Methanotrophic diversity in an agricultural soil as evaluated by denaturing gradient gel electrophoresis profiles of pmoA, mxaF and 16S rDNA sequences

Molecular methods were used to characterize the diversity of a methanotrophic population in an agricultural soil. For this purpose we have used DGGE analysis of functional and phylogenetic markers. Functional markers utilised comprised the pmoA-gene coding for the -subunit of the particulate methane monooxygenase (pMMO) present in all known methanotrophs and the mxaF-gene coding for the -subunit of methanol dehydrogenase (MDH) present in all Gram-negative methylotrophs. In addition, we have used 16S rDNA as a phylogenetic marker. DGGE patterns of an enrichment culture, and sequencing of major DGGE bands obtained with the bacterial specific primers showed that the community structure was dominated by methanotrophic populations related to Methylobacter sp. and Methylomicrobium sp. The PCR products amplified with the functional primer sets were related to both type I and type II methanotrophs. We also designed a new pmoA-targeting primer set which could be used in a nested protocol to amplify PCR-products from DNA extracted directly from the soil.     

利用变性梯度凝胶电泳分析微生物的多样性

综述了不依赖于培养的变性梯度凝胶电泳技术 (DGGE)分析微生物多样性的原理 ,并列举它的应用实例。DGGE和传统方法相比有很多优点 ,若将DGGE和其他方法结合起来 ,效果更好 ,应用更广泛。     

DGGE method for analyzing 16S rDNA of methanogenic archaeal community in paddy field soil

A denaturing gradient gel electrophoresis (DGGE) method for analyzing 16S rDNA of methanogenic archaeal community in paddy field soil is presented. Five specific primers for 16S rDNA of methanogenic archaea, which were modified from the primers for archaea, were first evaluated by polymerase chain reaction and DGGE using genomic DNAs of 13 pure culture strains of methanogenic archaea. The DGGE analysis was possible with two primer pairs (0348aF-GC and 0691R; 0357F-GC and 0691R) of the five pairs tested although 16S rDNA of some non-methanogenic archaea was amplified with 0348aF-GC and 0691R. These two primer pairs were further evaluated for use in analysis of methanogenic archaeal community in Japanese paddy field soil. Good separation and quality of patterns were obtained in DGGE analysis with both primer pairs. A total of 41 DNA fragments were excised from the DGGE gels and their sequences were determined. All fragments belonged to methanogenic archaea. These results indicate that the procedure of DGGE analysis with the primer pair 0357F-GC and 0691R is suitable for investigating methanogenic archaeal community in paddy field soil.     

Impacts of 2,4-D application on soil microbial community structure and on populations associated with 2,4-D degradation

The effect of 2,4-dichlorophenoxyacetic acid (2,4-D) application rate on microbial community structure and on the diversity of dominant 2,4-D degrading bacteria in an agricultural soil was examined using cultivation-independent molecular techniques coupled with traditional isolation and enumeration methods. Fingerprints of microbial communities established under increasing concentrations of 2,4-D (0-500 mg kg-1) in batch soil microcosms were obtained using denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA gene segments. While a 2,4-D concentration of at least 100 mg kg-1 was required to obtain an apparent change in the community structure as visualized by DGGE, the greatest impact of 2,4-D concentration occurred in the 500 mg kg-1 treatment, resulting in significantly reduced diversity of the dominant populations and enrichment by Burkholderia-like populations. The greatest diversity of 2,4-D degrading isolates was cultivated from the 10 mg kg-1 treatment, indicating that under these conditions, cultivation was more sensitive than DGGE for detecting changes in community structure. Most of these isolates harbored homologs of Ralstonia eutrophus JMP134 and Burkholderia cepacia tfdA catabolic genes. Results from this study revealed that agriculturally relevant application rates of 2,4-D may provide a temporary selective advantage for organisms capable of utilizing 2,4-D as a carbon and energy source.     

