阿特拉津降解菌ADH-2的分离、鉴定及其特性研究

从长期施用阿特拉津的玉米地中采集土样,通过富集培养的方法分离出一株能以阿特拉津为唯一碳、氮源生长的细菌ADH-2,结合生理生化特陛及16SrRNA基因的相似性分析将其初步鉴定为节杆菌属（Arthrobacter sp．）。该菌在10h内对100mg·L-1阿特拉津的降解率为99．9％。外加氮源能促进菌株的生长,但对阿特拉津的降解有轻微的抑制作用。外加蔗糖和葡萄糖能显著促进菌株的生长,但对阿特拉津的降解表现出显著的抑制。而淀粉既能促进菌株的生长又能促进阿特拉津的降解。对其降解基因的初步研究显示,该菌含有trzN、atzB和atzC3个阿特拉津降解相关基因。通过与本实验室另外两株阿特拉津降解菌比较,菌株ADH-2具有更好的应用潜力。     

阿特拉津降解菌FM326（Arthrobacter sp.）的分离筛选、鉴定和生物学特性

采集除草剂阿特拉津污染的土壤,通过直接涂布法和富集驯化培养分离法,分别获得6株和5株能够降解阿特拉津的细菌。通过降解效率和降解动态试验,筛选到1株高效降解阿特拉津的菌株FM326,该菌株能以阿特拉津为唯一的碳源和氮源生长,培养96h后对1000mg·L-1阿特拉津降解效率达到97%。通过生理生化鉴定和16SrDNA序列分析,菌株FM326鉴定为节杆菌属（Arthrobacter sp.）细菌。该菌株表现出最适生长温度30～35℃,最适生长pH值5～9,好氧生长的生长特性。     

阿特拉津降解细菌的筛选和鉴定

从营口农药厂排污口、药厂周围受污染土壤及未受污染农田分别采集活性污泥和土样,共富集分离到以阿特拉津作为唯一氮源生长的28个菌株。对所分离到的菌株进行降解能力的测定,筛选到降解能力相对较高的2个菌株,其降解率分别为62.7%、58.3%,分别编号为AT1、AT3;对AT1、AT3菌株进行初步鉴定,分别为芽孢杆菌(Bacillus sp.)、假单胞菌(Pseudom onas sp.)。     

污染土壤中原位阿特拉津降解菌的分离和鉴定

为克服传统富集培养分离降解菌的局限性,直接将长期受阿特拉津污染的土壤稀释后,涂布于加有土壤浸出液和阿特拉津农药的平板,分别从两个采自不同地区的污染土壤中各分离了一株高效广谱降解菌AG1和ADG1：它们能以阿特拉津为唯一碳源、氮源和能源生长,能分别在44h和48 h内降解1 000mg L^-1的阿特拉津,降解率100%;它们还能以扑草净、西玛津等三嗪类除草剂为唯一氮源生长.16S rDNA核苷酸序列分析结果表明菌株AG1与ADG1都与节杆菌属（Arthrobacter）的细菌有高度同源性,结合两株菌的形态特征及生理生化特征,将它们鉴定为Arthrobacter spp..PCR扩增两株菌的降解基因,结果表明它们的降解基因都是trzN和atzBC的组合,这是国内首次报道具有该基因类型的阿特拉津降解菌.     

