Physiological and biochemical characteristics and capacity for polyhydroxyalkanoates synthesis in a glucose-utilizing strain of hydrogen-oxidizing bacteria, Ralstonia eutropha B8562

The physiological, biochemical, genetic, and cultural characteristics of the glucose-utilizing mutant strain Ralstonia eutropha B8562 were investigated in comparison with the parent strain R. eutropha B5786. The morphological, cultural, and biochemical characteristics of strain R. eutropha B8562 were similar to those of strain R. eutropha B5786. Genetic analysis revealed differences between the 16S rRNA gene sequences of these strains. The growth characteristics of the mutant using glucose as the sole carbon and energy source were comparable with those of the parent strain grown on fructose. Strain B8562 was characterized by high yields of polyhydroxyalkanoate (PHA) from different carbon sources (CO2, fructose, and glucose). In batch culture with glucose under nitrogen limitation, PHA accumulation reached 90% of dry weight. In PHA, beta-hydroxybutyrate was predominant (over 99 mol %); beta-hydroxyvalerate (0.25-0.72 mol %) and beta-hydroxyhexanoate (0.008-1.5 mol %) were present as minor components. The strain has prospects as a PHA producer on glucose-containing media.     

The Autotrophic Synthesis of Polyhydroxyalkanoate by Alcaligenes eutrophusin the Presence of Carbon Monoxide

The CO-resistant strain B5786 of the hydrogen-oxidizing bacterium Alcaligenes eutrophuswas found to be able to synthesize polyhydroxyalkanoates (PHAs) under the conditions of growth limitation by nitrogen deficiency (the factor that promotes PHA synthesis) and growth inhibition by carbon monoxide. The gas mixtures that contained from 5 to 20 vol % CO did not inhibit the key enzymes of PHA synthesis–-ketothiolase, acetoacetyl-CoA reductase, hydroxybutyrate dehydrogenase, and PHA synthase. In the presence of CO, cells accumulated up to 70–75 wt % PHA (with respect to the dry biomass) without any noticeable increase in the consumption of the gas substrate. Chromatographic–mass spectrometric analysis showed that the PHA synthesized by A. eutrophusis a copolymer containing more than 99 mol % -hydroxybutyrate and trace amounts of -hydroxyvalerate. The PHA synthesized under the conditions described did not differ from that synthesized by A. eutrophuscells from electrolytic hydrogen.     

金枪鱼肉中组胺菌的分离及其理化性质分析

目的：对金枪鱼肉中的组胺生成菌进行生化实验和功能酶检测,进而对鱼肉中组胺菌的存在提供可参考数据。方法：利用组氨酸肉汤培养液,对金枪鱼肉中的组胺生成菌进行筛选,然后通过生物化学实验和形态学实验,进而对组胺生成菌进行分析。结果：分离得到2株组胺菌,菌种Ⅰ在5℃~30℃的培养温度范围内,随温度升高,组胺生成量逐渐增加,在30℃有最高组胺生成量,约2 700ppm。菌种Ⅱ在25℃有组胺最高生成量约为2 400ppm。结论：依据两种组胺菌的特征和生理生化特征,分离菌可能分别属于弧菌属、埃希氏菌属。     

一株降解DON毒素真菌的鉴定及其生理特性研究

为了对实验室已分离的1株高效降解脱氧雪腐镰刀菌烯醇毒素(DON)的真菌(NJA-1)鉴定,对该菌进行了形态学观察以及rDNA ITS区基因序列的扩增、测定。形态学观察发现该菌属于曲霉属黑色组曲霉黑曲霉集合体。进一步分别用引物对BMB-CR和ITS2及ITS1和ITS4扩增rDNA的ITSⅠ-5.8S rDNA-ITSⅡ,得到总长为738 bp的基因序列,与GENBank中已有的基因序列比对和亲缘关系比较分析发现该菌rDNA ITS区基因序列与塔宾曲霉的同源性为100%,最终确定NJA-1为1株塔宾曲霉。菌株NJA-1 rDNA ITS区基因序列收录入GENBank,登陆号为EF178271。还对NJA-1的生长特性、炭源和氮源的利用、适宜生长的培养基pH等生理特性进行了测定,为该菌的大量培养提供数据基础。     

