异育银鲫肠道蛭弧菌的分离及其生物学特性的研究

以一株具有致病力的温和气单胞菌作为筛选宿主菌,从异育银鲫肠道中分离到一株蛭弧菌,暂命名为BDF-H16。通过光学显微镜、相差显微镜、电子显微镜对蛭弧菌BDF-H16进行形态观察,并研究了其部分生物学特性。结果表明:蛭弧菌BDF-H16为革兰氏阴性菌,杆形或弧杆形,端生一根鞭毛,菌体大小多为0．2μm～0．5μm×0．8μm～1．2μm;蛭弧菌BDF-H16对实验所选用的革兰氏阴性菌和部分革兰氏阳性菌均有裂解作用;以大肠杆菌为宿主菌,其最佳生长条件为宿主菌浓度6．75×10~9cfu/mL、pH7．0～7．5、温度28℃;在NaCl含量0．85%～5．00%的培养基中能够生长;恩诺沙星、诺氟沙星对其有抑制作用。     

苯甲酸1，2-双加氧酶高活力菌株的筛选和发酵条件的研究

从176株细菌中,筛选出苯甲酸1,2-双加氧酶的高活力菌株假单胞菌(Pseudomonas)137。进行了该菌产酶的发酵条件试验。产酶的最适温度为32℃,最适起始pH为6．5—7．0。葡萄糖、麦芽糖和甘油对产酶有明显的抑制作用,苯甲酸钠对产酶有促进作用。氨态氮对菌体生长和产酶是必需的。琥珀酸钠是酶形成的有效诱导物,采用0．1％苯甲酸钠和0．2％琥珀酸钠培养基(pH6．5—7．0),于32℃振荡培养72小时,可获得高活力的苯甲酸1,2-双加氧酶,每克菌体酶活力可达5-8单位。     

一株硫酸盐还原菌的分离鉴定及生物学特性研究

从处理硫酸盐废水厌氧折流板反应器（Anaerobic battqe reactor,ABR）的污泥中分离到1株硫酸盐还原菌,对该菌株进行了形态、生理生化特性方面的研究,并对16S rDNA序列进行了分析。该菌株为杆状或弧状,大小为（0．5～0．7）μm×（1．4～1．9）μm,革兰染色阴性,芽胞染色阴性,能运动,具有硫酸盐还原功能。菌株最适生长pH为7．0～8．0,喜中性偏碱环境;初始[SO4^2-]为2000mg／L,OD600nm。值为1．206,SO4^2-去除率达到71％;该菌株能分别利用乳酸、丙酮酸、丁酸、乙酸、乙醇、甲醇、葡萄糖作为电子供体,进行硫酸盐异化还原,乳酸最有利于该菌SO4^2-的去除,SO4^2-去除率为91．4％,其次为丙酮酸,达到51．2％。基于16SrDNA序列同源性构建了系统发育树,结果表明此株菌是属于脱硫弧菌属（Desulfovibrio）的硫酸盐还原菌,与Desulfovibrio具有96．0％的序列相似性。     

高效反硝化菌aHD7的筛分、脱氮特性及厌氧氨氧化性

从活性污泥中筛选出一株高效反硝化菌aHD7,30℃静置培养3d,脱氮率可达91.7％,且反应过程中亚硝酸盐积累量较低,3d后亚硝酸盐氮浓度基本稳定在1.8mg.L-1.显微镜观察显示,菌株为革兰氏阴性杆菌,大小为0.5 μm×(1.5～2.5) μm.通过生理生化特性及16S rDNA同源性分析,初步推断该菌株为门多萨假单胞菌(Pseudomonas mendocina).考察了碳源、C/N、氮初始浓度、pH等因素对菌株反硝化性能的影响.结果表明:对中低浓度硝酸盐(硝酸盐氮浓度≤276.95 mg.L-1),脱氮率接近100％,硝酸盐氮浓度高达553.59 mg·L-1时,脱氮率可达66.8％,且亚硝酸盐积累量甚微;最适碳源为乙醇;C/N为6～8和偏中性条件有利于反硝化反应.aHD7具有较强的厌氧氨氧化性,平均氨利用率达4.56 mg·L-1·d-1.     

