多环芳烃（PAHs）是土壤环境中常见的一类持久性有机污染物。土壤中PAHs可通过挥发、光解、微生物降解等方式而去除，也可通过植物吸收积累作用而进入食物链，危害生态安全和人群健康。自然界中大部分细菌是以生物膜方式存在的，植物根表细菌生物膜可以减低土壤环境中污染物对植物的毒害作用并促进宿主植物生长。从植物根表筛选具有PAHs降解功能的细菌并将其定殖在植物根表，可在根表形成PAHs降解细菌生物膜；该生物膜对土壤中PAHs有阻隔和降解作用，这不仅有望降低植物吸收积累PAHs的污染风险，还可提高根际土壤中PAHs的去除效率。 本研究从PAHs污染场地健康植物根表筛选获得了两株具有PAHs降解功能的细菌（以下简称功能细菌），鉴定了功能细菌的分类地位，并研究了其生长和降解特性；明确了不同环境条件对功能细菌降解PAHs的影响，优化了菌株降解PAHs的环境条件；分别用抗生素抗性标记和绿色荧光蛋白基因（gfp）标记对功能细菌进行标记，将其定殖在植物根表，研究了功能菌株在植物根表的定殖分布及其对植物吸收积累PAHs的影响；探讨了表面活性剂作用下根表细菌生物膜对植物吸收积累PAHs的影响。主要研究成果如下： （1）从PAHs污染区健康植物牛筋草（Eleusine indica (L.) Gaertn.）根表分离获得了两株分别具有菲和芘降解功能的细菌。经生理生化测定及16S rRNA基因序列同源性分析，确定两株菌分别为具有菲降解能力的Diaphorobacter sp. Phe15和具有芘降解能力的Mycobacterium sp. Pyr9。优化了两株功能菌株降解PAHs的环境条件。其中，菌株Phe15降解菲的最适温度为30℃，最适pH值为7.0~10.0，该条件下2 d内其对初始浓度为100 mg·L-1<上标!>菲的降解率可达到97%以上。菌株Phe15对150 mg·L-1<上标!>浓度范围内的菲都具有良好的降解效果，且随着接种量的增加，降解逐渐加快。菌株Pyr9降解芘的最适温度为30℃，最适pH值为7.0~8.0，该条件下10 d能将初始浓度为 50 mg·L-1<上标!>的芘全部降解。芘浓度范围为50~200 mg·L-1<上标!>时，芘浓度越高，Pyr9对芘的降解率越低；供试接种量范围内（5~20%），接种量越高，Pyr9对芘的降解越快。 （2）分别用抗生素抗性标记和绿色荧光蛋白基因（gfp）标记对功能细菌Phe15和Pyr9进行了标记，并将其定殖在植物根表，研究了功能菌株在植物根表的定殖分布及其对植物吸收PAHs的影响。其中，菌株Phe15对氨苄青霉素（75 mg·L-1<上标!>）和低浓度的氯霉素（25 mg·L-1<上标!>）有抗性。利用这两种抗性作为菌株Phe15的筛选标记，采用灌根和浸种两种定殖方式将菌株Phe15定殖到三叶草根表。结果表明，菌株Phe15在植物根表的定殖促进了三叶草的生长，同时降低了菲在三叶草体内的浓度和积累量。两种定殖方式均可降低三叶草体内菲含量，且灌根方式要优于浸种。同时，两种定殖方式都能够提高污染土壤中菲的去除率，且灌根方式下土壤中菲去除率更高。 用绿色荧光蛋白基因（gfp）对菌株Pyr9进行了标记，标记菌株（Pyr9-gfp）对芘的降解能力没有显著变化。扫描电子显微镜和激光扫描共聚焦显微镜照片表明，菌株Pyr9-gfp可以在三叶草根表定殖并形成细菌生物膜。采用灌根方式将Pyr9-gfp定殖在三叶草根表，定殖在根表的Pyr9-gfp可以进入植物根部组织，并在植物体内增殖，且数量随土壤中芘污染浓度的增加而增大。功能细菌Pyr9-gfp在三叶草根表的定殖可以显著提高植物茎叶和根部生物量，降低植物体内芘浓度、积累量及富集系数，同时显著提高了土壤中芘的去除率。 （3）向PAHs污染土壤中添加表面活性剂Tween 80，探究了Tween 80作用下菌株Pyr9在三叶草根表的定殖及其对植物吸收PAHs和根际PAHs去除的影响。供试Tween 80浓度范围（100~1000 mg·kg-1<上标!>）内，随着Tween 80浓度升高，三叶草体内Pyr9的数量变化不明显，植物根部和茎叶生物量先增加后减小，Tween 80添加量为250 mg·kg-1<上标!>时，植物生物量最大。相同处理条件下，灌根组植物生物量大于不接菌组，表明菌株Pyr9在植物根表的定殖促进了植物生长。100~1000 mg·kg-1<上标!> Tween 80促进了植物对芘的吸收。Tween 80作用下，菌株Pyr9在三叶草根表的定殖同样可以降低三叶草体内芘含量。 添加Tween 80增大了三叶草根际土壤中芘的去除率。添加量为0~500 mg·kg-1<上标!>时，随着Tween 80添加量增加，土壤中芘的残留量降低。同一Tween 80添加量下，灌根组（CPR）土壤中芘含量显著低于不接种菌的植物组（CP）和接菌但未种植物组（CB），例如，Tween 80添加量为100 mg·kg-1<上标!>时，CPR处理比CP和CB处理土壤中芘残留量分别低12.8%和19.2%。Tween 80添加量为500 mg·kg-1<上标!>时，CPR处理土壤中芘残留量与污染土壤对照相比降低了49.4%。

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants in contaminated soils. The effect and fate of PAHs in soil is of great environmental and human health concern because of the carcinogenic, mutagenic, and teratogenic properties of PAHs, as well as their high concentrations and frequent observation in soil. PAHs accumulate in a variety of plant species. As the basis of human and animal food webs, plants could be routes by which harmful PAHs enter human and animal populations. There are numerous bacterial cells existing in the rhizosphere soil and many of them usually form biofilms on root surfaces, thus protecting plants from harsh external environments and promoting plant growth. PAH-degrading bacteria with the ability to form biofilms on root surfaces assist in reducing the plant PAH contamination risk and enhance the PAH-removal efficiency