL). carbons, such as viscosity, diffusion, and volatilization, which The biodegradation ratio of diesel oil at different initial changes the oil composition and bioavailability of the water- oil concentrations are shown in Fig. 4. The biodegradation soluble components. At low temperatures, the viscosity of the Fig. 7 The chromatogram of diesel oil remaining in Acinetobacter sp. diesel oil concentration of 2 % (v/v), and an initial concentration of strain Y2 culture (b) and abiotic control (a) media after 10 days of inoculated bacteria was 3×10 cells/mL incubation. The incubation conditions were pH 7.5, 30 °C, an initial Ann Microbiol (2013) 63:633–640 639 oil increases, while the volatility of toxic low-molecular- environment, a study of the effect of environmental conditions weight hydrocarbons decreases (Atlas and Bartha 1972), there- on the degradation of petroleum was required. Therefore, we by explaining why the ratio of degradation generally decreases studied such environmental factors as salinity, dissolved oxy- with decreasing temperature. gen, nutrient salts, and the mechanisms of degradation of The relationship between bacterial growth and biodegra- strain Y2 in the marine environment in order to determine dation ratio is shown in Fig. 6. The degradation ratio and the effect of these factors on the degradation of petroleum. The cell density (OD ) increased sharply up to 42 % and 2.0, degradation capabilities of diesel oil by Acinetobacter sp. respectively, on the first day of culture. The ratio of degra- strain Y2 suggest that this strain can be exploited further for dation subsequently increased with increasing cell density the development of effective bioremediation technology for on the first 4 days. Cell density started to decline from the the remediation of hydrocarbon pollution in the marine fifth day onwards, and the ratio of degradation increased environment. slowly, probably because the nutrient condition was rich at the beginning, so bacteria grew massively. Beginning on the Acknowledgments This work was supported by a grant from Shanghai fifth day, the degradation ratio increased slowly and the Natural Science Foundation of China (No. 08ZR1401300) bacteria growth volume began to decrease due to limitations imposed by metabolic wastes and available nutrients. Based on our experimental results, we determined the References optimal culture conditions for strain Y2 to be an initial pH ranging from 7.5 to 8.0, an incubation temperature of 30 °C, Atlas RM, Bartha R (1972) Biodegradation of petroleum in seawater at an initial diesel oil concentration of 2 % (v/v), and an initial low temperatures. Can J Microbiol 18:1851–1855 inoculating bacteria concentration of 3×10 cells/mL. The Bossert I, Bartha R (1984) The fate of petroleum in soil ecosystems. In: Atlas RM (ed) Petroleum microbiology. Macmillan , New York, degradation ratio of diesel oil reached as high as 80 % under pp 435–476 these optimal conditions after 10 days of incubation. Camilli R, Reddy CM, Yoerger DR, Van Mooy BA, Jakuba MV, Kinsey JC, McIntyre CP, Sylva SP, Maloney JV (2010) Tracking Gas chromatography–mass spectrometry analysis hydrocarbon plume transport and biodegradation at Deepwater Horizon. Science 330(6001):201–204 Cerniglia CE (1992) Biodegradation of polycyclic aromatic hydrocarbons. Diesel oil in the culture medium of Acinetobacter sp. strain Biodegradation 3:351–368 Y2 was analyzed by GC–MS after 10 days of culture Dibble JT, Bartha R (1979) Effect of environmental parameters on (Fig. 7). The components of diesel oil identified on the biodegradation of oil sludge. Appl Environ Microbiol 37:729–739 El-Naas MH, Al-Muhtaseb SA, Makhlouf S (2009) Biodegradation of chromatogram were mixed hydrocarbons (C11–C21), and phenol by Pseudomonas putida immobilized in polyvinyl alcohol all components of the diesel oil had been degraded by (PVA) gel. J Hazard Mater 164:720–725 Acinetobacter sp. strain Y2. Acinetobacter is one of the Foght JM, Westlake DWS (1987) Biodegradation of hydrocarbons in representative genera capable of growing on n-alkanes (Tani freshwater. In: Vandermeulen JH, Hrudey SE (eds) Oil in fresh- water: chemistry, biology, countermeasure technology. Pergamon et al. 2002). They are therefore frequently detected in waste Press, New York, pp 217–230 water treatment processes, seawater, and sewage sludge Gauthier MJ, Lafay B, Christen R, Fernandez L, Acquaviva M, Bonin (Gottsching and Schmidt. 