Comparison of molecular fingerprinting methods for analysis of soil microbial community structure

Denaturing gradient gel electrophoresis (DGGE), terminal-restriction fragment length polymorphism (T-RFLP) analysis, and automated ribosomal intergenic spacer analysis (ARISA) have been widely used as molecular fingerprinting methods for analysis of microbial communities. To find suitable methods, we compared the three fingerprinting methods by analyzing soil fungal communities in four differing land-use types: bare ground, crop fields, grasslands, and forests. We also examined optimal primer pairs for DGGE analysis by comparing single and mixed DNA samples of cultured fungal populations. Principal coordinate analysis (PCO), nonmetric multidimensional scaling method (NMDS), and analysis of similarities (ANOSIM), which are major multivariate statistical analyses for quantifying fingerprint patterns, were compared. All three fingerprinting methods yielded clear discrimination of soil fungal communities among the four land-use types, irrespective of statistical methods. The advantages and disadvantages of the three fingerprinting methods were discussed.     

Alteration and resilience of the soil microbial community following compost amendment: effects of compost level and compost-borne microbial community

Compost amendment has been reported to impact soil microbial activities or community composition. However, little information is available on (i) to what extent compost amendment concurrently affects the activity, size and composition of soil microbial community, (ii) the relative effect of the addition of a material rich in organic matter versus addition of compost-borne microorganisms in explaining the effects of amendment and (iii) the resilience of community characteristics. We compared five treatments in microcosms: (i) control soil (S), (ii) soil + low level of compost (Sc), (iii) soil + high level of compost (SC), (iv) sterilized soil + high level of compost [(S)C] and (v) soil + high level of sterilized compost [S(C)]. The actual C mineralization rate, substrate-induced respiration, size of microbial community (biomass and heterotrophic cells number), and structure of total microbial (phospholipid fatty acids) and bacterial (automated ribosomal intergenic spacer analysis, A-RISA) communities were surveyed during 6 months after amendment. Our results show that (i) compost amendment affected the activity, size and composition of the soil microbial community, (ii) the effect of compost amendment was mainly due to the physicochemical characteristics of compost matrix rather than to compost-borne microorganisms and (iii) no resilience of microbial characteristics was observed 6-12 months after amendment with a high amount of compost.     

Comparison of methods for total community DNA extraction and purification from compost

The differences on DNA yield and purity of three different DNA extraction protocols were compared with regard to the use for PCR and other molecular analyses. Total DNA was extracted from compost by the three protocols, and then was purified by spin-bind cartridges after being precipitated by PEG8000. The detection performed on a nucleic acid and protein analyzer showed that all three methods produced high DNA yields. The agarose gel electrophoresis showed that the fragments of crude and purified DNA had a length of about 23 kb. A eubacterial 16S rRNA gene-targeted primer pair was used for PCR amplification, and full length 16S rDNAs were amplified from all the purified DNA samples. After being digested by restriction endonucleases, the restriction map of amplified rDNA showed identical genetic diversity. The products of PCR using primer pair GC341F and 907R were also used for denaturing gradient gel electrophoresis analysis. The results indicated that high-quality DNA was extracted from compost by the three protocols, and each of the protocols is adapted to extract microbial genome DNA from compost expediently and cheaply.     

不同灌溉方式下3种土壤微生物活性测定方法比较

探究不同灌溉方式下土壤微生物活性,对维持土壤稳定和提高水资源利用效率具有重要意义。以沈阳农业大学长期定位灌溉试验基地为平台,采用土壤酶活性、土壤呼吸和微量热法,研究节水灌溉组覆膜滴灌、渗灌及对照组沟灌下的土壤微生物活性并比较3种微生物活性测定方法。结果表明:不同灌溉方式下土壤脲酶、转化酶、脱氢酶活性无显著差异,土壤呼吸在3个主要生长季也没有明显变化规律;而微量热法得到的热功率时间曲线呈现了典型的微生物生长特征趋势,覆膜滴灌的生长速率较大,且与沟灌的总热量、最大热功率相差不大。因此,从可持续农业观点出发,覆膜滴灌是保证土壤微生物活性较高的一种节水灌溉方式;微量热法也为传统方法下不易检测的微生物活性提供了新思路。     

