磷脂脂肪酸分析方法在微生物生态学中的应用

磷脂脂肪酸分析方法(PLFA)是基于生物化学手段的一种微生物生态学研究新技术,它具有对细胞生理活性没有特殊的要求,对样品保存时间也要求不高等优点,由样品中所有微生物提供信息,是一种快捷、可靠的分析方法。本文介绍了PLFA在微生物生态学研究中的应用,主要包括对微生物群落生物量、群落结构和功能及其变化,指示特定微生物以及营养状况方面的研究。     

Phospholipid,ester-linked fatty acid profiles as reproducible assays for changes in prokaryotic community structure of estuarine sediments

Phospholipid, ester-linked fatty acid profiles showed changes in benthic prokaryotic community structure reflecting culture manipulations that were both quantitative and statistically significant. Fatty acid structures, including the position and cis/ trans geometry of double bonds, were chemically verified by GC/MS after appropriate derivatization. The fatty acid profiles of independent flasks showed reproducible shifts when manipulated identically and significant differences when manipulated with different treatments. The absence of polyunsaturated fatty acids indicated that the consortia were predominantly prokaryotic. The prokaryotic consortia of different treatments could be differentiated by the proportions of cyclopropyl fatty acids and the proportions and geometry of monounsaturated fatty acids.     

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     

Phospholipid composition and fatty acid peroxidation during ageing of Acer platanoides seeds

Seeds of Norway maple ( Acer platanoides L.) differing in water content (10, 20 and 30%) were stored for 6 weeks at 20 to 30°C. During this period changes in phospholipids and fatty acids as well as in seed viability and germination capacity were studied. A considerable decrease in the phospholipid content was observed, which depended on the water content in the seeds and was related to the decrease of the seed germination capacity. The level of linoleic (18:2) and linolenic (18:3) acids in the phospholipid fraction decreased considerably in the course of the accelerated seed ageing. The results obtained suggest that phospholipid degradation and peroxidation of unsaturated fatty acids, followed by membrane destruction, play a considerable role in maple seed ageing.     

The use of phospholipid fatty acids (PL-FA) in the determination of rhizosphere specific microbial communities (RSMC) of two wheat cultivars

To determine differences in microbial community structure, phospholipid fatty acids (PL-FA) from rhizosphere bacteria of two different wheat cultivars Triticum aestivum L. (cv. Bohouth-6 and cv. Salamouni) were extracted and analyzed by gas chromatography. This approach was used to overcome the methodological underestimation of microbial densities obtained with isolation, culture techniques and microscopic observations. Our objective was to verify differences in PL-FA profiles from two wheat cultivars grown under controlled environmental conditions. Principal component analysis (PCA) and cluster analysis were used to detect dissimilarities between rhizosphere microbial communities of the two wheat cultivars and signature fatty acids (FA) were used to determine specific differences in the community structures. PCA of the two cultivars explained 79.18% of the variance on principal component 1 (PC1), which accounted for Bohouth-6 rhizosphere soil. The rhizosphere soil of Salamouni accounted for 11.66% of the variance on principal component 2 (PC2). The results demonstrated repeatedly the clustering of the samples into two distinct groups; each group belonging specifically to one of the two wheat cultivars. Profiles of Bohouth-6 showed higher amounts of cyclopropane acid 19:0cy and Sif 7 (Sum in feature 7) than Salamouni. Those FA are known as signature molecules for Gram-negative bacteria. This was also reflected by the higher bacterial counts (cfu g^–1 fresh root weight) of Gram-negative bacteria from the rhizosphere of the former than the latter. The results indicated that under controlled environmental conditions, wheat cultivars of different genotypes exhibit distinct microbial colonization in their rhizosphere.     

Discrimination of microbial diversity by fatty acid profiles of phospholipids and lipopolysaccharides in differently cultivated soils

The effects of long-term management practices on the diversity of the microbial community were examined by analyzing the composition of fatty acids (FAs) in phospholipids (PL) and lipopolysaccharides (LPS). According to the Principal Component Analysis (PCA) of total fatty acids the soils were divided in two groups: a) Black fallow soil (1) and soils cropped with potatoes (3, 4), and b) green fallow soil (2), soils cropped with wheat (5, 6), crop rotation (7) and grassland (8). The PCA for saturated FAs and for hydroxy FAs of both PL and LPS shows that the green fallow soil (2) can be distinguished from the other soils. For monounsaturated FAs the grassland soil (8) and for polyunsaturated FAs the wheat with vetch soil (6) clearly differed from the other soils. Fatty acids with biomarker quality such as 15:0 for bacteria and 18:26 for fungi were used for determining the ratio between bacteria and fungi: the black fallow soil (1) and the soil managed with crop rotation (7) contained significantly higher proportions of bacteria than the other soils. The largest proportion of the indicator fatty acid il5:0 for Gram-positive bacteria was measured in the black fallow soil (1), while the-hydroxy FAs indicative of Gram-negative bacteria most frequently occurred in manured potato cropped soil (4). Both indicator fatty acids 18:26 for fungi and cy19:0 for anaerobic bacteria had their highest concentrations in the manured potato cropped soil (4).     