异育银鲫肠道蛭弧菌的分离及其生物学特性的研究

以一株具有致病力的温和气单胞菌作为筛选宿主菌,从异育银鲫肠道中分离到一株蛭弧菌,暂命名为BDF-H16。通过光学显微镜、相差显微镜、电子显微镜对蛭弧菌BDF-H16进行形态观察,并研究了其部分生物学特性。结果表明:蛭弧菌BDF-H16为革兰氏阴性菌,杆形或弧杆形,端生一根鞭毛,菌体大小多为0．2μm～0．5μm×0．8μm～1．2μm;蛭弧菌BDF-H16对实验所选用的革兰氏阴性菌和部分革兰氏阳性菌均有裂解作用;以大肠杆菌为宿主菌,其最佳生长条件为宿主菌浓度6．75×10⁹cfu/mL、pH7．0～7．5、温度28℃;在NaCl含量0．85%～5．00%的培养基中能够生长;恩诺沙星、诺氟沙星对其有抑制作用。     

一个降解染料的希瓦氏菌新种——中国希瓦氏菌

从细胞形态、生理生化特性和16S rRNA基因和gyrB基因序列同源性分析等方面对一株广谱高效染料降解菌D14进行了鉴定.菌株D14的细胞壁革兰氏染色为阴性,细胞为杆状,大小为0.6μm～1.0μm×1.0μm～4.0μm,周身纤毛,极生单鞭毛,其生长pH范围为pH 7.0～10.0,最适生长pH为8.0,生长温度范围为4℃～40℃,最适生长温度为20℃～30℃.该菌株具有还原三价铁,液化明胶、Tween40和Tween 80,产生H2S的能力.在乳酸钠存在的条件下,能还原硝酸盐、亚硝酸盐、铁氧化物和硫代硫酸钠.可利用D-半乳糖、D-葡萄糖、蔗糖、丙酸钠、L-亮氨酸等多种有机物为碳源.BIOLOG细菌自动鉴定系统鉴定结果表明该菌株应归属于希瓦菌属.16S rDNA和gyrB基因序列分析结果表明,菌株D14与其亲缘关系最近的菌株Shewanella putrefacien ATCC8071T的16S rDNA序列相似值为97%(小于97.7%),gyrB基因序列相似值为87%(小于90%).菌体所含有的主要脂肪酸为iso-15:0,16:1ω7c,15:0和16:0,DNA中(G C)mol%含量为49.3.结合菌株D14的生理生化特征和分子生物学特性,将菌株定为希瓦氏菌属的一个新种,命名为中国希瓦氏菌(Shewanella cinica)D14T.     

一株中度嗜热嗜酸硫氧化杆菌的分离和系统发育分析

从云南腾冲温泉酸性水样分离得到一株中度嗜热嗜酸硫氧化杆菌MTH 0 4 ,对分离菌株进行了形态、生理生化特性研究及 1 6SrDNA序列分析。该菌株为革兰氏阴性细菌 ,短杆状 ,菌体大小 (0 6～ 0 8) μm× (1～ 2 ) μm ,化能自养 ,可利用硫磺、四硫酸盐、硫代硫酸盐为能源生长 ,不能利用蛋白胨、葡萄糖、酵母粉 ,也不能进行混合型生长。最适生长温度在 4 0℃～ 4 5℃之间 ,最适生长pH 2 0～ 3 0 ,代时 8h。以 1 6SrDNA序列同源性为基础构建了包括 1 3株相关种属在内的系统发育树 ,结果表明 ,MTH 0 4与喜温硫杆菌 (Thiobacilluscaldus)处于同一进化树分支中 ,相似性达 99 5 %以上     

一株产碱性蛋白酶菌株的选育及其发酵条件的研究

从土壤中分离到的553株芽孢杆菌中选出一株产蛋白酶活力较高菌株,经紫外线和亚硝基胍诱变,获得一株产碱性蛋白酶的菌株,酶活力可达10000u／m1,此菌株经鉴定为地衣状芽孢杆菌(Bacillus licheniformis)。适宜的发酵条件：培养基由6％玉米粉(山芋粉或葡萄糖),4％豆饼粉(或其水解液),Na,HP0．·12Hz0 0．4％,KH：P0·0．03％,Na zc0,0．1％组成,pH7．O,37℃旋转摇床振荡培养{2一q6小时。酶作用的最适条件：60℃,pH9．0一10．5,在pH6．0—10．5范围内稳定。酶受DFP抑制。     

