Pseudomonas alcaliphila AD-28的脱氮性能及其关键酶活性

【背景】异养硝化-好氧反硝化菌由于能够同时实现硝化反硝化作用而备受关注,但由于菌的种类不同,其脱氮途径不尽相同,研究菌株脱氮关键酶的种类及其活性可以推测菌株的脱氮途径,从而为菌株在生产上的应用提供技术支撑。【目的】研究Pseudomonas alcaliphila AD-28的脱氮性能及其关键酶的活性,为菌株脱氮分子机理研究奠定基础。【方法】以柠檬酸钠为碳源,以硫酸铵、亚硝酸钠、硝酸钾为氮源,研究菌株AD-28的脱氮性能并检测其关键酶氨单加氧酶(AMO)、羟胺氧化还原酶(HAO)、亚硝酸盐还原酶(NIR)、硝酸盐还原酶(NAR)的酶活性。【结果】菌株AD-28培养24h的菌密度(OD600)可达1.971,对初始浓度为18.85mg/L的氨氮、26.13mg/L的硝酸盐氮、19.47mg/L的亚硝酸盐氮、66.11 mg/L的总氮去除率均达到96%以上;关键酶AMO、HAO、NIR和NAR的比活力分别为0.028、0.003、0.011、0.027 U/mg。【结论】Pseudomonas alcaliphila AD-28能同时进行异养硝化-好养反硝化作用,该菌在AMO作用下将NH4+-N氧化为羟胺,然后由HAO氧化为NO2--N,NO2--N和NO3--N在NIR、NAR等酶的催化作用下脱氮。     

代谢工程强化脱氮副球菌DYTN-1去除氮素污染物

【目的】脱氮副球菌(Paracoccus denitrificans)是一种环境友好的α-变形菌纲菌株,在有氧条件下也可进行反硝化过程,具有较好的脱氮能力。本研究以脱氮副球菌DYTN-1为底盘细胞,筛选氮素诱导型启动子用于强化硝化和反硝化途径,进而达到代谢工程强化脱氮副球菌DYTN-1去除氮素污染物的目的。【方法】通过接合转移的方法分别将过表达amoA、amoB、hao和nirS基因的重组质粒导入脱氮副球菌DYTN-1细胞中。经过荧光定量检测和氮素定量检测对脱氮副球菌DYTN-1的基因元件和氮去除能力进行表征。【结果】从基因组中挖掘了6个受NO₂^‒、NO₃^‒和NH₄⁺诱导的启动子,诱导差异为2‒26倍;且过表达nirS的菌株用2 g/L KNO₃处理24 h后培养基中NO₃^‒的残余量为野生型菌株的67%。同时过表达hao和nirS基因的菌株在用1 g/L NH₄Cl和2 g/L KNO₃处理12 h后,其NO₃^‒的剩余量仅为野生型菌株的50%,且最终总氮的降解效率达79.5%,剩余总氮仅为野生型菌株的一半。【结论】上述研究表明,利用筛选获得的启动子工具在P. denitrificans DYTN-1中进行代谢工程改造强化氮素污染物的去除具有可行性。     

基于响应面法对一株好氧反硝化菌脱氮效能优化

【目的】水体富营养化是当今我国水环境面临的重大水域环境问题,氮素超标排放是主要的引发因素之一。好氧反硝化菌构建同步硝化反硝化工艺比传统脱氮工艺优势更大。获得高效的好氧反硝化菌株并通过生长因子优化使脱氮效率达到最高。【方法】经过序批式生物反应器(Sequencing batch reactor,SBR)的定向驯化,筛选获得高效好氧反硝化菌株,采用响应面法优化好氧反硝化过程影响总氮去除效率的关键因子(碳氮、溶解氧、pH、温度)。【结果】从运行稳定的SBR反应器中定向筛选高效好氧反硝化菌株Pseudomonas T13,采用响应面法对碳氮比、pH和溶解氧关键因子综合优化获得在18 h内最高硝酸盐去除率95%,总氮去除率90%。该菌株的高效反硝化效果的适宜温度范围为25?30 °C;最适pH为中性偏碱;适宜的COD/NO3?-N为4:1以上;最佳溶解氧浓度在2.5 mg/L。【结论】从长期稳定运行的SBR反应器中筛选获得一株高效好氧反硝化菌Pseudomonas T13,硝酸盐还原酶比例占脱氮酶基因的30%以上,通过运行条件优化获得硝氮去除率达到90%以上,对强化废水脱氮工艺具有良好应用价值。     