from contaminated soil. In this work, two PAH-degrading bacterial strains, Diaphorobacter sp. Phe15 and Mycobacterium sp. Pyr9, were isolated from the root surface of healthy plants (Eleusine indica (L.) Gaertn.) grown in a PAH-polluted site. The influences of environmental conditions on PAH biodegradation and bacterial growth were investigated.Strain Phe15 was marked with antibiotic resistance, and strain Pyr9 was marked with the gfp gene. The colonization on root surface of clover and PAH-degradation performance of the two strains were elucidated with greenhouse experiments. The effects of a nonionic surfactant Tween 80 on the uptake of test PAHs by plant were evaluated. The main results are as follows. (1) Two PAH-degrading bacterial strains, Phe15 and Pyr9, were isolated from the root surface of healthy plants (Eleusine indica (L.) Gaertn.) grown in a PAH-polluted site. The phenanthrene-degrading bacterial strain Phe15 was identified as Diaphorobacter sp. and the pyrene-degrading strain Pyr9 was identified as Mycobacterium sp. based on physiological characteristics and 16S rRNA gene sequence homology analysis. Strain Phe15 can use phenanthrene as the sole carbon source, and strain Pyr9 can use pyrene as the sole carbon source. At 30℃, pH 7.0 and 150 r·min-1<上标!>, 93% of phenanthrene (initial concertration of 100 mg·L-1<上标!>) in mineral medium is degraded by strain Phe15 in 24 hours, and all the phenanthrene in medium can be degraded totally within 48 hours. The optimum temperature for bacterial growth and phenanthrene degradation by strain Phe15 is 30℃, and the optimum pH is between 7.0~10.0. With the inoculum density increases, the phenanthrene-degrading rate increases correspondingly. While for strain Pyr9, the pyrene in mineral medium (initial concertration of 50 mg·L-1<上标!>) can be degraded completely after 10 d at 30°C, pH 7.0 and 150 r·min-1<上标!>. The optimum temperature for bacterial growth and pyrene degradation by strain Pyr9 is 30℃, and the optimum pH is 7.0~8.0. When the inoculum density increases, the pyrene-degrading rate increases correspondingly. (2) Strain Phe15 and strain Pyr9 are marked with antibiotic resistance and gfp gene, respectively, and then the colonization on root surface of clover and PAHs degradation performance of the two strains were investigated with greenhouse experiments. Strain Phe15 is resistant to ampicillin (75 mg·L-1<上标!>) and chloramphenicol (25 mg·L-1<上标!>), so the two antibiotics are selected to marke strain Phe15 after colonization on root surface of clover with root irrigation or seed soaking. Strain Phe15 can promote the plant growth and reduce the phenanthrene concertration and accumulation inner plant significantly. Furthermore, after root irrigation or seed soaking, strain Phe15 can also enhance the phenanthrene removel from the rhizosphere soil. In addition, compared to seed soaking, more phenanthrene removel from soil was detected with root irrigation by strain Phe15. Strain Pyr9 is resistant to ampicillin (75 mg·L-1<上标!