Diversity of Kenyan soda lake alkaliphiles assessed by molecular methods

DNA was extracted from water and sediment samples taken from soda lakes of the Kenyan-Tanzanian Rift Valley. DNA was also extracted from microbial enrichment cultures of sediment samples. 16S rRNA genes were amplified by the polymerase chain reaction and microbial diversity was studied using denaturing gradient gel electrophoresis (DGGE) of 16S rDNA amplicons. Cloning and sequencing of single DGGE bands showed that they usually contained mixed amplicons. Several of the amplicon sequences had high identities, up to 99%, with 16S rRNA genes of organisms previously isolated from soda lakes, while others were only distantly related, with identities as low as 82%. Phylogenetic analysis of the sequenced amplicons indicated that sequences were related to the haloarchaeal, Bacillus/Clostridium, Rhodobacterium/Thioalcalovibrio/ Methylobacter, and Cytophaga/Flavobacterium/Bacteroides (CFB) groups and the enterobacteria/Aeromonas/Vibrio part of the 3 subdivision of the Proteobacteria.Communicated by K. Horikoshi     

Characterization of alkalotolerant bacterial community by 16S rDNA-based denaturing gradient gel electrophoresis method for degradation of dibenzofuran in soil microcosm

An alkalotolerant bacterial community was developed by continuous enrichment in the chemostat in presence of dibenzofuran (DF) as sole carbon source. Six different types of bacterial isolates were cultured on nutrient broth agar plates together with six operational taxonomic units (OTUs) at pH 7.0 and pH 8.0 by 16S rDNA-DGGE method. However, isolates of microbial community was declined from three OTUs (pH 9.0) to two at pH 10.0 after enrichment in alkaline condition. Among the six isolates tested for degradation of DF, Pseudomonas sp. and Bacillus sp. the members of alkalotolerant bacterial community had better potency to degrade dibenzofuran. Alkalotolerant bacterial community introduced in soil microcosm for evaluation of survival of most suitable isolates and degradation of dioxin-like compound indicated more than 90% degradation of dibenzofuran after 45 days by the bacterial community enriched for 180 days in the chemostat at pH 10, however, microbial community was not competent to utilize even 50% DF after day 30, not enriched in the chemostat. The survival of competent bacteria monitored by DGGE method in soil microcosm indicated presence of two major alkalotolerant isolates for utilization of dibenzofuran, substantiated the results and significance of alkalotolerant bacteria for in situ bioremediation of dioxin-like compounds in the environment.     

Bacterial and fungal communities of wilted Italian ryegrass silage inoculated with and without Lactobacillus rhamnosus or Lactobacillus buchneri

Aims: To understand the effects of lactic acid bacteria (LAB) inoculation on fermentation products, aerobic stability and microbial communities of silage. Methods and Results: Wilted Italian ryegrass was stored in laboratory silos with and without inoculation of Lactobacillus rhamnosus and Lactobacillus buchneri. The silos were opened after 14, 56 and 120 days and then subjected to aerobic deterioration for 7 days. Intensive alcoholic fermentation was found in untreated silage; the sum of ethanol and 2,3‐butanediol content at day 14 was about 7 times higher than that of lactic and volatile fatty acids. Alcoholic fermentation was suppressed by L. rhamnosus and L. buchneri inoculation and lactic acid and acetic acid became the dominant fermentation products, respectively. Silages were deteriorated in untreated and L. rhamnosus‐inoculated silages, whereas no spoilage was found in L. buchneri‐inoculated silage. Enterobacteria such as Erwinia persicina, Pantoea agglomerans and Rahnella aquatilis were detected in untreated silage, whereas some of these bacteria disappeared or became faint with L. rhamnosus treatment. When silage was deteriorated, Lactobacillus brevis and Bacillus pumilus were observed in untreated and L. rhamnosus‐inoculated communities, respectively. The inoculated LAB species was detectable in addition to untreated bacterial communities. Saccharomyces cerevisiae and Pichia anomala were the main fungi in untreated and L. rhamnosus‐inoculated silages; however, P. anomala was not visibly seen in L. buchneri‐inoculated silage either at silo opening or after exposure to air. Conclusion: Inoculation with L. rhamnosus can suppress alcoholic fermentation of wilted grass silage with elimination of enterobacteria at the beginning of fermentation. Addition of L. buchneri may improve aerobic stability, with distinct inhibitory effect observed on P. anomala after silo opening. Significance and Impact of the Study: Bacterial and fungal community analyses help us to understand how inoculated LAB can function to improve the fermentation and aerobic stability of silage.     