耐冷亚硝酸盐型反硝化菌Pseudomonas tolaasii Y-11的鉴定及其脱氮特性

摘要:【目的】反硝化细菌在生物脱氮中具有重要作用,而耐冷亚硝酸盐型反硝化细菌研究较少,本文从长期淹水的冬水田泥土分离获得一株耐冷高效去除亚硝酸盐氮和总氮的好氧反硝化细菌Y-11,明确其分类地位以及除氮特性,以期为后续利用该菌在初冬到春末处理亚硝酸盐水体污染奠定基础。【方法】通过形态学特征、特异性磷脂脂肪酸以及16S rRNA基因测序分析对该菌株进行鉴定;在好氧条件下以亚硝酸钠为唯一氮源,分别研究不同初始温度、转速、pH、碳源、接种量以及亚硝酸盐氮浓度对该菌去除亚硝酸盐氮和总氮的影响,确定最适降解条件。【结果】分离得到的菌株Y-11,经鉴定归于托拉斯假单胞菌(Pseudomonas tolaasii);在国内外尚无该种菌具有反硝化作用的报道,是对亚硝酸盐型反硝化细菌的进一步补充。Y-11菌株的最适脱氮条件为15 ℃,200 r/min,pH7.0,100 mL反硝化培养基中最适接种量为1.5×108 CFU,最佳碳源为乙酸 钠,亚硝酸盐氮为10 mg/L;以乙酸钠为电子供体,15 ℃、初始pH为7.2、150 r/min 振荡培养,48 h对亚硝酸盐氮和总氮的去除率分别为100%和61.28%。【结论】Y-11是一株具有较高反硝化能力的托拉斯假单胞菌,能高效地去除亚硝酸盐氮和总氮,其最适温度是15 ℃左右,是一株耐冷反硝化细菌。     

耐高浓度氨氮的异养硝化好氧反硝化菌株U1的鉴定及其脱氮特性

【背景】城市垃圾渗滤液是一种成分复杂的有机废水,含氮量高,如果未经处理直接排放到环境中会造成严重的环境污染。【目的】筛选可以耐受垃圾渗滤液中高浓度氨氮并高效去除污水中氮素的异养硝化好氧反硝化菌株,为解决垃圾渗滤液的氮素污染提供功能菌株。【方法】从垃圾渗滤液中筛选分离能耐受高氨氮浓度的菌株,通过测定各菌株的脱氮能力,筛选到一株脱氮能力最强的菌株,命名为U1,通过测定16S rRNA基因序列和生理生化特性确定该菌株为铜绿假单胞菌。进一步研究了菌株U1在不同初始氨氮浓度、碳源、转速、初始pH、碳氮比等单因素变量下的脱氮能力,并结合L₉（3⁴）正交试验研究了菌株U1的最佳脱氮条件。【结果】分离出一株铜绿假单胞菌并命名为U1。该菌株的最优脱氮条件为:初始氨氮浓度为1 000 mg/L,红糖和柠檬酸三钠的混合碳源,pH 6.0,C/N为10,转速为130 r/min,菌株U1的最大总氮去除率为64.37%,最大氨氮去除率为76.73%。对于总氮和氨氮含量分别是2 345 mg/L和1 473.8 mg/L的垃圾渗滤液,菌株U1最大总氮去除率为27.86%...     

