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1 盐田嗜盐单胞杆菌的分离鉴定及其 PHB 积累的研究隋丽英, 张娇娇, 许高超, 邓元告 (, ) 摘要 : 微生物聚合物聚 - - 羟基丁酸酯 (PHB) 具有良好的生物降解性生物相容性和热塑性, 在环保医药和农业领域具有广阔的应用前景高 PHB 积累菌株的获得及其发酵条件的研究具有很好的实用价值作者从渤海湾日晒盐场盐田卤水中分离出一株杆状细菌, 经 16S rdna 序列测序比对, 确定为盐杆菌属 (Halomonas), 命名为 Halomonas , 其最适生长盐度为 50~100, 最适生长 ph 为 7~8, 属中度嗜盐菌在酸水解酪蛋白 (7.5 g/l) 的基础培养基中分别添加和 30 g/l 葡萄糖, 研究盐度 100 条件下不同葡萄糖添加量对 Halomonas 生长和 PHB 含量的影响结果表明, 随着葡萄糖浓度的增加, 该菌株的生长和 PHB 积累显著提高葡萄糖质量浓度为 30 g/l 时, 细胞干质量达 8.60 g/l, PHB 含量达到 5.57 g/l( 即达到细胞干质量的 65%) 本研究为渤海湾盐田微生物资源的开发和利用提供了有价值的基础数据关键词 : 嗜盐细菌 ; 盐杆菌属 (Halomonas); PHB 积累 ; 葡萄糖中图分类号 : Q 文献标识码 : A 文章编号 : (2015) doi: /hykx (Polyhydroxyalkanoates, PHA),, (Poly- -hydroxybutyrate, PHB) PHA, ( 0.2~0.5 m), PHB -, (Dicentrarchus labrax) 3 (Macrobrachium rosenburgii) 4 (Eriocheir sinensis) 5-6 7, 8, PHB 9, ( : (Alcaligenes latus), (Cupriavidus necator), (Bacillus cereus), (Ralstonia eutropha) (Escherichia coli) ) 10 ( : (Halomonas boliviensis) ( : (Haloferax mediterranei) (Haloarcula sp.) , 50 14,, 15 ;, PHB PHB Halomonas sp.,, PHB, 1 材料与方法 1.1 嗜盐细菌的分离纯化和鉴定, 100(Atago Refractometer- S28M) CM 16, ( ,,, ph 7.2~7.4(Mettler FE20K) 100 L, 37 7 d, r/min 3~4 d : ; : : ( ); ( ) : (1966-),,,,,, suily@hotmail.com 16 / 2015 / 39 / 5

2 ,, 3 d, DNA(Axygen), 16S rdna, 94 5 min; 94 1 min, 60 1 min, 72 2 min, 29 ; min : F27: 5 -AGAGTTTGATCCTGGCTCAG-3, R1462: 5 -ATCCAGCCGCAGATTCCCCT-AC-3 16S rdna 1500 bp, ( ), GenBank BLAST GenBank (>97%), CLUSTAL X MEGA 5.0 Neighborjoining,, (Hitachi SU 1510) (pH 7.2~7.4) ph ( 100), h, OD 600 nm, ph 7 d NaCl (100 g/l), API 20E (Biomérieux), d, 1.2 葡萄糖对嗜盐细菌生长和 PHB 积累的影响 4, 3 (7.5 g/l), 0, 10, g/l 300 ml 100 ml, 2%(v/v), r/m 11 d 24 h PHB 3 ml, r/min 10 min,, 2,, h 12 PHB mg/l PHB (Goodfellow, UK),, PHB mg/l 5 ml, min, 4 10 min, OD 235 nm, Excel, y= x (R 2 =0.9989) PHB 1mL, PHB 2 结果 2.1 Halomonas 的分离纯化和鉴定 16S rdna Genbank BLAST, (Halomonas alkaliphila)(nr042256), Halomonas , Genbank KF697249( 1),, (0.5~0.8) m (2.0~3.0) m, ( 2) ph 7~8, 50 ~100( 3), API 20E, β-, 1 16S rrna Fig. 1 Phylogenetic tree based on partial 16S rrna gene sequence Marine Sciences / Vol. 39, No. 5 /

图2 Fig. 2 电子显微镜下 Halomonas 100-16-2 细胞( 5000) Cells of Halomonas 100-16-2 under electronic microscope ( 5000) 图3 Halomonas 100-16-2 在不同 ph 和盐度条件下培养 Fig.