Fifteen years of climate change manipulations alter soil microbial communities in a subarctic heath ecosystem

Soil microbial biomass in arctic heaths has been shown to be largely unaffected by treatments simulating climate change with temperature, nutrient and light manipulations. Here, we demonstrate that more than 10 years is needed for development of significant responses, and that changes in microbial biomass are accompanied with strong alterations in microbial community composition. In contrast to slight or nonsignificant responses after 5, 6 and 10 treatment years, 15 years of inorganic NPK fertilizer addition to a subarctic heath had strong effects on the microbial community and, as observed for the first time, warming and shading also led to significant responses, often in opposite direction to the fertilization responses. The effects were clearer in the top 5 cm soil than at the 5–10 cm depth. Fertilization increased microbial biomass C and more than doubled microbial biomass P compared to the non-fertilized plots. However, it only increased microbial biomass N at the 5–10 cm depth. Fertilization increased fungal biomass and the relative abundance of phospholipid fatty acid (PLFA) markers of gram-positive bacteria. Warming and shading decreased the relative abundance of fungal PLFAs, and shading also altered the composition of the bacterial community. The long time lag in responses may be associated with indirect effects of the gradual changes in the plant biomass and community composition. The contrasting responses to warming and fertilization treatments show that results from fertilizer addition may not be similar to the effects of increased nutrient mineralization and availability following climatic warming.     

Quantitative comparisons ofin situ microbial biodiversity by signature biomarker analysis

Microscopic examinations have convinced microbial ecologists that the culturable microbes recovered from environmental samples represent a tiny proportion of the extant microbiota. Methods for recovery and enzymatic amplification of nucleic acids from environmental samples have shown that a huge diversity existsin situ, far exceeding any expectations which were based on direct microscopy. It is now theoretically possible to extract, amplify and sequence all the nucleic acids from a community and thereby gain a comprehensive measure of the diversity as well as some insights into the phylogeny of the various elements within this community. Unfortunately, this analysis becomes economically prohibitive if applied to the multitude of niches in a single biome let alone to a diverse set of environments. It is also difficult to utilize PCR amplification on nucleic acids from some biomes because of coextracting enzymatic inhibitors. Signature biomarker analysis which potentially combines gene probe and lipid analysis on the same sample, can serve as a complement to massive environmental genome analysis in providing quantitative comparisons between microniches in the biome under study. This analysis can also give indications of the magnitude of differences in biodiversity in the blome as well as provide insight into the phenotypic activities of each community in a rapid and cost-effective manner. Applications of signature lipid biomarker analysis to define quantitatively the microbial viable biomass of portions of an Eastern USA deciduous forest, are presented.     

Influence of intercropping and intercropping plus rhizobial inoculation on microbial activity and community composition in rhizosphere of alfalfa (Medicago sativa L.) and Siberian wild rye (Elymus sibiricus L.)

Alfalfa–Siberian wild rye intercropping is the predominant cropping system used to produce forage in China. In this study, the effects of intercropping and intercropping-rhizobial inoculation on soil enzyme activities, microbial biomass and bacterial community composition in the rhizosphere were examined. In both treatments, the yield of alfalfa, microbial biomass and activities of soil urease, invertase and alkaline phosphatase in the alfalfa rhizosphere were markedly increased, whereas there was a slight increase in the yield of Siberian wild rye, few impacts on soil microbial biomass, and decreased enzyme activities (except for urease) in the Siberian wild rye rhizosphere. Terminal restriction fragment length polymorphism (T-RFLP) of 16S rRNA genes indicated that Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Deltaproteobacteria, Firmicutes, Actinobacteria and Bacteroidetes were the major bacterial groups in the rhizosphere of both plants. However, intercropping and rhizobial inoculation induced some shifts in the relative abundance of them. Nitrosomonas and Nitrosospira groups were detected in all treatments by the T-RFLP patterns of ammonia monooxygenase subunit A ( amoA ) gene, but the relative abundance of Nitrosomonas increased and that of Nitrosospira decreased in the intercropping-rhizobial inoculation treatment. Both treatments tended to increase the diversity of amoA . Conclusively, the two treatments clearly affected soil microbial composition and soil enzyme activities, which might be reflected in changes in yield.     