好氧反硝化细菌SY-D-22的分离、优化及脱氮机理

【背景】在处理含硝酸盐氮的废水中,常见微生物不能同时高效去除硝酸盐氮和总氮,导致处理废水时往往使用多种微生物复合菌剂或者使用复杂的工艺。【目的】高效、安全地去除水中的硝酸盐氮和总氮。方法】富集筛选出一株新型高效好氧反硝化细菌,对其进行16S rRNA基因鉴定。利用响应面法对其影响脱氮因素进行优化并探索其最佳脱氮条件。研究其对含硝酸盐氮废水的反硝化能力及脱氮特性。【结果】从活性污泥中筛选获得一株新型高效好氧反硝化细菌SY-D-22,经鉴定为葡萄球菌属(Staphylococcus)。响应面法优化其最佳反硝化条件为：pH 8.18,C/N为13.39,温度31.43°C,摇床转速130 r/min。当以最佳碳源柠檬酸钠为唯一碳源时,对于100 mg/L浓度的NO₃^--N去除率可达100%,同时对于总氮(total nitrogen,TN)的去除率为95.34%,具有高效脱氮能力。【结论】从活性污泥中筛选出一株新型好氧反硝化细菌Staphylococcus SY-D-22,通过响应面法条件优化,菌株的硝酸盐氮去除率达到100.00%,总氮去除率达到...     

短程反硝化菌株FDN-1的分离鉴定及其脱氮性能

从富集的活性污泥菌群中,筛选得到反硝化菌株FDN-1。通过对菌株形态观察、生理生化鉴定以及16SrDNA序列分析,确定该菌株为黄杆菌属（Havobacterium mizutaii）。以N02---N为氮源、甲醇为碳源进行驯化,亚硝酸盐氮浓度在200-400mg／L范围时,总氮去除率均在90％以上;脱氮能力验证结果表明,该菌株能够处理含亚硝酸盐的污水,进水亚硝酸盐氮浓度不能超过600mg／L。     

一株海洋好氧反硝化细菌的鉴定及其好氧反硝化特性

【目的】从处理海洋养殖循环水的生物滤器生物膜中分离到1株具有好氧反硝化活性的细菌(菌株2-8),并进一步研究了该菌的分类地位及反硝化特性。【方法】采用16S rRNA基因序列分析对菌株进行初步鉴定,采用好氧培养技术,探讨了碳源种类、起始pH、NaCl浓度、C/N、温度和摇床转速对菌株2-8好氧反硝化活性的影响。【结果】该菌株的16S rRNA基因序列与Pseudomonas segetis FR1439T(AY770691)的相似性最高,达到99.9%,因此初步鉴定菌株2-8属于假单胞菌属(Pseudomonas sp.2-8)。碳源类型和C/N对其好氧反硝化作用的影响最为显著,以柠檬酸钠为唯一碳源,C/N为15时脱氮效率最高,低C/N导致亚硝酸盐的积累;其好氧反硝化的最适温度和pH分别为30℃和7.5;菌株2-8在摇床转速为160r/min下脱氮效果最好;NaCl浓度对其反硝化活性的影响不明显。【结论】在初始硝酸氮浓度为140mg/L,以柠檬酸钠为唯一碳源、C/N为15、pH为7.5、NaCl浓度为30g/L,30℃以及160r/min摇床培养的条件下,菌株2-8在48h内脱氮率可达92%且无亚硝酸盐积累。     

深海沉积物高效脱氮菌筛选分离及其脱氮特性研究

【背景】深海海域具有高压、低温、无光等环境条件,蕴含着丰富而独特的微生物资源。【目的】从深海沉积物中定向分离、筛选脱氮效率高的好氧脱氮菌株资源,并揭示其脱氮特性,为开发水体脱氮微生物技术提供物质基础。【方法】以东太平洋、南大西洋、西南印度洋共10个站位的深海沉积物为研究材料,在28°C下使用无机氮源连续进行两轮富集培养,然后定性筛选可以脱除氨氮、亚硝态氮和硝态氮的菌株,并通过形态学和16S rRNA基因序列分析进行初步分类鉴定;对优选得到的功能菌株,分别采用以氨氮、亚硝态氮、硝态氮为唯一氮源的培养基定量研究其生长和脱氮性能。【结果】从10份大洋深海沉积物样品中共分离得到49株好氧反硝化菌,其中3株在有氧条件下反硝化效率较高,分别命名为Pseudomonassp.G111、Pseudomonassp.G112和Dietziamaris W023a,其中菌株G111和G112与模式菌株博岑假单胞菌Pseudomonas bauzanensis BZ93T的16S rRNA基因序列相似度为99.2%,菌株W023a与模式菌株海洋迪茨氏菌DietziamarisATCC35013T的16SrRNA基因序列相似度为99.9%。菌株G111、G112和W023a培养48h后,对氨氮的脱除率分别为98.0%、85.2%和97.6%;对亚硝态氮的脱除率分别为71.9%、67.5%和34.7%;对硝态氮的脱除率分别为66.0%、52.6%和56.3%。菌株G111、G112和W023a均为异养硝化-好氧反硝化菌,可通过好氧反硝化作用将亚硝态氮和硝态氮还原为含氮气体,也可通过异养硝化-好氧反硝化作用将氨氮转化为含氮气体。【结论】从深海沉积物中分离筛选得到3株高效好氧反硝化菌,所获得的菌株在水体净化、污水处理、生态系统修复等领域具有应用潜力。     