3 图2 Fig. 2 电子显微镜下 Halomonas 细胞( 5000) Cells of Halomonas under electronic microscope ( 5000) 图3 Halomonas 在不同 ph 和盐度条件下培养 Fig h 的细胞生长 Growth of Halomonas cultured for 72 h at different ph and salinities 化或发酵 D-葡萄糖肌醇 D-山梨醇 D-甘露醇 L-鼠李糖 D-蜜二糖苦杏仁苷 D-蔗糖和 L-阿拉不同葡萄糖质量浓度条件下 Halomonas 的 Fig. 4 生长 Growth of Halomonas at different concentrations of glucose 图5 不同葡萄糖质量浓度下 Halomonas 细胞中 PHB 含量 Fig. 5 PHB content in cells of Halomonas at different concentrations of glucose 糖质量浓度为 20 g/l 时, PHB 含量在第 7 天达到最高(2.54 g/l), 而后基本维持在该水平; 但当葡萄糖质量浓度为 30 g/l 时, PHB 含量在第 11 天达到最高, 为 5.57 g/l 3 伯糖等产酸 2.2 图4 讨论葡萄糖对 Halomonas 生长和 PHB 积累的影响 PHA 的能力较强 9, 已报道有 30 多个种属, 其中利随葡萄糖添加量的增加, Halomonas 的用玻利维亚盐单胞菌生产 PHB 的研究最为广泛, 该细胞干质量显著提高(图 4) 葡萄糖质量浓度为 0 菌株可以利用葡萄糖木糖蔗糖麦芽低聚糖 10 和 20 g/l 各组均在培养 5~6 d 时进入稳定期, 第 6 乙酸钠和丁酸等为底物合成 PHB, 以农业副产品水天细胞干质量分别达到和 4.90 g/l 葡萄糖质解物为底物生产 PHB, 含量可达细胞干质量的量浓度为 30 g/l 时, 虽然前期生长较 20 g/l 组慢, 但在 50%~56% 玻利维亚盐单胞菌培养盐度一般为 50 12, 15, 其他组别进入生长稳定期后, 该组别细胞干质量持较高盐度下细胞生长较为缓慢, 原因是高盐度下大续增加, 第 11 天细胞干质量达到 8.60 g/l 量 ATP 用于积累胞内渗透压调节物质, 使细胞生长在嗜盐细菌中, 盐杆菌科(Halomonadaeae)合成 Halomonas 细胞中 PHB 含量的增长趋受到限制 14 本研究结果表明, 添加碳源对 PHB 的势与细胞干质量的变化基本一致(图 5) 当葡萄糖质量积累具有明显的促进作用, 在盐度 100 条件下, 细胞浓度为 0 g/l 时, PHB 含量在第 3 天达到最高(0.18 g/l), 生长和细胞中 PHB 积累随葡萄糖浓度的增加显著提而后下降; 但葡萄糖质量浓度为 10 g/l 时, PHB 含量高葡萄糖质量浓度为 30 g/l 时 Halomonas 在第 4 天达到最高(0.60 g/l), 而后开始下降; 葡萄细胞干质量最高达 8.60 g/l, PHB 含量达到海洋科学 / 2015 年 / 第 39 卷 / 第 5 期

4 g/l( 65%), Tan g/l, Halomonas sp. 6 g/l PHB 69% Quillaguaman 12, 4.5% NaCl LC1, PHB 55% Quillaguaman 19, 0.4%NH 4 Cl, 0.22%K 2 HPO 4 2%,, LC1 44 g/l, PHB 81%, PHB 1.1g/(L h) PHA : PHB,, Protomonas extorquens, Protomonas oleovorans;,,, PHB g/l, Halomonas , PHB, 3~4 20 g/l,, PHB 7,,,,, PHB 30 g/l, PHB,, PHB PHB, PHB,, PHB ;, PHB -, 21 Halomonas ,, 22, ( ),,, : [1] Lauzier G C, Revol J F, Debzi E M. Hydrolytic degradation of isolated poly- -hydroxybuytrate (PHB) [J]. Polymers, 1994, 35(19): [2],. - (PHB) [J]., 2000, 2(4): [3] De Schryver P, Sinha A K, Kunwar P S, et al. Poly-βhydroxybutyrate (PHB) increases growth performance and intestinal bacterial range-weighted richness in juvenile European sea bass, Dicentrarchus labrax[j]. Applied Microbiology and Biotechnology, 2010, 86(5): [4] Nhan D T, Wille M, De Schryver P, et al. The effect of poly-β-hydroxybutyrate on larviculture of the giant fresh water prawn Macrobrachium rosenbergii[j]. Aquaculture, 2010, 302(1-2): [5] Sui L Y, Cai J L, Sun H X, et al. Effect of poly-βhydroxybutyrate on Chinese mitten crab Eriocheir sinensis larvae challenged with pathogenic Vibrio anguillarum[j]. Journal of Fish Diseases, 2012, 35(5): [6] Sui L Y, Liu Y, Sun H X, et al. The effect of poly-β-hydroxybutyrate on the performance of Chinese mitten crab (Eriocheir sinensis Milne-Edwards) zoea larvae[j]. Aquaculture Research, 2014, 45(3): [7],,,. [J]., 2013, 44(5): [8] Defoirdt T, Sorgeloos P, Bossier P, et al. Alternatives to antibiotics for the control of bacterial disease in aquaculture [J]. Current Opinion in Microbiology, 2011, 14(3): [9] Quillaguaman J, Guzman H, Van-Thuoc D, et al. Synthesis and production of polyhydroxyalkanoates by halophiles: current potential and future prospects[j]. Appllied Microbiology and Biotechnology, 2010, 85(6): [10] Verlinden R A J, Hill D T, Kenward M A, et al. Bacterial synthesis of biodegradable polyhydroxyalkanates[j]. Journal of Applied Microbiology, 2007, 102(6): [11] Chen C W, Don T M, Yen H F. Enzymatic extruded starch as a carbon source for the production of poly (3-hydroxybutyrate-co-3-hydroxyvalerate) by Haloferax mediterranei[j]. Process Biochemistry, 2006, 41(11): [12] Quillaguaman J, Delgado O, Mattiasson B, et al. Poly ( -hydroxybutyrate) production by a moderate halophile Halomonas boliviensis LC1[J]. Enzyme and Microbial Technology, 2006, 38(1): Marine Sciences / Vol. 39, No. 5 /

