盐田嗜盐单胞杆菌的分离鉴定及其PHB 积累的研究

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 r DNA序列测序比对,确定为盐杆菌属(Halomonas),命名为Halomonas 100-16-2,其最适生长盐度为50~100,最适生长p H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0、10、20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 100-16-2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。     

中国九个内陆盐湖卤虫的品系特征

分析了中国９个内陆盐湖卤虫的生物学测定值,孵化特性,蛋白质含量和脂肪酸组成,不同品系的卤虫在卵径,无节幼体体长,脱壳卵和无节幼体干重等生物学测定值方面存在着极其显著的差异（Ｐ〈０．０１）。     

Impact of brine acidification on hatchability,survival and reproduction of Artemia parthenogenetica and Artemia franciscana in salt ponds,Bohai Bay,China

We studied the effect of pH(pH 5, 6, 7 and 8) on the hatching percentage, survival and reproduction of Artemia strains in Bohai Bay salt ponds. Strains included parthenogenetic Artemia from Bohai Bay(BHB), Artemia franciscana from San Francisco Bay, and A. franciscana artifi cially produced in salt ponds in Vietnam. The latter was included as a potential inoculum for biological management of salt ponds. The hatching percentage of cysts after 24 h and the survival rate of the tested Artemia strains were signifi cantly reduced when exposed to a culture medium at pH 5 for 18 d( P 0.05). The tolerance of Artemia to 48 h acid exposure varied with developmental stage, increasing in the following order: juvenile, nauplii, pre-adult, with maximum tolerance in adults. All strains of Artemia tested could not reproduce at pH 5. At pH levels from pH 6–8, a higher pH generally resulted in a shorter brood interval and enhanced ovoviviparity. Hence, we suggest that brine acidifi cation has a negative impact on Artemia populations in the Bohai Bay saltworks. Inoculation of Artemia with either local parthenogenetic Artemia or exotic A. franciscana should be feasible at pH 7–8.     

Characterization of halophilic C50 carotenoid-producing archaea isolated from solar saltworks in Bohai Bay,China

Halophilic archaea comprise the majority of microorganisms found in hypersaline environments. C50 carotenoids accumulated in archaea cells are considered potential biotechnological products and possess a number of biological functions. Ten red colordes were isolated from brine water in a saltem crystaltizer pond of the Hangu Saltworks, China. 16S rRNA gene sequence analysis showed that the colonies belonged to the extremely halophilic archaea genera Halobacterium and Halorubrum. Two representative strains, Halobacterium strain SP-2 and Halorubrum strain SP-4, were selected for further study on the phenotypic characteristics and effects of salinity and pH on accumulation and composition of pigments in their cells. The archaeal strains were isolated and grown in a culture medium prepared by dissolving yeast extract （10 g/L） and acid-hydrolyzed casein （7.5 g/L） into brine water obtained from a I.ocal salt pond. Their optimum salinity and pH for growth were 250 and 7, respectively, although pigment accumulation （OD490/ mL broth） was highest at pH 8. In addition, at 150-300 salinity, increasing salinity resulted in decreasing pigment accumulation. Analysis of the UV-Vis spectrum, TLC and HLPC chromatograms showed that C50 carotenoid bacterioruberin is the major pigment in both strains.     

