土壤微生物生态学研究中的非培养方法

土壤微生物种类和数量是评价土壤健康质量的重要指标之一。然而环境中90%以上的微生物不能够通过传统的培养基培养方法获得。最近发展起来的分析方法如磷脂脂肪酸(PLFA)、BIOLOG微孔板和分子生物学的方法可从不同方面对土壤微生物群落进行更为详尽的分析。论文就土壤微生物生态学研究中使用的主要方法进行了综述。     

Tree species influences soil microbial community diversity but not biomass in a karst forest in southwestern China

中国西南喀斯特森林树种对土壤微生物群落多样性和生物量的影响 陆地生态系统中植物种对土壤微生物群落结构的影响不一，而喀斯特生态系统中植物种对土壤微生物群落结构影响的研究尚未见报道。本研究利用磷酸脂肪酸(PLFA)法，分析了黔中高原型喀斯特常绿落叶阔叶混交林5个优势树种—窄叶石栎(Lithocarpus confinis Huang)、圆果化香(Platycarya longipes Wu)、滇鼠刺(Itea yunnanensis Franch.)、安顺润楠(Machilus cavaleriei H. Lév.)、云贵鹅耳枥(Carpinus pubescens Burkill)—与土壤理化性质对土壤微生物群落组成和生物量的影响。在测试的212个土壤样品 中共检测出132种PLFA，每个样品土壤微生物平均PLFA数量和生物量分别为65.97和11.22 µg g^–1。土壤表层(0–10 cm)的土壤微生物PLFA数量与下层(10–20 cm)土壤接近，但前者土壤微生物生物量显著高于后者(P < 0.05)。树种影响土壤微生物PLFA数量，但对土壤微生物生物量没有影响。云贵鹅耳枥附近的土壤微生物PLFA数量显著高于其他树种(P < 0.05)，而其他树种土壤微生物PLFA数量接近。土壤微生物 生物量与表层土壤的理化性质无显著相关，但与下层土壤的有机碳、全氮和全磷含量呈显著正相关 (P < 0.05)。总之，黔中高原型喀斯特森林真菌/细菌生物量比率低，微生物总生物量低，但微生物群落多样性高。树种对土壤微生物群落多样性产生影响。     

磷脂脂肪酸法在土壤微生物群落分析中的应用

土壤微生物群落的组成一直是土壤学、微生物学和生态学研究的热点问题。我国在这方面的研究处于国际前列,越来越多的研究成果在国际重要刊物上发表。而磷脂脂肪酸(PLFA)法在土壤微生物群落分析中占有举足轻重的地位,国内外学者都热衷于使用该方法。但是PLFA法的使用仍存在一些不足的地方,需要研究学者们慎重使用。本文综述了国际上相关研究,概述了PLFA方法使用的发展历史,应用及挑战。总结了使用和数据解读时需要注意的问题,整理了PLFA法相关的生物标记以及与新方法结合的设想,方便以后研究的开展。     

采煤塌陷地不同施肥处理对土壤微生物群落结构的影响

采用磷脂脂肪酸(PLFA)分析方法,对山西长治襄垣五阳煤矿采煤沉陷区复垦土壤的微生物群落结构进行了研究。结果表明:施肥处理能够不同程度的增加土壤微生物的PLFA总量、细菌PLFA量和真菌PLFA量,其中化肥+有机肥处理下的土壤微生物PLFA总量,细菌PLFA量和真菌PLFA量增加作用比较明显,差异显著于对照处理;对不同施肥处理的复垦土壤微生物群落PLFA进行主成分分析也可得出化肥+有机肥处理下的土壤微生物的群落结构变化比较大,从因子载荷图上进一步分析可知,化肥+有机肥处理下的土壤中代表真菌的不饱和脂肪酸C18∶2ω6t、C18∶3ω6、18∶1ω9t、18∶3ω3的含量较高,这些脂肪酸将化肥+有机肥处理与单施化肥和对照处理区分开来,产生了明显的优势种群。土壤PLFA总量与土壤有机质、碱解氮、速效磷和速效钾含量都有很好的相关性,相关系数分别为0.76,0.85,0.67和0.67。     