Analysis of fungal diversity of grapes by application of temporal temperature gradient gel electrophoresis - potentialities and limits of the method

AIMS: Fungi could be responsible for several problems in wines but the fungal ecosystem of grapes remains little known. The use of traditional methods does not allow to describe quickly this ecosystem. Therefore, we need to improve the knowledge about these fungi to prevent defects in wine. This study aims at evaluating the potentialities of the temporal temperature gradient gel electrophoresis (TTGE) method to describe the fungal ecosystem of grapes. METHODS AND RESULTS: The internal transcribed spacer (ITS) region was amplified and analysed using TTGE. A reference database of 56 fungal species was set up to evaluate the discrimination power of the method. The database was used for the direct identification of the fungal species present in complex samples. The sensitivity of the method is below 10(4) spores per ml. CONCLUSIONS: This method allows to describe the fungal diversity of grapes, but does not always allow to directly identify all fungal species, because of the taxonomic resolution of the ITS sequences. However, this identification strategy is less time consuming than traditional analysis by cloning and sequencing the bands. SIGNIFICANCE AND IMPACT OF THE STUDY: With this method, it will be possible to compare the fungal species present in different vineyards and to connect the presence of some fungi with particular defects in wine.     

Ammonia biofiltration and community analysis of ammonia-oxidizing bacteria in biofilters

Biological removal of ammonia was investigated using compost and sludge as packing materials in laboratory-scale biofilters. The aim of this study is to characterize the composition of ammonia-oxidizing bacteria (AOB) in two biofilters designed to remove ammonia. Experimental tests and measurements included analysis of removal efficiency and metabolic products. The inlet concentration of ammonia applied was 20–100 mg m⁻³. Removal efficiencies of BFC and BFS were in the range of 97–99% and 95–99%, respectively. Periodic analysis of the biofilter packing materials showed ammonia was removed from air stream by nitrification and by the improved absorption of NH₃ in the resultant acidity. Nitrate was the dominant product of NH₃ transformation. Changes in the composition of AOB were examined by using nested PCR, denaturing gradient gel electrophoresis (DGGE) and sequencing of DGGE bands. DGGE analysis of biofilter samples revealed that shifts in the community structure of AOB were observed in the experiment; however, the idle phase did not cause the structural shift of AOB. Phylogenetic analysis revealed the population of AOB showed Nitrosospira sp. remains the predominant population in BFC, while Nitrosomonas sp. is the predominant population in BFS.     

Effect of disodium fumarate on ruminal metabolism and rumen bacterial communities as revealed by denaturing gradient gel electrophoresis analysis of 16S ribosomal DNA

The aim of the present study was to examine the effects of feeding diets with addition of disodium fumarate (DF) to goats on ruminal metabolism and changes of rumen bacterial communities. Four cannulated goats were used in a 4 × 4 Latin square design. The results showed that ruminal pH increased linearly (P<0.01)as the amount of DF added increased, while lactate production decreased linearly (P<0.01). DF addition did not affect the production of acetate, propionate, butyrate, TVFA and NH₃-N. The effect of DF on the changes in rumen bacterial-community structure of goats was analyzed using 16S rDNA-based approaches. Amplicons of the V6-V8 variable regions of bacterial 16S rDNA were analyzed by denaturing gradient gel electrophoresis (DGGE), cloning and sequencing. Differences in rumen bacterial community structure were determined based on the Shannon index of diversity for pairwise comparison of the DGGE fingerprints and revealed significant changes in rumen microbiota after DF addition. As compared with those fed with the control diet, goats fed on the diets with DF addition showed a higher bacterial diversity. The sequences of seven amplicons in total 11 clones showed less than 97% similarity with those of previously identified or unidentified bacteria, suggesting that most bacteria in the gastrointestinal tract have not been cultured or identified. Amplicons related to Succinivibrio dextrinosolvens species were found in most DGGE fingerprints derived from goats on the diet containing DF, but not in goats on the control diet. These results demonstrated the ability of DF to improve the metabolism of rumen lactate fermentation and to influence the bacterial composition of the rumen in goats.     

