微生物分子生态学进展[综述]

现代分子生物学技术在生态学研究中的应用大大推动了微生物生态学的发展,导致了微生物分子生态学的产生．微生物分子生态学方法弥补了传统的微生物生态学方法的不足,使人们可以避开传统的分离培养过程而直接探讨自然界中微生物的种群结构及其与环境的关系．微生物生态学研究中采用的分子生物学方法主要有核酸探针技术、ＰＣＲ扩增技术、ｒＲＮＡ序列同源性分析方法、剃度凝胶电泳方法等．这些技术方法的采用,取得了一系列重要的成果和微生物生态学研究中的突破［１～６］,使得对某些微生物的研究成为可能,并在分子水平上阐述了生态问题…     

Design and use of group-specific primers and probes for real-time quantitative PCR

Real-time quantitative polymerase chain reaction (qPCR) has gained popularity as a technique to detect and quantify a specific group of target microorganisms from various environmental samples including soil, water, sediments, and sludge. Although qPCR is a very useful technique for nucleic acid quantification, accurately quantifying the target microbial group strongly depends on the quality of the primer and probe used. Many aspects of conducting qPCR assays have become increasingly routine and automated; however, one of the most important aspects, designing and selecting primer and probe sets, is often a somewhat arcane process. In many cases, failed or non-specific amplification can be attributed to improperly designed primer-probe sets. This paper is intended to provide guidelines and general principles for designing group-specific primers and probes for qPCR assays. We demonstrate the effectiveness of these guidelines by reviewing the use of qPCR to study anaerobic processes and biologic nutrient removal processes. qPCR assays using group-specific primers and probes designed with this method, have been used to successfully quantify 16S ribosomal Ribonucleic Acid (16S rRNA) gene copy numbers from target methanogenic and ammonia-oxidizing bacteria in various laboratory- and full-scale biologic processes. Researchers with a good command of primer and probe design can use qPCR as a valuable tool to study biodiversity and to develop more efficient control strategies for biologic processes.     

溴系阻燃剂毒理效应的计算模拟预测与环境风险评估进展

溴系阻燃剂(brominated flame retardants,BFRs)作为生产和使用量巨大的有机阻燃剂,在环境介质和生物体内被广泛检出。BFRs及其代谢产物会对生物体造成神经毒性、遗传毒性、发育毒性及内分泌干扰效应等,环境和健康风险得到日益广泛的关注。国内外学者通过最小二乘法(LS)和类似马尔可夫链蒙特卡罗算法(AMCMC)等统计分析方法建立了BFRs在环境中转化和归趋的预测模型,采用分子对接、分子动力学模拟和定量结构-活性关系(QSAR)模型等方法分析了BFRs及其代谢产物毒性作用的微观机制。综合近年来国内外的相关研究,发现BFRs的结构(取代基的类型和数量)会影响其环境行为和毒理效应,在总结研究成果的基础上,对BFRs的生态和健康风险进行评估,为其未来的研究和发展方向提供参考。     

高分子量多环芳烃--苯并[a]芘的生物降解研究进展

多环芳烃(PAHs)是一类广泛分布于海洋环境中的含有两个以上苯环的有机化学污染物.苯并[a]芘(BaP)是一种由5个苯环组成的分布广泛、致癌性极强的多环芳烃化合物,人们将其作为多环芳烃的指示物,通过研究其在环境中的产生、迁移、转化、降解及毒理作用来判断多环芳烃的污染情况.BaP已成为国内外环境监测的重要指标之一.本文综述了不同环境中BaP的来源与分布,BaP在环境中的行为,微生物对BaP的代谢途径,以及微生物降解BaP的相关限制因子,并结合作者在这方面研究的部分工作与目前国际研究的热点,提出值得进一步探究的有关问题.图4表3参51     

Development of pretreatment protocol for DNA extraction from biofilm attached to biologic activated carbon (BAC) granules

The biologic activated carbon (BAC) process is widely used in drinking water treatments. A comprehensive molecular analysis of the microbial community structure provides very helpful data to improve the reactor performance. However, the bottleneck of deoxyribonucleic acid (DNA) extraction from BAC attached biofilm has to be solved since the conventional procedure was unsuccessful due to firm biomass attachment and adsorption capacity of the BAC granules. In this study, five pretreatments were compared, and adding skim milk followed by ultrasonic vibration was proven to be the optimal choice. This protocol was further tested using the vertical BAC samples from the full-scale biofilter of Pinghu Water Plant. The results showed the DNA yielded a range of 40 μg·g^-1 BAC (dry weight) to over 100 μg·g^-1 BAC (dry weight), which were consistent with the biomass distribution. All results suggested that the final protocol could produce qualified genomic DNA as a template from the BAC filter for downstream molecular biology researches.     

