: (Acinetobacter sp. BZ-15) (Acinetobacter) [6]. [7],, ( )., [6],., : [8]..,,., : alk [9,10], P450 CYP153 [11]..,.,,.,., [12]., [13~15],, [16]., [17],

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1 中国科学 : 生命科学 2016 年第 46 卷第 9 期 : 1091 ~ 1100 SCIENTIA SINICA Vitae life.scichina.com 中国科学杂志社 SCIENCE CHINA PRESS 高效石油烃降解菌不动杆菌 (Acinetobacter sp. BZ-15) 的筛选鉴定及其降解性能研究刘玉华 12 1*, 胡晓珂,, ;, *, xkhu@yic.ac.cn : ; : ; : ( : , ) ( : ) 本研究旨在从土壤中筛选高效石油烃降解菌株, 并对其系统分类和降解特性进行研究, 为石油污染的原位修复奠定基础. 该研究从滨州油井溢油污染土壤样品中分离得到一株高效石油烃降解菌株 BZ-15, 对菌株 BZ-15 进行形态观察 16S rrna 基因序列分析及系统发育树分析 ; 对该菌株的生长特性进行研究 ; 通过 GC-MS 分析其对原油组分中不同碳原子饱和烃的降解特性 ; 同时研究吐温 -20 对其生长及降解特性的影响 ; 对该菌株中的烷烃羟化酶基因 alkm 进行了克隆. 结果表明, 菌株 BZ-15 为不动杆菌属 (Acinetobacter sp.) 细菌, 在 LB 培养基中其代时为 3.25 h, 添加吐温 -20 代时为 2.67 h, 吐温 -20 可促进菌株 BZ-15 生长 ; 该菌株可降解 C 13 ~C 28 碳链长度饱和烃, 饱和烃降解率为 61.0%, 添加吐温 -20 饱和烃降解效率为 52.2%, 吐温 -20 可抑制菌株 BZ-15 降解饱和烃 ; 菌株 BZ-15 存在烷烃羟化酶基因 alkm, 通过末端氧化途径对饱和烃进行降解. 不动杆菌, 石油烃, 烷烃羟化酶, 吐温 -20,,.,.,.. ( ), [1]., [2]. [3], [4].,,.,, [5]. :,. (Acinetobacter sp. BZ-15). :, 2016, 46: Liu Y H, Hu X K. Microbial degradation of petroleum hydrocarbons by Acinetobacter sp. BZ-15, isolated from contaminated soil. Sci Sin Vitae, 2016, 46: , doi: /N

2 : (Acinetobacter sp. BZ-15) (Acinetobacter) [6]. [7],, ( )., [6],., : [8]..,,., : alk [9,10], P450 CYP153 [11]..,.,,.,., [12]., [13~15],, [16]., [17],,.,.,,, [15,18]., [19].,, 0.05~1.0 g/l [20], [21].,, -20( ),,. 1 材料与方法 1.1 试剂和培养基., - (gas chromatography-mass spectrometer, GC-MS). (1). BHB(Bushnell-Haas Broth) : MgSO g/l, CaCl g/l, KH 2 PO 4 1 g/l, K 2 HPO 4 1 g/l, NH 4 NO 3 1 g/l, FeCl g/l, NaCl 25 g/l, ph 7.0±0.2. (2). BHB 0.5%, ph 7.0±0.2. (3). 1.5%, ph 7.0±0.2. (4) (LB ). 10 g/l, 5 g/l, 10 g/l, ph 7.0±0.2. (5). 1.5%, ph 7.0± 石油烃降解菌的筛选. 10 g 100 ml, 180 r/min, 30 7., h., 3, LB,. 1.3 石油烃降解菌 DNA 提取 BZ-15 LB (30, 150 r/min)16 h, 1.8 ml, DNA UltraClean TM Microbial DNA Isolation Kit (MO-BIO, ) DNA. DNA 20, DNA. 1.4 石油烃降解菌的鉴定. 16S rrna. 16S rrna PCR DNA, 27F 1492R [22] ( 1). PCR 50 μl, : 1 PCR Buffer, 0.2 mmol/l dntps, 0.2 mmol/l 27F, 0.2 mmol/l 1492R 1 U Taq. PCR 1092