山医群体近交系中国地鼠血液生理生化指标的测定分析

目的对山医群体中国地鼠近交A、E家系主要血液生理、生化和血清电解质指标进行检测分析。方法对山医群体中国地鼠近交A、E家系成年动物进行空腹采血,常规方法测定13项血液生理、12项生化以及4项电解质指标,分别对相同性别不同家系间和同一家系不同性别间指标进行统计分析。结果相同性别A、E家系间动物血小板（PLT）、中间细胞数（MID）、谷丙转氨酶（ALT）、总胆固醇（TC）、总胆红素（TBiL）、肌酐（Cr）和血钾（K）差异有显著性;相同家系雌、雄动物间PLT、TBiL和尿酸（UA）差异有显著性。结论 A、E家系动物间产生了一定的血液生理、生化性状的分离。     

Biochemical and molecular characterization of a succinate semialdehyde dehydrogenase involved in the catabolism of 4-hydroxybutyric acid in Ralstonia eutropha

A succinate semialdehyde dehydrogenase gene (gabD) was identified to be disrupted in a transposon-induced mutant of Ralstonia eutropha exhibiting the phenotype 4-hydroxybutyric acid-leaky. The native gabD gene was cloned by colony hybridization using a homologous gabD-specific DNA probe. DNA sequencing revealed an 1452-bp open reading frame, and the deduced amino acid sequence showed strong similarities to NADP(+)-dependent succinate semialdehyde dehydrogenases from Escherichia coli, Rhizobium sp., Homo sapiens and Rattus norvegicus. The gabD gene was heterologously expressed in a recombinant E. coli strain harboring plasmid pSK::EE6.8. Similar to the molecular organization of the gab cluster in E. coli, additional genes encoding enzymes for the degradation of gamma-aminobutyrate are closely related to gabD in R. eutropha. Enzymatic studies indicated the existence of a second NAD(+)-dependent succinate semialdehyde dehydrogenase in R. eutropha.     

大豆根际土壤中氢氧化细菌的分离、筛选和基本特征

土壤中的氢氧化细菌能够利用土壤中的H2为能源并同化CO2,增加其种群数量并促进作物生长.采用气体循环培养体系(持续通氢气装置),通过电解水的方式产生H2,与通入的空气混合,形成流速为280ml.min-1、含H2量为41.6～125μmol.L-1的混合气体.采用矿质盐固体培养基,在适当的H2、O2和CO2下以H2作为唯一能源分离氢氧化细菌.采用此方法从大豆根际土壤样品中分离出40余株细菌,对其进行耗氢能力测定表明,有20株菌具有氧化氢功能和自养生长能力,初步判断这20株菌为氢氧化细菌,并测定了菌落形态及生理生化特征.     

Neural and Hybrid Optimizations of the Fed-Batch Synthesis of Poly-β-Hydroxybutyrate by Ralstonia eutropha in a Nonideal Bioreactor

Poly-β-hydroxybutyrate (PHB) is synthesized by some microorganisms under stressful conditions. Despite its properties being comparable to those of synthetic polymers, and its biocompatibility and biodegradability, low productivities have dampened commercial interest in microbial PHB production. To increase production efficiency, a fed-batch fermentation with Ralstonia eutropha was optimized recently through a neural-cum-dispersion model (D-model) incorporating incomplete dispersion and noise in the feed streams. The approach described in the work has been improved in two ways: first by a model comprising neural networks only (N-model) and then by a hybrid neural model (H-model) with a mathematical component. At optimum dispersion, PHB production through the N-model optimization was 35% more than by the D-model, and this was enhanced by a further 58% using hybrid optimization. Recognizing that the D-model itself more than doubled the PHB production compared to a noise-free fully dispersed bioreactor, the present results establish hybrid neural optimization as a viable method for PHB production improvement under realistic conditions.     

