丁草胺降解菌的分离鉴定及降解特性的研究

从处理农药生产废水的膜生物反应器中分离到一株能以丁草胺为惟一碳源和能源生长的细菌BD-1,经鉴定为施氏假单孢菌(Pseudomonas stutzeri).在纯培养的条件下测定了BD-1对丁草胺的降解性能.结果表明,在接种量为菌浓度OD415=0.2,pH7.0、30℃条件下,BD-l对丁草胺的降解符合一级动力学特征,1.0、10.0和100.0 rag·L的丁草胺的降解半衰期分别为0.11、0.60和0.96d.BD-1在不同pH及温度下对丁草胺的降解作用为pH 7.0>pH6.0>pH 8.0,30℃>20℃>40℃.GC/MS初步分析结果表明,丁草胺的主要微生物降解产物为2-氯-2',6'-二乙基乙酰苯胺和2,6-二乙基苯胺.     

生物除磷的研究

以巢湖底泥为原料进行梯度驯化,利用平板分离技术,从底泥中分离到三株聚磷菌株,参照伯杰氏手册进行菌种分类鉴定,初步确定均属假单胞菌属(Pseudomonasspp)。通过进行相应的单因子优化实验,初步确定其最佳生长条件为:三株菌的最适生长温度为30℃;K1菌最适pH为8,K2、K3菌最适pH为7;K1、K3菌最适菌量为OD=0.4,K2菌最适菌量为OD=0.6。同时研究了氧气、碳源对这3株菌聚磷能力的影响,在厌氧条件下出现放磷现象,好氧条件下过量摄磷现象;以乙酸钠为碳源时,其聚磷率高于以乳糖、甲醇、乙醇为碳源时的摄磷量。     

固氮施氏假单胞菌RpoN蛋白对鞭毛合成的影响

选择性σ因子σ~(54)(又称为RpoN)参与到许多细菌特定基因的转录起始过程,如氮同化基因、四碳二羧酸转运基因以及鞭毛基因。为了研究施氏假单胞菌A1501(Pseudomonas stutzeri,A1501)中σ因子RpoN对鞭毛合成的影响,构建了A1501的rpoN缺失突变株与功能回补株,并对野生型、突变株与回补株的鞭毛结构、swimming运动、生物膜形成、根际定殖能力以及鞭毛基因的表达水平进行了比较分析。结果表明:rpo N突变株丧失了鞭毛结构与swimming运动能力,根际定殖与生物膜形成能力相对野生型分别下降了25倍与10倍;此外,rpoN缺失后A1501中大量鞭毛基因发生显著下调;启动子分析发现RpoN除了可能参与调控Ⅱ级、Ⅲ级鞭毛基因外,还可能调控一级基因fli A以及四级基因fliC。以上结果说明RpoN在不同调控等级影响A1501鞭毛的合成,进而影响该菌运动、生物膜形成、根际定殖这些与水稻建立起联合固氮体系息息相关的过程。     

Impact of the use of biofertilizers on cotton ( Gossypium hirsutum ) crop under irrigated agro-ecosystem

High nitrogen fixing, phosphate solubilizing, phytohormones producing isolates of Azotobacter, Azospirillum, Acetobacter and Pseudomonas were used as inoculants for cotton. Important cultures were selected on the basis of their effect on root/shoot length and chemotactic behaviour. Selected bioinoculants were earlier tested for their beneficial properties like nitrogen fixation (ARA), ammonia excretion, IAA production etc. These bio-inoculants were further tested for phosphate solubilization property. Various chosen strains were tested with Desi (HD 123) and American (H 1098) cotton under field conditions (as for wheat). Plant height and boll weight were determined at the time of harvesting whereas survival rate of inoculated bacteria was determined after 30, 80 and 130 days respectively. In the year 2000–01, on the basis of boll number and boll weight plant^?1 AVK 51 (36; 76.2?g plant^?1), HT 57 (27; 56.9?g plant^?1), AC18 (33; 61.5?g plant^?1), Ala 27 (36; 61.4?g plant^?1) and Pseudomonas (34; 71.3?g plant^?1) were identified as significant both for American and desi cotton varieties. Highest survival rate was observed with Mac 68 (33.4 × 10⁵) followed by HT54 (31.5 × 10⁵) after 30 days of sowing, which decreased after 80 days and remained constant up to 130 days. This trend was observed with all the cultures. Similar results were observed in 2001–02. 25?kg ha^?1 N saving was observed with A. chroococcum (AVK51) bioinoculant for cotton crop.     

