Bands In Denaturing Gradient Gel Electrophoresis Profiles Research Articles

Variation of Bacterial Community Diversity in Rhizosphere Soil of Sole-Cropped versus Intercropped Wheat Field after Harvest.

As the major crops in north China, spring crops are usually planted from April through May every spring and harvested in fall. Wheat is also a very common crop traditionally planted in fall or spring and harvested in summer year by year. This continuous cropping system exhibited the disadvantages of reducing the fertility of soil through decreasing microbial diversity. Thus, management of microbial diversity in the rhizosphere plays a vital role in sustainable crop production. In this study, ten common spring crops in north China were chosen sole-cropped and four were chosen intercropped with peanut in wheat fields after harvest. Denaturing gradient gel electrophoresis (DGGE) and DNA sequencing of one 16S rDNA fragment were used to analyze the bacterial diversity and species identification. DGGE profiles showed the bacterial community diversity in rhizosphere soil samples varied among various crops under different cropping systems, more diverse under intercropping system than under sole-cropping. Some intercropping-specific bands in DGGE profiles suggested that several bacterial species were stimulated by intercropping systems specifically. Furthermore, the identification of these dominant and functional bacteria by DNA sequencing indicated that intercropping systems are more beneficial to improve soil fertility. Compared to intercropping systems, we also observed changes in microbial community of rhizosphere soil under sole-crops. The rhizosphere bacterial community structure in spring crops showed a strong crop species-specific pattern. More importantly, Empedobacter brevis, a typical plant pathogen, was only found in the carrot rhizosphere, suggesting carrot should be sown prudently. In conclusion, our study demonstrated that crop species and cropping systems had significant effects on bacterial community diversity in the rhizosphere soils. We strongly suggest sorghum, glutinous millet and buckwheat could be taken into account as intercropping crops with peanut; while hulled oat, mung bean or foxtail millet could be considered for sowing in wheat fields after harvest in North China.

Imidacloprid induces changes in the structure, genetic diversity and catabolic activity of soil microbial communities

This is the first report describing the effect of imidacloprid applied at field rate (FR, 1 mg/kg of soil) and 10 times the FR (10*FR, 10 mg/kg of soil) on the structural, genetic and physiological diversity of soil bacterial community as determined by the phospholipid fatty acid (PLFA), the denaturing gradient gel electrophoresis (DGGE), and the community level physiological profile (CLPP) approaches. PLFA profiles showed that imidacloprid significantly shifted the microbial community structure and decreased the biomass of the total, bacterial and fungal PLFAs, however, this effect was transient at the FR dosage. The alterations in DGGE patterns caused by imidacloprid application, confirmed considerable changes in the overall richness and diversity of dominant bacteria. Although, as a result of imidacloprid application, the metabolic activity of microbial communities was generally lower, the richness and functional biodiversity of the soil microbial community were not negatively affected. In general, the analysis of the variance indicated that the measured parameters were significantly affected by treatment and the incubation time, however, the incubation time effect explained most of the observed variance. Imidacloprid degradation and the appearance of some new bands in DGGE profiles suggest the evolution of bacteria capable of degrading imidacloprid among indigenous microflora.

An optimized DNA extraction and purification method from dairy manure compost for genetic diversity analysis

An unbiased DNA extraction protocol is necessary for analysis of genetic diversity, particularly, of genes in complex environmental samples by nucleic acid techniques. In the present study, three manual extraction methods and two commonly used commercial kits, which were accompanied by two DNA purification strategies, were compared based on cell lysis efficiency, DNA and humic acid yields, PCR amplification and denaturing gradient gel electrophoresis (DGGE) analysis. The results show that in spite of higher cell lysis efficiencies of the two commercial kits, the purified DNA yields were only one-third of that obtained by the two manual methods of FTSP (Freeze-thaw-SDS-Protein K) and FTSPP (Freeze-thaw-SDS-Protein K-Polyvinylpolypyrrolidone). The purified DNA from all five methods was pure enough for successful PCR and real-time PCR amplifications in the presence of 1 μg μL(-1) BSA. However, the FTSPP extraction method with DNA purification by a Wizard(®) kit yielded the largest number of 16S rRNA gene copies and ribotypes or bands in DGGE profiles, which indicated a superiority over the other four methods. The development of this optimized DNA extraction and purification method may provide a valuable tool for further molecular analysis of compost.

