Pseudomonas putida GM6多聚磷酸盐激酶(ppk)基因的克隆及表达

以一株高效聚磷菌Pseudomonas putida GM6为研究材料.为获得其多聚磷酸盐激酶（polyphosphate kinase,ppk）基因,并验证该基因在磷酸盐转运系统中的作用,根据已报道的ppk基因保守区域设计引物,从其总DNA中成功扩增到ppk基因的部分片段（约528 bp）.随后采用快速染色体步移方法（Self-formed adaptor PCR,SEFA-PCR）技术扩增片段的上下游基因序列,将三个序列拼接,用OMIGA软件分析其ORFs,推测ppk基因全长为2 220 bp（GenBank accession number DQ133537）.构建的多聚磷酸盐激酶表达菌株E. coli BL21（DE3）/ pET29a-ppk经IPTG诱导后3 h时,明显出现分子量约为81 kDa的表达产物.且表达菌株在12 h时的磷去除率高达80%（对照菌株的磷去除率仅为18%）,远高于已报道的40%的去除率.这表明ppk基因在E. coli中的过量表达,导致了E. coli菌体中poly-P的大量聚集,从而大大去除了培养基中的磷酸盐.     

Pseudomonas putida GM6在活性污泥中的生物强化除磷

The enhanced biological phosphorus removal （EBPR） method is widely adopted for phosphorus removal from wastewater, yet little is known about its microbiological and molecular mechanisms. Therefore, it is difficult to predict and control the deterioration of the EBPR process in a large-scale municipal sewage treatment plant. This study used a novel strain isolated in the laboratory, Pseudomonas putida GM6, which had a high phosphate accumulating ability and could recover rapidly from the deteriorated system and enhance the capability of phosphorus removal in activated sludge. Strain GM6 marked with gfp gene, which was called GMTR, was delivered into a bench-scale sequencing batch reactor （SBR） of low efficiency, to investigate the colonization of GMTR and removal of phosphorus. After 21 days, the proportion of GMTR in the total bacteria of the sludge reached 9.2%, whereas the phosphorus removal rate was 96%, with an effluent concentration of about 0.2 mg L^-1. In the reactor with the addition of GMTR, phosphorus was removed quickly, in 1 h under anaerobic conditions, and in 2 h under aerobic conditions. These evidences were characteristic of EBPR processes. Field testing was conducted at a hospital sewage treatment facility with low phosphorus removal capability. Twentyone days after Pseudomonas putida GM6 was added, effluent phosphorus concentration remained around 0.3 mg L^-1, corresponding to a removal rate of 96.8%. It was therefore demonstrated that Pseudomonas putida GM6 could be used for a quick startup and enhancement of wastewater biological phosphorus removal, which provided a scientific basis for potential large-scale engineering application.     

Pseudomonas putida GM6的gfp基因标记及在活性污泥中的定殖

强化生物除磷（EBPR）工艺在废水生物除磷中广为应用,但由于对EBPR工艺的微生物学和分子机理尚不了解,对大规模生活污水处理厂EBPR工艺处理效果突然恶化和运行不稳定等状况难以预测和控制。本研究利用本实验室分离鉴定的高效聚磷菌Pseudomonas putida GM6,来进行投菌试验以快速恢复和强化活性污泥的除磷能力。为了解GM6菌株在装置中的定殖情况,首先,通过三亲结合对Pseudomonas putida GM6成功进行了gfp基因标记;其次,将标记菌株GMTR投至实验室规模除磷效果不好的序批式反应器（SBR）装置中,研究GMTR在活性污泥中的定殖情况,投菌初期,GMTR占污泥细菌总数的1.5%～3%;至21d时,GMTR所占比例升至9.2%,GMTR在活性污泥中得以定殖。与此同时,研究了投菌后R2的聚磷能力变化,投菌5d后,磷的去除率逐渐提高,运行21d时磷的去除率升至96%,28d后出水浓度稳定在0.2mgL^-1左右。结果表明,Pseu-domonas putida GM6能快速启动强化废水生物除磷功能。这为下一步的大规模的工程化应用提供了科学依据。     