一株产碱极端嗜盐杆菌

从青海省大柴旦盐湖中分离到一株极端嗜盐杆菌。该菌株在培养过程中产碱,革兰氏阴性,细胞杆状,0．7-1.0×2-3μm,极生鞭毛运动,绝对好氧,以氨基酸作唯一碳源。不利用碳水化合物,生长所需盐(NaCl)浓度在1 2％以上,最适盐浓度为l 8％。生长pH范围6-l0,最适pH9。MgSO4·7H2Oo-3％对生长无明显影响。细胞蛋白质酸性,不含二氨莲庚二酸和胞壁酸,含甘油二醚键化合物,不具有色素。根据以上特征,将该菌株定为一个新种,定名为产碱嗜盐杆菌(Halobocierium haloalcaligenum n.sp.)。     

一个可降解直链烷基苯磺酸盐的新种

本文鉴定了一株可完全降解直链烷基苯磺酸盐（Linear Alkylbenzene Sulphonate,简称LAS）的菌株GZ6。革兰氏染色阴性,细胞为杆状或短杆状,大小为0.5μm～0.8 μm×1.0μm～2.0 μm,其生长pH范围为pH6.0～10.0,最适生长pH为7.0,生长温度范围为4℃～40℃,最适生长温度为30℃。生化特征测定除过氧化氢酶、尿酶、精氨酸脱羧酶反应为阳性,其它均为阴性。可利用Chloridazon、安替比林（antipyrin）以及LAS等为碳源。不能利用大多数糖醇。醌组分以泛醌Q10为主。菌体脂肪酸主要为C18∶1、C16∶0及C16∶1。DNA中G+C mol％含量为7010。16S rRNA 序列分析表明菌株GZ6 与其亲缘关系最近的菌株Phenylobacterium immobile DSM1986T序列相似值为9749％,DNADNA杂交率为40%。菌株GZ6具极生鞭毛,可运动,两者在细胞形态有很大差异。故将菌株GZ6定为苯基杆菌属的新种可动苯基杆菌（Phenylobaterium mobile） GZ6。     

光合细菌H3菌株的分离及其生物学特性研究

H3菌株系由盐田微生物垫中分离获得的光合细菌株。革兰氏阴性杆菌,0.5-0.7×15-2.5μm,在培养条件改变时呈多形态,其膨大细胞可达4.0-7.0μm.单根极生鞭毛,二均分裂。菌落及液体培养物呈深紫红色。菌体蛋白质含量约50%,各种必需氨基酸齐全,并含有丰富的细菌叶绿素a和类胡萝卜素。H3菌株在光照和黑暗条件下均可生长,能利用多种有机和无机碳、氮源,并能固N2.在含0.5-7.0%NaCI培养基中均可生长。但光照、盐浓度、温度、pH和通气等条件对其生长量有明显的影响。作者对其分类地位和应用潜力作了讨论。       

Autotrophic ammonia oxidation at low pH through urea hydrolysis.

Ammonia oxidation in laboratory liquid batch cultures of autotrophic ammonia oxidizers rarely occurs at pH values less than 7, due to ionization of ammonia and the requirement for ammonium transport rather than diffusion of ammonia. Nevertheless, there is strong evidence for autotrophic nitrification in acid soils, which may be carried out by ammonia oxidizers capable of using urea as a source of ammonia. To determine the mechanism of urea-linked ammonia oxidation, a ureolytic autotrophic ammonia oxidizer, Nitrosospira sp. strain NPAV, was grown in liquid batch culture at a range of pH values with either ammonium or urea as the sole nitrogen source. Growth and nitrite production from ammonium did not occur at pH values below 7. Growth on urea occurred at pH values in the range 4 to 7.5 but ceased when urea hydrolysis was complete, even though ammonia, released during urea hydrolysis, remained in the medium. The results support a mechanism whereby urea enters the cells by diffusion and intracellular urea hydrolysis and ammonia oxidation occur independently of extracellular pH in the range 4 to 7.5. A proportion of the ammonia produced during this process diffuses from the cell and is not subsequently available for growth if the extracellular pH is less than 7. Ureolysis therefore provides a mechanism for nitrification in acid soils, but a proportion of the ammonium produced is likely to be released from the cell and may be used by other soil organisms.     