Evaluation of extraction and purification methods for obtaining PCR-amplifiable DNA from compost for microbial community analysis

Analysis of microbial community structure in complex environmental samples using nucleic acid techniques requires efficient unbiased DNA extraction procedures; however, humic acids and other contaminants complicate the isolation of PCR-amplifiable DNA from compost and other organic-rich samples. In this study, combinations of DNA extraction and purification methods were compared based on DNA yield, humic acid contamination, PCR amplifiability, and microbial community structure assessed by terminal restriction fragment length polymorphisms (TRFLP) of amplified 16S rRNA genes. DNA yield and humic acid contamination, determined by A230, varied significantly between extraction methods. Humic acid contamination of DNA obtained from compost decreased with increasing salt concentration in the lysis buffer. DNA purified by gel permeation chromatography (Sepharose 4B columns) gave satisfactory PCR amplification with universal eubacterial 16S rRNA gene primers only when A260/A280 ratios exceeded 1.5. DNA purified with affinity chromatography (hydroxyapatite columns), and showing A260/A280 ratios as high as 1.8, did not show consistently satisfactory PCR amplification using the same 16S rRNA primers. Almost all DNA samples purified by agarose gel electrophoresis showed satisfactory PCR amplification. Principal components analysis (PCA) of TRFLP patterns differentiated compost types based on the presence/absence of peaks and on the height of the peaks, but differences in TRFLP patterns were not appreciable between extraction methods that yielded relatively pure DNA. High levels of humic acid contamination in extracted DNA resulted in TRFLP patterns that were not consistent and introduced a bias towards lower estimates of diversity.     

健康儿童与轮状病毒感染儿童肠道菌群结构的比较研究

目的比较分析轮状病毒感染个体与健康个体肠道菌群结构的差异。方法采集11例轮状病毒感染个体及6例健康个体的粪便样品,提取粪便样品中细菌的混合DNA,先通过ERIC-PCR结合分子杂交的技术分析两组个体之间肠道微生物组成的相似性;再扩增粪便样品中菌群的16SrRNA基因,利用PCR—TGGE技术分析肠道菌群的组成情况。结果轮状病毒感染个体与健康个体相比,肠道菌群中GC含量较低的菌明显减少,同时肠道菌群有宿主专一性。结论轮状病毒感染会导致儿童肠道内菌群结构失调。     

Comparison of DNA extraction methods for human gut microbial community profiling

The human gut harbors a vast range of microbes that have significant impact on health and disease. Therefore, gut microbiome profiling holds promise for use in early diagnosis and precision medicine development. Accurate profiling of the highly complex gut microbiome requires DNA extraction methods that provide sufficient coverage of the original community as well as adequate quality and quantity. We tested nine different DNA extraction methods using three commercial kits (TianLong Stool DNA/RNA Extraction Kit (TS), QIAamp DNA Stool Mini Kit (QS), and QIAamp PowerFecal DNA Kit (QP)) with or without additional bead-beating step using manual or automated methods and compared them in terms of DNA extraction ability from human fecal sample. All methods produced DNA in sufficient concentration and quality for use in sequencing, and the samples were clustered according to the DNA extraction method. Inclusion of bead-beating step especially resulted in higher degrees of microbial diversity and had the greatest effect on gut microbiome composition. Among the samples subjected to bead-beating method, TS kit samples were more similar to QP kit samples than QS kit samples. Our results emphasize the importance of mechanical disruption step for a more comprehensive profiling of the human gut microbiome.     

Investigation of the microbial community structure and activity as indicators of compost stability and composting process evolution

In a bid to identify suitable microbial indicators of compost stability, the process evolution during windrow composting of poultry manure (PM), green waste (GW) and biowaste was studied. Treatments were monitored with regard to abiotic factors, respiration activity (determined using the SOUR test) and functional microflora. The composting process went through typical changes in temperature, moisture content and microbial properties, despite the inherent feedstock differences. Nitrobacter and pathogen indicators varied as a monotonous function of processing time. Some microbial groups have shown a potential to serve as fingerprints of the different process stages, but still they should be examined in context with respirometric tests and abiotic parameters. Respiration activity reflected well the process stage, verifying the value of respirometric tests to access compost stability. SOUR values below 1 mg O₂/g VS/h were achieved for the PM and the GW compost.     