Development and applications of functional gene microarrays in the analysis of the functional diversity, composition, and structure of microbial communities

Functional gene arrays (FGAs) are a special type of microarrays containing probes for key genes involved in microbial functional processes, such as biogeochemical cycling of carbon, nitrogen, sulfur, phosphorus, and metals, biodegradation of environmental contaminants, energy processing, and stress responses. GeoChips are considered as the most comprehensive FGAs. Experimentally established probe design criteria and a computational pipeline integrating sequence retrieval, probe design and verification, array construction, data analysis, and automatic update are used to develop the GeoChip technology. GeoChip has been systematically evaluated and demonstrated to be a powerful tool for rapid, specific, sensitive, and quantitative analysis of microbial communities in a high-throughput manner. Several generations of GeoChip have been developed and applied to investigate the functional diversity, composition, structure, function, and dynamics of a variety of microbial communities from different habitats, such as water, soil, marine, bioreactor, human microbiome, and extreme ecosystems. GeoChip is able to address fundamental questions related to global change, bioenergy, bioremediation, agricultural operation, land use, human health, environmental restoration, and ecological theories and to link the microbial community structure to environmental factors and ecosystem functioning.     

环境微生物群落分析的T-RFLP技术及其优化措施

末端限制性酶切片段长度多态性分析(terminal restriction fragment length polymorphism, T-RFLP)是近年来发展起来的、不依赖于培养的微生物群落分析方法之一.由于其在微生物群落结构分析方面的特点,包括分辨率高、易于实现自动化及互联网海量数据共享等优势,自1997年最先被报道以来得到了广泛的应用,成为环境微生物群落分析的最强有力的工具之一.本文详细介绍了T-RFLP技术的原理,并从环境样品群落DNA的提取、引物设计和PCR扩增、限制性酶切、电泳分离检测和T-RFLP图谱解析等5个方面讨论了用该技术解析环境微生物群落的方法和技巧,简述了近8 a来国外T-RFLP技术在群落分析中的研究进展.类似于其他的分子微生物生态学技术, T-RFLP也有自身的缺陷,因此重点分析了该技术的局限性及相应的解决办法.图2表1参62     

分子技术在湿地微生物群落解析中的应用

人工湿地的研究和应用近年来受到了广泛重视。微生物是人工湿地系统的重要组成部分,其群落结构对于湿地的净化功能的发挥具有重要影响。与传统的微生物分析技术相比,分子生物学技术在解析人工湿地微生物群落结构时无需纯培养,具有高效、快速、简便的特点,使其广泛应用于环境微生物的研究。文章综述了近年来在聚合酶链反应技术（Polymerase Chain Reaction,PCR）基础上发展起来的几种新的分子生物技术,包括PCR-DGGE、LH-PCR、T-RFLP、PCR-SSCP和ARDRA,以及其在人工湿地微生物研究中的应用现状。通过这些分子技术,可以分析湿地处理特定废水过程中微生物的数量、丰度、多样性及优势种;鉴定湿地中特定功能菌群（如氨氧化细菌、反硝化细菌、除硫菌等）的数量、活动分布、空间变更及与污染物去除的关系;判断各种系统条件（如不同基质、植物、水力负荷等）的设置对微生物多样性和稳定性的影响。最后,对分子技术在湿地领域的未来发展进行了展望。     