Plant-mediated effects of elevated ultraviolet-B radiation on peat microbial communities of a subarctic mire

Elevated ultraviolet‐B (UVB) radiation has been reported to have few effects on plants but to alter the soil microbial community composition. However, the effects on soil microorganisms have to be mediated via plants, because direct radiation effects are only plausible on the uppermost millimeters of soil. Here, we assessed secondary effects of UVB on soil microbes. The responses in the dominant plant Eriophorum russeolum, peat pore water and microbial communities in the peat were recorded at a subarctic mire in the middle of the third growing season under field exposure simulating 20% depletion in the ozone layer. The UVB treatment significantly reduced the sucrose and the total soluble sugar (sucrose+glucose+fructose) concentration of the plant leaves while increasing the sucrose concentration in the belowground storage organ rhizome. The starch concentration of the leaves was also slightly reduced by elevated UVB. In the plant roots, carbohydrate concentrations remained unaffected but the total phenolics concentration increased under elevated UVB. We suggest that the simultaneously observed decrease in bacterial growth rate and the altered bacterial community composition are due to UVB‐induced changes in the plant photosynthate allocation and potential changes in root exudation. There were no effects of elevated UVB on microbial biomass, peat pore water or nutrient concentrations in the peat. The observed responses are in line with the previously reported lower ecosystem dark respiration under elevated UVB, and they signify that the changed plant tissue quality and lower bacterial activity are likely to reduce decomposition.     

Effects of three herbicides on soil microbial biomass and activity

Three post-emergence herbicides (2,4-D, picloram and glyphosate) were applied to samples of an Alberta agricultural soil at concentrations of 0, 2, 20, and 200 μg g⁻¹. The effects of these chemicals on certain microbial variables was monitored over 27 days. All herbicides caused enhancement of basal respiration but only for 9 days following application, and only for concentrations of 200 μg g⁻¹. Substrate-induced respiration was temporarily depressed by 200 μg g⁻¹ picloram and 2,4-D, and briefly enhanced by 200 μg g⁻¹ glyphosate. It is concluded that because changes in microbial variables only occurred at herbicide concentrations of much higher than that which occurs following field application, the side-effects of these chemicals is probably of little ecological significance.     

Relative importance of the effect of 2,4-D,glyphosate, and environmental variables on the soil microbial biomass

Two post-emergence herbicides (glyphosate and 2,4-D) were applied at field application levels to tilled field plots in a mixed cropping area in south-central Alberta. The effects of these chemicals on certain variables associated with microbial biomass and activity were monitored in these plots (as well as corresponding control plots) for 45 days. Glyphosate did not influence any of the microbial variables tested. Addition of 2,4-D significantly influenced all microbial variables investigated but these effects were transient, being detectable only within the first 1–5 days of herbicide addition. The effects of 2,4-D addition on the microbial variables tested, even when significant, were typically small and probably of little ecological consequence especially when spatial and temporal variation in these variables is taken into account.     

Development of compost maturity and Actinobacteria populations during full-scale composting of organic household waste

AIMS: This study investigates changes in microbiological and physicochemical parameters during large-scale, thermophilic composting of a single batch of municipal organic waste. The inter-relationships between the microbial biomass and community structure as well as several physicochemical parameters and estimates of maturation were evaluated. METHODS AND RESULTS: Analyses of signature fatty acids with the phospholipid fatty acid and ester-linked methods showed that the total microbial biomass was highest during the early thermophilic phase. The contribution of signature 10Me fatty acids from Actinobacteria indicated a relatively constant proportion around 10% of the microbial community. However, analyses of the Actinobacteria species composition with a PCR-denaturing gradient gel electrophoresis approach targeting 16S rRNA genes demonstrated clear shifts in the community structure. CONCLUSIONS: This study demonstrates that compost quality, particularly maturity, is linked to the composition of the microbial community structure, but further studies in other full-scale systems are needed to validate the generality of these findings. SIGNIFICANCE AND IMPACT OF THE STUDY: The combination of signature lipid and nucleic acid-based analyses greatly expands the specificity and the scope for assessing the microbial community composition in composts. The results presented in this study give new information on how the development of the compost microbial community is connected to curing and maturation in the later stages of composting, and emphasizes the role of Actinobacteria in this respect.     

