构建全细胞生物传感器测定农田土壤中有机磷农药甲基对硫磷含量

土壤中污染物的生物有效性评估对于准确评价环境污染风险至关重要,而全细胞生物传感器是此类评估的重要工具之一。本研究旨在使用新型全细胞生物传感器建立土壤中甲基对硫磷(methyl parathion,MP)的检测方法。首先,使用筛选出的甲基对硫磷水解酶基因(methyl parathion degrading gene,mpd)和pUC19质粒骨架以及已有的特异性诱导元件pobR为材料,构建全细胞生物传感器。然后,以96孔的酶标板为载体和以5种全细胞生物传感器为指示细胞,建立了土壤提取液样品中甲基对硫磷的分析方法,并应用于实际测试和田间土壤样品中甲基对硫磷的检测。以检测性能的最佳大肠杆菌(Escherichia coli)DH5α/pMP-AmilCP为例,其检测限为6.21−6.66μg/L,线性范围为10−10000μg/L。E.coli DH5α/pMP-RFP和E.coli DH5α/pMP-AmilCP的方法用于分析土壤提取液样品中甲基对硫磷的浓度,具有较好的检测性能。这种全细胞生物传感器方法有助于快速评估土壤中甲基对硫磷的生物有效性强弱,从而有效判断有机磷农药甲基对硫磷对土壤的污染风险。     

可食用文蛤中氧化性胁迫的生物标记物与甲基毒死蜱和草甘膦的生物富集

近几十年期间,在种群和个体受干扰后,对作为早期指示剂的生物标记物的研究受到越来越多的关注。我们用对水生生态系统污染敏感的生物标记物双壳类软体动物(文蛤)来评估两种有机磷杀虫剂(甲基毒死蜱、草甘膦)的影响。文蛤是水生生态系统污染的一种敏感的指示物种。在不同时间段测定文蛤中不同组织的非酶的(谷胱苷肽)和酶的(过氧化氢酶)抗氧化剂,作为文蛤中生物标记物的反应。在实验室条件下,测定了脂质过氧化作用、蛋白羰基含量、总蛋白含量、总脂质含量以及胆碱酯酶的活性。对不同的生物标记与杀虫剂的生物体内积累的相互关系进行了研究。甲基毒死蜱在文蛤组织中具有最大的诱导氧化胁迫的潜能,导致脂质过氧化反应增加并抑制抗氧化剂。而且,鳃是对该反应最敏感的器官。文蛤是一种极好的甲基毒死蜱的积聚者,因为暴露60天后,可以测定到其组织中浓度为824.0 mg/kg w.w的甲基毒死蜱。随着在草甘膦中暴露时间的增加,与背景水平相比,组织中草甘膦的浓度增加大约8×103mg/kg w.w。可以得出这样的结论:在一种生物中测定几种生物标记物是有用的。在双壳类中,蛋白质的羰基诱导可用于双壳类中化学污染物诱导的氧化胁迫的生物指示剂。抗氧化剂的防御成分是敏感的参数,是评估污染的水生生态系统的有用的生物标记物。辅以蛤组织的化学分析,生物标记参数能够提供一种有力的监测工具。     

蚯蚓生物标记物在土壤生态风险评价中的应用

蚯蚓在土壤中行使了很多重要的生态功能,蚯蚓生物标记物常用作土壤污染风险评价研究。这篇综述的目的是探讨当前蚯蚓生物标记物研究是否可以应用到实际的土壤污染风险评价。1)讨论了蚯蚓生物标记物在土壤污染风险评价体系中的重要性,认为它是化学分析方法的有益补充,可以提供更为全面和客观的土壤污染信息;2)综述了相关研究中所使用的蚯蚓类型,土壤类型和生物标记物类型,及其它试验设计要素和最后结果的变异,认为目前蚯蚓生物标记物研究以实验室基础研究为主,筛选出了大量的生物标记物,一定程度上揭示了生物标记物的对各类典型污染物及其组合的应答机制;同时也认为,未来的蚯蚓生物标记物研究应该重点探讨将其应用到实际的土壤污染风险评价中的可行性及如何应用;3)目前不同研究之间从试验设计到结果都具有很大的变异,难以通过综合比较获得完全可靠的具有实践意义的结论和成果,因此,有必要通过建立标准化的蚯蚓生物标记物研究方法,推动生物标记物的研究工作;4)提出了蚯蚓生物标记物研究方法标准化的具体建议,推荐了蚯蚓生物标记物走向实际应用所需要解决的问题。     