恶臭假单胞菌(Pseudomonas putida)GM6的聚磷特性研究

从城市污水处理厂好氧池活性污泥中分离获得一株高效聚磷菌株GM6，经生理生化和16SrDNA初步鉴定为恶臭假单胞菌（Pseudomonas putida）。GM6生长pH在5．5至8．5之间，最适生长pH为6．5；当pH值为7．0时，聚磷能力最强，pH值小于5．5或大于7．5时，除磷能力明显下降。通气量试验表明，装液量对GM6的生长影响较小；装液量为100ml时，磷的去除效果最好，当装液量大于150ml时磷的去除效果变差。GM6的最适生长温度为27℃，当温度小于5℃或大于37℃时生长较慢；当温度为20℃其除磷效果最好，温度大于33℃或小于5℃时，除磷效果明显变差。好氧条件下GM6在合成废水、MOPS培养基、LB及YG培养基中培养时磷的绝对去除量分别为9．87、12．1、87．3和67．1mgL^-1，磷的去除率分别为96．6％、85％、71．6％和63％，磷的绝对去除量和去除率远高于E．coli。好氧培养时菌体吸磷能力测定结果表明，GM6在合成废水、MOPS、LB及YG培养基中培养24h的菌体干重含磷量在6．80％～9．32％之间，而对照菌体含磷量在0．98％～2．31％之间，聚磷菌的聚磷效果远高于对照菌。用序批式反应器对GM6进行厌氧好氧纯培养时，厌氧末的上清液磷浓度为16．8mgL^-1，CODCr为230mgL^-1，放磷速率为4．5mgL^-1h^-1；好氧末的上清液磷浓度为2．56mgL^-1，CODCr为40mgL^-1，好氧吸磷速率为2．73mgL^-1h^-1，菌株GM6表现出了明显的好氧吸磷、厌氧放磷现象，具有聚磷菌的典型特征。     

球形红杆菌磷耐受能力和除磷特性的研究

通过纯种培养试验，考察了废水生物除磷优势菌球形红杆菌的磷耐受能力和除磷特性。结果表明，球形红杆菌具有较强的磷耐受能力，且磷酸盐含量会对其除磷特性产生一定的影响。缺磷环境会使菌体产生过量摄磷现象；而在高磷环境下，菌体摄磷量的变化趋势与直接转入富磷培养类似：磷酸盐含量为1％时，菌体摄磷量基本未发生变化，当磷酸盐含量达到2％时，由于菌体的生长受到影响，摄磷量略有下降。结果还表明，球形红杆菌在微好氧培养过程中能达到与厌氧－－微好氧培养过程相似的除磷效果，从而提示只要废水中存在足够的碳源，即使全部是微好氧过程，该菌也能有效地摄取磷酸盐而达到较好的除磷效果，由此可见，将球形红杆菌应用于高浓度含磷废水的实际处理工艺中前景看好。     

一株聚磷菌GP44 的筛选、鉴定及其聚磷特性研究

采用纯培养结合蓝白斑筛选法从巢湖和南淝河底泥中分离筛选出能聚磷(P)的 11 株解 P 细菌,好氧培养时菌体吸 P 能力测定结果表明,GP44 的菌体含 P 量达到 11.92%,具有较高的聚 P 能力,对其初步鉴定为鉴定菌株 GP44 属肠杆菌科中的克雷伯氏菌属土生克雷伯氏菌(K.terrigena).GP44 在废水合成培养基上最佳聚 P 温度 30℃、初始 pH 为 7.5、最佳装液量为 120 ml/250 ml 和最适 C 源是葡萄糖,Mg2+、K+ 和 Fe3+ 有利于菌株 GP44 的生物除 P.     

污水处理反硝化除磷-诱导结晶磷回收工艺中除磷微生物特性

为探究反硝化除磷-诱导结晶磷回收工艺中缺氧池污泥释磷、吸磷以及微生物特征,利用荧光原位杂交(fluorescence in situ hybridization,FISH)技术、电子扫描显微镜(scanning electron microscope,SEM)观察了微生物的数量、分布和形态;通过批次试验考察了污泥在厌氧/好氧和厌氧/缺氧2种模式下的释磷和吸磷特征。结果表明:该双污泥系统缺氧池中聚磷菌占总细菌比例的69.7%,明显高于单污泥系统中富集的聚磷菌比例,污泥中的微生物多呈杆状;厌氧/好氧、厌氧/缺氧模式下单位污泥浓度(mixed liquor suspended solids,MLSS)总吸磷量(以PO43--P计)分别为22.84、18.60 mg/g,反硝化聚磷菌(denitrifying polyphosphate-accumulating organisms,DPAO)占聚磷菌(polyphosphate-accumulating organisms,PAO)的比例为81.44%,表明在长期的厌氧/缺氧运行条件下可以富集到以硝酸盐为电子受体的反硝化聚磷菌,同时还存在着仅以氧气为电子受体的聚磷菌;通过pH值和氧化还原电位(oxidation reduction potential,ORP)的实时监测可以快速地了解污水生物处理系统中各类反应的进程,对调控工艺参数有着重要的意义。综上所述,为保证污水生物处理工艺的正常稳定运行,将微生物分析与常规的化学参数分析结合起来考察将是未来发展的必然趋势。     