Identification of cellulolytic bacteria in soil by stable isotope probing

Plant residues, mainly made up of cellulose, are the largest fraction of organic carbon material in terrestrial ecosystems. Soil microorganisms are mainly responsible for the transfer of this carbon to the atmosphere, but their contribution is not accurately known. The aim of the present study was to identify bacterial populations that are actively involved in cellulose degradation, using the DNA-stable isotope probing (DNA-SIP) technique. (13)C-cellulose was produced by Acetobacter xylinus and incubated in soil for 7, 14, 30 and 90 days. Total DNA was extracted from the soil, the (13)C-labelled (heavy) and unlabelled (light) DNA fractions were separated by ultracentrifugation, and the structure of active bacterial communities was analysed by bacterial-automated ribosomal intergenic spacer analysis (B-ARISA) and characterized with denaturing gradient gel electrophoresis (DGGE). Cellulose degradation was associated with significant changes in bacterial community structure issued from heavy DNA, leading to the appearance of new bands and increase in relative intensities of other bands until day 30. The majority of bands decreased in relative intensity at day 90. Sequencing and phylogenetic analysis of 10 of these bands in DGGE profiles indicated that most sequences were closely related to sequences from organisms known for their ability to degrade cellulose or to uncultured soil bacteria.

Assemblages of ericoid mycorrhizal and other root‐associated fungi from Epacris pulchella (Ericaceae) as determined by culturing and direct DNA extraction from roots

Ericoid mycorrhizal fungal endophytes form mycorrhizal associations with Ericaceae plant taxa and are regarded as essential to the ecological fitness of the plants in extremely nutrient-poor soils worldwide. We isolated fungi from roots of Epacris pulchella (Ericaceae) in a south-eastern Australian sclerophyll forest and compared rDNA internal transcribed spacer (ITS) restriction fragment length polymorphisms (RFLPs) and sequences for the cultured isolate assemblage with fungi identified in DNA extracted directly from the same root systems by cloning or denaturing gradient gel electrophoresis (DGGE). The most abundant RFLP types in the cultured isolate assemblage were identified as putative ericoid mycorrhizal ascomycete endophytes, and these also represented the most abundant RFLP types in the cloned assemblage and the most intense bands in DGGE profiles. Each method identified unique taxa, notably putative basidiomycetes in the DNA extracted directly from E. pulchella roots. However, the relative abundance of these was low.

Activity and Diversity of Methanogens in a Petroleum Hydrocarbon-Contaminated Aquifer

Methanogenic activity was investigated in a petroleum hydrocarbon-contaminated aquifer by using a series of four push-pull tests with acetate, formate, H(2) plus CO(2), or methanol to target different groups of methanogenic Archaea. Furthermore, the community composition of methanogens in water and aquifer material was explored by molecular analyses, i.e., fluorescence in situ hybridization (FISH), denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes amplified with the Archaea-specific primer set ARCH915 and UNI-b-rev, and sequencing of DNA from dominant DGGE bands. Molecular analyses were subsequently compared with push-pull test data. Methane was produced in all tests except for a separate test where 2-bromoethanesulfonate, a specific inhibitor of methanogens, was added. Substrate consumption rates were 0.11 mM day(-1) for methanol, 0.38 mM day(-1) for acetate, 0.90 mM day(-1) for H(2), and 1.85 mM day(-1) for formate. Substrate consumption and CH(4) production during all tests suggested that at least three different physiologic types of methanogens were present: H(2) plus CO(2) or formate, acetate, and methanol utilizers. The presence of 15 to 20 bands in DGGE profiles indicated a diverse archaeal population. High H(2) and formate consumption rates agreed with a high diversity of methanogenic Archaea consuming these substrates (16S rRNA gene sequences related to several members of the Methanomicrobiaceae) and the detection of Methanomicrobiaceae by using FISH (1.4% of total DAPI [4',6-diamidino-2-phenylindole]-stained microorganisms in one water sample; probe MG1200). Considerable acetate consumption agreed with the presence of sequences related to the obligate acetate degrader Methanosaeata concilii and the detection of this species by FISH (5 to 22% of total microorganisms; probe Rotcl1). The results suggest that both aceticlastic and CO(2)-type substrate-consuming methanogens are likely involved in the terminal step of hydrocarbon degradation, while methanogenesis from methanol plays a minor role. DGGE profiles further indicate similar archaeal community compositions in water and aquifer material. The combination of hydrogeological and molecular methods employed in this study provide improved information on the community and the potential activity of methanogens in a petroleum hydrocarbon-contaminated aquifer.