土壤线虫对环境污染的指示作用

线虫作为土壤中型动物区系的一部分,在土壤生态系统中发挥着重要的作用,因其所特有的生物特性,使其广泛地应用于土壤健康状况的指示研究中．根据国内外的研究现状,分别对典型线虫在土壤污染指示与诊断中的应用和线虫群落的各种指数指标法(如成熟指数、多样性指数、相似度指数、关键种法、N／C比值和生理指标法)及其特点进行概述,论述了土壤动物线虫及其对污染环境的生物指示作用。提出土壤线虫作为一种有效的生态毒理诊断指标具有良好的发展前景．     

环境质量评价中的生物指示与生物监测

通过比较生物指示与生物监测技术和仪器分析技术的分析过程及原理,结合北京市土壤中土壤脲酶与土壤重金属含量之间的相关关系的案例,及生物指示与生物监测技术在水生生态系统及大气污染研究中的应用状况,系统阐述了生物指示与生物监测技术研究的发展历程、研究前沿及发展方向.指出定量校正及不同学科与地区的科学团队的合作是生物指示与生物监测技术研究中需要克服的关键问题,并提出生物指示与生物监测技术研究的目标是整合不同学科的研究方向,为人类健康与环境安全的保护与预测提供技术支持.     

DNA指纹技术在污染土壤生态毒理诊断中的应用

生物标记物能在细咆或分子水平上指示暴露．效应关系,是进行污染土壤生态毒理诊断的主要技术手段之一。随着分子生物学技术的飞速发展,出现了一系列以聚合酶链式反应为基础的、在分子水平上检测污染物质导致的生物体DNA损伤的DNA指纹技术。DNA指纹技术的主要类型有：随机扩增多态性DNA（RAPD）、聚合酶链式反应．单链构象多态性（PCR-SSCP）、扩增片段长度多态性（AFLP）、任意引物聚合酶链式反应（AP—PCR）、差异显示反转录聚合酶链式反应（DDRT）、短DNA重复序列（SSR）及限制片段长度多态性（RFLP）等。这些技术与检测基因突变、染色体畸变和损伤为主的一系列经典研究方法如彗星分析、微核实验等相比具有简便、快速、灵敏等优点。本文着重介绍了随机扩增多态性DNA、聚合酶链式反应．单链构象多态性、扩增片段长度多态性3种重要的DNA指纹技术在污染土壤诊断中的应用。     

土壤污染的生态毒理诊断研究进展

土壤生态毒理学是污染土壤生态风险评价以及土壤污染控制的理论依据,土壤环境中有毒物质的生态毒理效应及其分子机制的研究是土壤生态毒理学的核心内容.土壤污染生态毒理诊断集合了土壤污染的全部信息,提供了土壤的整体毒性效应.因此,开展土壤污染生态毒理诊断研究有重要的理论意义和现实意义.本文在参考国内外土壤污染生态毒理诊断研究成果的基础上,介绍了常用土壤污染生态毒理效应诊断方法和指标,并探讨了不同方法的利弊.传统毒理诊断方法往往只能表征胁迫的程度,不能解释损伤和响应发生分子机理.污染土壤毒性评估的生物标记物和土壤污染分子诊断技术,可深入探讨外源胁迫下生物的解毒机制.本文对其进行了较为系统的介绍,并对未来的研究趋势进行展望.     

基于文献计量的细菌-生物炭协同修复重金属污染土壤研究进展

为提高重金属污染土壤可持续修复效能,研究生物炭与细菌对重金属污染土壤的协同修复作用。基于文献计量学分析及重金属污染土壤修复背景,总结了细菌与生物炭对土壤重金属的稳定化特征及菌炭间的相互作用,分析了单一生物炭或细菌对重金属污染土壤修复的局限性,强调了细菌-生物炭协同修复技术的优势,阐述了细菌与生物炭主要通过离子交换、固定作用、氧化还原作用和迁移作用等重要机制有效修复重金属污染土壤,揭示了细菌-生物炭协同作用在重金属污染土壤修复中的巨大应用价值。文献计量学研究表明,生物炭与细菌对重金属污染土壤的协同修复已得到广泛关注。目前认为:生物炭与细菌的协同作用可有效改良土壤理化性质及提高土壤修复效率,也可促进植物生长及植物修复进程;生物炭对细菌影响具有双重性质,可促进细菌生长,也可能对细菌产生毒害;细菌可改变生物炭的理化性质,进而强化生物炭的重金属固定性能;细菌协同生物炭联合修复重金属污染土壤过程中,生物炭主导吸附和固定,细菌则发挥活化和解毒等功能;优化细菌-生物炭组合形式,发展混合细菌与多种类生物炭协同技术,是复合重金属污染土壤可持续修复亟待解决的重要问题;进一步揭示细菌与生物炭对重金属污染土壤的耦合作用及长效作用机制,规避生物炭生产和应用中的潜在生态健康风险,研发新型高效能细菌与生物炭复合体是细菌协同生物炭可持续修复重金属污染土壤应用领域面临的挑战。     