产荧光假单胞菌分离鉴定及其质粒DNA复制区的克隆

从国内多个地区的65份土样中分离纯化了产荧光假单胞菌株146株,其中菌株DSC3和DKXC1含有质粒,细胞形态,菌落特征的观察和检测证明DKXC1菌株为恶臭假单胞菌（Pseudomonas putida),以其为出发菌株,克隆了该菌质粒DNA复制子1.7kb片段,序列分析结果表明该DNA片段由3个ORF组成,分别编码311、130、115个氨基酸;它与已知的Pf-E.coli穿梭质粒pUCP19的复制区同源性只有49%,该复制子DNA序列已在GenBank登记,登记号为AF273219,这是国内分离克隆的第一个Pseudomonas质粒DNA复制子,以此复制子构建了4.7kb的E.coli-Pf穿梭载体pYQSPE,该质粒在荧光假单胞菌P303中能够稳定复制和遗传。     

少动鞘氨醇单胞菌ZX4儿茶酚2,3-双加氧酶基因的克隆与序列分析

利用PCR扩增分离得到了少动鞘氨醇单胞菌(Sphingomonas paucimobotis)ZX4菌株的儿茶酚2，3-双加氧酶基因(c230-ZX4)。核苷酸序列测定与分析表明，该基因片段全长924bp且具有一个完整的阅读框，编码306个氨基酸残基。序列分析结果表明，该基因与已报道的来自菌株Sph.yanoitcuyae B1和Sphingomonas sp．KMG425的xylE基因(编码产物均为C23O)具有较高的核酸序列同源性，分别为94．37％和94．05％；与研究较多的Pseudomonas putida的TOL型质粒pWWO上的xylE基因同源性仅为57．79％。与其同源性较高的xylE基因编码的多肽氨基酸序列比较发现，c23O-ZX4编码产物的第86、92、113、125、126、182、185、186、216及218位的氨基酸残基均有所不同。     

草莓多聚半乳糖醛酸酶抑制蛋白基因（PGIP）的克隆及组织特异性表达

以草莓(Fragaria×ananassa)叶片为试材,采用PCR技术,克隆到1条多聚半乳糖醛酸酶抑制蛋白基因,命名为FaPGIP.基因组DNA片段包含一个长为168 bp的内含子;cDNA编码区全长999 bp,编码332个氨基酸残基.预测该蛋白质分子量为37.1 kD,等电点为7.67,N端24个氨基酸残基构成的疏水区域为信号肽,具有5个潜在的N-糖基化位点.序列同源比对结果表明,所克隆的FaPGIP基因与GenBank中登录的蔷薇科及其它物种PGIP基因具有较高的同源性.采用SyPred3D软件预测了该基因所编码的蛋白质的三维结构,表明FaPGIP蛋白质的三维模型类似马蹄的结构,含12段α-螺旋和21段β-折叠,中心LRR结构域由10个串联的LRRs基序组成.半定量RT-PCR分析显示,FaPGIP在草莓根、茎、叶、花和果五种组织中均有表达,其表达量为果>叶>花>根>茎,相对表达量分别为0.246 0、0.166 4、1.371 2、0.872 1和3.415 4.     

水稻秸秆和玉米秸秆在好气和厌氧条件下的腐解规律

采用室内模拟培养的方法,研究了水稻秸秆和玉米秸秆在好气和厌氧条件下的腐解规律。结果表明:在0~3个月的培养时间内,水稻秸秆和玉米秸秆腐解较快,腐解率达55%以上。在好气培养条件下,水稻秸秆和玉米秸秆质量减少50%所需要的时间(t_(1/2))分别为59.2 d和52.9 d,而在厌氧培养条件下的t_(1/2)分别为72.6 d和79.9 d。水稻秸秆和玉米秸秆在好气培养条件下的碳释放速率常数k(0.61~0.6月~(-1))高于其在厌氧培养条件下的碳释放速率常数k(0.55~0.57月~(-1))。水稻秸秆和玉米秸秆在好气培养条件下的氮释放速率常数k(0.25~2.36月~(-1))也高于其在厌氧培养条件下的氮释放速率常数k(0.16~2.32月~(-1))。水稻秸秆和玉米秸秆中纤维素、半纤维素和木质素含量在好气培养条件下的减少速率高于其在厌氧培养条件下的减少速率。综上所述,好气培养条件有利于作物秸秆降解和营养物质的释放。     