Composition and diversity of ammonia-oxidising bacterial communities in wastewater treatment reactors of different design treating identical wastewater

Autotrophic ammonia-oxidising bacteria (AOB) are a crucial component of the microbial communities of nitrifying wastewater treatment systems. Nitrification is known to occur in reactors of different configuration, but whether AOB communities are different in reactors of different design is unknown. We compared the diversity and community structure of the betaproteobacterial AOB in two full-scale treatment reactors - a biological aerated filter (BAF) and a trickling filter - receiving the same wastewater. Polymerase chain reaction (PCR) of 16S ribosomal RNA (rRNA) gene fragments with AOB-selective primers was combined with denaturing gradient gel electrophoresis (DGGE) to allow comparative analysis of the dominant AOB populations. The phylogenetic affiliation of the dominant AOB was determined by cloning and sequencing PCR-amplified 16S rRNA gene fragments. DGGE profiles were compared using a probability-based similarity index (Raup and Crick). The use of a probability-based index of similarity allowed us to evaluate if the differences and similarities observed in AOB community structure in different samples were statistically significant or could be accounted for by chance matching of bands in DGGE profiles, which would suggest random colonisation of the reactors by different AOB. The community structure of AOB was different in different sections of each of the reactors and differences were also noted between the reactors. All AOB-like sequences identified, grouped within the genus Nitrosomonas. A greater diversity of AOB was detected in the trickling filters than in the BAF though all samples analysed appeared to be dominated by AOB most closely related to Nitrosococcus mobilis. Numerical analysis of DGGE profiles indicated that the AOB communities in depth profiles from the filter beds were selected in a non-random manner.

Composition and diversity of ammonia-oxidising bacterial communities in wastewater treatment reactors of different design treating identical wastewater

Autotrophic ammonia-oxidising bacteria (AOB) are a crucial component of the microbial communities of nitrifying wastewater treatment systems. Nitrification is known to occur in reactors of different configuration, but whether AOB communities are different in reactors of different design is unknown. We compared the diversity and community structure of the betaproteobacterial AOB in two full-scale treatment reactors – a biological aerated filter (BAF) and a trickling filter – receiving the same wastewater. Polymerase chain reaction (PCR) of 16S ribosomal RNA (rRNA) gene fragments with AOB-selective primers was combined with denaturing gradient gel electrophoresis (DGGE) to allow comparative analysis of the dominant AOB populations. The phylogenetic affiliation of the dominant AOB was determined by cloning and sequencing PCR-amplified 16S rRNA gene fragments. DGGE profiles were compared using a probability-based similarity index (Raup and Crick). The use of a probability-based index of similarity allowed us to evaluate if the differences and similarities observed in AOB community structure in different samples were statistically significant or could be accounted for by chance matching of bands in DGGE profiles, which would suggest random colonisation of the reactors by different AOB. The community structure of AOB was different in different sections of each of the reactors and differences were also noted between the reactors. All AOB-like sequences identified, grouped within the genus Nitrosomonas. A greater diversity of AOB was detected in the trickling filters than in the BAF though all samples analysed appeared to be dominated by AOB most closely related to Nitrosococcus mobilis. Numerical analysis of DGGE profiles indicated that the AOB communities in depth profiles from the filter beds were selected in a non-random manner.

Relationships between microbial community structure and hydrochemistry in a landfill leachate-polluted aquifer.

Knowledge about the relationship between microbial community structure and hydrogeochemistry (e.g., pollution, redox and degradation processes) in landfill leachate-polluted aquifers is required to develop tools for predicting and monitoring natural attenuation. In this study analyses of pollutant and redox chemistry were conducted in parallel with culture-independent profiling of microbial communities present in a well-defined aquifer (Banisveld, The Netherlands). Degradation of organic contaminants occurred under iron-reducing conditions in the plume of pollution, while upstream of the landfill and above the plume denitrification was the dominant redox process. Beneath the plume iron reduction occurred. Numerical comparison of 16S ribosomal DNA (rDNA)-based denaturing gradient gel electrophoresis (DGGE) profiles of Bacteria and Archaea in 29 groundwater samples revealed a clear difference between the microbial community structures inside and outside the contaminant plume. A similar relationship was not evident in sediment samples. DGGE data were supported by sequencing cloned 16S rDNA. Upstream of the landfill members of the beta subclass of the class Proteobacteria (beta-proteobacteria) dominated. This group was not encountered beneath the landfill, where gram-positive bacteria dominated. Further downstream the contribution of gram-positive bacteria to the clone library decreased, while the contribution of delta-proteobacteria strongly increased and beta-proteobacteria reappeared. The beta-proteobacteria (Acidovorax, Rhodoferax) differed considerably from those found upstream (Gallionella, Azoarcus). Direct comparisons of cloned 16S rDNA with bands in DGGE profiles revealed that the data from each analysis were comparable. A relationship was observed between the dominant redox processes and the bacteria identified. In the iron-reducing plume members of the family Geobacteraceae made a strong contribution to the microbial communities. Because the only known aromatic hydrocarbon-degrading, iron-reducing bacteria are Geobacter spp., their occurrence in landfill leachate-contaminated aquifers deserves more detailed consideration.