【作者】 肖潇；

【导师】 罗胜联； 曾光明；

【作者基本信息】 湖南大学 ， 环境工程， 2011， 博士

【摘要】 随着人类活动,尤其是工业生产的发展,重金属污染变得日益严重。重金属是一类典型的环境污染物,具有致癌、致畸等危害,因而对人类健康和自然环境造成了极大威胁。近年来,利用物理化学方法治理重金属污染的的方法取得了很大发展,主要有淋洗法、化学沉淀法、电化学法、氧化还原法、离子交换法、膜技术法、反向渗透法、蒸发治理等方法。但这些方法治理费用高、效率低下、需要大量劳动力、且处理过程缺乏选择性。生物修复技术是指利用动植物和微生物等生物的某些特性来改良重金属污染的措施,相比于物理化学法,其具有有明显的安全性和经济性的优势,因此成为重金属污染修复的潜在技术之一。然而,现阶段制约生物修复这一技术进行实际应用的关键在于如何高效、快速地从自然界中筛选得到具有潜在修复作用的重金属抗性菌株。近期,关于内生菌,特别是重金属超累积植物内生菌与其宿主植物协同处理重金属污染的研究方面取得了明显进展。内生菌是指寄居在植物组织内而不对宿主植物产生伤害的一类微生物,其与宿主植物可能存在共生、互生等多种作用关系。大部分内生菌是通过植物的根围或叶部进入植物体内,部分可能一直存在于植物种子体内。超累积植物是指能在重金属污染土壤中生长良好并能累积大量重金属在其体内的一类特殊植物。重金属超累积植物的另一个功能是作为“过滤器”,自土壤微生物中筛选有潜在修复作用的重金属抗性菌株,让其定殖于体内以帮助其对抗重金属毒性。基于这一理论,本研究首次提出了快速、高效地从重金属超累积植物中筛选同时具有优良环境适应性和重金属修复能力的菌株用于实际重金属污染的生物修复。首先,本论文在传统植物内生菌分离方法的基础上建立了一种高效、快速的植物内生菌分离方法。与传统方法相比本方法主要应用表面活性剂十二烷基磺酸钠(SDS)和植物纤维素酶作为植物内生菌的提取增溶剂。应用传统方法和本方法从龙葵、齿果草、蚂蝗七、水鸡仔4种植物体内分别提取分离植物内生菌,结果表明SDS和植物纤维素酶能通过水解植物纤维组织有效地提高植物内生菌分离效果。随后,利用该方法从镉超累积植物龙葵体内分离获得了97株内生菌,其中根内生菌34株,茎内生菌36株,叶内生菌27株。除从茎中分离到的唯一一株内生菌是真菌以外,其他所有内生菌都是细菌。通过检测所有龙葵植物体内重金属镉含量和龙葵内生菌对重金属抗性发现:不同部位分离得到的内生菌对重金属镉的抗性程度不一,从镉含量较高部位分离得到的内生菌其相应的重金属抗性要高于从镉含量相对较低部位分离的内生菌的重金属抗性。这表明不同种类的内生菌有可能寄居于植物的不同部位。内生真菌LSE10是从镉超累积植物龙葵的茎中分离得到的唯一真菌。分子生物学鉴定表明它属于Microsphaeropsis sp.。在体外培养条件下,LSE10的生物量是同等条件下培养的非内生真菌Rhizopus cohnii.的2倍以上。利用LSE10作为生物吸附剂直接吸附废水中的重金属镉,结果表明其理论最大吸附能力高达247.5 mg/g (2.2 mmol/g),高于绝大多数已经报道的生物吸附剂或者其他类型吸附剂。红外光谱分析发现LSE10表面存在的羧基、氨基、巯基、羟基等化学基团在镉的生物吸附中起关键作用。具有多种优良重金属抗性的内生细菌EB L14是从镉超累积植物龙葵叶部分离得到的内生菌。生理生化鉴定以及16S rDNA的鉴定都表明它是杆菌属(Bacillus sp.)细菌。在相对较低二价重金属离子存在条件下EB L14的生物量反而高于无重金属存在条件下的生物量,即:表现出毒物兴奋效应。研究发现,该现象是ATP酶活性异常增高所带来的副作用。EB L14在二价重金属离子存在条件下通过提高ATP酶的活性向胞外排出有毒的重金属离子,而能量利用的异常增多导致了其生物量的过度增长。在24 h培养时间内,EB L14能特异性地从含浓度为10 mg/L不同重金属的培养基中分别去除75.78%的镉,80.48%的铅,以及21.25%的铜。在相同条件下EB L14对重金属铬几乎没有任何吸收和去除作用。对EB L14去除重金属机理研究表明:能够通过抑制其ATP酶的活力来提高它的重金属修复效率。EB L14优良的环境适应性和其高效的重金属去除能力,表明它在低浓度重金属生物修复领域具有较好的应用前景。在工业代谢抑制剂N,N′-二环己基碳二亚胺(DCC)或2,4-二硝基酚(DNP)存在的条件下,内生菌EB L14对镉的生物修复效果研究表明:虽然,在DCC或DNP存在的条件下,EB L14的生物量受到很大的抑制。但是其对重金属镉的去除率却分别从73.6%升高至93.7%和80.8%。在培养24小时后,通过分别分析其镉的总吸收去除浓度和细胞内镉浓度,发现DCC和DNP都是通过抑制EB L14的向胞外运输镉离子从而有效地增强了镉去除率。EB L14的这一特性强有力地证明在工业代谢抑制剂存在的情况下,内生菌EB L14具有实践应用于镉的生物修复的优势。本论文是首次尝试利用重金属超累积植物内生菌这一不但具有良好环境适应能力而且具有多种重金属去除机制的微生物直接用于重金属生物修复的研究。本研究有助于生物修复这一更安全、更经济的重金属污染处理方法能够走出实验室最终应用于实际重金属污染处理。更多还原