阿特拉津降解菌LY-2的分离鉴定及其对污染土壤的修复

除草剂阿特拉津(2-氯-4-乙胺基-6-异丙胺基-1,3,5-三嗪,2-chloro-4-ethylamino-6-isopropylamino-1,3,5-triazine,Atrazine)在很多国家普遍使用,对自然环境和人类健康造成威胁。本研究利用富集法,从黑龙江省哈尔滨市巴彦县施用多年阿特拉津的玉米地表层土中,分离出高效降解阿特拉津的菌株LY-2。LY-2以阿特拉津为唯一氮源,初步鉴定属于肠杆菌属(Enterobacter sp.)。PCR检测结果表明,LY-2含有阿特拉津降解相关的基因—阿特拉津氯水解酶基因(atrazine chlorohydrolase gene,atzA)、羟基阿特拉津脱乙胺基水解酶基因(hydroxyatrazine N-ethylaminohydrolase gene,atzB)和N-异丙基氰尿酰氨异丙氨基水解酶基因(N-isopropylammelide isopropylaminohydrolase gene,atzC)。LY-2在48 h内对100 mg/L阿特拉津降解率为98.7%;适宜温度为25~35℃,适宜酸碱度为p H 6~9;外加氮源没有降低该菌株对阿特拉津的降解率。土壤修复实验结果显示,培养7 d后,LY-2菌株对阿特拉津污染土壤(100 mg/kg)的降解率高达86.7%,培养14 d后,降解率高达90.1%。LY-2菌株在相对较短的时间内表现出良好的修复效果,在修复阿特拉津污染的土壤方面具有潜在的应用价值。     

粘土矿物固定化微生物对土壤中阿特拉津的降解研究

以粘土矿物为载体,采用吸附挂膜法对已筛选的阿特拉津降解菌株进行固定化,并应用固定化微生物降解土壤中的阿特拉津。结果表明,该菌株在粘土矿物上生长良好,根据菌种生理生化特性、环境扫描电镜图片以及16S rDNA基因的相似性分析初步鉴定该菌株为Ochrobactrum sp.。接种降解菌能明显加快阿特拉津在土壤中的降解速率,粘土矿物固定化微生物的降解效果要明显优于游离菌,粘土矿物粒径越小,固定化微生物的降解效果越好,纳米粘土矿物固定化微生物的降解效果要好于原粘土矿物。用一级动力学方程描述阿特拉津在土壤中的降解过程,不同土壤中阿特拉津的降解速率不同。阿特拉津在红壤、砂姜黑土、黄褐土中的降解半衰期（t1/2）分别为36.9、49.1、55.0 d,投加纳米蒙脱石固定化降解菌后的半衰期则分别为16.3、25.3、21.7 d。     

4株苯磺隆降解菌的分离鉴定及其生长特性

从长期受农药苯磺隆污染的土壤中通过采用富集培养分离技术得到4株以苯磺隆为唯一碳源生长的细菌,分别将其命名为B1、B2、B3和B4。通过观察这4种菌株的形态学特征,研究其生理生化特性以及分析其16S rDNA序列,初步鉴定菌株B1为铜绿假单胞菌(Pseudomonas aeruginosa),B2为戴尔福特菌(Delftia sp.),B3为微杆菌(Microbacterium sp.),B4为产碱杆菌(Alcaligenes sp.)。并通过研究温度、初始pH值、接种量、苯磺隆初始浓度、培养基体积、氮源、碳源、Mg2+浓度等因素对4种菌株生长情况的影响,确定了菌株的最佳生长条件。结果显示,B1菌株的最适温度为35℃,其他3株菌株均为30℃。菌株B3最适pH为8.0,其余3株菌株均为pH7.0。B1和B3菌株最适接种量为15%,B2和B4最适接种量为10%。菌株B3最适苯磺隆初始浓度为100mg·L-1,其余菌株最适苯磺隆初始浓度均为200mg·L-1。4株菌株最适培养基体积均为75mL,最适氮源均为硝酸铵,最适碳源均为葡萄糖。B2菌株最适Mg2+浓度为100mg·L-1,其余3株菌株均为200mg·L-1。B1和B4菌株最适NaCl浓度为20g·L-1,B2菌株NaCl浓度为5~30g·L-1,B3菌株最适NaCl浓度为50g·L-1。该结果为利用微生物对农药苯磺隆污染的土壤进行原位生物修复提供理论依据。     

一株红霉素降解菌红圆酵母的选育及其降解特性

选取长期堆放红霉素药渣的土壤,经驯化富集后筛选到1株能高效降解红霉素的菌株,根据表型特征、生理生化特性及18S rDNA序列同源性分析,鉴定为红酵母属的粘性红圆酵母（Rhodotorula mucilaginosa）。该菌的18S rDNA序列片段长度为1 562 bp,在GenBank登录号为EU294522。该菌可以利用红霉素作为碳源生长,适用碳源与氮源分别为蔗糖和NH4Cl。该菌降解红霉素的最适温度为30 ℃,最适pH 5.0～5.5,最适接菌量6%,降解率与通气量成正相关。在最适反应条件下培养48 h, 该菌株对红霉素的降解率可达100%。     