两株异养硝化细菌的分离鉴定及其脱氮特性

【目的】利用异养硝化培养基,从华中农业大学实验猪场污水中筛选得到2株具有较高脱氮效率的细菌。【方法】通过形态学特征及16S rDNA序列的系统发育分析,对分离菌株进行了鉴定。且对菌株P2和P9降解氨氮的相关特性也作了研究。此外,将菌株单独或混合接种于猪场污水,检测其处理实际污水的脱氮效果。【结果】初步判断菌株P2为副球菌属(Paracoccus sp.),P9为申氏杆菌属(Shinella sp.)。2株细菌能在有机物存在下进行异养硝化作用,经24h培养,菌株P2和P9对氨氮的去除率可达80%左右,同时未发现亚硝酸盐、硝酸盐积累;但菌株P2,P9不能以NO 3-或NO 2-为唯一氮源发生好氧反硝化作用。菌株P2和P9异养硝化的最适碳源为丁二酸钠,最适C/N比为9,且脱氮过程中pH值从6.8到8.9一直呈上升趋势。菌株对小分子碳源具有较强的依赖性,在加入小分子碳源的情况下,其对污水具有较强的脱氮能力,且这两个菌株混合施用较单独作用氨氮去除效果更好。【结论】菌株P2和P9脱氮能力较强,其在污水处理行业具有重要的应用前景。     

Comparative study of Bifidobacteriumanimalis, Escherichiacoli, Lactobacilluscasei and Saccharomycesboulardii probiotic properties

The present work investigates some probiotic properties of four different microorganisms (Bifidobacterium animalis var. lactis BB-12, Escherichia coli EMO, Lactobacillus casei and Saccharomyces boulardii). In vitro and in vivo tests were carried out to compare cell wall hydrophobicity, production of antagonistic substances, survival capacity in the gastrointestinal tract of germ-free mice without pathological consequence, and immune modulation by stimulation of Küpffer cells, intestinal sIgA and IL-10 levels. In vitro antagonism against pathogenic bacteria and yeast was only observed for the probiotic bacteria B. animalis and L. casei. The hydrophobic property of the cell wall was higher for B. animalis and E. coli EMO, and this property could be responsible for a better ability to colonize the gastrointestinal tract of germ-free mice. Higher levels of sIgA were observed mainly for S. boulardii, followed by E. coli EMO and B. animalis, and only S. boulardii induced a significant higher level of IL-10. In conclusion, for a probiotic use, S. boulardii presented better characteristics in terms of immunomodulation, and B. animalis and L. casei for antagonistic substance production. The knowledge of the different probiotic properties could be used to choice the better microorganism depending on the therapeutic or prophylactic application.     

Non-specificity of a colorimetric method for the estimation of N-acetyl-d-glucosamine

The colorimetric method of Reissig et al. for the estimation of N-acetylamino sugars, is often used as a specific method for the quantification of the N-acetyl-d-glucosamine. Although this assay is more sensitive to the monomer, it recognizes all soluble N-acetyl-d-glucosamine oligomers. This result is very important because this method is extensively used in biology for the estimation of chitinolytic activity.     