Extraction and Analysis of Microbial Phospholipid Fatty Acids in Soils

Phospholipid fatty acids (PLFAs) are key components of microbial cell membranes. The analysis of PLFAs extracted from soils can provide information about the overall structure of terrestrial microbial communities. PLFA profiling has been extensively used in a range of ecosystems as a biological index of overall soil quality, and as a quantitative indicator of soil response to land management and other environmental stressors.The standard method presented here outlines four key steps: 1. lipid extraction from soil samples with a single-phase chloroform mixture, 2. fractionation using solid phase extraction columns to isolate phospholipids from other extracted lipids, 3. methanolysis of phospholipids to produce fatty acid methyl esters (FAMEs), and 4. FAME analysis by capillary gas chromatography using a flame ionization detector (GC-FID). Two standards are used, including 1,2-dinonadecanoyl-sn-glycero-3-phosphocholine (PC(19:0/19:0)) to assess the overall recovery of the extraction method, and methyl decanoate (MeC10:0) as an internal standard (ISTD) for the GC analysis.     

Negative feedback on a perennial crop: Fusarium crown and root rot of asparagus is related to changes in soil microbial community structure

The dynamic equilibrium of an ecosystem is driven by mutual feedback interactions between plants and soil microorganisms. Asparagus exerts a particularly strong influence on its soil environment through abundant production of persistent phenolic acids, which impact selectively soil microorganisms and may be involved in Fusarium crown and root rot (FCRR) of asparagus. In a survey of 50 asparagus plantations of the province of Québec, we found that FCRR was associated with a profound cultivar-specific, reorganization of the soil microbial community, as revealed by phospholipid fatty acid (PLFA) profiling. According to PLFA indicators, microbial biodiversity as well as bacterial and fungal abundance dropped sharply with the onset of FCRR in fields planted with the cultivar Guelph Millenium. This drop was followed by a similar drop in the arbuscular mycorrhizal population. Biodiversity and microbial population size then increased to finally reach a new equilibrium. Discriminant analysis of PLFA profiles obtained from soil samples also indicated a shift in soil microbial community structure associated with FCRR development in fields planted with the cultivar Jersey Giant. Different soil biological conditions, as indicated by microbial biomass C and N and soil enzyme activities, were associated with different cultivars. Preceding crop, manure application, geographical location and tillage depth also influenced the structure of soil microbial communities in asparagus plantations, as determined by PLFA profiling. If higher FCRR incidence is a consequence of the soil microbial community reorganization, means to reduce FCRR incidence in asparagus plantations may be found among practices such as soil organic fertilization, soil tillage and intercropping strategies that would dilute the negative influence of asparagus on the soil microbial community. Finally, FCRR outbreaks were generally promoted by a previous crop of maize. It seems that maize and asparagus host a F. proliferatum teleomorph (Gibberella fujikoroi) of the same mating type.     

Fungal and bacterial growth in soil with plant materials of different C/N ratios

Fungal (acetate-in-ergosterol incorporation) and bacterial (leucine/thymidine incorporation) growth resulting from alfalfa (C/N=15) and barley straw (C/N=75) addition was studied in soil microcosms for 64 days. Nitrogen amendments were used to compensate for the C/N difference between the substrates. Fungal growth increased to a maximum after 3–7 days, at five to eight times the controls, following the addition of straw, and three to four times the controls following the addition of alfalfa. After 20–30 days, the fungal growth rate converged with the controls, resulting in a cumulative fungal growth two to three times the controls following straw addition and about 20% higher than the controls following alfalfa addition. The bacterial growth rate reached rates five times the controls following alfalfa addition and twice that of the controls following straw addition after 3–7 days. It remained elevated after 64 days. The cumulative bacterial growth was two and four times the controls following straw and alfalfa addition, respectively. A negative correlation was found between N addition and bacterial growth, while N stimulated fungal growth. Thus, the C/N ratio of the additions (substrate and extra N) could not entirely explain the different results regarding fungal and bacterial growths. Respiration was not always related to the combined growth of the microorganisms, emphasizing the requirement for a better understanding of growth efficiencies of fungi and bacteria.     

Biotechnological production and applications of microbial phenylalanine ammonia lyase: a recent review

Abstract

Phenylalanine ammonia lyase (PAL) catalyzes the nonoxidative deamination of l-phenylalanine to form trans-cinnamic acid and a free ammonium ion. It plays a major role in the catabolism of l-phenylalanine. The presence of PAL has been reported in diverse plants, some fungi, Streptomyces and few Cyanobacteria. In the past two decades, PAL has gained considerable significance in several clinical, industrial and biotechnological applications. Since its discovery, much knowledge has been gathered with reference to the enzyme’s importance in phenyl propanoid pathway of plants. In contrast, there is little knowledge about microbial PAL. Furthermore, the commercial source of the enzyme has been mainly obtained from the fungi. This study focuses on the recent advances on the physiological role of microbial PAL and the improvements of PAL biotechnological production both from our laboratory and many others as well as the latest advances on the new applications of microbial PAL.