溶解氧等环境因素对Alafia河表层沉积物磷释放影响的模拟研究

在实验室条件下,采用混合均匀的Alafia河表层沉积物并以蒸馏水为上覆水进行沉积物磷释放模拟试验,研究了溶解氧、有机碳和金属铝对沉积物磷（PO4-P）释放的影响。结果表明,试验期间厌氧条件下沉积物磷释放量明显高于好氧条件下。厌氧条件下,未添加金属铝、添加有机碳源的试验组中沉积物释放平衡时水中磷浓度为5.93mg.L-1;好氧条件下,平衡时水中磷浓度为2.39mg.L-1。研究还发现,未添加金属铝的试验组,无论好氧还是厌氧条件下,未添加有机碳源时,沉积物磷释放量高于添加碳源条件;而在添加金属铝后,添加有机碳源的试验组中磷释放量略高于未添加碳源的试验组。添加金属铝能够明显抑制沉积物中磷的释放。未添加金属铝,厌氧条件下,平衡时水中磷浓度为6.91mg.L-1,好氧条件下,释放平衡时水中磷浓度为3.70mg.L-1;而添加金属铝的试验组,无论厌氧还是好氧条件下,上覆水中磷浓度均很小,接近于0mg.L-1。     

上海郊区河流沉积物N、P释放的初步研究

A laboratory study was conducted to observe the release of nitrogen and phosphorus from the sedimentsunder both anaerobic and aerobic conditions. The samples used were five creek sediments and a fish-pondsediment (as a comparison) obtained from suburban Shanghai. High loads of nitrogen and phosphoruswere found in the creek sediments. Total nitrogen of the sediments ranged from 1.17 to 5.95 g kg^-1; totalphosphorus from 608.63 to 2 033.95 mg kg^-1. Making up more than 90% of the total nitrogen, organicnitrogen was the dominant nitrogen fraction in the sediments; where as inorganic phosphorus was the dominant phosphorus fraction, which made up more than 85 percent of the total phosphorus. Cabound phosphorus fraction dominated inorganic phosphorus, which occupied more than 50% of the total. A large amount of ammonium was released from the sediments under both aerobic and anaerobic conditions, and the anaerobic releases were slightly greater than the aerobic. In addition, ammonium contents in the aerobic waters decreased sharply after reaching the peaks because of strong nitrification, compared with the relatively maintained ammonium peaks in the anaerobic waters. Anaerobic phosphate releases were much greater than the aerobic and the released ortho-phosphate was mainly from Fe-bound phosphorus. Ammonium and ortho-phosphate releases from the sample of the dredged creek were the lowest, showing that creek dredging could effectively remove contaminants from the surface of sediments and weaken the release potentials of nitrogen and phosphorus. Ammonium and ortho-phosphate releases from the fish-pond sediment were greater than those from the creek sediments though its total nitrogen and phosphorus were not the highest, which was probably due to the larger amount of biologically degraded organic matter in the fish-pond sediment.     

五氯酚在长期定位施肥土壤中的残留动态

研究好氧和厌氧条件下五氯酚 (PCP) 在长期不施肥 (CK),施无机肥尿素 (N),施有机肥 (OM)和无机有机肥配施 (N+OM) 4 种处理土壤中的残留动态.结果表明,无论在好氧或厌氧条件下,PCP 在土壤中的消解均遵循一级动力学方程.在好氧条件下,PCP 在 CK、N、OM 和 N+OM 4 种处理土壤中半衰期分别为 26.9、27.7、21.5、20.6 天,在厌氧条件下,PCP 的半衰期分别为 30.8、32.2、27.1、25.9 天.表明无论在好氧或厌氧条件下,长期单施有机肥或无机有机肥配施显著加速 PCP 在土壤中的消解,原因可能是长期单施有机肥或无机有机肥配施能显著提高土壤有机质含量和微生物活性,加速 PCP 在土壤中的消解.好氧条件下 PCP 的消解速率显著高于厌氧条件.     