Autotrophic Ammonia Oxidation at Low pH through Urea Hydrolysis

Ammonia oxidation in laboratory liquid batch cultures of autotrophic ammonia oxidizers rarely occurs at pH values less than 7, due to ionization of ammonia and the requirement for ammonium transport rather than diffusion of ammonia. Nevertheless, there is strong evidence for autotrophic nitrification in acid soils, which may be carried out by ammonia oxidizers capable of using urea as a source of ammonia. To determine the mechanism of urea-linked ammonia oxidation, a ureolytic autotrophic ammonia oxidizer, Nitrosospira sp. strain NPAV, was grown in liquid batch culture at a range of pH values with either ammonium or urea as the sole nitrogen source. Growth and nitrite production from ammonium did not occur at pH values below 7. Growth on urea occurred at pH values in the range 4 to 7.5 but ceased when urea hydrolysis was complete, even though ammonia, released during urea hydrolysis, remained in the medium. The results support a mechanism whereby urea enters the cells by diffusion and intracellular urea hydrolysis and ammonia oxidation occur independently of extracellular pH in the range 4 to 7.5. A proportion of the ammonia produced during this process diffuses from the cell and is not subsequently available for growth if the extracellular pH is less than 7. Ureolysis therefore provides a mechanism for nitrification in acid soils, but a proportion of the ammonium produced is likely to be released from the cell and may be used by other soil organisms.     

耐低温甲烷杆菌SHB的分离鉴定与生长特性分析

利用亨盖特厌氧操作技术,从承德塞罕坝湿地污泥中分离出1株耐低温的产甲烷菌SHB。该菌株宽约0.31μm,长约2.87μm,细杆,形成长丝。能够利用H2/CO2和甲钠盐生长,不利用甲醇、三甲胺、乙酸和二级醇类。最适生长温度范围为16～40℃,最适pH为6.0～9.0,能在0%～5%盐浓度范围内生长。该菌株对青霉素、链霉素、罗红霉素、利福平、新霉素有抗性,对氯霉素、四环素、卡那霉素、庆大霉素敏感。经生理、生化及16S rDNA分析,确定该菌株与甲烷杆菌属(Methanobacterium sp.)的亲缘关系最近。该菌株的生长温度和pH范围都很宽,在沼气生产方面有潜在的应用前景。     

一株河流污泥中产甲烷杆菌的生物学特性研究

目的:筛选产甲烷菌并对其进行鉴定和特性分析.方法:利用亨盖特厌氧操作技术从河流污泥中分离出的一株产甲烷杆菌.通过形态特征、生理生化、16S rRNA基因序列分析确定菌株的分类地位,利用气相色谱仪测定甲烷含量.结果:该菌株杆状,产芽孢,极端严格厌氧,宽约0.36μm,长约3.20μm,有时观察成链状,在液体培养的过程中,聚集成絮状沉积在管底.能够利用H2/CO2和甲钠盐生长,不利用甲醇、三甲胺、乙酸和二级醇类.最适生长温度范围为37℃,最适pH为7.5 ～8.0,能在0～1％盐浓度范围内生长.结论:菌株AG的最适温度较高,最适pH偏碱,在沼气发酵中具有重要应用前景.     

Optimal operational factors for nitrite accumulation in batch reactors

The environmental factors that affected the accumulation of nitrite in nitrifying reactors were investigated using a mixed culture. A batch reactor with 50 mg-N/l of ammonia was used. The pH, temperature and dissolved oxygen concentration were varied. The concentration of unionized free ammonia also changed with the oxidation of ammonia and the variation of pH and temperature. The accumulation of nitrite was affected sensitively by pH and temperature. A higher nitrite concentration was observed at pH 8-9 or temperature around 30 °C. The dissolved oxygen also affected, giving the highest nitrite accumulation at around 1.5 mg/l. These were the favoredconditions for nitrite production. The free ammonia concentration influenced thenitrite accumulation also, by inhibiting nitrite oxidation. The inhibition becameapparent at a concentration of approximately 4 mg/l or above, but insignificant atbelow 1 mg/l. Thus, simultaneously high free ammonia concentration and maximumspecific ammonia-oxidation rate (above 15 × 10^-3 mg-N/mg-VSSh)were needed for a significant nitrite accumulation. When the two conditions were met, thenthe highest accumulation was observed when the ratio of the maximum specific oxidationrate of ammonia to the maximum specific oxidation rate of nitrite (k_a/k_n) was highest.Under the optimal operating conditions of pH 8, 30 °C and 1.5 mg/l of dissolvedoxygen, as much as 77% of the removed ammonia accumulated in nitrite.     