Effects of MULTIFOLIATE-PINNA, AFILA, TENDRIL-LESS and UNIFOLIATA genes on leafblade architecture in Pisum sativum

In order to dissect the genetic regulation of leafblade morphogenesis, 16 genotypes of pea, constructed by combining the wild-type and mutant alleles of MFP, AF, TL and UNI genes, were quantitatively phenotyped. The morphological features of the three domains of leafblades of four genotypes, unknown earlier, were described. All the genotypes were found to differ in leafblade morphology. It was evident that MFP and TL functions acted as repressor of pinna ramification, in the distal domain. These functions, with and without interaction with UNI, also repressed the ramification of proximal pinnae in the absence of AF function. The expression of MFP and TL required UNI function. AF function was found to control leafblade architecture multifariously. The earlier identified role of AF as a repressor of UNI in the proximal domain was confirmed. Negative control of AF on the UNI-dependent pinna ramification in the distal domain was revealed. It was found that AF establishes a boundary between proximal and distal domains and activates formation of leaflet pinnae in the proximal domain.     

Presence of Rhizobium Etli bv . Phaseoli and Rhizobium Gallicum bv . Gallicum in Egyptian Soils

Seven bean rhizobial strains EBRI 2, 3, 21, 24, 26, 27 and 29 identified as Rhizobium etli, and EBRI 32 identified as Rhizobium gallicum, isolated from Egyptian soils and which nodulated Phaseolus vulgaris efficiently, were subjected to hybridization with a nifH probe in order to estimate the copy number of this gene. Seven strains (EBRI 2, 3, 21, 24, 26, 27 and 29) which were only able to nodulate Phaseolus vulgaris, contained three copies of the nifH gene, consistent with their identification as Rhizobium etli bv. phaseoli. Only one strain (EBRI 32) which nodulated both Phaseolus vulgaris and Leucaena leucocephala, had one copy of nifH gene. This confirmed the classification of this strain as Rhizobium gallicum bv. gallicum.     

Characterisation of cell wall polysaccharides, arabinogalactans-proteins (AGPs) and phenolics of Cola nitida, Cola acuminata and Garcinia kola seeds

Many Cola plant species are endemic to West and Central Africa. Cola acuminata and Cola nitida are used as masticatory when fresh, while the dried nuts are used for beverages and pharmaceutical purposes in Europe and North America. Garcinia kola seeds, that serve as a substitute for the true kola nuts, are used in African traditional medicine for the treatment of various diseases, including colic, headache and liver cirrhosis. Seeds extracts of G. kola are also known for their anti-inflammatory, antimicrobial and antiviral properties. To gain information on the chemical properties of the kolas, we have isolated and analyzed cell wall polysaccharides, arabinogalactan-proteins and phenolic substances from the seeds of the three kola species. The sugar composition of cell wall material of C. acuminata, C. nitida and G. kola revealed that Gal (up to 30%), Ara, GalA and Glc as the predominant monosaccharides, representing approximately 90% by mol of the total hydrolysable sugar present in this material. In Ammonium oxalate cell wall fraction, GalA was found to be the major sugar present in all kola species. In the alkali-soluble fraction, there were significant differences in the level of Glc and Gal. The level of Glc was high in C. acuminata and C. nitida while the level of Gal and Xyl were high in C. nitida and G. cola. Isolation and quantification of arabinogalactan-proteins demonstrate that G. kola seeds contained four to eight times more of these proteoglycans than the seeds of the other two species. Finally, analysis of soluble phenolic substances shows that caffeine and catechin were largely represented in C. acumina and C. nitida seeds, with caffeine accounting for 50% of all soluble phenolics. These findings indicate that the three Kola seeds are highly enriched in pectins and proteoglycans and that C. acuminata and C. nitida can be used as a possible source of caffeine and catechin.     

转Cry1Ac/1Ab基因棉花对异色瓢虫生长发育及其捕食功能的影响

【目的】为探究转Cry1Ac/1Ab基因棉花对异色瓢虫生长发育及其捕食功能的影响。【方法】以转Cry1Ac/1Ab基因棉与其亲本常规棉为实验材料,利用取食不同棉花品种叶片的棉铃虫饲喂异色瓢虫幼虫。【结果】与常规亲本棉相比,取食饲喂转基因棉花叶片的初孵棉铃虫幼虫的异色瓢虫幼虫从1龄发育至化蛹期时间延长0.77 d,但差异不显著;除1龄幼虫体重增加(0.0773 mg)外,其余各龄期幼虫体重均有所下降,但差异均不显著;异色瓢虫1、2、3、4龄幼虫对初孵棉铃虫捕食量均随棉铃虫密度的增加而增加,捕食功能反应均符合HollingⅡ圆盘方程。【结论】转Cry1Ac/1Ab基因棉花对异色瓢虫生长发育无显著影响,饲喂取食转Cry1Ac/1Ab基因棉花的棉铃虫对异色瓢虫捕食功能无显著差异。     