葡萄糖和盐度对卤虫生长、养殖系统生物絮团形成及其微生物多样性的影响

在零换水卤虫养殖系统中,研究葡萄糖和盐度对卤虫Artemia franciscana生长、卤水中生物絮团形成、水质及其微生物多样性的影响。实验设四组,每组三个平行,分别在盐度为50、100和150的养殖水体中添加葡萄糖(添加量随着卤虫的生长逐渐增加从每天12mg/L到37mg/L),对照组盐度为50且不添加葡萄糖。单位养殖水体为1L,卤虫起始密度约为1200ind/L,投喂浓缩杜氏藻Dunaliella viridis,养殖周期为20天。结果显示,添加葡萄糖可以显著提高生物絮团体积,降低水中的铵盐、亚硝酸盐、硝酸盐和总氮水平,提高卤虫总生物量(P0.05)。但随着盐度升高,卤虫生物量显著降低(P0.05),亚硝酸盐和总氮水平显著升高(P0.05),絮团体积有所升高。另外,葡萄糖添加和盐度对卤虫粗蛋白和脂肪酸含量无显著影响(P0.05)。PCR-DGGE分析表明,γ-变形菌纲(γ-proteobacterium)为生物絮团中优势菌群且以海洋细菌为主。与对照组相比,添加葡萄糖和升高盐度显著降低菌群丰富度指数(P0.05),环境承载力下降。本研究结果为高盐环境条件下生物絮团的形成机理及其在卤虫人工养殖中的应用提供了参考。     

添加碳源和不同盐度对卤虫生长、养殖系统生物絮团形成及其微生物多样性的影响

在零换水卤虫养殖系统中,研究碳源添加和不同盐度对卤虫生长、卤水中生物絮团形成、水质及其微生物多样性的影响。实验设四组,每组三个平行,分别在盐度为50、100和150的养殖水体中添加葡萄糖,对照组盐度为50且不添加碳源。单位养殖水体为1L,卤虫起始密度约为1200个/L,投喂浓缩杜氏藻,养殖周期为2周。结果显示,添加葡萄糖显著提高生物絮团体积,降低水中的铵盐、亚硝酸盐、硝酸盐和总氮水平,提高卤虫总生物量(P<0.05)。盐度升高,卤虫生物量显著降低(P<0.05),亚硝酸盐和总氮水平显著升高(P<0.05),絮团体积有所升高。添加葡萄糖和不同盐度对卤虫粗蛋白和脂肪酸含量无显著影响(P>0.05)。PCR-DGGE分析表明,γ-变形菌纲为生物絮团中优势菌群且以海洋细菌为主。与对照组相比,添加葡萄糖和升高盐度显著降低菌群丰富度指数(Rr) (P<0.05),环境承载力下降。本研究结果为高盐环境条件下生物絮团的形成机理及其在卤虫人工养殖中的应用提供了参考。     

无效基因fruitless参与卤虫Artemia franciscana的生殖调控研究

fruitless （fru）是在昆虫与甲壳动物求偶、交配行为以及生殖发育过程中发挥关键作用的基因。卤虫不仅是水产育苗的重要开口饲料,而且是生物学研究的重要实验生物。本实验通过对卤虫Artemia franciscana转录组的筛选,获得了fru基因的开放阅读框（ORF）并进行生物信息学分析,利用qPCR技术研究了它在卤虫生殖腺发育的不同阶段的表达情况,最后利用RNAi显微注射技术,深入探索了它的功能。经过生物信息学分析,fru基因的ORF长度为1 215 bp,其中包含了404个氨基酸,其分子量和等电点分别为45.19 kDa和5.28,属于酸性亲水性蛋白,且不含信号肽和跨膜结构;结构域预测显示fru存在BTB_POZ和HTH两个结构域,二级结构主要由α-螺旋和无规则卷组成,三级结构与其对应。qPCR结果显示,fru基因的表达量在卵囊的晚期胚胎中表现出明显的增加趋势,其表达量显著高于其他发育阶段（P < 0.01）;而在精巢的未成熟期,fru基因的表达量也有所增加,其表达量显著高于成熟早期、中期和晚期（P < 0.01）。经过RNA干扰,我们发现fru基因的表达量有了显著的下降（P < 0.01）,并且经过受到干扰的雌虫后代都为休眠卵。这表明fru基因对于A. franciscana的生殖和发育有着重要的影响,甚至可能是影响卤虫繁殖方式的关键因素。通过本次研究,我们获得了关于fru基因在卤虫生殖发育中的作用及其相关分子机制的重要信息,可以帮助我们更好地理解这一重要的生物学过程。     