硅藻重金属污染生态学研究进展

硅藻是水生生态系统健康的指示生物之一,对环境变化极为敏感,某些典型硅藻已应用于指示水体重金属污染.本文围绕地表水重金属污染,从毒性效应、生物吸附和累积、生态适应机制及生物指示与生态修复作用等方面,综述了硅藻重金属污染生态学研究进展,阐述重金属污染下硅藻的生长趋势和硅壳形态的变化,硅藻对重金属生物吸附和生物累积的差别,硅藻对重金属的表面络合和离子交换等生态适应机制,以及硅藻对水体重金属污染的指示作用和生态修复作用,为水生生态系统的重金属污染防治与预警技术提供科学依据.     

菌根真菌对石油污染土壤修复作用的研究进展

生物修复技术是处理石油污染土壤最简单有效的方法之一。本文在系统概述土壤石油污染的环境危害及多种土壤修复技术的基础上,着重介绍了菌根生物修复技术,论述了菌根真菌对土壤中石油污染物的降解效果,探讨了菌根真菌降解污染物的可能机制：酶作用、根际作用、共代谢作用、基因调控;讨论了石油污染土壤的菌根生物修复前景和发展趋势。     

Importance of soil-water relations in assessing the endpoint of bioremediated soils

This study was conducted to investigate water movement in hydrocarbon contaminated soils. Three soils were studied, a hydrocarbon contaminated soil, the same soil after 3 years of bioremediation, and a control soil from the same site. There was a critical soil water content around 18% (bioremediated soil) and 20% (contaminated soil), above which the sorptivity of the contaminated soil was near that of the control soil. For soils with water contents below this value, there was a strong divergence in sorptivity between contaminated and control or bioremediated soils. Results suggest that water availability in contaminated soils will be highly dependent on soil water properties as water potential approaches the permanent wilting point (-1.5 MPa matrix potential).Infiltration of water into air dry (2% m.c. w/w) hydrocarbon contaminated soils was up to three orders of magnitude slower than for the control soil. For air dried soils, the infiltration rate of the contaminated and bioremediated soils was constant with time. This was in contrast to the control soil where infiltration rate was a function of the reciprocal of the square root of time.     

Germination and development of Mimosa pilulifera in petroleum-contaminated soil and bioremediated soil

In 2000 there was an oil spill at the Getúlio Vargas Refinery (REPAR/PETROBRÁS) in Paraná, Brazil. Nearly five years after contamination and the use of bioremediation, a study was carried out to identify the effects of the contaminated soil and the bioremediated soil on the germination and initial growth of Mimosa pilulifera seedlings. The experiment consisted of three treatments: petroleum-contaminated soil, bioremediated soil and uncontaminated soil, with five repetitions each. The following measurements were taken after 30, 60 and 90 days of planting: the percentage of germination, biomass and leaf area of the eophylls, biomass and length of the shoot and the roots in addition to the shoot/root ratio. The percentage of germination and the root biomass were not affected by the contaminated soil or by the bioremediated soil. On both the contaminated soil and the bioremediated soil biomass and leaf area of the eophyll were reduced. Plant length and shoot biomass were lower in the contaminated soil. Furthermore, the effect of the contaminated soil and the bioremediated soil was greater in the shoot than in the root system, since the bioremediation reduced the toxicity of the petroleum-contaminated soil.     

污染土壤生态修复的理论内涵、方法及应用

讨论了污染土壤生态修复的理论内涵、修复方法的优化组合及其应用案例.将污染土壤生态修复的目标拓展为:目标污染物降低到可接受程度、土壤生态毒理性降低到可接受程度、部分恢复或全部恢复土壤的生态服务功能.归纳出污染土壤生态修复的5个特点、5个原则.初步提出污染土壤生态修复方法优化组合的原则.污染土壤生态修复方法优化组合表现出以...     

两种镉积累型小麦根际微生物群落结构及功能多样性

为探究不同积累型小麦品种对根际微生物群落结构及功能多样性的影响,以镉低积累型小麦济麦22和镉高积累型小麦冀5265为研究材料,采用分离培养法和Biolog-Eco微平板法分析根际细菌数量、可培养优势群落结构以及微生物群落功能多样性。结果表明：污染土壤济麦22根际总细菌数量和抗Cd细菌数量均显著高于冀5265,而非污染土壤中两品种间无差异。污染土济麦22根际发现较多产脲酶和高镉抗性菌株(200 mg/L)。污染土济麦22根际优势菌多为Arthrobacter sp.和Bacillus sp.,冀5265根际优势菌主要为Streptomyces sp.;非污染土济麦22与冀5265根际优势菌群相似,均以Bacillus sp.为主。Biolog试验结果表明,两个小麦品种根际微生物群落对碳源的利用能力存在差异,济麦22根际微生物AWCD值、Mc Intosh指数、Shannon-Wiener指数、Simpson指数在污染土和无污染土中均显著高于冀5265。因此,污染土壤中不同积累型小麦品种根际微生物群落结构及功能多样性均存在差异,该研究结果对于揭示高低积累型小麦根际微生物机制提供了重要参考依...     