竹炭固定化微生物对土壤中阿特拉津的降解研究

采用环境友好材料竹炭为主要载体,壳聚糖和海藻酸钠为辅助载体,固定从污泥中分离出的阿特拉津降解菌株,研究不同固定材料对降解菌生长的影响,以及固定化微生物对土壤中阿特拉津的降解效果.结果表明,竹炭对阿特拉津降解菌具有较强的吸附固定能力,且竹炭粒径越小,固定化效果越好.利用壳聚糖和海藻酸钠交联并加固阿特拉津降解菌,增大了固定化空间,显著增加了降解菌的生物量,并提高了阿特拉津的降解效率.1％壳聚糖+5％海藻酸钠+竹炭+降解菌颗粒对阿特拉津降解菌的固定化效果最佳,施用该微生物固定化颗粒28天后,砂姜黑土及红壤中阿特拉津残留率分别为48.07％和47.23％.     

结晶纤维素降解细菌的筛选、分离与鉴定

采用以结晶纤维素（Avicel）为唯一碳源的平板活性筛选法,从60份森林腐殖土、腐术样品中分离得到2株在pH5．0、37℃条件下高效降解结晶纤维素的细菌菌株GXN152和GXN153。以菌株的总DNA基因为模板,用细菌的16S rRNA基因的通用引物扩增得到菌株GXN152和GXN153的16S rRNA基因序列。测序分析表明菌株GXN152的16S rRNA基因序列和洋葱假单胞菌（Burkholderia cepacia）菌株CNR22的16S rRNA基因序列的同源性最高,具有98％的同源性,生理生化特性检测表明菌株GXN152具有洋葱假单胞菌的鉴别特征,将结晶纤维素降解菌GXN152鉴定为洋葱假单胞菌。菌株GXN153的16S rRNA基因序列和类芽孢杆菌（Paenibacillus favisporus）菌株GMP01的16S rRNA基因序列的同源性最高,具有99％的同源性,生理生化特性检测表明菌株GXN153具有类芽孢杆菌的鉴别特征,将纤维素降解菌GXN153鉴定为类芽孢杆菌。     

A comparison of three atrazine-degrading bacteria for soil bioremediation

The ability of three atrazine-degrading bacteria, Pseudomonas sp. strain ADP, a Pseudaminobacter sp., and a Nocardioides sp., to degrade and mineralize this herbicide in a loam soil was evaluated in laboratory microcosms. These bacteria all hydrolytically dechlorinate atrazine, and degrade atrazine in pure culture with comparable specific activities. The Pseudaminobacter and Nocardioides can utilize atrazine as sole carbon and nitrogen source, whereas the Pseudomonas can utilize the compound only as a nitrogen source. The Pseudomonas and Pseudaminobacter mineralize the compound; the end product of atrazine metabolism by the Nocardioides is N-ethylammelide. At inoculum densities of 10⁵ cells/g soil, only the Pseudaminobacter and Nocardioides accelerated atrazine dissipation. The Pseudaminobacter mineralized atrazine rapidly and without a lag, whereas atrazine was mineralized in the Nocardioides-inoculated soil but only after a lag of several days. The Pseudaminobacter remained viable longer than did the Pseudomonas in soil. PCR analysis of recovered bacteria indicated that the genes atzA (atrazine chlorohydrolase) and atzB (hydroxyatrazine ethylaminohydrolase) were less stable in the Pseudaminobacter than the Pseudomonas. In summary, this study has revealed important differences in the ability of atrazine-hydrolyzing bacteria to degrade this compound in soil, and suggests that the ability to utilize atrazine as a carbon source is important to establish "enhanced degradation" by ecologically meaningful inoculum densities.     