Design and Experiments of the High Voltage Pulsed Electric Fields Sterilization System

This experiment designed the pulsed electric fields （PEF） of high intensity of 100 kV· cm^-1 sterilization system. Fluorescent pseudomonas as target cell was operated 180 s in the PEF. By observing the difference of the bacteria before and after the disposal by TEM, it is found that the cell walls of the treated bacteria were broken. Irreversible perforations were formed on the cell membrane. The cell inclusions and cell fragments were leaked. The cell died as a result. The results showed that the PEF sterilization system designed can be used for liquid food sterilization experiments.     

节杆菌与施氏假单胞菌对甲基对硫磷的共降解研究

为实现对有机磷农药甲基对硫磷的彻底降解,研究利用有机磷农药降解菌施氏假单胞菌YC-YH1与对硝基苯酚降解菌CN2对甲基对硫磷进行共降解试验,同时克隆了参与降解过程的相关基因,并对2株菌株在对甲基对硫磷污染土壤的原位修复中的效果进行研究。结果表明,与YC-YH1单独降解相比,YC-YH1和CN2共降解在处理前32 h无较大差异;但之后,甲基对硫磷和对硝基苯酚的降解速度明显加快,处理48 h后甲基对硫磷即被完全降解,比单独用YC-H1降解时提前了16 h,处理64 h时对硝基苯酚就被完全降解。而土壤修复的研究结果表明,菌株YC-YH1与CN2能够高效降解甲基对硫磷,72 h对土壤中100 mg/kg甲基对硫磷的降解率接近100%,尤以未灭菌土壤中降解效果更好。     

Pseudomonas stutzeri|A1501基因组结构及功能注释

采用全基因组“shotgun”方法完成了固氮斯氏假单胞菌A1501的全基因组序列测定,并进行了基因组结构与功能注释分析。A1501基因组全长4 567 418 bp,含有4 146个ORFs。该基因组中已鉴定了42个编码转座酶的重复序列,这些序列的存在预示着转座现象在A1501菌中非常活跃,预示该菌与其他生物之间基因交流可能比较频繁。比较基因组表明,为了适应特定的生存环境,假单胞菌在基因组结构和遗传信息容量上产生了明显的分化。此外,基因组分析鉴定了A1501环境适应的遗传基础,包括物质转运、信号传导和趋化系统等,这些系统是细菌能够在根际土壤环境中保持竞争力以及能够与水稻形成高效联合固氮体系的关键。A1501基因组的完成为进一步开展功能基因组学和蛋白质组学研究奠定了基础。     

施氏假单胞菌Na+/H+逆向转运蛋白基因nhaA的克隆与表达分析

Na+/H+逆向转运蛋白是生物耐盐的关键因子,能够维持高盐胁迫下生物体的正常生长代谢。利 用PCR技术从施氏假单胞菌（Pseudomonas stutzeri）中克隆质膜Na+/H+逆向转运蛋白基因nhaA。序列分 析表明,该基因全长1167 bp,编码388 个氨基酸,含有12 个跨膜结构域和保守的功能氨基酸残基位点： Asp-133、Asp-163、Asp-164、His-225、Gly-338。与大肠杆菌nhaA 基因核苷酸和编码氨基酸序列的同源 性分别为99%和99.2%。将该基因与pET-28a 构建成原核表达载体pET-nhaA,转化E.coli BL21,经IPTG 诱导后获得相对分子量约为41 kD的蛋白。平板法和生长曲线法检测表明,重组菌在800 mmol/L NaCl 胁迫下仍能正常生长,耐盐能力显著提高。以上结果证实克隆到的基因是施氏假单胞菌nhaA 基因家族 的成员,具有盐胁迫下将Na+逆向转运至胞外的功能,为进一步利用该基因进行作物耐盐改良奠定了基 础。该基因的GenBank登录号为EU545468。     

雏鸡绿脓杆菌病的诊断

报告了３个疫点肉用仔鸡群中自发的败血型绿脓杆菌病．总共３８５０羽３～５日龄雏鸡中，在２０ｄ流行期间，９１０羽发病，其中５７６羽死亡．从病死鸡的心血和肝中分离到绿脓杆菌而证实了临床诊断．     

施氏假单胞菌JHY01菌株毒死蜱降解酶的定位及其提取条件的优化

对具有降解有机磷农药毒死蜱作用的施氏假单胞菌JHY01菌株的降解酶进行定位,测得其胞外酶、胞内酶、细胞碎片中残留酶对毒死蜱的降解率分别为8.41%、79.85%、77.14%.由此确定该菌株的有机磷降解酶主要为胞内酶.采用超声波破碎方法提取降解酶,对降解酶提取条件进行正交优化,结果显示,最佳超声波(超声波碎仪Sonics-VCX500,功率500W,频率20kHz)破碎条件是:菌体重量:缓冲液体积为1:4(g/ml),总辐射次数30次,每次辐射4s,间隔48.在此条件下所提取的粗酶液酶活力最高,对毒死蜱的降解效率可达84.47%.