Use of green fluorescent protein and luciferase biomarkers to monitor survival and activity of Arthrobacter chlorophenolicus A6 cells during degradation of 4-chlorophenol in soil

The recently isolated novel species Arthrobacter chlorophenolicus A6 is capable of growth on and degradation of high concentrations of 4-chlorophenol (up to 350 μg ml⁻¹) as the sole carbon and energy source. This strain shows promise for bioremediation of environmental sites contaminated with high levels of chlorophenols. In this study, green fluorescent protein ( gfp ) or luciferase ( luc ) genes were used as biomarkers for monitoring cell number and activity, respectively, during degradation of 4-chlorophenol by A. chlorophenolicus cells. The individual marked strains, Arthrobacter chlorophenolicus A6L ( luc -tagged) and Arthrobacter chlorophenolicus A6G ( gfp -tagged), were monitored during degradation of 250 μg ml⁻¹ 4-chlorophenol in pure culture and 175 μg g⁻¹ 4-chlorophenol in soil microcosms. Both gene-tagged strains were capable of cleaning up the contaminated soil during 9 d incubation. During the bioremediation experiments, the luc -tagged cells were monitored using luminometry and the gfp -tagged cells using flow cytometry, in addition to selective plate counting for both strains. The cells remained at high population levels in the soil (evidenced by GFP-fluorescent cell counts) and the A. chlorophenolicus A6L population was metabolically active (evidenced by luciferase activity measurements). These results demonstrate that the  Arthrobacter chlorophenolicus A6 inoculum is effective for cleaning-up soil containing high concentrations of 4-chlorophenol.     

Structural diversity of microorganisms in chemically perturbed soil assessed by molecular and cytochemical approaches

Until recently, our understanding of microbial community development in soil ecosystems exposed to different inorganic and organic pollutants has been limited to culturable microorganisms because of the techniques available. The discovery that most soil microorganisms are non-culturable but potentially viable and metabolically active accelerated the application of different culture-independent methods for structural diversity assessments of the microbial community. This review examines the results of recent studies on the impact of heavy metals and organic pollutants on the diversity of the microflora obtained with methods based on analyses of signature biomarkers such as nucleic acids and fatty acids. The application of these techniques allowed researchers to pinpoint reduction of microbial diversity in contaminated soil, and significant shifts in the community structure, leading to the dominance of only a few populations (species) and the disappearance of others, some of which were never isolated by conventional methods (e.g. an increase in Acidobacterium or a decrease in terrestrial non-thermophilic Crenarchaeota). Although the new techniques are not free from limitations, they allow the monitoring of the virtual impact of stressors on soil microorganisms and the direction of resuscitation of the microbial community during natural or induced bioremediation, especially when using combined approaches.     

土壤重金属污染生态过程、效应及修复

本文探讨了重金属在土壤中的行为特性及其生态化学过程作用的特点,不同的重金属元素在土壤胶体颗粒表面的吸附特性及不同元素间的交互作用类型有所不同,自然对重金属迁移积累的影响有所不同。土壤重金属污染影响了种子萌发、幼苗生长、作物产量及体内重金属含量,探讨了污染土壤的酶学评价方法。阐述了重金属污染土壤生物修复的特点,展望了重金属污染生态过程研究的未来发展趋势。     

Microwave-Assisted Process (MAPTM) for the Extraction of Contaminants from Soil

Laboratory-scale tests were performed to evaluate the use of Environment Canada's patented Microwave-Assisted Process (MAP_TM) for the extraction of petroleum hydrocarbons from contaminated soil. The purpose of these tests was to determine the potential for using the process for large-scale processing of contaminated soil. Tests were performed using three soil types: a certified sediment and certified soil, both contaminated with polycyclic aromatic hydrocarbons (PAHs), and spiked peat soil contaminated with long-chain petroleum hydrocarbons. The test methods used were based on existing MAP techniques that have been proven for the sample preparation of contaminated soils for analytical purposes. The parameters evaluated concentrated on those that are amenable to a continuous large-scale process running at atmospheric pressures. This meant using solvents that are inexpensive and readily available in large volumes, low solvent to material ratios, and optimized energy inputs. In general, it was found that microwaves could be used to enhance the solvent extraction of the contaminants from the soil and that the properties of the soil greatly affected the extent to which the contaminants were removed.