节杆菌AD26的分离鉴定及其与假单胞菌ADP对阿特拉津的联合降解

用富集培养法,从农药厂的工业废水中分离到高效降解除草剂阿特拉津的AD26菌株,通过16SrRNA基因序列分析,该菌株被鉴定为节杆菌（Arthrobacter sp.）。降解基因的PCR分析表明,AD26含有阿特拉津降解基因trzN和atzBC,它能以阿特拉津为唯一氮源、蔗糖或柠檬酸钠为碳源生长,将阿特拉津降解成氰尿酸,降解速度快但降解不完全。假单胞菌（Pseudomonas sp.）ADP是Wackett实验室分离的阿特拉津降解菌株,含有阿特拉津降解基因atzABCDEF,能以阿特拉津为唯一氮源、柠檬酸钠为碳源（不能以蔗糖为碳源）生长,将阿特拉津降解成NH3和CO2,降解完全但降解速度慢。在阿特拉津浓度为200mg·L^-1的无机盐培养基中进行的AD26和ADP混合培养表明,它们对阿特拉津的降解发生了互补和增强作用,两个菌株能在以阿特拉津为唯一氮源、蔗糖为碳源的培养基中生长,而且生长和降解速率都好于单个菌株,培养72h后阿特拉津去除率达到99.9%,其中76.7%的阿特拉津被降解成NH3和CO2。这表明由节杆菌AD26和假单胞菌ADP组成的混合菌株在阿特拉津废水处理和污染土壤的生物修复中有很好的应用潜力。     

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。     

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。     

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。     

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％.     

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.)。     

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。     

Arthrobacter sp. AG1菌株降解土壤中阿特拉津研究

在阿特拉津浓度为50mg/kg干土的黄棕壤、潮土和红壤接种1.5×106CFU/g干土的降解菌Arthrobacter sp. AG1,10天后土壤中的阿特拉津分别降解至1.5、6.6和10mg/kg干土。阿特拉津的降解速率受到土壤性质的影响,但AG1仍能在不满足其生长繁殖要求的pH值的土壤中有效降解酸性土壤中阿特拉津;土壤中水分含量对降解效果影响较大,>20%时降解效果较好;土壤低含水量和低pH值会导致AG1降解阿特拉津的活力下降。不同的接种量对降解效果有一定影响,但105～107CFU/g干土接种量的AG1都能有效发挥降解作用。AG1降解完土壤中的阿特拉津后,在土壤含水量分别为5%和15%的情况下能长期保持降解活性,对60天后第2次施入黄棕壤和潮土中的50mg/kg阿特拉津4天时降解效率在65%以上。     

丛枝菌根(AM)真菌对土壤中阿特拉津降解的影响

于盆栽高粱(Sorghum,龙杂一号)条件下研究了丛枝菌根(AM)真菌根内球囊霉(Glomus intraradices,GI)和摩西球囊霉(Glomus mosseae,GM)降解土壤中阿特拉津的效用。结果表明,阿特拉津(浓度为50 mg/kg)污染土壤中,供试AM真菌都能够侵染高粱根系形成菌根,而且GM比GI侵染效果好,最高侵染率可达到90.5%,显著提高了植株的生物量。接种AM真菌后土壤中阿特拉津的残留浓度显著低于不接种对照处理,并且接种GM比GI对阿特拉津的降解效果显著。接种GM处理的土壤中阿特拉津最高降解率达到了91.6%,其中菌根效应占22.6%。接种AM真菌的宿主植物根际土壤中微生物数量多于不接种处理,且GM优于GI处理,说明AM真菌能促进根际微生物的繁殖。此外,接种AM真菌后能显著增加土壤中脲酶活性,但对过氧化氢酶活性影响不显著。认为GM是一株比较理想的修复阿特拉津污染土壤的AM真菌。     

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.     

除草剂莠去津和灭草松单用和混用在土壤中的降解

The application of a mixture of bentazone （3-isopropyl-1H-2,1,3-benzothiadiazin-4（3H）-one-2,2-dioxide） and atrazine （6-chloro-N2-ethyl-N4-isopropyl-1,3,5-triazine-2,4-diamine） is a practical approach to enhance the herbicidal effect. Laboratory incubation experiments were performed to study the degradation of bentazone and atrazine applied in combination and individually in maize rhizosphere and non-rhizosphere soils. After a lag phase, the degradation of each individual herbicide in the non-autoclaved soil could be adequately described using a first-order kinetic equation. During a 30-d incubation, in the autoclaved rhizosphere soil, bentazone and atrazine did not noticeably degrade, but in the non-autoclaved soil, they rapidly degraded in both non-rhizosphere and rhizosphere soils with half-lives of 19.9 and 20.2 d for bentazone and 29.1 and 25.7 d for atrazine, respectively. The rhizosphere effect significantly enhanced the degradation of atrazine, but had no significant effect on bentazone. These results indicated that biological degradation accounted for the degradation of both herbicides in the soil. When compared with the degradation of the herbicide applied alone, the degradation rates of the herbicides applied in combination in the soils were lower and the lag phase increased. With the addition of a surfactant, Tween-20, a reduced lag phase of degradation was observed for both herbicides applied in combination. The degradation rate of bentazone accelerated, whereas that of atrazine remained nearly unchanged. Thus, when these two herbicides were used simultaneously, their persistence in the soil was generally prolonged, and the environmental contamination potential increased.     