Characterization of recA genes and recA mutants of Rhizobium meliloti and Rhizobium leguminosarum biovar viciae

Summary DNA fragments carrying the recA genes of Rhizobium meliloti and Rhizobium leguminosarum biovar viciae were isolated by complementing a UV-sensitive recA ^– Escherichia coli strain. Sequence analysis revealed that the coding region of the R. meliloti recA gene consists of 1044 by coding for 348 amino acids whereas the coding region of the R. leguminosarum bv. viciae recA gene has 1053 bp specifying 351 amino acids. The R. meliloti and R. leguminosarum bv. viciae recA genes show 84.8% homology at the DNA sequence level and of 90.1% at the amino acid sequence level. recA ^– mutant strains of both Rhizobium species were constructed by inserting a gentamicin resistance cassette into the respective recA gene. The resulting recA mutants exhibited an increased sensitivity to UV irradiation, were impaired in their ability to perform homologous recombination and showed a slightly reduced growth rate when compared with the respective wild-type strains. The Rhizobium recA strains did not have altered symbiotic nitrogen fixation capacity. Therefore, they represent ideal candidates for release experiments with impaired strains.The accession numbers: X59956 R. LEGUMINOSARUM REC A ALAS-DNA; X59957 R. MELITOTI REC A ALAS-DNA     

Application of petG-trnP sequence to systematic study of Chinese Cupressus species

Chinese Cupressus L. includes five species. The molecular phylogenetic relationship of the Cupressus species and Chamaecyparis L. were determined by comparing 417–479 bp of chloroplast petG-trnP intergenic spacer sequence. In PAUP^* analysis, Platycladus orientalis was used as the functional out group. By using the maximum likelihood method 1 077 trees were examined and the result showed that one tree had a best score of -Ln=2 232.47. The phylogenetic tree clearly showed that Chamaecyparis nootkatensis was diverged from other Chamaecyparis species. Based on the results, together with evidences from other aspects, we consider that Cupressus funebris and Chamaecyparis nootkatensis should be placed in the genus Cupressus. The use of cpDNA intergenic spacer petG-trnP in Cupressus was also discussed. __________ Translated from Journal of Sichuan University (Natural Science Edition), 2005, 42(5): 1033–1037 [译自: 四川大学学报 (自然科学版) 2005, 42(5): 1033–1037]     

Hydroxylation of steroids by Fusarium oxysporum, Exophiala jeanselmei and Ceratocystis paradoxa

The potential of Fusarium oxysporum var. cubense UAMH 9013 to perform steroid biotransformations was reinvestigated using single phase and pulse feed conditions. The following natural steroids served as substrates: dehydroepiandrosterone (1), pregnenolone (2), testosterone (3), progesterone (4), cortisone (5), prednisone (6), estrone (7) and sarsasapogenin (8). The results showed the possible presence of C-7 and C-15 hydroxylase enzymes. This hypothesis was explored using three synthetic androstanes: androstane-3,17-dione (9), androsta-4,6-diene-3,17-dione (10) and 3α,5α-cycloandrost-6-en-17-one (11). These fermentations of non-natural steroids showed that C-7 hydroxylation was as a result of that position being allylic. The evidence also pointed towards the presence of a C-15 hydroxylase enzyme.The eleven steroids were also fed to Exophialajeanselmei var. lecanii-corni UAMH 8783. The results showed that the fungus appears to have very active 5α and 14α-hydroxylase enzymes, and is also capable of carrying out allylic oxidations.Ceratocystis paradoxa UAMH 8784 was grown in the presence of the above-mentioned steroids. The results showed that monooxygenases which effect allylic hydroxylation and Baeyer–Villiger rearrangement were active. However, redox reactions predominated.