Isolation and characterization of halophilic bacteria and archaea from salt ponds in Hangu Saltworks,Tianjin, China

A total of 26 isolates were obtained from solar salt ponds of different salinities(100, 150, 200, and 250) in Hangu Saltworks Co. Ltd., Tianjin, China. Phylogenetic analysis of 16 SrRNA gene sequences indicated that five bacteria genera H alomonas, Salinicoccus, Oceanobacillus, Gracibacillus, and Salimicrobium and one archaea genera H alorubrum were present. The genus H alomonas was predominant with eight strains distributed in a salinity range of 100–200, followed by H alorubrum with six strains in salinity 250. Based on the genus and original sampling salinity, eight bacterial and two archaeal isolates were selected for further morphological, physiological, and biochemical characterization. All of the bacterial strains were moderately halophilic with the optimal salinity for growth being either 50 or 100, while two archaeal strains were extremely halophilic with an optimal growth salinity of 200. Additionally, we put forth strain SM.200-5 as a new candidate S alimicrobium species based on the phylogenic analysis of the 16 SrRNA gene sequence and its biochemical characteristics when compared with known related species.     

盐度对不同卤虫品系存活和生殖性能的影响

作者采用中国渤海湾盐田固有的渤海湾孤雌生殖卤虫(Bohai Bay Artemia parthenogenetica,BHB)和分别源自美国旧金山湾盐田(SFB)与越南Vinh Chau 人工养殖(VSFB)的两性生殖卤虫(A. franciscana),研究盐度(分别为70, 125 和180)对上述品系卤虫存活和生殖力的影响。结果表明, 在实验盐度范围内, 所有品系卤虫存活率随盐度升高而显著降低, 但与BHB 卤虫相比, SFB 和VSFB 卤虫对高盐度的耐受力较强。盐度显著影响BHB 卤虫性成熟时间和产后代间隔时间, 并随盐度的升高而延长,但对SFB 和VSFB 卤虫没有显著影响。所有品系卤虫每窝产幼体或产卵数量均随盐度升高而有降低的趋势。较低盐度(70)下, BHB 卤虫以产幼体占绝对优势, VSFB 产幼体较多, SFB 产卵较多; 而在较高盐度(125 和180)下, VSFB 以产幼体占绝对优势, SFB 和BHB 也以产幼体居多。总之, 与SFB 和BHB 相比, VSFB 在高盐环境下存活率较高, 且以产幼体占绝对优势, 表明VSFB 卤虫更适应于高盐盐田环境,有利于该品系卤虫在盐场建立优势种群。     

聚-羟基丁酸酯(PHB)对中华绒螯蟹(Eriocheir sinensis)幼蟹生长和肝胰腺酶活力的影响

分别在饲料中添加0%、1%和3%聚?-羟基丁酸酯(PHB), 研究PHB对中华绒螯蟹(Eriocheir sinensis)幼蟹生长和肝胰腺酶活力的影响。将初始体重为0.8—1.0g的幼蟹(每组60只)在循环水养殖系统单独培养60d后, 对幼蟹蜕皮间隔、个体增重和肝胰腺酶活力等指标进行测定。结果表明, 随饲料中PHB添加量的增加, 幼蟹个体体重增加, 蜕皮间隔缩短, 3% PHB添加组显著高于未添加PHB的对照组(P<0.05)。肝胰腺中总超氧化物歧化酶(T-SOD)活力下降, 碱性磷酸酯酶(ALP)活力增加, 酸性磷酸酯酶(ACP)活力相对稳定, 谷草转氨酶(GOT)和谷丙转氨酶含量(GPT)活力总体较低且显著下降(P<0.05)。PHB对中华绒螯蟹幼蟹生长发育的促进作用, 除了与?-羟基丁酸提供能量物质有关外, 还与非特异免疫防御能力的提高和肝胰腺细胞良好的生理状态密切相关。