乙草胺降解菌Y-4的分离鉴定及降解特性研究

从长期经乙草胺污染的污泥中分离到一株能以乙草胺为唯一碳源和能源生长的菌株Y-4,通过生理生化实验和16S rDNA同源性序列分析,鉴定为申氏杆菌属（Shinella sp.）。采用室内培养方法,研究了Y-4对乙草胺的降解特性。结果表明,Y-4能有效地降解浓度为5～200 mg.L-1的乙草胺,在48 h内对50 mg.L-1乙草胺的降解率达到83.3%。菌株Y-4降解乙草胺的最适pH值为8.0,最适温度为30℃,其对丙草胺和丁草胺等农药也有良好的降解效果。     

接种丛枝菌根真菌对玉米秸秆降解的影响

为研究接种丛枝菌根真菌(AMF)对玉米秸秆降解的影响,利用玉米秸秆为材料制成网袋,采用盆栽试验,以玉米(Zea mays L.)为宿主植物,分别接种Glomus intraradices和Glomus mosseae,于30,40,50,60d时收获后分析玉米秸秆降解量和C、N释放量,并运用Olson的指数模型Bt/B0=e-kt计算玉米秸秆及C、N的降解系数。数据表明,接种G.intraradices、G.mosseae显著提高了玉米秸秆降解量和降解系数,与不接种处理相比,分别高出5.21%,6.26%。C释放量、碳素降解系数也明显增加。接种处理减少了N释放量,且氮素降解系数随时间延长而下降。接种处理玉米秸秆的C、N降解系数不同直接反映了其降解速度的差异,进而影响了玉米秸秆的C/N,使秸秆更易于降解。研究结果显示出丛枝菌根真菌在生态系统氮循环中具有重要意义。     

Interactions between 14C-labelled atrazine and the soil microbial biomass in relation to herbicide degradation

A laboratory incubation experiment was set up to determine the effects of atrazine herbicide on the size and activity of the soil microbial biomass. This experiment was of a factorial design (0, 5, and 50 g g^–1 soil of non-labelled atrazine and 6.6×10³ Bq g^–1 soil of ¹⁴C-labelled atrazine) x (0, 20, and 100 g g^–1 soil of urea-N) x (pasture or arable soil with a previous history of atrazine application). Microbial biomass, measured by substrate-induced respiration and the fumigation-incubation method, basal respiration, incorporation of ¹⁴C into the microbial biomass, degradation of atrazine, and ¹⁴C remaining in soil were monitored over 81 days. The amount of microbial biomass was unaffected by atrazine although atrazine caused a significant enhancement of CO₂ release in the non-fumigated controls. Generally, the amounts of atrazine incorporated into the microbial biomass were negligible, indicating that microbial incorporation of C from atrazine is not an important mechanism of herbicide breakdown. Depending on the type of soil and the rate of atrazine application, 18–65% of atrazine was degraded by the end of the experiment. Although the pasture soil had twice the amount of microbial biomass as the arable soil, and the addition of urea approximately doubled the microbial biomass, this did not significantly enhance the degradation of atrazine. This suggests that degradation of atrazine is largely independent of the size of the microbial biomass and suggests that other factors (e.g., solubility, chemical hydrolysis) regulate atrazine breakdown. A separate experiment conducted to determine total amounts of ¹⁴C-labelled atrazine converted into CO₂ by pasture and arable soils showed that less than 25% of the added ¹⁴C-labelled atrazine was oxidised to ¹⁴CO₂ during a 15-week period. The rate of degradation was significantly greater in the arable soil at 24%, compared to 18% in the pasture soil. This indicates that soil microbes with previous exposure to atrazine can degrade the applied atrazine at a faster rate.     

How increasing availabilities of carbon and nitrogen affect atrazine behaviour in soils

 The effect of increasing amounts of glucose and mineral N on the behaviour of atrazine was studied in two soils. One had been exposed to atrazine under field conditions (adapted soil), the other had not (non-adapted soil), resulting, respectively, in an accelerated degradation of atrazine in the adapted soil and in a slow degradation of the herbicide in the non-adapted soil. The dissipation of ¹⁴C-atrazine via degradation and formation of non-extractable "bound" residues was followed during laboratory incubations in soils supplemented or not with increasing amounts of glucose and mineral N. In both soils, glucose added at rates of up to 16 g C kg^–1 soil did not modify atrazine mineralization but increased the formation of bound residues; this was probably due to the retention of atrazine by the growing microbial biomass. Atrazine dealkylation was enhanced when a large amount of glucose was added. In both soils, the addition of the largest dose of mineral N (2.5 g N kg^–1 soil) decreased atrazine mineralization. The simultaneous addition of glucose and mineral N enhanced their effects. When the largest doses of mineral N and glucose were added, atrazine mineralization stopped in both soils, and the proportion of bound residues increased. Glucose and mineral N additions influenced atrazine mineralization to a greater extent in the adapted soil than in the non-adapted one, as revealed by ANOVA, although glucose addition had a greater effect than N. The competition for space and nutrients between atrazine-degrading microorganisms and the total heterotrophic microflora probably contributed to the decrease in atrazine mineralization. Received: 9 June 1998