3 : : 94 5 min, min, 55 1 min, 72 2 min, min. PCR 1%, Invitrogen ( ). NCBI(National Center for Biotechnology Information) BLAST, Mega. 1.5 菌株生长曲线测定 BZ-15, 1 ml 100 ml 100 ml 0.5% -20, 180 r/min, 30. UV-7100 ( ), 600 nm,.,,. : t2 t1 G, (lgw2 lg W1) / lg 2 t 1, t 2, W 1, W 2 A 菌株降解石油烃效果测定 10%(v/v), 30, 180 r/min 5,,.,, 25 ml. ( 3 g 2 g 1 g), 1 ml, 20 ml 15 ml (2 1) 15 ml,, ( ) ( ) ( ) 3 [23]. GC-MS. : HP-5MS (30 m 320 μm 0.25 μm) %., 30 ml/min, 350 ml/min. FID ml/min, 5 1, 1 μl [24]. 1.7 石油烃降解功能基因的克隆 DNA, alkb-1f 1 PCR 27F 5 -AGAGTTTGATCCTGGCTCAG R 5 -TGACTGACTGAGGYTACCTTGTTACGACTT-3 alkb-1f 5 -AAYACNGCNCAYGARCTNGGNCAYAA-3 alkb-1r 5 -GCRTGRTGRTCNGARTGNCGYTG-3 alkb-1r [25] ( 1) alkb PCR. PCR 50 μl, : 1 PCR Buffer, 0.2 mmol/l dntps, 0.2 mmol/l alkb-1f, 0.2 mmol/l alkb-1r 1 U Taq. PCR : min; 94 1 min, 55 1 min, 72 1 min, 40 ; min. PCR 1%, Invitrogen. NCBI(National Center for Biotechnology Information) BLAST,, BZ-15, Mega. 2 结果 2.1 菌株的筛选与鉴定,, BZ-15.,, 0.5~1.0 mm. LEICA DM500 ( ),,. 16S rrna PCR, BLAST,,, Acinetobacter psychrotolerans strain Ths 97%. 16S rrna,. MEGA5.0 BZ 菌株生长特性 BZ h, 2. BZ ,, 4 h,. 30 h., -20 BZ

: (Acinetobacter sp. BZ-15) 1 Acinetobacter sp. BZ-15 16S rrna 3 Acinetobacter sp. BZ-15 2 Acinetobacter sp. BZ-15 A: Acinetobacter sp. BZ-15 ; B:, DAPI Acinetobacter sp. BZ-15., 3.25 h, -20 2.67 h.

4 : (Acinetobacter sp. BZ-15) 1 Acinetobacter sp. BZ-15 16S rrna 3 Acinetobacter sp. BZ-15 2 Acinetobacter sp. BZ-15 A: Acinetobacter sp. BZ-15 ; B:, DAPI Acinetobacter sp. BZ-15., 3.25 h, h. -20 ( 17.8%), BZ 菌株对石油烃的降解效果, BZ-15,, ( 3A). 4, (4,6-diamidino-2- phenylindole, DAPI),, BZ-15( ), ( 3B). 10%(v/v), BZ-15 5,,,. 5 GC-MS, 4., %., BZ-15 C 13 ~C 28 ( 5A), ( 5B). C 17, C 19, BZ-15 65%, C 13, C %, C24~C28 80% ( 2). 2.4 吐温 -20 对石油烃降解效果的影响 -20, BZ %, BZ % -20, ( 6). 0.3% -20 BZ-15,,, %. -20, BZ-15 C 13, C %,, C 24 ~C 28 80% ( 2). 1094

5 : Acinetobacter sp. BZ-15 GC-MS A: ; B: 5, 10% 2 Acinetobacter sp. BZ-15 (%) C 13 C 14 C 15 C 16 C 17 C 18 C 19 C C 21 C 22 C 23 C 24 C 25 C 26 C 27 C 石油烃降解功能基因的克隆 BZ-15 ( alk). alkb, PCR, 500 bp. PCR, BLAST, Acinetobacter sp. M-1 alkane hydroxylase A (82%). MEGA5.0 ( 7). 3 讨论, [2]., ( ph,, )., [26]., 1095

6 : (Acinetobacter sp. BZ-15) 5 Acinetobacter sp. BZ-15. BZ-15,, 16S rrna, BZ-15.,, BZ-15 C 13 ~C 28, 61%. [27] Acinetobacter sp. 1 60%, Acinetobacter sp. 2 67%., BZ-15,,., [28]., [29]. -20,. [30] %. -20 BZ-15, 15%., -20 BZ-15, BZ-15 ( -20). 0.3% -20 BZ

7 : Acinetobacter sp. BZ-15 GC-MS A: ; B: 5, 10% 7 Acinetobacter sp. BZ-15 alkm, BZ-15-20,,., BZ-15,.,,,, - A [31].,, [32,33]., BZ-15., Acinetobacter calcoaceticus ADP1, alkm 1097