微生物苯丙氨酸解氨酶(PAL)/肉桂酸途径生物转化制L-苯丙氨酸酶源菌种选育

Ｌ苯丙氨酸（Ｌｐｈｅｎｙｌａｌａｎｉｎｅ,ＬＰｈｅ）是生化营养的必需氨基酸之一,作为临床医药用氨基酸输液、食品、饲料添加剂的必需成份和一些抗癌药物苯丙氨酸氮芥、甲酰溶肉瘤素等合成的中间体而得到广泛应用．自１９６５年天冬甜肽（Ａｓｐａｒｔａｍｅ,ＡＰＭ,阿斯巴甜）发现并开发生产以来,这一新型甜味剂日渐风行,导致对高光学纯度ＬＰｈｅ这一主要构成原料需求量激增,人们纷纷开展ＬＰｈｅ的研制生产．在众多生物合成途径中,肉桂酸酶法转化生产ＬＰｈｅ的研究开发因其原料易得价廉,倍受国内外的许多公司和…     

Characterization of the dissolved organic matter in sewage effluent of sequence batch reactor: the impact of carbon source

Dissolved organic matter (DOM) transformation in sequence batch reactor (SBR) fed with carbon sources of different biodegradability was investigated. During the biologic degradation process, the low molecular weight (MW) fraction (< 1 kDa) gradually decreased, while the refractory compounds with higher aromaticity were aggregated. Size exclusion chromatography (SEC) and fluorescence of excitation emission matrices (EEM) demonstrated that more biopolymers (polysaccharides or proteins) and humic-like substances were presented in the extracellular polymeric substance (EPS) extracted from the SBR fed with sodium acetate or glucose, while the EPS from SBR fed with slowly biodegradable dissolved organic carbon (DOC) substratestarch had relatively less biopolymers. Comparing the EfOM in sewage effluent of three SBRs, the effluent from SBR fed with starch is more aromatic. Organic carbon with MW>1 kDa as well as the hydrophobic fraction in DOM gradually increased with the carbon sources changing from sodium acetate to glucose and starch. The DOC fractionation and the EEM all demonstrated that EfOM from the effluent of the SBR fed with starch contained more fulvic acid-like substances comparing with the SBR fed with sodium acetate and glucose.     

分子微生物生态学技术在中国油田开发中的应用

分子微生物生态学技术近年来在研究油藏环境中微生物群落和生态系统等方面得到了广泛的应用,已经成为指导微生物采油技术开发和应用的一个不可或缺的工具.本文较系统地总结了我国利用分子微生物生态学方法研究油藏微生物群落结构的进展,其中包括大庆油田和西北部油田的16SrRNA基因序列分析,胜利油田16SrDNA的荧光标记末端多形态限制性片断分析技术(T-RFLP),以及吉林油田应用的DNA基因跟踪检测技术,并指出了存在问题和研究方向.图1参18     

现代分子生物学技术在动物肠道微生物多样性研究中的应用

现代分子生物技术的蓬勃发展解决了不可培养微生物研究的难题,使得肠道微生物的研究进入一个新的阶段.本文主要介绍了基于16SrRNA的分子分析技术,包括DGGE(变性梯度凝胶电泳),TGGE(温度梯度凝胶电泳),SSCP(单链构象多态性),RFLP(限制性片段长度多态性)等肠道微生物研究工作中常用的分子生物学技术方法、适用范围及可能的发展方向,为动物肠道微生物区系的进一步研究及开发应用提供帮助.表1参42     

Environmental controls on denitrifying communities and denitrification rates: insights from molecular methods.

The advent of molecular techniques has improved our understanding of the microbial communities responsible for denitrification and is beginning to address their role in controlling denitrification processes. There is a large diversity of bacteria, archaea, and fungi capable of denitrification, and their community composition is structured by long-term environmental drivers. The range of temperature and moisture conditions, substrate availability, competition, and disturbances have long-lasting legacies on denitrifier community structure. These communities may differ in physiology, environmental tolerances to pH and O2, growth rate, and enzyme kinetics. Although factors such as O2, pH, C availability, and NO3- pools affect instantaneous rates, these drivers act through the biotic community. This review summarizes the results of molecular investigations of denitrifier communities in natural environments and provides a framework for developing future research for addressing connections between denitrifier community structure and function.     

