PCR-denaturing Gradient Gel Electrophoresis Analysis Research Articles

Phytoplankton community succession shaping bacterioplankton community composition in Lake Taihu, China

PCR-denaturing gradient gel electrophoresis (DGGE) and canonical correspondence analysis (CCA) were used to explore the relationship between succession of phytoplankton community and temporal variation of bacterioplankton community composition (BCC) in the eutrophic Lake Taihu. Serious Microcystis bloom was observed in July–December 2008 and Bacillariophyta and Cryptophyta dominated in January–June 2009. BCC was characterized by DGGE of 16S rRNA gene with subsequent sequencing. The DGGE banding patterns revealed a remarkable seasonality which was closely related to phytoplankton community succession. Variation trend of Shannon–Wiener diversity index in bacterioplankton community was similar to that of phytoplankton community. CCA revealed that temperature and phytoplankton played key roles in structuring BCC. Sequencing of DGGE bands suggested that the majority of the sequences were affiliated with common phylogenetic groups in freshwater: Alphaproteobacteria, Betaproteobacteria, Bacteroidetes and Actinobacteria. The cluster STA2-30 (affiliated with Actinobacteria) was found almost across the sampling time at the two study sites. We observed that the family Flavobacteriaceae (affiliated with Bacteroidetes) tightly coupled to diatom bloom and the cluster ML-5-51.2 (affiliated with Actinobacteria) dominated the bacterioplankton communities during Microcystis bloom. These results were quite similar at the two sampling sites, indicating that BCC changes were not random but with fixed pattern. Our study showed insights into relationships between phytoplankton and bacterioplankton communities at species level, facilitating a better understanding of microbial loop and ecosystem functioning in the lake.

Long-term defaunation increases the abundance of cellulolytic ruminococci and methanogens but does not affect the bacterial and methanogen diversity in the rumen of sheep1

Protozoa are commensal eukaryotes in the rumen of herbivores. Protozoa are large producers of hydrogen, which is utilized by methanogenic archaea to produce methane, a greenhouse gas. The removal of protozoa from the rumen (defaunation) decreases methanogenesis, but also negatively affects fiber digestion, which is the main function of the rumen. The aim of this study was to examine the effect of long-term defaunation on the structure of the microbiota and particularly methanogenic archaea and fibrolytic bacteria to better understand the microbial mechanisms responsible for the decrease in methanogenesis and fibrolysis. The trial was conducted in 5 adult sheep subjected successively to long-term defaunation (2 yr), refaunation (12 wk), and short-term defaunation (10 wk). Methanogens were enumerated by quantitative PCR targeting the rrs (16S ribosomal RNA subunit) and mcrA (methyl coenzyme-M reductase) genes. The rrs gene was used to quantify the 3 major culturable rumen cellulolytic bacterial species (i.e., Fibrobacter succinogenes, Ruminococcus albus, and Ruminococcus flavefaciens) and total bacteria. Bacterial and methanogen diversity was also examined by PCR-DGGE (PCR-denaturing gradient gel electrophoresis) analysis targeting the rrs and mcrA genes, respectively. Total rumen bacterial density estimated as rrs copies per gram of DM of rumen content increased in response to long- and short-term defaunation (+1 log, P < 0.001), but without noticeable shifts in diversity. Defaunation increased the rrs copies per gram of DM of rumen content of R. albus and R. flavefaciens (+2 log, P < 0 0.001), but did not affect that of F. succinogenes. Despite a 20% reduction in methane emission in the 2 defaunated periods, the mcrA and rrs copies of methanogens per gram of DM of rumen content increased (+1 log, P < 0.001) in the absence of protozoa, whereas the diversity of the dominant methanogenic community was not modified. This study shows no major difference between long- and short-term defaunation in abundance and diversity of bacteria and archaea. It also provides evidence that monitoring the abundance and diversity of methanogens is not sufficient to comprehend the microbial mechanisms leading to a reduction in methane emissions by ruminants. This study also reports for the first time in sheep a selective effect of defaunation on the abundance of cellulolytic bacterial species.