Oxidation of pyrimidine nucleosides and nucleotides by osmium tetroxide

1. Pyrimidine nucleosides such as thymidine, uridine or cytidine are oxidized readily at 0° by osmium tetroxide in ammonium chloride buffer. There is virtually no oxidation in bicarbonate buffer of similar pH. Oxidation of 1-methyluracil yields 5,6-dihydro-4,5,6-trihydroxy-1-methyl-2-pyrimidone. 2. Osmium tetroxide and ammonia react reversibly in aqueous solution to form a yellow 1:1 complex, probably OsO₃NH. A second molecule of ammonia must be involved in the oxidation of UMP since the rate of this reaction is approximately proportional to the square of the concentration of unprotonated ammonia. 3. 4-Thiouridine reacts with osmium tetroxide much more rapidly than does uridine. The changes of absorption spectra are different in sodium bicarbonate buffer and in ammonium chloride buffer. They occur faster in the latter buffer and, under suitable conditions, cytidine is a major product. 4. Polyuridylic acid is oxidized readily by ammoniacal osmium tetroxide, but its oxidation is inhibited by polyadenylic acid. Pyrimidines of yeast amino acid-transfer RNA are oxidized more slowly than the corresponding mononucleosides, especially the thymine residues. Appreciable oxidation can occur without change of sedimentation coefficient.     

Rescue of the orphan enzyme isoguanine deaminase

Cytosine deaminase (CDA) from Escherichia coli was shown to catalyze the deamination of isoguanine (2-oxoadenine) to xanthine. Isoguanine is an oxidation product of adenine in DNA that is mutagenic to the cell. The isoguanine deaminase activity in E. coli was partially purified by ammonium sulfate fractionation, gel filtration, and anion exchange chromatography. The active protein was identified by peptide mass fingerprint analysis as cytosine deaminase. The kinetic constants for the deamination of isoguanine at pH 7.7 are as follows: k(cat) = 49 s(-1), K(m) = 72 μM, and k(cat)/K(m) = 6.7 × 10(5) M(-1) s(-1). The kinetic constants for the deamination of cytosine are as follows: k(cat) = 45 s(-1), K(m) = 302 μM, and k(cat)/K(m) = 1.5 × 10(5) M(-1) s(-1). Under these reaction conditions, isoguanine is the better substrate for cytosine deaminase. The three-dimensional structure of CDA was determined with isoguanine in the active site.     

Metabolic profiling reveals growth related FAME productivity and quality of Chlorella sorokiniana with different inoculum sizes

Inoculum size strongly affects cell growth and lipid accumulation of microalgae, one of the most potential biodiesel feedstock, however, the metabolic mechanism(s) of the lipid biosynthesis upon inoculum size has not been fully explored yet. The effects of inoculum size on cell growth, lipid accumulation, and metabolic changes of a green microalga Chlorella sorokiniana were investigated. In our experimental range of inoculum size, the productivity and the cetane number (CN) of fatty acid methyl esters (FAME) increased with increasing initial cell density, and the inoculum of 1 × 10(7) cells mL(-1) processed much higher productivity (up to 2.02-fold) and CN (up to 1.19-fold) of the FAME than the others. A significant correlation between the metabolic profile and quantity and quality of lipid production was revealed by partial least-squares to latent structures (PLS) analysis, and 15 key metabolites were identified. Most of those metabolites were involved in the photosynthetically fixed carbon metabolism. Furthermore, light intensity as one of the vital limitation factors for the high inoculum size cultivation was evaluated by illumination assay and the results revealed that increasing light intensity could improve the polyunsaturated fatty acids composition and lipid accumulation of C. sorokiniana. The lipid productivity of the culture was improved by 71.21% with the light intensity of 110 μmol m(-2) s(-1), compared to that under the irradiance of 65 μmol m(-2) s(-1).