Identifying human-induced influence on microbial community: A comparative study in the effluent-receiving areas in Hangzhou Bay

Microbial compositions showed high differences in two study areas. COD was the key anthropogenic indicator in the coastal wastewater disposal area. Distinctive microbes capable of degrading toxic pollutants were screened. Microbial communities in effluent-receiving areas followed “niche theory”. Microbial community structure is affected by both natural processes and human activities. In coastal area, anthropegenetic activity can usually lead to the discharge of the effluent from wastewater treatment plant (WWTP) to sea, and thus the water quality chronically turns worse and marine ecosystem becomes unhealthy. Microorganisms play key roles in pollutants degradation and ecological restoration; however, there are few studies about how the WWTP effluent disposal influences coastal microbial communities. In this study, sediment samples were collected from two WWTP effluent-receiving areas (abbreviated as JX and SY) in Hangzhou Bay. First, based on the high-throughput sequencing of 16S rRNA gene, microbial community structure was analyzed. Secondly, several statistical analyses were conducted to reveal the microbial community characteristics in response to the effluent disposal. Using PCoA, the significant difference of in microbial community structure was determined between JX and SY; using RDA, water COD and temperature, and sediment available phosphate and ammonia nitrogen were identified as the key environmental factors for the community difference; using LDA effect size analysis, the most distinctive microbes were found and their correlations with environmental factors were investigated; and according to detrended beta-nearest-taxon-index, the sediment microbial communities were found to follow “niche theory”. An interesting and important finding was that in SY that received more and toxic COD, many distinctive microbes were related to the groups that were capable of degrading toxic organic pollutants. This study provides a clear illustration of eco-environmental deterioration under the long-term human pressure from the view of microbial ecology.     

Effect of acetate and propionate on the production and characterization of soluble microbial products (SMP) in aerobic granular sludge system

It is well known that soluble microbial products (SMP) derived from biological systems found in wastewater treatment plant effluent are responsible for chemical oxygen demand (COD). Aerobic granular sludge (AGS) is recognized as an efficient and innovative approach for wastewater treatment. Acetate and propionate as two dominant organic substances in wastewater need to be effectively removed before discharge. The aim of this study was to investigate the effectiveness of acetate and propionate to interfere with the accumulation, molecular weight (MW) distribution and composition of SMP in aerobic granular sludge (AGS) system using two identical sequencing batch reactors (SBR) named R1 and R2, respectively, thus reducing the COD. The results demonstrated that more SMP accumulated in R1 in presence of acetate compared to R2 treated with propionate. A positive correlation for SMP formation was detected as evidenced by aeration rates, but a negative correlation with hydraulic retention time (HRT). The MW distribution analysis suggested that small molecules (MW <3?kDa) were the dominant fraction of SMP in R1 and R2, accounting for 57–79% and 39–61%, respectively. In addition, gas chromatography-mass spectrometry (GC-MS) demonstrated that esters, alkanes, alkenes and alcohols were the predominant low-MW SMP in R1 and R2. More peaks were present in R2 (61) than R1 (46). Esters were the predominant SMP (39%) in R1, while alkanes were the predominant SMP (31%) in R2. Further, microbial community analysis indicated that more α- and γ-Proteobacteria groups which readily utilize low-MW SMP were found in R2 compared to R1-granules which may have contributed to less SMP accumulated in R2. The abundant genera in the granules were Zoogloea and Azoarcus, in particular, a greater quantity of Azoarcus was detected in R2 than R1-granules, which may be associated with higher degradation of aromatics in SMP.     

氯/溴稳定同位素分析技术及其在环境科学研究中的应用

稳定同位素分析被认为是环境污染物溯源和转化途径探究的有效工具.针对氯/溴稳定同位素研究已经开发了一些较为可靠的分析技术,被广泛应用于氯乙烯、氯苯、溴酚、多溴二苯醚和有机氯农药等有机污染物的研究.本文综述了近年来氯/溴同位素分析技术的最新进展,介绍了稳定同位素分析技术在含氯/溴有机污染物的溯源分析和降解途径识别等方面的应用实例,分析了现有分析技术在仪器测定、分析策略、理论知识等方面的不足,展望了该技术的发展方向及其在环境科学领域内的应用前景.     