Bacterial and Archaeal Community Structure of Two Adjacent Calcite Speleothems in Kartchner Caverns, Arizona, USA

Information concerning the bacterial and archaeal communities present on calcite speleothems in carbonate caves is of interest because the activity of these microbes has been implicated as a potential biogenic component in the formation of secondary mineral deposits. In addition, these speleothems may harbor unique, previously unidentified microbes. The current study presents a comparative analysis of the superficial bacterial and archaeal community structure of multiple stalactites from two different cave formations located in close proximity to each other in a nonhuman-impacted area of Kartchner Caverns, Arizona, USA. PCR-denaturing gradient gel electrophoresis analysis (PCR-DGGE) revealed that microbial communities sampled from stalactites of a single speleothem are more similar to each other than to the communities sampled from stalactites of an adjacent speleothem, suggesting that both bacterial and archaeal communities are speleothem-specific. SR-XRD analysis confirmed that both speleothems sampled were primarily calcite, but subtle differences were detected in the elemental composition profiles obtained from ICP-MS analysis indicating that substrate geochemistry was also speleothem-specific. PhyloChip analysis of composite samples from both speleothems revealed a broad diversity of phyla represented in the bacterial communities, while bacterial and archaeal bands sequenced from the DGGE profiles confirmed the presence of unique phylotypes not closely related ( < 96% similarity) to any sequences deposited in the GenBank database.

Characterization of halotolerant and oligotrophic bacterial communities in Asian desert dust (KOSA) bioaerosol accumulated in layers of snow on Mount Tateyama, Central Japan

Microbial particles transported by Asian desert dust (KOSA) possibly impact ecosystems and human health in downwind environments and are commonly called “bioaerosols.” The microbial communities associated with KOSA mineral particles (KOSA bioaerosol), which were collected from the snow cover on Mt. Tateyama, were investigated by means of a culture-amendment technique combined with denaturing gradient gel electrophoresis (DGGE) analysis using 16S rRNA genes. After the stratigraphy of the snow layer formed on the walls of a snow pit on Mt. Tateyama, samples were collected from 2 layers, which included KOSA particles and one which did not. The snow samples with KOSA particles indicated microbial growth in the 100 and 10−1 dilution media and in the medium with NaCl below 10%, while the snow sample without KOSA particles showed no microbial growth in the culture media. The PCR–DGGE analysis revealed that the bacterial compositions in the snow samples including KOSA mineral particles were mainly composed of the members of the phyla Actinobacteria, Firmicutus, and Proteobacteria. In particular, the 2 phylotypes appeared in the microbial cultures were similar to the members of the B. subtilis group, which has been detected in bioaerosol samples collected from the atmosphere over KOSA arrival (Suzu City) and source (Dunhuang City) regions. Presumably, halotolerant and oligotrophic bacterial communities are associated with the KOSA particles that descend to the snow cover on Mt. Tateyama.

Bacterial species associated with sound and Botrytis-infected grapes from a Greek vineyard

Grape bacterial microbiota plays central roles in the quality of grapes and wine, yet its diversity remains poorly described. In the present study, bacterial species associated with sound and Botrytis-infected grapes of two cultivars originating from the same vineyard were assessed. Isolates were identified by PCR-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) and sequence analysis of partial 16S rRNA gene. Comparable counts were recorded between Botrytis-infected and sound grape samples. In all cases, the majority of isolates belonged to different species of Enterobacteriaceae. The dominant species in the vineyard was Klebsiella oxytoca that was found in different combinations with Citrobacter freundii, Enterobacter spp., Erwinia sp., Pantoea dispersa, Tatumella ptyseos or other species. In fermenting musts, those populations declined while other species evolved, like Lactobacillus plantarum and Enterobacter ludwigii. Populations in botrytised samples persisted longer during spontaneous fermentations. Present study suggests that bacterial diversity on grapes may be wider than previously described.