一株具有多途径氮代谢功能的荧光假单胞菌

为研究全自养脱氮过程，采用斯凯尔曼亚硝化单胞杆菌培养基，从南京某城市污水处理厂序批式处理系统（SBR）中分离得到一株具有多途径氮代谢功能的荧光假单胞菌Pseudomonas fluorescens dN13。该菌株在有氧条件下，无有机碳源存在，能进行氧化NH3-N产能代谢生长；有有机碳源存在且NO2^- -N和NO3^- -N的浓度较高时，它能将NO2^- -N和NO3^- -N还原为NH3-N。在厌氧条件下，只有乙酸钠作碳源时，该菌株仍能进行硝酸亚硝酸还原（反硝化）作用。在这个具有多途径氮代谢功能的菌株细胞内，硝化反硝化过程耦合成为可能。     

Application of low‐phosphorus‐containing legume residues reduces extractable phosphorus in a tropical Ultisol

Application of legume green manure (GM) is suggested to be effective in increasing the availability of native soil phosphorus (P) and the dissolution and utilization of phosphate rock (PR)‐P by food crops. Experiments were conducted to study the dynamics of extractable P (P extracted by Bray‐1‐extracting solution) of an Ultisol amended with or without GM residues of contrasting P concentrations in the absence of growing plants. In two separate experiments, GM residues of Aschynomene afraspera (a flood‐tolerant legume) and of Crotalaria micans (upland) with varying P concentrations were added to an acidic soil amended with PR‐P or triple superphosphate (TSP) in plastic bottles. Soil moisture was brought to field capacity of the soil in the upland experiment and saturated with distilled water in the lowland setup. This was done to simulate aerobic upland and anaerobic lowland soil conditions in the relevant plastic bottles. Only P concentration of the residues added varied, while lignin and C : N ratios were similar. A temperature of 25°C was maintained throughout the experiment. Changes in soil extractable Bray‐1‐P were measured at the end of the incubation period (60 or 80 d). In the aerobic soils, extractable P in the combined PR+GM or TSP+GM treatments was significantly lower than in the PR‐ or TSP‐ treated soils. The amendment with GM residues alone significantly increased Bray‐1‐P over the unamended control in the case of the inorganic P‐fertilized GM residues. The trend in extractable P was similar in the soils incubated under anaerobic conditions. However, in the case of PR, concentrations of P extracted by Bray‐1 solution did not significantly change in the presence or absence of GM. The results suggest that the incorporation of GM residues with low P concentration does not lead to a net P release in upland or lowland soils. These results have implications for nutrient cycling in farming systems in W Africa as most of the soils are poor and very low in available P.     

沉积物中核酸态有机磷及其矿化过程研究

水体沉积物界面中磷的释放机制非常复杂,生物作用导致的pH值和氧化还原条件变化对湖泊沉积物矿化能力有显著影响。普遍认为沉积物中有机磷的矿化过程是对水体富营养化具有重要贡献的环节之一,而核酸态有机磷可从一定程度上表征微生物水平,反应微生物对矿化作用的影响。据此采用DNA提取的方法对沉积物中核酸态有机磷的含量进行测定,并模拟沉积物中核酸在不同pH和溶解氧条件下的矿化过程。结果表明,DNA提取方法可以快速、准确提取沉积物中核酸磷;沉积物中核酸磷含量为0．43～0．61μg·g^-1,占有机磷总量的0．27％～0．37％,精确度为1．58％～3．63％。模拟实验结果表明,在两种情况下上覆水中总磷浓度均呈波动式上升,在pH接近7时沉积物中核酸磷较低,有氧条件下核酸磷呈下降趋势。     

转柽柳金属硫蛋白基因（MT1）烟草的获得及对重金属镉的抗性分析

将柽柳（Tamarix. sp）金属硫蛋白基因（MT1）插入到植物表达载体pBI121，利用农杆菌（Agrobactrium tumefaciens）介导法将MT1导入烟草基因组。对转基因T1植株的卡那霉素抗性分析表明，多数转基因植株后代表现为3:1的分离比例，只有少数转基因植株的抗性分离表现为15:1或不符合孟德尔遗传的分离比例。说明整合到烟草基因组的外源基因多为单拷贝基因，也有少数为多拷贝基因。对具有卡那霉素抗性的转基因植株进行PCR-Southern检测和Northern杂交分析表明，外源MT1基因已整合到烟草基因组，并且得到了正确表达。转基因植株对重金属镉的抗性比对照显著提高，表现为转基因植株的株高和鲜重均明显优于非转基因株系。