喹啉与吲哚驯化的反硝化反应器的微生物群落结构分析比较

[目的]本研究旨在比较分析分别以喹啉和吲哚为底物,在相同条件下驯化的两个反硝化生物反应器的微生物群落结构.[方法]采用相同的种子污泥和相同的驯化条件,经过大约6周的驯化后,两个反应器均达到稳定而高效的污染物去除能力,通过16S rDNA克隆文库技术对两个反应器的微生物群落结构进行研究.[结果]研究发现,微生物群落结构表现出很大的差异.喹啉驯化的群落中所有的OTU都属于Betaproteobacteria,而吲哚驯化的群落中Betaproteobacteria占56.3%,吲哚驯化的群落具有更高的多样性.两个群落的优势OTU也不同,喹啉驯化群落中Thauera及其它Rhodocyclaceae科的微生物占整个群落的73%,而吲哚驯化群落中优势OTU为Comamonadaceae科、Alcaligenaceae科和Rhodocyclaceae科等类型的微生物,其中Comamonadaceae科的一个OTU占整个群落的28.7%.[结论]不同的驯化底物对微生物群落的组成具有较强的选择作用.首次报道并比较了可高效降解喹啉和吲哚的反硝化生物反应器的微生物群落结构.     

Response of microbial community structure to microbial plugging in a mesothermic petroleum reservoir in China

Microbial plugging, a microbial enhancement of oil recovery (MEOR) technique, has been applied in a candidate oil reservoir of Daqing Oil Field (China). The goal of this study is to monitor the survival of injected bacteria and reveal the response of microbial communities in field trial of microbial plugging through injection of selected microbial culture broth and nutrients. Culture-dependent enrichment and culture-independent 16S rDNA clone library methods were used. The results show that it was easy to activate targeted biopolymer-producing bacteria in a laboratory environment, and it was difficult for injected exogenous bacteria to survive. In addition, microbial communities in the oil reservoir also changed before and after the field trial. However, microbial communities, activated by fermentative medium for biopolymer-producing bacteria, appeared to show greater differences in the laboratory than in the natural reservoir. It was concluded that microbial populations monitoring was important to MEOR; results of response of microbial communities could provide a guide for the future field trials.     

Variations of soil structure and microbial population in a compost amended soil

Changes in soil structure and in microbial population were recorded in a long term field experiment over the growing season of maize (June–November). Determinations were made on samples from plots which had received, for two years, the following treatments: mineral fertilizers, farmyard manure and three rates of compost. Seasonal variations were observed for the stability of soil aggregates, total porosity, pore size distribution, mycorrhizal infection and aerobic cellulolytic microorganisms. The stability of the soil aggregates changed in a similar way to that found for both mycorrhizal infection and the number of aerobic cellulolytic microorganisms. Physical characteristics were not affected in any instance by the organic dressings and microbiological populations were generally influenced only by the higher doses of compost.     

Comparison of DNA extraction methods for microbial community profiling with an application to pediatric bronchoalveolar lavage samples

Barcoded amplicon sequencing is rapidly becoming a standard method for profiling microbial communities, including the human respiratory microbiome. While this approach has less bias than standard cultivation, several steps can introduce variation including the type of DNA extraction method used. Here we assessed five different extraction methods on pediatric bronchoalveolar lavage (BAL) samples and a mock community comprised of nine bacterial genera to determine method reproducibility and detection limits for these typically low complexity communities. Additionally, using the mock community, we were able to evaluate contamination and select a relative abundance cut-off threshold based on the geometric distribution that optimizes the trade off between detecting bona fide operational taxonomic units and filtering out spurious ones. Using this threshold, the majority of genera in the mock community were predictably detected by all extraction methods including the hard-to-lyse Gram-positive genus Staphylococcus. Differences between extraction methods were significantly greater than between technical replicates for both the mock community and BAL samples emphasizing the importance of using a standardized methodology for microbiome studies. However, regardless of method used, individual patients retained unique diagnostic profiles. Furthermore, despite being stored as raw frozen samples for over five years, community profiles from BAL samples were consistent with historical culturing results. The culture-independent profiling of these samples also identified a number of anaerobic genera that are gaining acceptance as being part of the respiratory microbiome. This study should help guide researchers to formulate sampling, extraction and analysis strategies for respiratory and other human microbiome samples.     