Bacterial communities in Asian dust-containing snow layers on Mt. Tateyama, Japan

Asian dust particles transported from Asian desert areas are trapped in spring snow on the western plateau of Mt. Tateyama, Japan. We investigated bacterial communities in snow layers containing Asian dust, and other underlying snow layers, on Mt. Tateyama during 2008 and 2009. Several genera of bacteria were present among isolates obtained from Asian dust-containing snow layers, including the presumed dust-associated bacterial genus Bacillus. In particular, we detected isolates with identical 16S rDNA sequences (AB500941) over 2 consecutive years, and these isolates were most closely related to Bacillus subtilis. PCR-denaturing gradient gel electrophoresis (DGGE) analysis showed that bacterial community profiles in the Asian dust-containing snow layers were similar over the 2 years. The bacterial communities differed between the Asian dust-containing layers and other layers. Total bacterial cell counts ranged from 6.85×10² to 2.39×105 cells ml-1, with higher values in the Asian dust-containing layers than in the other layers. A backward trajectory analysis showed that the possible source of Asian dust was similar each year. The bacterial community composition of the Asian dust-containing layers presumably reflected the long-distance transportation of microorganisms from dust source regions.

The Development and Stability of the Genus Bacteriodes from Human Gut Microbiota in HFA Mice Model

Human flora-associated (HFA) mice are frequently applied in studying the ecology and metabolism of human gut microbiota. However, the development and stability of the genus Bacteriodes, a prominent bacteria group of human gut microbiota, in HFA mice have not yet fully been examined. In this study, PCR-denaturing gradient gel electrophoresis (DGGE) analysis was employed to monitor the Bacteriodes community in the fecal microbiota of six HFA Kunming mice during a period of 3 weeks. Based on the DGGE banding patterns, the majority of prominent bands in the HFA mice DGGE profile were also typical bands in the human DGGE profile, despite the absence of three bands (corresponding to two different B. thetaiotaomicron strains and one B. intestinalis strain) from the human DGGE profile. The Dice coefficient of similarity for the fecal microbiota of HFA mice in comparison to the human donor sample ranged between 74 ± 6% and 81 ± 7%. The phylogeny of bands in the DGGE profile showed that the dominant Bacteriodes species in the fecal microbiota of HFA mice were B. thetaiotaomicron, representing 66.7% of all bands. Our results indicate that the genus Bacteriodes in the fecal microbiota of HFA mice was selected from the human donor and could remain relatively stable over time.

Temperature-dependent differences in community structure of bacteria involved in degradation of petroleum hydrocarbons under sulfate-reducing conditions

The aim of this study was to characterize the microbial community involved in anaerobic degradation of petroleum hydrocarbon under low- and moderate-temperature conditions. Sulfate-reducing enrichment cultures growing on crude oil and p-xylene were established at low and moderate temperatures. Bacterial community structures of the cultures were characterized by 16S rRNA gene-based analysis and organisms responsible for degradation of p-xylene were investigated by analysis of the bamA gene, involved in anaerobic degradation of aromatic compounds. The PCR-denaturing gradient gel electrophoresis analysis indicated significant differences in microbial community structures among the cultures, depending on the temperatures of incubation. Difference depending on the temperatures was also observed in the cloning analysis of the bamA gene performed on the p-xylene-degrading enrichment cultures. Majority of clones detected in the culture of moderate temperature were related to Desulfosarcina ovata, whereas more diverse bamA gene sequences were obtained from the culture incubated at low temperature. Temperature-dependent differences in microbial community were demonstrated by the analyses of two genes. It was suggested that sulfate-reducing bacteria of phylogenetically different groups might be involved in the degradation of petroleum hydrocarbons in different temperature environments. This study is the first report of p-xylene-degrading sulfate-reducing enrichment culture at low temperature. The results of the experiments at low temperature were distinctly different from those reported in previous studies performed at moderate temperatures.