一株新的多菌灵高效降解茵的筛选与降解特性分析

从长期施用多菌灵农药的土壤中,通过富集筛选,获得1株新的多菌灵高效降解菌株。通过生理生化实验和16SrDNA序列同源性分析鉴定该菌株,应用高效液相色谱法对纯培养条件下菌株的降解特性和粗酶提取液的降解性能进行了分析。结果表明,筛选所获得的菌株与Raoultella菌属的亲缘关系最近,将其命名为Raoultellasp．MBC,该菌株能在以多菌灵为唯-碳源的无机盐培养基中生长;25℃、pH7．0、200r·min。的最适生长条件下避光振荡培养72h,多菌灵的降解率达到100％;在最适培养条件下外加氮源和碳源在培养后期均可以提高多菌灵的降解率,外加氮源对多菌灵的降解效果优于外加碳源;该菌体的粗酶提取液具有降解多菌灵活性,且多菌灵降解酶为诱导酶。研究结果为多菌灵污染土壤的生物修复和酶修复提供了材料和理论依据。     

Influence of microbial inoculation (Pseudomonas sp. strain ADP), the enzyme atrazine chlorohydrolase,and vegetation on the degradation of atrazine and metolachlor in soil

The concentrations of atrazine in the freshly added soils and the soils that had been incubated for 50 days significantly decreased 1 day after the addition of the enzyme atrazine chlorohydrolase or the soil bacterium Pseudomonas sp. strain ADP as compared with those in the uninoculated soils. Atrazine chlorohydrolase or ADP had no effect on the degradation of metolachlor. The half-lives of atrazine in the freshly added soils and in the aged soils after the treatment with atrazine chlorohydrolase or ADP markedly decreased as compared with those in the uninoculated soils. The half-lives of metolachlor in the aged soils were much longer than those of freshly added metolachlor. The percentage atrazine degraded in the freshly treated soils was much higher than that in the aged soils. This indicates that aging significantly decreased the bioavailability of atrazine. Vegetation significantly decreased the concentration of metolachlor. However, vegetation showed no effect on the degradation of atrazine.     

Solvent extraction characterization of bioavailability of atrazine residues in soils

Characterization of pesticide bioavailability, particularly in aged soils, is of continued interest because this information is necessary for environmental risk assessment. The objective of this study was to correlate atrazine residue bioavailability in aged soils, as determined by solvent extraction methods, to atrazine mineralization by an atrazine-degrading bacterium. Webster clay loam and Zimmerman fine sand soils were treated with UL-ring-labeled [14C]atrazine and incubated for up to 8 weeks. At the end of each incubation period, soils were either not extracted, extracted with 0.01 M CaCl2, or extracted with 0.01 M CaCl2/aqueous methanol. Soils were then inoculated with the bacterium Pseudomonas sp. strain ADP, which is capable of rapidly mineralizing the atrazine ring. This allowed for the evaluation of the bioavailability of aged atrazine residues without the contribution of atrazine desorption from soil. Results of these studies indicated that the amounts of atrazine residues in aged soils extracted by 0.01 M CaCl2 and aqueous methanol were correlated to amounts of atrazine mineralized by Pseudomonas sp. strain ADP. Consequently, 0.01 M CaCl2/methanol extractable atrazine in aged soils may be used to estimate bioavailable residues, and this technique may be useful to determine the bioavailability of other compounds in soils, especially other triazine herbicides.     

灭多威降解菌mdw-1的分离和降解特性研究

Methomyl, an extremely toxic pesticide, is widely used in agriculture. A strain named mdw-1 capable of degrading methomyl rapidly was successfully isolated from activated sludge in this study. It could utilize methomyl as the sole carbon or nitrogen source. The optimal temperature and medium pH for its growth and methomyl biodegradation were 30-C and 7.0, respectively. It was identified as a Paracoccus sp. according to its morphological features, physiological and biochemical characteristics, and phylogenetic analysis based on the sequence of 16S rDNA. Gas chromatography-mass spectrometry (GC-MS) analysis showed that methomyl could be completely transformed to S-methyl-N-hydroxythioacetamidate in 10 h of incubation with the isolate mdw-1.     

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。