2007; Phrommanicha et al. P, Bertrand JC (1992) Marinobacter hydrocarbonoclasticus gen. 2009). Microorganisms are known to attack and degrade a nov., sp. nov., a new, extremely halotolerant, hydrocarbon-degrading marine bacterium. Int J Syst Bacteriol 42(4):568–576 specific component as compared with other components of Gottsching A, Schmidt S (2007) Productive degradation of the biocide petroleum. Obayori et al. (2009) observed that the same benzyl benzoate by Acinetobacter sp. strain AG1 isolated from compounds in different crude oil samples were degraded the River Elbe. Res Microbiol 158:251–257 to different extents by the same organisms. Acinetobacter Ilori MO, Amobi CJ, Odocha AC (2005) Factors affecting biosurfactant production by oil degrading Aeromonas spp. isolated from a tropical sp. strain Y2 has the unusual characteristic of being able to environment. Chemosphere 61:985–992 grow on a broad range of n-alkanes, which suggests that Jeong WG, Cho SM (2007) Long-term effect of polycyclic aromatic strain Y2 could potentially be useful for the remediation of hydrocarbon on physiological metabolisms of the Pacific oyster, hydrocarbon pollution in the marine environment. Crassostrea gigas. Aquaculture 265:343–350 Kasai Y, Kishira H, Harayama S (2002) Bacteria belonging to the genus Cycloclasticus play a primary role in the degradation of aromatic hydrocarbons released in a marine environment. Appl Conclusion Environ Microbiol 68:5625–5633 Lane DJ (1991) 16S/23S rRNA sequencing. In: Goodfellow M, Stack- ebrandt E (eds) Nucleic acid techniques in bacterial systematics. Acinetobacter sp strain Y2 has a high ability to degrade Wiley, Chichester, pp 115–175 diesel oil in a wide pH, temperature and initial diesel oil Li JL, Chen BH (2009) Effect of nonionic surfactants on biodegrada- concentration range. Given the complexity of oil products tion of phenanthrene by a marine bacteria of Neptunomonas and environmental conditions in a contaminated natural naphthovorans. J Hazard Mater 162:66–73 640 Ann Microbiol (2013) 63:633–640 Liu YC, Li LZ, Wu Y et al (2010) Isolation of an alkane-degrading contaminants, 4th edn. China Environmental Science Press, Bei- Alcanivorax sp. strain 2B5 and cloning of the alkB gene. Bioresour jing, pp 491–495 Technol 101:310–316 Swannell RPJ, Lee K, McDonagh M (1996) Field evaluations of Obayori OS, Adebusoye SA, Adewale AO, Oyetibo GO, Oluyemi OO, marine oil spill bioremediation. Microbiol Rev 60:342–365 Amokun RA, Ilori MO (2009) Differential degradation of crude Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: Molecular oil (Bonny Light) by four Pseudomonas strains. J Environ Sci Evolutionary Genetics Analysis (MEGA) software version 4.0. 21:243–248 Mol Biol Evol 24:1596–1599 Olivera NL, Nievas ML, Lozada M (2009) Isolation and characterization Tani A, Ishige T, Sakai Y, Kato N (2002) Two acyl-CoA dehydro- of biosurfactant-producing Alcanivorax strains: hydrocarbon acces- genases of Acinetobacter sp. strain M-1 that uses very long-chain sion strategies and alkanehydroxylase gene analysis. Res Microbiol n-alkanes. J Biosci Bioeng 94(4):326–329 160:19–26 Tomohisa K, Miyanaga A, Kanaya S, Morikawa M (2010) Gene Pagnout C, Frache G, Poupin P, Maunit B, Muller JF, Ferard JF (2007) cloning and characterization of an aldehyde dehydrogenase from Isolation and characterization of a gene cluster involved in PAH long-chain alkane-degrading Geobacillus thermoleovorans B23. degradation in Mycobacterium sp. strain SNP11: Expression in Extremophiles 14:33–39 Mycobacterium smegmatis mc2155. Res Microbiol 158:175– Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG 186 (1997) The CLUSTAL_X windows interface: flexible strategies Phrommanicha S, Suanjitb S, Upathamc S, Gramsd SV, Kruatrachued for multiple sequence alignment aided by quality analysis tools. M, Pokethitiyookd P, Korgee G, Hofmanne A (2009) Quantitative Nucleic Acids Res 25:4876–4882 detection of the oil-degrading bacterium Acinetobacter sp. strain Venosa AD, Zhu XQ (2003) Biodegradation of crude oil contaminating MUB1 by hybridization probe based real-time PCR. Microbiol marine shorelines and freshwater wetlands. Spill Sci Technol B 8 Res 164:486–492 (2):163–178 Quan ZX, Bae HS, Baek JH, Chen WF, Im WT, Lee ST (2005) Xiu ZD, Miao YC (2001) Manual of determinative bacteriology. Science Rhizobium daejeonense sp. nov. isolated from a cyanide treatment Press, Beijing bioreactor. Int J Syst Evol Microbiol 55:2543–2549 Yamamoto T, Nakaoka M, Komatsu T, Kawai H, Ohwada K, Marine Seksan P, Sudarat S, Suchart U, Suksiri VG, Maleeya K, Prayad P, Gunter Life Research Group of Takeno (2003) Impacts by heavy-oil spill K, Annemarie H (2009) Quantitative detection of the oil-degrading from the Russian tanker Nakhodka on intertidal ecosystems: re- bacterium Acinetobacter sp. strain MUB1 by hybridization probe covery of animal community. Mar Pollut Bull 47:91–98 base dreal-time PCR. Microbiol Res 164:486–492 Yang X, Sun Y, Qian S (2004) Biodegradation of seven polychlorinated Sun ZG, Huang YR et al (2002) Determination methods for examina- biphenyls by a newly isolated aerobic bacterium (Rhodococcus sp. tion of water and wastewater. In: Wei FS (ed) Organic R04). J Ind Microbiol. Biotechnol 31:415–420

Journal

Annals of Microbiology – Springer Journals

Published: Sep 16, 2012

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