Effects of autochthonous microbial community on the die-off of fecal indicators in tropical beach sand

The recently observed high levels of fecal indicators in beach sand confound beach water monitoring efforts. The high levels of fecal indicators may be caused by the loss or the reduced activities of common environmental stresses controlling die-off in the sand. Microcosm experiments were conducted to compare the effects of biotic stresses from autochthonous sand bacteria, protozoa, and viruses on Escherichia coli and Enterococcus faecalis in two tropical beach sands. The inhibition of protozoan activities by cycloheximide did not significantly affect the die-off of E. coli, indicating that protozoan predation played a limited role in beach sand. The contribution from phage infection to E. coli die-off was also negligible. Consequently, autochthonous bacteria were identified as the predominant biotic stress to the die-off of E. coli in beach sand. Subsequent experiments demonstrated that the beach sand had a very low protozoan concentration and low protozoan growth potential when compared with various environmental samples. Co-culturing of E. coli with autochthonous sand bacterial isolates significantly enhanced E. coli die-off. PCR-denaturing gradient gel electrophoresis analysis revealed a complex sand bacterial community, suggesting that bacterial antagonistic effects may be widespread. The study also found that E. faecalis exhibited a much longer survival in beach sand compared with E. coli.

Evaluation of the specificity and effectiveness of selected oral hygiene actives in salivary biofilm microcosms

The microbiological effects of biocidal products used for the enhancement of oral hygiene relate to the active compound(s) as well as other formulation components. Here, we test the specificities of selected actives in the absence of multiple excipients. Salivary ecosystems were maintained in tissue culture plate-based hydroxyapatite disc models (HDMs) and modified drip-flow biofilm reactors (MDFRs). Test compounds stannous fluoride (SF), SDS, triclosan (TCS), zinc lactate (ZL) and ZL with SF in combination (ZLSF) were delivered to the HDMs once and four times daily for 6 days to MDFRs. Plaques were characterized by differential viable counting and PCR-denaturing gradient gel electrophoresis (DGGE). TCS and SDS were the most effective compounds against HDM plaques, significantly reducing total viable counts (P<0.05), whilst SF, ZL and ZLSF were comparatively ineffective. TCS exhibited specificity for streptococci (P<0.01) and Gram-negative anaerobes (P<0.01) following a single dosing and also on repeated dosing in MDFRs. In contrast to single exposures, multiple dosing with ZLSF also significantly reduced all bacterial groups, whilst SF and ZL caused significant but transient reductions. According to PCR-DGGE analyses, significant (P<0.05) reductions in eubacterial diversity occurred following 6 day dosing with both TCS and ZLSF. Concordance of MDFR eubacterial profiles with salivary inocula ranged between 58 and 97%. TCS and ZL(SF) exhibited similar specificities to those reported for formulations. TCS was the most potent antibacterial, after single and multiple dosage regimens.

Correlation of Particular Bacterial PCR-Denaturing Gradient Gel Electrophoresis Patterns with Bovine Ruminal Fermentation Parameters and Feed Efficiency Traits

The influence of rumen microbial structure and functions on host physiology remains poorly understood. This study aimed to investigate the interaction between the ruminal microflora and the host by correlating bacterial diversity with fermentation measurements and feed efficiency traits, including dry matter intake, feed conversion ratio, average daily gain, and residual feed intake, using culture-independent methods. Universal bacterial partial 16S rRNA gene products were amplified from ruminal fluid collected from 58 steers raised under a low-energy diet and were subjected to PCR-denaturing gradient gel electrophoresis (DGGE) analysis. Multivariate statistical analysis was used to relate specific PCR-DGGE bands to various feed efficiency traits and metabolites. Analysis of volatile fatty acid profiles showed that butyrate was positively correlated with daily dry matter intake (P < 0.05) and tended to have higher concentration in inefficient animals (P = 0.10), while isovalerate was associated with residual feed intake (P < 0.05). Our results suggest that particular bacteria and their metabolism in the rumen may contribute to differences in host feed efficiency under a low-energy diet. This is the first study correlating PCR-DGGE bands representing specific bacteria to metabolites in the bovine rumen and to host feed efficiency traits.