羊草基因型数目与氮添加对土壤微生物群落的交互影响

物种多样性(或同一物种遗传多样性)减少和氮富集都是影响陆地生态系统进程的主要因素,它们之间的交互作用是否对土壤微生物群落产生显著影响已成为研究者关心的主要科学问题。研究羊草基因型数目(1、2、4三种基因型数目组合)和氮添加(无氮添加、低氮添加和高氮添加3种水平)对土壤微生物群落的总磷脂脂肪酸(PLFA,Phospholipid Fatty Acid)含量、细菌PLFA生物标记含量、真菌PLFA生物标记含量、真菌/细菌比、以及基于每个PLFA生物标记相对含量百分比所得微生物群落的Shannon-Wiener多样性指数和Simpson优势度指数的影响。结果表明:氮添加对细菌PLFA生物标记含量,以及土壤微生物PLFA生物标记的Shannon-Wiener多样性指数和Simpson优势度指数具有显著影响(P0.05);基因型数目对所测变量无显著影响(P0.05),但基因型数目和氮添加的交互作用对细菌PLFA生物标记含量和真菌/细菌比具有显著影响(P0.05)。研究结果为全球变化背景下氮沉降及重要物种种群数量减少对土壤微生物群落的影响提供了科学数据,为合理解释群落动态变化提供了数据支持。     

浓香型白酒窖池微生物群落结构特征

窖池是中国白酒,尤其是浓香型大曲酒生产颇具特色的固态生物反应器,窖龄与微生物群落结构关系密切且复杂,对产品质量影响非常显著.本研究以微生物细胞膜的特征组分磷酸脂肪酸(PLFA)为指标,研究了不同窖龄(5年、100年和300 年)窖池窖泥、糟醅和黄水的微生物群落结构特征.结果表明:窖泥中总PLFA含量最高,糟醅次之,黄水最低.PLFA的组成因窖龄而异,黄水中总PLFA含量随窖池窖龄的增长而减小.窖泥中直链饱和脂肪酸含量最高,为PLFA总量的50.7%～73.9%,其中300年窖池窖泥最高.窖泥中表征革兰氏阳性(G+)厌氧细菌的PLFA含量较高,而糟醅和黄水中均以表征革兰氏阴性(G-)厌氧菌的PLFA含量较高.100年窖泥中表征G+菌、G-菌和厌氧菌的PLFA含量高于其他窖龄相应样品.5年窖窖泥、糟醅和黄水中真菌PLFA含量均高于其他窖龄相应样品.经主成分分析,5年窖和100年窖中主要变异菌群是G+菌和真菌,300年窖中主要变异菌群是细菌.描述窖池微生物群落特征的多样性指数可以选用PLFA的频次、Simpson优势度指数和Shannon多样性指数.     

磷脂脂肪酸谱图分析方法及其在微生物生态学领域的应用

应用磷脂脂肪酸谱图分析技术对微生物群落进行定量分布,克服了传统的微生物培养方法和显微技术的局限性。介绍了磷脂脂肪酸谱图分析方法及其在微生物生态学领域中的应用,包括对微生物群落的生物量、群落结构、营养状况和新陈代谢活动等方面的研究。     

磷脂脂肪酸方法在土壤微生物分析中的应用*

磷脂脂肪酸(PLYA)是活体微生物细胞膜的重要组分,不同类群的微生物能通过不同的生化途径合成不同的PLFA,部分PLFA可以作为分析微生物量和微生物群落结构等变化的生物标记。在土壤微生物分析中,越来越多地采用了PLFA方法。主要对PLFA方法在土壤微生物分析中的应用做一综述。     

闽江河口红树林土壤微生物群落对互花米草入侵的响应

采用磷脂脂肪酸标记法(PLFA)研究外来入侵植物互花米草对闽江河口湿地红树林土壤微生物群落结构的影响,并探讨其主要影响因素。结果表明:从3种不同植被群落土壤(红树林群落MC、红树林-互花米草混生群落MS、互花米草群落SC)共检测到22种PLFA生物标记,MS土壤微生物PLFA生物标记总量明显高于其他植被群落,3种植被群落土壤理化性质和酶活性的变化趋势为:MCMSSC,表明互花米草入侵后土壤微生物量增加,而理化性质和酶活性均有明显下降,红树林湿地土壤质量发生了明显退化。3种植被群落土壤中含量最高的PLFA生物标记是16:0,16:1w7c,9Me15:0w,18:1w12c。土壤中特征微生物相对生物量存在明显差异,细菌分布量最大,其次是真菌和放线菌,原生动物分布量最小。群落多样性指数呈相似规律,MS土壤微生物类群多样性指数均小于MC,表明互花米草入侵后土壤微生物群落多样性指数均有下降。通过主成分分析,基本能区分出3种不同植被群落微生物群落的特征。土壤理化性质、酶活性间存在相关性,有机碳、全氮、蔗糖酶、过氧化氢酶与革兰氏阴性菌、放线菌呈显著或极显著正相关。研究结果表明互花米草入侵在一定程度上具有影响红树林群落土壤营养代谢循环的潜力,特别是关于碳、氮、磷等的循环及酶活性,改变部分有利于自身生长的土壤环境相关的微生物类群含量,竞争有利环境,迅速扩张实现入侵。     