乳酸菌受体菌株的筛选、鉴定及特性研究

从乳酸菌保健品中分离筛选到一株能作为受体菌的乳酸菌菌株COCC101,经鉴定为粪肠球菌(Enterococcusfaecali)。抗药性实验显示这株粪肠球菌对多数药物敏感或中度敏感;粪肠球菌中没有质粒存在,转化效率和电场强度有对应的正相关,最高达到2×104转化子/μgDNA,并且能广泛接受不同来源的质粒;在Nisin诱导下可表达外源的绿色荧光蛋白(GFP)。这些结果显示COCC101菌株在乳酸菌基因工程研究中有望成为受体菌。     

甲基营养菌甲醇脱氢酶基因的克隆及表达

目的:从甲基营养菌MP681中扩增甲醇脱氢酶(MDH)基因,在大肠杆菌中表达并检测其活性,同时在MP681中考察该基因对吡咯喹啉醌(PQQ)产生的影响。方法:根据MP681基因组序列设计引物,PCR扩增靶基因mdh,构建表达载体,考察活性,利用接合转移转化至MP681,考察PQQ的合成。结果:扩增得到甲基营养菌MP681甲醇脱氢酶基因,在大肠杆菌中的表达产物能够催化甲醇脱氢;将携带mdh基因的质粒转入MP681后,PQQ产量略有提高。结论:获得编码MDH的基因,该基因能够在大肠杆菌中表达,且表达产物具有生物活性;甲醇脱氢酶基因表达对宿主菌的PQQ合成可能有一定影响。     

Physiological,Biochemical, and Cytological Characteristics of a Haloalkalitolerant Methanotroph Grown on Methanol

The halotolerant alkaliphilic methanotroph Methylomicrobium buryatense 5B is capable of growth at high methanol concentrations (up to 1.75 M). At optimal values of pH and salinity (pH 9.5 and 0.75% NaCl), the maximum growth rate on 0.25 M methanol (0.2 h^–1) was twice as high as on methane (0.1 h^–1). The maximum growth rate increased with increasing medium salinity and pH. The growth of the bacterium on methanol was accompanied by a reduction in the degree of development of intracytoplasmic membranes, the appearance of glycogen granules in cells, and the accumulation of formaldehyde, formate, and an extracellular glycoprotein at concentrations of 1.2 mM, 8 mM, and 2.63 g/l, respectively. The glycoprotein was found to contain 23% protein and 77% carbohydrates, the latter being dominated by glucose, mannose, and aminosugars. The major amino acids were glutamate, aspartate, glycine, valine, and isoleucine. The glycoprotein content rose to 5 g/l when the concentration of potassium nitrate in the medium was augmented tenfold. The activities of sucrose-6-phosphate synthase, glycogen synthase, and NADH dehydrogenase in methanol-grown cells were higher than in methane-grown cells. The data obtained suggest that the high methanol tolerance of M. buryatense 5B is due to the utilization of formaldehyde for the synthesis of sucrose, glycogen, and the glycoprotein and to the oxidation of excess reducing equivalents through the respiratory chain.     

一株轻度嗜盐反硝化细菌的分离鉴定和反硝化特性初探

从处理高盐度废水的成熟活性污泥中分离筛选得到1株轻度嗜盐反硝化细菌GYL, 通过对该菌株的形态观察、生理生化实验以及16S rDNA序列分析, 确定该菌株为盐单胞菌(Halomonas sp.)。该菌株能在盐度为10%的培养液中生长, 最适盐度为2%~7%, 最适pH为7.5~8.5, 最佳碳源为蔗糖, 在25°C~30°C的温度范围内脱氮效率达到80%以上。对该菌株的异养硝化能力进行了测定, 其对氨氮的去除率可达98.3%, 说明该菌株可实现同步硝化反硝化, 即该菌可以独立完成生物脱氮的全部过程。     

Bioenergetic Conditions of Butyrate Metabolism by a Syntrophic, Anaerobic Bacterium in Coculture with Hydrogen-Oxidizing Methanogenic and Sulfidogenic Bacteria