【Abstract】 Heavy metals pollution by human activities, especially industrial activities, is posing significant threats to human health and the environment due to their high occurrence as a contaminant, low solubility in biota, and the classification of several heavy metals as carcinogenic and mutagenic.Many physicochemical strategies, such as filtration, chemical precipitation, electrochemical treatment, oxidation/reduction, ion exchange, membrane technology, reverse osmosis, and evaporation recovery, have been developed to remove heavy metals. However, most strategies appear to be expensive, inefficient, and labor-intensive, or the treatment process lacks selectivity. Bioremediation which involves the use of potential microbes to remove heavy metals has been considered to be a safe and economic alternative to physicochemical strategies. The crucial constraint of this technology is to obtain promising and useful metal-resistant strains from enormous microbes on earth.Recently, the benefits of endophytes, especially hyperaccumulator endophytes, alone and/or combining with plants have been successfully tried for toxic heavy metal removal from contaminated water and soil. Endophytes refer to the microbes which inhabit in the interior of plant tissues and form a range of different relationships including symbiotic, mutualistic, commensalistic and trophobiotic without causing any harm to the host. Most endophytes are originally from the rhizosphere or phyllosphere, while some may get entrance through the seed. Hyperaccumulator, a plant species that lives in the heavy metals contaminated soil can accumulate exceptionally with high quantities of certain heavy metal. It could also be functioned as‘filters’while selecting those promising and useful metal-resistant strains from soil microbes. Based on this theory, an novel technology for obtaining hose promising and useful metal-resistant strains from heavy metal hyperaccumulator endophytes (HMHEs) was developed and operated.In this study, a highly effective new method for isolation of endophytes was established, firstly. Surfactant, SDS and cellulase were applied for extraction endophytes from from their plant hosts. The endopytes of four tested plants (Solanum Nigrum L., alomonia Lour, Chirita fimbrisepala, Schizocapsa plantaginea) were isolated via traditional methods and new methods, respectively. The results indicated that surfactant and cellulase could effectively facilitate the isolation of endophytes by hydrolyzing the cellulose of the plants hosts.Several cadmium hyperaccumulator (Solanum Nigrum L.) plants were collected from the sewage discharge canal bank of Zhuzhou Smeltery (27°52′N, 113°05′E). By using the established isolation method, a total number of 97 endophytes were isolated from Solanum Nigrum L. 34, 36 and 27 endophytes were isolated from the roots, stems and leaves of the plants, respectively. Most of they were bacterial endopytes, only one fungus was isolated from the stem of the plants. The heavy metal resistances (minimal inhibitory concentration, MIC) of all the endophytes were tested. The endophytes from shoot and root demonstrated different degrees of cadmium resistance. These results suggest that different microbial communities may inhabit in the different compartments of the plant.Endophytic Fungus (EF) LSE10 was isolated from the cadmium hyperaccumulator Solanum Nigrum L. It was identified as Microsphaeropsis sp. When cultured in vitro, the biomass yield of this EF was more than twice that of None-endophytic fungus (NEF) Rhizopus cohnii. Subsequently, it was used as a biosorbent for biosorption of cadmium from the aqueous solution. The results showed that the maximum biosorption capacity was 247.5 mg/g (2.2 mmol/g) which was much higher than those of other adsorbents, including biosorbents and activated carbon. Carboxyl, amino, sulphonate and hydroxyl groups on EF LSE10 surface were responsible for the biosorption of cadmium.Heavy metal bioremediation by a multi-metal resistant endophytic bacteria L14 (EB L14) isolated from the cadmium hyperaccumulator Solanum Nigrum L. was characterized for its potential application in metal treatment. 16S rDNA analysis revealed that this endophyte belonged to Bacillus sp. The hormesis of EB L14 were observed in presence of divalent heavy metals (Cu, Cd and Pb) at a relatively lower concentration (10 mg/L). Such hormesis was the side effect of abnormal activities increases of ATPase which was planned to provide energy to help EB L14 reduce the toxicity of heavy metals by exporting the cations. Within 24 h incubation, EB L14 could specifically uptake 75.78%, 80.48%, 21.25% of cadmium, lead and copper at the initial concentration of 10 mg/L. However, nearly no chromium uptake was observed. The mechanism study indicated that its remediation efficiencies may be greatly promoted through inhibiting the activities of ATPase. The excellent adaptation abilities and promising remediation efficiencies strongly indicated the superiority of this endophyte in low concentrations heavy metal bioremediation, which could be useful for developing efficient metal removal system.Bioremediations of cadmium by endophytic bacterium (EB) L14 (Bacillus sp.) in the presence of industrially used metabolic inhibitors (DCC or DNP) were investigated. In the presence of DCC or DNP, the biomass population of EB L14 was greatly inhibited. However, the cadmium removal of EB L14 increased from 73.6% (in the absence of DCC or DNP) to 93.7% and 80.8%, respectively. The analysis of total and intracellular cadmium concentrations during 24 h of incubation indicated that this enhanced cadmium removal was the inhibition effect of DCC or DNP on the cations export resistance system of EB L14. This unique property strongly indicated the superiority of this endophyte for practical application in cadmium bioremediation in the presence of industrially used metabolic inhibitors.This dissertation was the first attempt to directly use heavy metal hyperaccumulator endophytes (HMHEs) which not only possess the excellent adaptability but also have been equipped with various metal-sequestration systems for heavy metal bioremediation. This study offers the ability to fully achieve the application of bioremediation in practical heavy metal decontamination.更多还原