8 : (Acinetobacter sp. BZ-15), [34,35]. BZ-15 alkm, alkm., BZ-15,,,,,., BZ-15. Throne-Holst [36], Acinetobacter venetianus 6A2 C 10 ~C 18., BZ-15 C 13 ~C 28,,., BZ-15,,,. 4 结论 (1) 1 BZ-15, 16S rrna, Acinetobacter sp. BZ-15. (2) C 17, C 19, BZ-15 65%, C 13, C %,. (3) -20, BZ %. -20, 52%. (4) BZ-15 alkm,. 参考文献 1 Madsen E L. Determining in situ biodegradation: facts and challenges. Environ Sci Technol, 1991, 25: Head I M, Jones D M, Roling W F. Marine microorganisms make a meal of oil. Nat Rev Microbiol, 2006, 4: ,,.., 2009, 18: Gentili A R, Cubitto M A, Ferrero M, et al. Bioremediation of crude oil polluted seawater by a hydrocarbon-degrading bacterial strain immobilized on chitin and chitosan flakes. Int Biodeter Biodegr, 2006, 57: Sugimori D, Utsue T. A study of the efficiency of edible oils degraded in alkaline conditions by Pseudomonas aeruginosa SS-219 and Acinetobacter sp. SS-192 bacteria isolated from Japanese soil. World J Microbiol Biotechnol, 2012, 28: ,,,.., 2011, 5: Colwell R R. Ecological aspects of microbial degradation of petroleum in the marine environment. Crit Rev Microbiol, 1977, 5: Ji Y, Mao G, Wang Y, et al. Structural insights into diversity and n-alkane biodegradation mechanisms of alkane hydroxylases. Front Microbiol, 2013, 4: 58 9 Ratajczak A, Geißdörfer W, Hillen W. Alkane hydroxylase from Acinetobacter sp. strain ADP1 is encoded by alkm and belongs to a new family of bacterial integral-membrane hydrocarbon hydroxylases. Appl Environ Microbiol, 1998, 64: Ratajczak A, Geißdörfer W, Hillen W. Expression of alkane hydroxylase from Acinetobacter sp. Strain ADP1 is induced by a broad range of n-alkanes and requires the transcriptional activator AlkR. J Bacteriol, 1998, 180: Maier T, Forster H H, Asperger O, et al. Molecular characterization of the 56-kDa CYP153 from Acinetobacter sp. EB104. Biochem Biophys Res Commun, 2001, 286: ,.., 2004, 23: Lanzon J B, Brown D G. Partitioning of phenanthrene into surfactant hemi-micelles on the bacterial cell surface and implications for surfactant-enhanced biodegradation. Water Res, 2013, 47: Li F, Zhu L, Zhang D. Effect of surfactant on phenanthrene metabolic kinetics by Citrobacter sp. SA01. J Environ Sci, 2014, 26: Santanu P. Surfactant-enhanced remediation of organic contaminated soil and water. Adv Colloid Interface Sci, 2008, 138: ,,,.., 2012, 43: ,,.., 2001, 20: Kang S, Jeong H Y. Sorption of a nonionic surfactant Tween 80 by minerals and soils. J Hazard Mater, 2015, 284:

9 : ,. Bacillus pumilus strain Bap9 [a]., 2012, 6: ,,. Tween., 2014, 8: Yanto D H, Tachibana S. Enhanced biodegradation of asphalt in the presence of Tween surfactants, Mn 2+ and H 2 O 2 by Pestalotiopsis sp. in liquid medium and soil. Chemosphere, 2014, 103: Enticknap J J, Kelly M, Peraud O, et al. Characterization of a culturable alphaproteobacterial symbiont common to many marine sponges and evidence for vertical transmission via sponge larvae. Appl Environ Microbiol, 2006, 72: Bastow T P, Aarssen B G K V, Alexander R, et al. Biodegradation of aromatic land-plant biomarkers in some Australian crude oils. Organic Geochem, 1999, 30: Bost F D, Frontera-Suau R, McDonald T J, et al. Aerobic biodegradation of hopanes and norhopanes in Venezuelan crude oils. Organic Geochem, 2001, 32: Wasmund K, Burns K A, Kurtbo D I, et al. Novel alkane hydroxylase gene (alkb) diversity in sediments associated with hydrocarbon seeps in the timor sea, Australia. Appl Environ Microbiol, 2009, 75: Townsend G T, Prince R C, Suflita M. Anaerobic biodegradation of alicyclic constituents of gasoline and natural gas condensate by bacteria from an anoxic aquifer. FEMS Microbiol Ecol, 2004, 49: ,,,.., 2014, 34: Zheng G, Selvam A, Wong J W C. Enhanced solubilization and desorption of organochlorine pesticides (OCPs) from soil by oil-swollen micelles formed with a nonionic surfactant. Environ Sci Technol, 2012, 46: Tobe S, Majima T, Tadenuma H, et al. Nonionic surfactants enhancing bactericidal activity at their critical micelle concentrations. J Oleo Sci, 2015, 64: ,,,.., 2004, 23: Van Hamme J D, Singh A, Ward O P. Recent advances in petroleum microbiology. Microbiol Mol Biol Rev, 2003, 67: Maeng J H, Sakai Y, Tani Y, et al. Isolation and characterization of a novel oxygenase that catalyzes the first step of n-alkane oxidation in Acinetobacter sp. strain M-1. J Bacteriol, 1996, 178: Sakai Y J, Maeng J H, Kubota S, et al. A nonconventional dissimilation pathway for long chain n-alkanes in Acinetobacter sp. M-1 that starts with a dioxygenase reaction. J Ferment Bioeng, 1996, 81: Geissdörfer W, Frosch S C, Haspel G, et al. Two genes encoding proteins with similarities to rubredoxin and rubredoxin reductase are required for conversion of dodecane to lauric acid in Acinetobacter calcoaceticus ADP1. Microbiology, 1995, 141: Parche S, Geissdörfer W, Hillen W. Identification and characterization of xcpr encoding a subunit of the general secretory pathway necessary for dodecane degradation in Acinetobacter calcoaceticus ADP1. J Bacteriol, 1997, 179: Throne-Holst M, Markussen S, Winnberg, A, et al. Utilization of n-alkanes by a newly isolated strain of Acinetobacter venetianus: the role of two AlkB-type alkane hydroxylases. Appl Microbiol Biotechnol, 2006, 72:

10 : (Acinetobacter sp. BZ-15) Microbial Degradation of Petroleum Hydrocarbons by Acinetobacter sp. BZ-15, Isolated from Contaminated Soil LIU YuHua 1,2 & HU XiaoKe 1 1 Key Laboratory of Coastal Biology and Bioresouce Utilization, Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences, Yantai , China; 2 University of Chinese Academy of Sciences, Beijing , China One of the major environmental problems is the contamination of petroleum hydrocarbons as a result of activities related to the petrochemical industry. This study aimed to isolate petroleum hydrocarbon-degrading bacteria from soil, and to study their systemic classification and degradation characteristics. A bacterial strain BZ-15 capable of degrading petroleum hydrocarbons was isolated from the oil-spilled soils in Binzhou, China. The bacterium was identified as belonging to the genus Acinetobacter through morphological and 16S rrna gene homological analysis. Its growth rate and biodegradation ability were investigated by cell culture on media supplemented with crude oil with or without the surfactant Tween-20. Non-ionic surfactant Tween-20 can promote the growth of BZ-15, reducing the generation time from 3.25 to 2.67 h. After 5 days of growth, GC-MS analysis showed that the degradation efficiency of this bacterial strain for saturated hydrocarbons could reach 61%. However, the degradation efficiency for saturated hydrocarbons decreased and was only 52% Tween-20. The strain BZ-15 could biodegrade saturated hydrocarbons in which carbon chains were of the length C 13 C 28. Alkane hydroxylase (alkm) was successfully cloned, which indicates that the bacterial strain could degrade alkane via the terminal oxidative pathway. Acinetobacter sp., petroleum hydrocarbons, alkane hydroxylase, Tween-20 doi: /N

[1] Nielsen [2]. Richardson [3] Baldock [4] 0.22 mm 0.32 mm Richardson Zaki. [5-6] mm [7] 1 mm. [8] [9] 5 mm 50 mm [10] [11] [12] -- 40% 50%

[1] Nielsen [2]. Richardson [3] Baldock [4] 0.22 mm 0.32 mm Richardson Zaki. [5-6] mm [7] 1 mm. [8] [9] 5 mm 50 mm [10] [11] [12] -- 40% 50% 38 2 2016 4 -- 1,2, 100190, 100083 065007 -- 0.25 mm 2.0 mm d 10 = 0.044 mm 640 3 300. Richardson--Zaki,,, O359 A doi 10.6052/1000-0879-15-230 EXPERIMENTAL STUDY OF FLUID-SOLID TWO-PHASE FLOW IN A VERTICAL