Microenvironments and microbial community structure in sediments

The aim of this study was to explore the potential of a combined chemical and microbiological approach as part of a study of organic carbon oxidation processes in sediments. An assessment of microbiological diversity using molecular techniques was carried out in combination with high resolution chemical measurements at the sediment-water interface of a coastal lagoon affected by eutrophication in autumn 2000. There was a 0.2 mm overlap between the O2 and H2S profiles. pH showed a maximum just above the sediment-water interface coinciding with an oxygen maximum, suggesting photosynthetic activity, and a minimum coinciding with the O2-H2S interface. The redox potential was high in bottom water and surface sediment, reflecting the presence of oxygen and oxides, and reached low values after a step-wise decrease at -18 mm. Reduction of Fe occurred within the biofilm at the O2-H2S interface and was mostly due to reduction by H2S. The elevated concentrations of dissolved Mn in the oxic water may have been caused either by in situ production within organic aggregates or lateral water flow from sites nearby at which Mn2+ diffuses out of the sediment. Sequences related to sulphur chemolitotrophs were retrieved from the biofilm samples, which is consistent with the small overlap between O2 and H2S observed in this biofilm. Although the resolution of techniques used was different, sequencing results were consistent with chemical data in delineating the same horizons according to redox, pH or ecological properties.     

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.     

Plant and soil properties determine microbial community structure of shared Pinus-Vaccinium rhizospheres in petroleum hydrocarbon contaminated forest soils

Rhizosphere communities are critical to plant and ecosystem function, yet our understanding of the role of disturbance in structuring these communities is limited. We tested the hypothesis that soil contamination with petroleum hydrocarbons (PHCs) alters spatial patterns of ecto- (ECM) and ericoid (ERM) mycorrhizal fungal and root-associated bacterial community structure in the shared rhizosphere of pine (Pinus contorta var. latifolia) and lingonberry (Vaccinium vitis-idaea) in reconstructed sub-boreal forest soils. Pine seeds and lingonberry cuttings were planted into containers with an organic (mor humus, FH or coarse woody debris, CWD) layer overlying sandy mineral horizons (Ae and Bf) of forest soils collected from field sites in central British Columbia, Canada. After 4 months, 219 mg cm^-2 crude oil was applied to the soil surface of half of the systems; systems were sampled 1 or 16 weeks later. Composition, relative abundance and vertical distribution of pine ECMs were assessed using light microscopy; community profiles were generated using LH-PCR of ribosomal DNA. Multivariate analysis revealed that plant and soil factors were more important determinants of community composition than was crude oil treatment. Fungal communities differed between pine and lingonberry roots; ECM communities were structured by soil layer whereas ERM communities varied between FH and CWD soil systems. Bacterial communities varied between plants and soil layers, indicating properties of ECM and ERM rhizospheres and the soil environment influence bacterial niche differentiation. This integration of mycorrhizal and bacterial community analysis contributes to a greater understanding of forest soil sustainability in forest ecosystems potentially contaminated with PHCs.     