Molecular community analysis of arbuscular mycorrhizal fungi associated with five selected plant species from heavy metal polluted soils

Arbuscular mycorrhizal fungi (AMF) are known to play an important role in plant tolerance to heavy metals (HMs) stress. This study aimed to understand the diversity of AMF communities associated with five selected plant species ( Phytolacca americana, Rehmannia glutinosa, Perilla frutescens, Litsea cubeba and Dysphania ambrosioides) from severely HMs polluted soils in Dabaoshan Mine region, China, using molecular methods. Plant roots and rhizospheric soils were sampled from four sites, respectively. Targeting the fungal small subunit (SSU) rRNA gene, PCR-denaturing gradient gel electrophoresis (DGGE) analysis indicated that varied AMF communities colonized different plant species, and the AMF communities in rhizospheric soils were different from those in plant roots. Total six SSU rRNA gene clone libraries including four root samples and two rhizospheric soil samples were constructed. Screening clone libraries by DGGE and sequence analysis revealed that Glomus dominated all of the samples except for the roots of D. ambrosioides, while Kuklospora and Ambispora dominated the roots of D. ambrosioides and the rhizosphere of P. americana. This study indicates that diverse AMF are associated with these selected plants, and they are potentially useful to promote the phytoremediation of this HMs polluted area.

Chemosynthetic bacteria found in bivalve species from mud volcanoes of the Gulf of Cadiz

As in other cold seeps, the dominant bivalves in mud volcanoes (MV) from the Gulf of Cadiz are macrofauna belonging to the families Solemyidae (Acharax sp., Petrasma sp.), Lucinidae (Lucinoma sp.), Thyasiridae (Thyasira vulcolutre) and Mytilidae (Bathymodiolus mauritanicus). The delta(13)C values measured in solemyid, lucinid and thyasirid specimens support the hypothesis of thiotrophic nutrition, whereas isotopic signatures of B. mauritanicus suggest methanotrophic nutrition. The indication by stable isotope analysis that chemosynthetic bacteria make a substantial contribution to the nutrition of the bivalves led us to investigate their associated bacteria and their phylogenetic relationships based on comparative 16S rRNA gene sequence analysis. PCR-denaturing gradient gel electrophoresis analysis and cloning of bacterial 16S rRNA-encoding genes confirmed the presence of sulfide-oxidizing symbionts within gill tissues of many of the studied specimens. Phylogenetic analysis of bacterial 16S rRNA gene sequences demonstrated that most bacteria were related to known sulfide-oxidizing endosymbionts found in other deep-sea chemosynthetic environments, with the co-occurrence of methane-oxidizing symbionts in Bathymodiolus specimens. This study confirms the presence of several chemosynthetic bivalves in the Gulf of Cadiz and further highlights the importance of sulfide- and methane-oxidizing symbionts in the trophic ecology of macrobenthic communities in MV.