微生物分子生态学发展历史及研究现状

微生物群落多样性是微生物生态学和环境学研究的重点之一。分子生物学方法应用于微生物群落结构分析使得对环境样品中占大多数的不可培养微生物的研究成为了可能。由于功能上高度保守,序列上的不同位置具有不同的变异速率,核糖体RNA（rRNA）是目前在微生物分子生态学上最为有用以及应用最广泛的分子标记,通过rRNA序列比对,可以分析不同分类水平的系统发育关系。元基因组学研究方法通过对环境样品中的各种微生物群落的总的基因组进行分析,充分展示了环境微生物代谢途径,极大地扩展了对微生物的认识。快速发展的高通量测序极大地促进了各项微生物生态学技术的发展,带来了新的突破。     

Linking microbial community composition and soil processes in a California annual grassland and mixed-conifer forest

To investigate the potential role of microbial community composition in soil carbon and nitrogen cycling, we transplanted soil cores between a grassland and a conifer ecosystem in the Sierra Nevada California and measured soil process rates (N-mineralization, nitrous oxide and carbondioxide flux, nitrification potential), soil water and temperature, and microbial community parameters (PLFA and substrate utilization profiles) over a 2 year period. Our goal was to assess whether microbial community composition could be related to soil process rates independent of soil temperature and water content. We performed multiple regression analyses using microbial community parameters and soil water and temperature as X-variables and soil process rates and inorganic N concentrations as Y-variables. We found that field soil temperature had the strongest relationship with CO₂ production and soil NH₄⁺ concentration, while microbial community characteristics correlated with N₂O production, nitrification potential, gross N-mineralization, and soil NO₃^– concentration, independent of environmentalcontrollers. We observed a relationship between specific components of the microbial community (as determined by PLFA) and soil processes,particularly processes tightly linked to microbial phylogeny (e.g. nitrification). The most apparent change in microbial community composition in response to the 2 year transplant was a change in relative abundance of fungi (there was only one significant change in PLFA biomarkers for bacteria during 2 years). The relationship between microbial community composition and soil processes suggests that prediction of ecosystem response to environmental change may be improved by recognizing and accounting for changes in microbial community composition and physiological ecology.     

The functional significance of the microbial biomass in organic and conventionally managed soils

In order to achieve sustainability in managed ecosystems we must understand management impacts on soil processes and clarify the regulatory role of the microbial community on these processes. Crop rotation and organic management practices are thought to have positive impacts on the microbial biomass; however, the specific impacts of crop rotation organic management on soil microbial ecology are largely unknown. The effect of organic management on soil microbial ecology was investigated using soils collected from the Rodale Institute Research Center's long-term Farming Systems Trial (FST) experiment. The FST, begun in 1981, included a manured and a cover cropped organic rotation and a conventionally managed grain based rotation. Soil respiration rates and¹³C-isotope fate in a companion study suggest that the biomass characteristics of the FST treatment soils were different in November 1991. However, direct measurement of the microbial community at this time using Phospholipid Fatty Acid Analysis (PLFA) did not identify statistically significant treatment based differences in soil biomass characteristics. Variability among the PLFA profiles of treatment replicates was as great as variability between farming systems. Treatment based trends were observed among selected PLFAs, particularly those present in large amounts, that were consistent with indirect biomass and biomass-dependent measures. Overall, PLFA profiles, soil respiration rates and¹³C-cycling suggested that the organic cover cropped soil had the Largest and most heterogeneous microbial population while the biomass of the organic-manure amended soil was the least heterogeneous, and the most metabolically active. Present address: University of Illinois, 11025. Goodwin ave. Urbana, IL 61801, USA     

Physiological Status and Community Composition of Microbial Mats of the Ebro Delta, Spain, by Signature Lipid Biomarkers