The butyrate-oxidizing, proton-reducing, obligately anaerobic bacterium NSF-2 was grown in batch cocultures with either the hydrogen-oxidizing bacterium Methanospirillum hungatei PM-1 or Desulfovibrio sp. strain PS-1. Metabolism of butyrate occurred in two phases. The first phase exhibited exponential growth kinetics (phase a) and had a doubling time of 10 h. This value was independent of whether NSF-2 was cultured with a methanogen or a sulfate reducer and likely represents the maximum specific growth rate of NSF-2. This exponential growth phase was followed by a second phase with a nearly constant rate of degradation (phase b) which dominated the time course of butyrate degradation. The specific activity of H₂ uptake by the hydrogen-oxidizing bacterium controlled the bioenergetic conditions of metabolism in phase b. During this phase both the Gibbs free energy (ΔG′) and the butyrate degradation rate (v) were greater for NSF-2-Desulfovibrio sp. strain PS-1 (ΔG′ = −17.0 kJ/mol; v = 0.20 mM/h) than for NSF-2-M. hungatei PM-1 (ΔG′ = −3.8 kJ/mol, v = 0.12 mM/h). The ΔG′ value remained stable and characteristic of the two hydrogen oxidizers during phase b. The stable ΔG′ resulted from the close coupling of the rates of butyrate and H₂ oxidation. The addition of 2-bromoethanesulfonate to a NSF-2-methanogen coculture resulted in the total inhibition of butyrate degradation; the inhibition was relieved when Desulfovibrio sp. strain PS-1 was added as a new H₂ sink. When the specific activity of H₂ consumption was increased by adding higher densities of the Desulfovibrio sp. to 2-bromoethanesulfonate-inhibited NSF-2-methanogen cocultures, lower H₂ pool sizes and higher rates of butyrate degradation resulted. Thus, it is the kinetic parameters of H₂ consumption, not the type of H₂ consumer per se, that establishes the thermodynamic conditions which in turn control the rate of fatty acid degradation. The bioenergetic homeostasis we observed in phase b was a result of the kinetics of the coculture members and the feedback inhibition by hydrogen which prevents butyrate degradation rates from reaching their theoretical V_max.     

超大型烟草突变株的生理生化特征和分子生物学鉴定

超大型烟草突变株再生植株高度是野生型的2．2倍,叶片数是野生型的3．3倍,呈现晚花发育特征;叶片气孔保卫细胞中叶绿体数是野生型的1．3倍,叶绿素a、b和叶绿素总量均高于野生型,可溶性蛋白质含量是野生型的1．18倍,过氧化物酶、细胞色素氧化酶同工酶电泳图谱上有一定差异;可溶性蛋白SDS-PAGE电泳图谱上比野生型少4条谱带;RAPD结果表明突变体在DNA水平上确实发生了变化,DDRT-PCR结果显示出两者在基因表达上有差异。突变株再生植株可以开花结实,植株高大、叶数多,晚花的特征可以稳定遗传。     

一株脱氮细菌鉴定及其效果研究

针对传统人工湿地治理污染河湖水工艺中存在的脱氮效率低、环境安全性差等问题，从污水处理厂生化池污泥中分离纯化获得菌株TY（CGMCC1.18865，该菌种已于2020年在中国普通微生物菌种保藏中心入库），通过配水试验，验证菌株脱氮特性，并进一步以火山岩为填料，采用湿地模拟系统装置对比附配TY菌的湿地系统与传统湿地系统对污染水体的脱氮效果的差异；通过16S rRNA基因序列分析，鉴定获得的菌株TY，同时对其表现出的反硝化作用进行分析研究。结果表明：分离获得的TY菌株为假单胞菌属（Pseudomonas sp.），配水试验中对氨氮和总氮的去除率分别为84.2%和93.6%。通过对模拟湿地系统出水氨氮、总氮和化学需氧量（COD）监测，发现30 d平均去除率分别可提升至94.3%、88.0%和82.3%，较传统湿地分别提高27.9%、28.8%和7.3%。研究表明，TY菌脱氮过程中有少量的硝态氮和亚硝态氮的积累，可降低对环境的不利影响、有利于脱氮反应连续进行，从而提高人工湿地脱氮效率，具有进一步探究的前景。     