黑土微生物量和酶活性对邻苯二甲酸二丁酯污染的响应

邻苯二甲酸二丁酯(di-n-butyl phthalate,DBP)是一种在环境中广泛存在的有毒有机化合物,已被我国列为优先控制污染物之一。本研究探讨了不同浓度的DBP污染对黑土呼吸、微生物量以及黑土酶活性的影响。结果表明,DBP污染处理的黑土呼吸速率和微生物量碳较对照均显著增加;微生物氮在DBP污染过程中呈"降低-升高-降低"波动性变化;微生物磷与DBP污染浓度呈显著负相关;DBP对黑土多酚氧化酶表现为先促进后抑制,对转化酶和蛋白酶活性表现为低浓度促进而高浓度抑制;在DBP污染过程中脲酶呈现被激活状态;黑土过氧化氢酶和酸性磷酸酶均受到DBP污染的显著抑制。通过相关性分析发现,土壤微生物量、土壤酶活性与DBP污染浓度之间存在着高度的相关性。由此可推断,DBP污染改变了黑土呼吸、微生物量和酶学活性的代谢特征,进而有可能影响了黑土的生态系统功能,威胁到黑土的可持续利用。     

大气颗粒物暴露与健康效应研究进展

大气颗粒物一直是影响我国大多数城市空气质量的首要污染物,且呈现出与欧美不同的煤烟、机动车尾气以及开放源复合型污染并存的高浓度污染态势,已有研究发现颗粒物的短期或长期暴露均会对人体产生不良的健康效应。本文从环境科学、暴露科学、环境流行病学和环境毒理学研究等方面系统综述了大气颗粒物健康效应研究的方法和进展,可为我国的大气颗粒物健康效应研究与大气颗粒物环境质量标准的修订提供方法学参考和经验借鉴。目前我国PM10污染尚未得到有效控制,细颗粒物(PM2.5)的污染也已引起关注,建议在不同区域开展空气污染健康效应的系统研究。     

Structural equation-based latent growth curve modeling of watershed attribute-regulated stream sensitivity to reduced acidic deposition

Little information is available about the effects of watershed attributes on long-term stream chemical responses to reduced acidic deposition. Long-term chemical data (1980-2006) for 21 stream sites from the Appalachian Mountain region and data of 8 watershed attributes of the corresponding watersheds were analyzed to study such effects. Latent growth curve modeling (LGM), a structural equation modeling (SEM) approach, was conducted to quantify stream sensitivity to reduced acidic deposition in the region, and to model the initial chemical conditions (intercepts) and changing rates (slopes) in three time periods: 1980s, 1990s, and 2000s. The modeled chemical trends were generally consistent with those trends which were detected by trend analyses in a previous study. Watershed attributes including area, mean elevation, percentage of developed land, percentage of grassland, percentages of shale and sandstone, percentage of barren land, and percentage of soil type Gilpin-Upshur-Vandalia (GUV) were found to affect stream sensitivity to the reduced acidic deposition with their importance in the respective order. The stream sensitivity in turn regulated streams’ chemical initial conditions and their changing rates in the studied watersheds. This innovative application of LGM for modeling long-term chemical trends advanced our understanding of the role of watershed attributes in regulating streams’ responses to reduced acidic deposition. This approach can be applied for evaluating streams’ responses to acidic deposition in other regions and, more broadly, for studying causal effects on long-term behaviors in other ecological and environmental science subjects.     

Conservation mycology in Australia and the potential role of citizen science

Fungi are undoubtedly important for ecosystem functioning; however, they have been omitted or given scant attention in most biodiversity policy documents, management plans, and formal conservation schedules throughout the world. This oversight may be due to a general lack of awareness in the scientific community and compounded by a scarcity of mycology‐associated curricula at the tertiary level and a lack of mycologists in research institutions. Although molecular techniques advance the systematic cataloging of fungi and facilitate insights into fungal communities, the scarcity of professional mycologists in the environmental sciences hampers conservation efforts. Conversely, citizen science initiatives are making significant contributions to the mycology discipline by increasing awareness and extending the scope of fungal surveys. Future research by professional and amateur mycologists into the distribution of fungi and their function in ecosystems will help identify wider and more effective conservation goals.