5 [13] Tanran M, Amirkhani H. Strategies of poly (3-hydroxybutyrate) synthesis by Haloarcula sp. IRU1 utilizing glucose as carbon source: Optimization of culture conditions by Taguchi methodology[j]. International Journal of Biological Macromolecules, 2010, 47(5): [14] Oren A. Microbial life at high salt concentrations: phylogenetic and metabolic diversity[j]. Saline Systems, 2008, 4: 2. [15] Tan D, Xue Y S, Aibaidula G, et al. Unsterile and continous production of polyhydroxybutyrate by Halomonas TD01[J]. Bioresource and Technology, 2011, 102(17): [16] Xu G C, Deng Y G, Song D H, et al. Isolation and characterization of a new moderately halophilic bacterium strain SM from solar saltern ponds[c]// Proceeding of the 2012 International Conference on Applied Biotechnology, China, Tianjin. Berlin, Heideberg : Springer-Verlag, 2014, 251 (2): [17],. β- [J]., 1997, 16(2): [18],. - (PHB) : PHB [J]., 2011, 20(3): [19] Quillaguaman J, Doan-Van T, Guzman H, et al. Poly ( -hydroxybutyrate) production by Halomonas boliviensis LC1 in fed bactch culture J]. Applied Microbiology and Biotechnogy, 2008, 78(2): [20] Lee S Y. Plastic bacteria? Progress and prospects for polyhydroxyalkanoate production in bacteria[j]. Trends in Biotechnology, 1996, 14(11): [21] Kadouri D, Jurkevitch E, Okon Y, et al. Ecological and agricutlural significance of bacteria polyhydroxyalkanoates [J]. Critical Review in Microbiology, 2005, 31(2): [22] Waise A C. Recovery of halophilic archaebacteria from natural environment[j]. FEMS Microbiology and Ecology, 1988, 53(3-4): Isolation and identification of Halomonas sp. from solar saltpond and study of PHB accumulation in its cells SUI Li-ying, ZHANG Jiao-jiao, XU Gao-chao, DENG Yuan-gao (Tianjin Key Laboratory of Marine Resources and Chemistry, College of Marine Science and Engineering, Tianjin University of Science and Technology, Tianjin , China) Received: Feb., 23, 2014 Key words: Halophilic bacteria; Halomonas sp.; PHB accumulation; glucose Abstract: Poly- -hydroxybutyrate (PHB) is the polyester reserved in cells of microorganisms. It has biodegradable, biocompatible and thermoplastic characteristics, thus has a great potential of commercial application in the field of environmental protection, medicine and agriculture. It is important to obtain high-phb accumulated bacteria and study their fermentation condition. A rod bacterial strain was isolated from brine water in saltpond of Bohai Bay coastal solar saltworks. Phylogenetic analysis of the 16S rrna gene sequence indicated that the isolate was Halomonas sp., namely Halomonas It is a moderately halophilic bacterium with optimal growth salinity of and ph of 7-8, respectively. The effect of increasing glucose supplementation (at level of 0, 10, 20 and 30 g/l, respectively) on growth and PHB content of Halomonas was investigated using basal culture medium containing 7.5 g/l acid-hydrolyzed casein. The results showed that glucose supplementation in the culture medium significantly improved the growth and PHB content of Halomonas sp. At a glucose concentration of 30 g/l, the cell dry weight and PHB content reached 8.60 g/l and 5.57 g/l (65% of cell dry weight). The current research has provided valuable data with respect of utilization of brine water bio-resources. ( 本文编辑 : 谭雪静 ) 20 / 2015 / 39 / 5

: 307, [], [2],,,, [3] (Response Surface Methodology, RSA),,, [4,5] Design-Expert 6.0,,,, [6] VPJ33 ph 3,, ph, OD, Design-Expert 6.0 Box-Behnken, VPJ3

: 307, [], [2],,,, [3] (Response Surface Methodology, RSA),,, [4,5] Design-Expert 6.0,,,, [6] VPJ33 ph 3,, ph, OD, Design-Expert 6.0 Box-Behnken, VPJ3 微生物学通报 FEB 20, 2008, 35(2) 306~30 Microbiology 2008 by Institute of Microbiology, CAS tongbao@im.ac.cn 生物实验室响应面分析法优化副溶血性弧菌生长条件刘代新宁喜斌 * 张继伦 2 (. 200090) (2. 20202) 摘要