Effects of the inoculant strain Pseudomonas putida KT2442 (pNF142) and of naphthalene contamination on the soil bacterial community

The naphthalene-degrading activity of a Pseudomonas sp. strain isolated from a creosote-contaminated soil was shown to be encoded by the IncP9 plasmid pNF142 by transfer to Pseudomonas putida KT2442. The effects of the inoculant strain KT2442 (pNF142) and of naphthalene contamination on the soil bacterial community were studied in microcosms with the following treatments: (I) soil, (II) soil with naphthalene, (III) soil with naphthalene and inoculated with KT2442 (pNF142). The inoculant became the dominant bacterial population in treatment (III) as evidenced by cultivation and denaturing gradient gel electrophoresis (DGGE) analysis. The bacterial DGGE profiles revealed drastically reduced complexity due to the numerical dominance of the inoculant. However, group-specific fingerprints (beta-proteobacteria, actinobacteria) that excluded KT2442 (pNF142) showed less severe changes in the bacterial community patterns. A major effect of naphthalene on the soil bacterial community was observed in treatment (II) after 21 days. Two dominant bands appeared whose sequences showed the highest similarity to those of Burkholderia sp. RP007 and Nocardia vinaceae based on 16S rRNA gene sequencing. These bands were less intense in treatment (III). The increased abundance of RP007-like populations due to naphthalene contamination was also confirmed by PCR amplification of the phnAc gene. The nahAc and nahH genes were detected in DNA and cDNA only in treatment III. Although the inoculant strain KT2442 (pNF142) showed good survival and expression of genes involved in naphthalene degradation, this study suggests that KT2442 (pNF142) suppressed the enrichment of indigenous naphthalene degraders.     

Monitoring impact of in situ biostimulation treatment on groundwater bacterial community by DGGE

Changes in bacterial diversity during the field experiment on biostimulation were monitored by denaturing gradient gel electrophoresis (DGGE) analysis of PCR-amplified 16S rDNA fragments. The results revealed that the bacterial community was disturbed after the start of treatment, continued to change for 45 days or 60 days and then formed a relatively stable community different from the original community structure. DGGE analysis of soluble methane monooxygenase (sMMO) hydroxylase gene fragments, mmoX, was performed to monitor the shifts in the numerically dominant sMMO-containing methanotrophs during the field experiment. Sequence analysis on the mmoX gene fragments from the DGGE bands implied that the biostimulation treatment caused a shift of potential dominant sMMO-containing methanotrophs from type I methanotrophs to type II methanotrophs.     

Species composition of an ectomycorrhizal fungal community along a local nutrient gradient in a boreal forest

Soil abiotic factors are considered to be important in determining the distribution of ectomycorrhizal (ECM) fungal species; however, there are few field data to support this. Here, we relate ECM species distributions to changes in soil chemistry along a short (90-m), natural nutrient gradient. The ECM community was characterized, using morphological and molecular techniques, in soil samples collected at 10-m intervals. There were pronounced changes in ECM fungal community structure along the transect, with many taxa showing discrete distributions. Although there was a change of host from Pinus to Picea along the gradient, host-specific fungi did not account for the observed change in community structure. Ordination analyses showed that community structure was strongly correlated with soil characteristics, in particular extractable ammonium and base saturation. However, autocorrelation among soil parameters makes it difficult to isolate the effects of individual parameters. The distinctive changes in soil and vegetation along the transect used in this study provided an exceptional opportunity to examine the local-scale impact of natural spatial heterogeneity on an ECM fungal community.     

Microbial community and biomass characteristics associated severe membrane fouling during start-up of a hybrid anoxic-oxic membrane bioreactor

In MBR, severe membrane fouling is often observed in the initial phase in which biomass is yet fully acclimated and stabilized in terms of microbial community structure and biomass characteristics. The focus of this study was to investigate the microbial community development and its influence on biomass characteristics and membrane fouling during start-up of a hybrid anoxic-oxic MBR. PCR-DGGE analysis indicated that the microbial community shifted in start-up period when a severe membrane fouling was observed. Small particle size, high fractal dimension (DF) and high EPS production, which were closely associated with microbial community, were found to be the major contributors to the severe fouling. Microbial community development was most likely the ultimate factor responsible for the severe membrane fouling.     

转Bt基因玉米对根际土壤细菌群落结构的影响

利用聚合酶链式反应-变性梯度凝胶电泳(PCR-DGGE)技术及扩增产物序列分析方法研究了转Bt基因玉米对根际土壤细菌群落结构及系统发育的影响.结果表明:转基因玉米与其非转基因亲本根际共有的土壤细菌分别隶属于变形菌门、疣微菌门、放线菌门、拟杆菌门、厚壁菌门、芽单胞菌门和酸杆菌门.其中,变形菌门为主要优势类群,占43.5%...