Abstract Physiological status of microbial mats of the Ebro Delta (Tarragona, Spain) based on the extraction of lipids considered ``signature lipid biomarkers' (SLB) from the cell membranes and walls of microorganisms has been analyzed. Data from a day–night cycle show significant differences in viable cells countings (PLFA cells counts) ranging from 1.5 × 10¹⁰ to 5.0 × 10¹⁰ cells g⁻¹ of sediment. Minimum values were observed at 18:00 and 6:00, when physicochemical conditions change drastically. The diversity of the microbial community was assessed by GC/MS analysis of phospholipid fatty acids (PLFA). The ratio of PLFA, representative of Gram-negative bacteria, comprises 47.8% of the total PLFA of the microbial mat community. The remaining PLFA was representative of Gram-positive (10.0%), anaerobic (5.7%), and eukaryotic microorganisms (5.7%), and other common lipids. Two different approaches were used as a comparative study to assess the physiological status of the microbial mats. Two parameters (cyclopropane fatty acids/ω7c monoenoic fatty acids, and measurement of the trans/cis monoenoic PLFA ratio) showed a minimum at midnight, suggesting the highest microbial activity. Higher values were observed at 18:00 and 6:00, coinciding with lower PLFA cell counts. Received: 14 May 1999; Accepted: 6 September 1999; Online Publication: 24 March 2000     

Physiology and microbial community structure in soil at extreme water content

A sandy loam soil was brought to 6 water contents (13-100% WHC) to study the effects of extreme soil moistures on the physiological status of microbiota (represented by biomass characteristics, specific respiration, bacterial growth, and phospholipid fatty acid, PLFA, stress indicators) and microbial community structure (assessed using PLFA fingerprints). In dry soils, microbial biomass and activity declined as a consequence of water and/or nutrient deficiency (indicated by PLFA stress indicators). These microbial communities were dominated by G+ bacteria and actinomycetes. Oxygen deficits in water-saturated soils did not eliminate microbial activity but the enormous accumulation of poly-3-hydroxybutyrate by bacteria showed the unbalanced growth in excess carbon conditions. High soil water content favored G bacteria.     

Quantitative sampling of indoor air biomass by signature lipid biomarker analysis

Exposure to airborne biocontaminants may result in a multitude of health effects and is related to a pronounced increase in adult-onset asthma. Established culture-based procedures for quantifying microbial biomass from airborne environments have severe limitations. Assay of the phospholipid fatty acid (PLFA) components of airborne microorganisms provides a quantitative method to define biomass, community composition and nutritional/physiological activity of the microbial community. By collecting airborne particulate matter from a high volume via filtration, we collected sufficient biomass for quantitative PLFA analysis. Comparing high (filtration) and low (impaction) volume air sampling techniques at 26 locations within the Eastern United States, we determined that PLFA analysis provided a viable alternative to the established but flawed culture-based techniques for measuring airborne microbial biomass and community composition. Compared to the PLFA analysis, the culture techniques underestimated the actual viable airborne biomass present by between one to three orders of magnitude. A case study of a manufacturing plant at which there had been complaints regarding the indoor air quality is presented. Phospholipid fatty acid characterization of the biomass enabled contamination point source determination. In comparison with samples taken outdoors, increases in the relative proportion of trans PLFA, reflecting shifts in the physiological status of viable airborne Gram-negative bacteria, were detected in the indoor air samples at a majority of sampling sites. Received 29 September 1998/ Accepted in revised form 8 January 1999     

Leaf Litter Mixtures Alter Microbial Community Development: Mechanisms for Non-Additive Effects in Litter Decomposition

To what extent microbial community composition can explain variability in ecosystem processes remains an open question in ecology. Microbial decomposer communities can change during litter decomposition due to biotic interactions and shifting substrate availability. Though relative abundance of decomposers may change due to mixing leaf litter, linking these shifts to the non-additive patterns often recorded in mixed species litter decomposition rates has been elusive, and links community composition to ecosystem function. We extracted phospholipid fatty acids (PLFAs) from single species and mixed species leaf litterbags after 10 and 27 months of decomposition in a mixed conifer forest. Total PLFA concentrations were 70% higher on litter mixtures than single litter types after 10 months, but were only 20% higher after 27 months. Similarly, fungal-to-bacterial ratios differed between mixed and single litter types after 10 months of decomposition, but equalized over time. Microbial community composition, as indicated by principal components analyses, differed due to both litter mixing and stage of litter decomposition. PLFA biomarkers a15∶0 and cy17∶0, which indicate gram-positive and gram-negative bacteria respectively, in particular drove these shifts. Total PLFA correlated significantly with single litter mass loss early in decomposition but not at later stages. We conclude that litter mixing alters microbial community development, which can contribute to synergisms in litter decomposition. These findings advance our understanding of how changing forest biodiversity can alter microbial communities and the ecosystem processes they mediate.     