土壤微生物群落对枯落物输入的响应

枯落物分解在陆地生态系统物质循环能量流动中起着关键性作用,明确枯落物输入对土壤微生物群落的影响有助于理解土壤微生物生物多样性和陆地生态系统功能的相互关系。本文采用整合分析方法,以中国为研究区域,以不添加枯落物为对照组,探究土壤微生物(真菌、细菌、放线菌)及微生物生物量碳、生物量氮对枯落物输入的响应。结果表明:与不添加枯落物相比,添加枯落物后土壤微生物生物量碳、生物量氮分别显著增加3.9%和4.4%;土壤真菌PLFA、细菌PLFA及总微生物PLFA分别增加4.0%、3.1%和2.4%。枯落物输入对土壤微生物的影响受到气候条件、年降水量、植被类型及土壤酸碱度等因素的显著影响;不同气候类型下,土壤微生物对枯落物输入的响应呈现出亚热带季风气候区>温带季风气候区>温带大陆气候区的趋势,以及随着年降水量的增加呈现出先升高后降低的趋势;不同植被类型下,土壤微生物对枯落物输入的响应呈现出阔叶林>草地≈混交林>针叶林的趋势。     

Drivers of microbial community structure in forest soils

Applied Microbiology and Biotechnology - Forests are essential biomes for global biogeochemical cycles, and belowground microorganisms have a key role in providing relevant ecosystem services. To...     

The microbial community structure in petroleum-contaminated sediments corresponds to geophysical signatures

The interdependence between geoelectrical signatures at underground petroleum plumes and the structures of subsurface microbial communities was investigated. For sediments contaminated with light non-aqueous-phase liquids, anomalous high conductivity values have been observed. Vertical changes in the geoelectrical properties of the sediments were concomitant with significant changes in the microbial community structures as determined by the construction and evaluation of 16S rRNA gene libraries. DNA sequencing of clones from four 16S rRNA gene libraries from different depths of a contaminated field site and two libraries from an uncontaminated background site revealed spatial heterogeneity in the microbial community structures. Correspondence analysis showed that the presence of distinct microbial populations, including the various hydrocarbon-degrading, syntrophic, sulfate-reducing, and dissimilatory-iron-reducing populations, was a contributing factor to the elevated geoelectrical measurements. Thus, through their growth and metabolic activities, microbial populations that have adapted to the use of petroleum as a carbon source can strongly influence their geophysical surroundings. Since changes in the geophysical properties of contaminated sediments parallel changes in the microbial community compositions, it is suggested that geoelectrical measurements can be a cost-efficient tool to guide microbiological sampling for microbial ecology studies during the monitoring of natural or engineered bioremediation processes.     

Comparison of methods to determine the centre of resistance of teeth

In orthodontic treatment, the locations of the centre of resistance (CR) of individual teeth and the applied load system are the major determinants for the type of tooth movement achieved. Currently, CR locations have only been specified for a relatively small number of tooth specimen for research purposes. Analysing cone beam computed tomography data samples from three upper central incisors, this study explores whether the effort to establish accurate CR estimates can be reduced by (i) morphing a pre-existing simplified finite element (FE) mesh to fit to the segmented 3D tooth-bone model, and (ii) individualizing a mean CR location according to a small parameter set characterising the morphology of the tooth and its embedding. The FE morphing approach and the semi-analytical approach led to CR estimates that differ in average only 0.04 and 0.12 mm respectively from those determined by very time-consuming individual FE modelling (standard method). Both approaches may help to estimate the movement of individual teeth during orthodontic treatment and, thus, increase the therapeutic efficacy.     

Short-range RNA-RNA crosslinking methods to determine rRNA structure and interactions

We describe details of procedures to analyze RNA-RNA crosslinks made by far-UV irradiation (< 300 nm) or made by irradiation with near-UV light (320-365 nm) on RNA containing photosensitive nucleotides, in the present case containing 4-thiouridine. Zero-length crosslinks of these types must occur because of the close proximity of the participants through either specific interactions or transient contacts in the folded RNA structure, so they are valuable monitors of the conformation of the RNA. Procedures to produce crosslinks in the 16S ribosomal RNA and between the 16S rRNA and mRNA or tRNA are described. Gel electrophoresis conditions are described that separate the products according to their structure to allow the determination of the number and frequency of the crosslinking products. Gel electrophoresis together with an ultracentrifugation procedure for the efficient recovery of RNA from the polyacrylamide gels allows the purification of molecules containing different crosslinks. These separation techniques allow the analysis of the sites of crosslinking by primer extension and RNA sequencing techniques. The procedures are applicable to other types of RNA molecules with some differences to control levels of crosslinking and separation conditions.