一株[艹屈]高效降解菌的分离鉴定及其降解特性

以多环芳烃[艹屈] (Chrysene)为选择培养基的碳源, 从焦化污泥中筛选出一株[艹屈]的高效降解菌SQ-1, SQ-1可在以[艹屈]为唯一碳源的无机盐培养基中生长, 经过电镜形态学观察、生理生化和16S rDNA序列分析, 并基于16S rDNA序列结果, 构建了该菌株的系统发育树。最终确定菌株SQ-1为木糖氧化无色杆菌(Achromobacter xylosoxidans)。又考察了[艹屈]的初始浓度、投菌量、pH值对SQ-1菌株降解[艹屈]效果的影响, 确定了最佳降解条件。结果表明, 该菌对水中[艹屈]具有良好的降解特性, 在[艹屈]浓度为40 mg/L、接种量10% (V/V)、pH 7.0~7.5、温度30°C条件下, 接种5 d后对[艹屈]的降解效率达到80%以上。     

Novel precursor substrates for polythioesters (PTE) and limits of PTE biosynthesis in Ralstonia eutropha

A novel class of biopolymers referred to as polythioesters (PTE) was recently detected when the polyhydroxyalkanoate (PHA) accumulating bacterium Ralstonia eutropha was cultivated in the presence of 3-mercaptopropionic acid (3MP) or 3,3'-thiodipropionic acid (TDP). In this study, 3,3'-dithiodipropionic acid (DTDP) and 3-mercaptovaleric acid (3MV) were identified as two additional precursor carbon sources for in vivo biosynthesis of PTE in R. eutropha. Biosynthesis of copolymers of 3-hydroxybutyrate (3HB) and 3MP, which contributed 19-25% of cell dry matter, was compared referring to the different precursor substrates. Using DTDP as carbon source, which is probably cleaved into two molecules 3MP, yielded an about 2.3-fold higher molar 3MP content of the copolyester than TDP, which is probably cleaved into only one molecule 3MP. Furthermore, cultivation of R. eutropha in the presence of 3MV resulted in biosynthesis of copolymers consisting predominantly of 3HB with low amounts of 3MV and 3-hydroxyvalerate, each contributing less than 5 mol% of the constituents. In contrast, 4-mercaptobutyric acid could be not incorporated into PHAs, although - as documented in this study - five different strategies, various precursor substrates, R. eutropha and also a recombinant strain of Escherichia coli were employed. Therefore, this study not only extended the range of substrates suitable for PTE biosynthesis and also the range of PTE constituents in R. eutropha, it also demonstrates limits for PTE biosynthesis in this bacterium.     

Participation of a Specific Substrate Degrading Strain in a Mixed Bacteria Culture as a Result of Ciliate Grazing

Participation of nitrilotriacetic acid degrading bacterial strain NTA-1 in the continuous-cultivated mixed culture was studied under different conditions including predation pressure of the ciliate Dexiostoma campyla (STOKES , 1886). From the viewpoint of dispersed/flocculated biomass distribution, significant relationships between NTA-1 and total bacteria ratio, and dispersed and total biomass ratio were proved in the systems without high concentrations of ciliates. The ciliate concentrations reaching 10⁴ ml⁻¹ stabilized flocculated biomass growth without directly affecting NTA-1 portion. Using fluorescently labelled NTA-1 bacteria, filter feeding rates of ciliates were evaluated (maximum individual uptake rate upon NTA-1 bacteria as a number of bacteria per ciliate per hour being 120 h⁻¹ and 260 h⁻¹ under ciliate division rate of 0.3 day⁻¹ and 1 day⁻¹, respectively). Biomass balance showed that dispersed NTA-1 bacteria should not serve as the sole feeding source for these free-swimming ciliates. The role of diversity of mixed bacterial diet in ciliate growth and the role of ciliate predation in stabilizing bacterial assemblage structure was proved.     

Regulation Culture on Cytological，Biochemical and Physiological Characteristics of Somatic Carrot Em

ＲｅｇｕｌａｔｉｏｎＣｕｌｔｕｒｅｏｎＣｙｔｏｌｏｇｉｃａｌ,ＢｉｏｃｈｅｍｉｃａｌａｎｄＰｈｙｓｉｏｌｏｇｉｃａｌＣｈａｒａｃｔｅｒｉｓｔｉｃｓｏｆＳｏｍａｔｉｃＣａｒｒｏｔＥｍｂｒｙｏｓＨＵＡＮＧＭｅｉ－ｊｕａｎ;（黄美娟）,ＨＵＡＮＧＳｈａｏ－...