Root controls on soil microbial community structure in forest soils

We assessed microbial community composition as a function of altered above- and belowground inputs to soil in forest ecosystems of Oregon, Pennsylvania, and Hungary as part of a larger Detritus Input and Removal Treatment (DIRT) experiment. DIRT plots, which include root trenching, aboveground litter exclusion, and doubling of litter inputs, have been established in forested ecosystems in the US and Europe that vary with respect to dominant tree species, soil C content, N deposition rate, and soil type. This study used phospholipid fatty-acid (PLFA) analysis to examine changes in the soil microbial community size and composition in the mineral soil (0–10 cm) as a result of the DIRT treatments. At all sites, the PLFA profiles from the plots without roots were significantly different from all other treatments. PLFA analysis showed that the rootless plots generally contained larger quantities of actinomycete biomarkers and lower amounts of fungal biomarkers. At one of the sites in an old-growth coniferous forest, seasonal changes in PLFA profiles were also examined. Seasonal differences in soil microbial community composition were greater than treatment differences. Throughout the year, treatments without roots continued to have a different microbial community composition than the treatments with roots, although the specific PLFA biomarkers responsible for these differences varied by season. These data provide direct evidence that root C inputs exert a large control on microbial community composition in the three forested ecosystems studied.     

Sheep-urine-induced changes in soil microbial community structure

Soil microbial communities play an important role in nutrient cycling and nutrient availability, especially in unimproved soils. In grazed pastures, sheep urine causes local changes in nutrient concentration which may be a source of heterogeneity in microbial community structure. In the present study, we investigated the effects of synthetic urine on soil microbial community structure, using physiological (community level physiological profiling, CLPP), biochemical (phospholipid fatty acid analysis, PLFA) and molecular (denaturing gradient gel electrophoresis, DGGE) fingerprinting methods. PLFA data suggested that synthetic urine treatment had no significant effect on total microbial (total PLFA), total bacterial or fungal biomass; however, significant changes in microbial community structure were observed with both PLFA and DGGE data. PLFA data suggested that synthetic urine induced a shift towards communities with higher concentrations of branched fatty acids. DGGE banding patterns derived from control and treated soils differed, due to a higher proportion of DNA sequences migrating only to the upper regions of the gel in synthetic urine-treated samples. The shifts in community structure measured by PLFA and DGGE were significantly correlated with one another, suggesting that both datasets reflected the same changes in microbial communities. Synthetic urine treatment preferentially stimulated the use of rhizosphere-C in sole-carbon-source utilisation profiles. The changes caused by synthetic urine addition accounted for only 10-15% of the total variability in community structure, suggesting that overall microbial community structure was reasonably stable and that changes were confined to a small proportion of the communities.     

Microbial Biomass and Community Structure in a Sequence of Soils with Increasing Fertility and Changing Land Use

Abstract The microbial biomass and community structure of eight Chinese red soils with different fertility and land use history was investigated. Two community based microbiological measurements, namely, community level physiological profiling (CLPP) using Biolog sole C source utilization tests and phospholipid fatty acid (PLFA) profiles, were used to investigate the microbial ecology of these soils and to determine how land use alters microbial community structure. Microbial biomass-C and total PLFAs were closely correlated to organic carbon and total nitrogen, indicating that these soil microbial measures are potentially good indices of soil fertility in these highly weathered soils. Metabolic quotients and C source utilization were not correlated with organic carbon or microbial biomass. Multivariate analysis of sole carbon source utilization patterns and PLFAs demonstrated that land use history and plant cover type had a significant impact on microbial community structure. PLFAs showed these differences more than CLPP methods. Consequently, PLFA analysis was a better method for assessing broad-spectrum community differences and at the same time attempting to correlate changes with soil fertility. Soils from tea orchards were particularly distinctive in their CLPP. A modified CLPP method, using absorbance readings at 405 nm and different culture media at pH values of 4.7 and 7.0, showed that the discrimination obtained can be influenced by the culture conditions. This method was used to show that the distinctive microbial community structure in tea orchard soils was not, however, due to differences in pH alone. Received: 1 December 1999; Accepted: 6 June 2000; Online Publication: 28 August 2000