>) and spectinomycin (25 mg·L-1<上标!>), it is successfully marked with gfp gene by electric transformation and the positive transformant with gfp-label is named as Pyr9-gfp. The pyrene-degrading ability of Pyr9-gfp does not show any significant difference with the wild strain Pyr9. Pyr9-gfp can actively colonize on clover root surface by root irrigation and form bacterial biofilm observed by confocal laser scanning microscope (CLSM). Strain Pyr9-gfp can grow and propagate inner plant tissues, the number of Pyr9-gfp increased with the increase of pyrene concentration in contaminated soil. The biomass of plant in Pyr9-gfp-innoculated treatment was significantly improved compared to the Pyr9-gfp-free treatment. The colonization on root surface by strain Pyr9-gfp decreased the pyrene concentration, accumulation and plant concentration factor (PCF) in plant tissues, as well as the pyrene residual in rhizosphere soil. (3) Surfactant Tween 80 was added into the natural PAH-contaminated soil to improve the bio-availability of pyrene, and then the effects on pyrene uptake by clover and pyrene removel from soil after colonization on root surface by strain Pyr9 were investigated. Between the Tween 80 concentration of 100~1000 mg·kg-1<上标!>, with the concentration increased, the number of strain Pyr9 inner plant tissues doesn’t change significantly, while the number of strain Pyr9 in soil increases at first and then decreases when the concentration of Tween 80 is more than 500 mg·kg-1<上标!>. Similarly, the biomass of plants increases at first and then decreases when the concentration of Tween 80 is more than 250 mg·kg-1<上标!>. In addition, after root irrigation with strain Pyr9, the plant growth of clover improves siganificantly With the addition of Tween 80 (100~1000 mg·kg-1<上标!>), the plant uptake of pyrene is significantly promoted. For example, when 500 mg·kg-1 of Tween 80 is added, the pyrene concentration in shoot or root increases by 38% and 23%, respectively. Furthermore, root irrigation by strain Pyr9 can significantly reduce the pyrene concentration and accumulation in plant tissues. For example, under 500 mg·kg-1<上标!> of Tween 80, the concentration of pyrene in clover root is reduced by 19.1% after root irrigation. A certain amount of Tween 80 can enhance the pyrene removal from soil. In the range of 0~500 mg·kg-1<上标!>, the pyrene residual concentration in soil decreases gradually with the increase of Tween 80. In addition, the pyrene concentration in soil decreases significantly after root irrigation. For example, when the Tween 80 concentration is 100 mg·kg-1<上标!>, the pyrene concentration in soil of Pyr9-gfp-innoculated treatment decreases by 12.8% and 19.2%, respectively, compared to Pyr9-gfp-free treatment and Pyr9-gfp-soil treatment. When the added amount of Tween 80 was 500 mg·kg-1<上标!>, the final residue of pyrene in soil was 2.17 mg·kg-1 in Pyr9-gfp-innoculated treatment, reduced by 49.4% compared to the 4.28 mg·kg-1<上标!> of pyrene in the contaminated soil control.