Inactivation of food spoilage bacteria by high pressure processing: Evaluation with conventional media and PCR–DGGE analysis

Microbial diversity and dynamic changes of sliced vacuum-packed cooked ham during refrigerated storage (0–90 days) after high pressure processing (400 MPa at 22 °C for 10 min) was investigated by using culture-dependent and culture-independent approaches. Isolation of genome DNA and total RNA directly from meat samples, followed by PCR–denaturing gradient gel electrophoresis (DGGE) and RT-PCR–DGGE on 16S rDNA V3 region, was performed to describe the structure of the bacterial community and active species in pressurized sliced cooked ham. The DGGE profile showed that most spoilage bacteria including Lactococcus garvieae, Weissella cibaria, Lactobacillus sakei, Lactobacillus curvatus, Weissella paramesenteroides, Leuconostoc carnosum and Lactococcus lactis subsp. lactis were completely inactivated after high pressure processing (HPP), whereas Weissella viridescens and Weissella minor survived HPP and induced the final spoilage. The microbial diversity of HPP samples during the whole refrigerated storage period was extremely simple. Our results clearly indicated that HPP was an efficient method for avoiding the growth of the major spoilage bacteria and could be used to prolong the shelf-life of sliced vacuum-packed cooked ham.

Characterization of Variation in Rumen Methanogenic Communities under Different Dietary and Host Feed Efficiency Conditions, as Determined by PCR-Denaturing Gradient Gel Electrophoresis Analysis

Understanding ruminal methanogens is essential for greenhouse gas mitigation, as well as for improving animal performance in the livestock industry. It has been speculated that ruminal methanogenic diversity affects host feed efficiency and results in differences in methane production. This study examined methanogenic profiles in the rumen using culture-independent PCR-denaturing gradient gel electrophoresis (PCR-DGGE) analysis for 56 beef cattle which differed in feed efficiency, as well as diet (the cattle were fed a low-energy diet or a high-energy diet). The methanogenic PCR-DGGE profiles detected were greatly affected by diet, and the major pattern changed from a community containing predominantly Methanobrevibacter ruminantium NT7 with the low-energy diet to a community containing predominantly Methanobrevibacter smithii, Methanobrevibacter sp. AbM4, and/or M. ruminantium NT7 with the high-energy diet. For each diet, the methanogenic PCR-DGGE pattern was strongly associated with the feed efficiency of the host. Diet-associated bands for Methanobrevibacter sp. AbM4 and M. smithii SM9 and a feed efficiency-related band for M. smithii PS were identified. The abundance of total methanogens was estimated by determining the numbers of copies of the 16S rRNA genes of methanogens. However, the size of the methanogen population did not correlate with differences in feed efficiency, diet, or metabolic measurements. Thus, the structure of the methanogenic community at the species or strain level may be more important for determining host feed efficiency under different dietary conditions.

Effects of Plant Genotype and Growth Stage on the Betaproteobacterial Communities Associated with Different Potato Cultivars in Two Fields

Bacterial communities in the rhizosphere are dynamic and susceptible to changes in plant conditions. Among the bacteria, the betaproteobacteria play key roles in nutrient cycling and plant growth promotion, and hence the dynamics of their community structures in the rhizosphere should be investigated. Here, the effects of plant cultivar, growth stage, and soil type on the communities associated with potato cultivars Aveka, Aventra, Karnico, Modena, Premiere, and Désirée were assessed for two different fields containing sandy soil with either a high or low organic compound content. Thus, bacterial and betaproteobacterial PCR-denaturing gradient gel electrophoresis analyses were performed to analyze the effects of plant cultivar and growth on the rhizosphere community structure. The analyses showed that in both fields all cultivars had a rhizosphere effect on the total bacterial and betaproteobacterial communities. In addition, the plant growth stage strongly affected the betaproteobacterial communities in both fields. Moreover, the community structures were affected by cultivar, and cultivars differed in physiology, as reflected in their growth rates, root development, and estimated tuber starch contents. Analyses of betaproteobacterial clone libraries constructed for two selected cultivars (one cultivar that produced low-starch-content tubers and one cultivar that produced high-starch-content tubers), as well as bulk soil, revealed that the rhizospheres of the two cultivars selected for specific bacteria, including plant-growth-promoting bacteria, such as Variovorax and Achromobacter spp. In addition, quantitative PCR-based quantification of the Variovorax paradoxus-specific functional gene asfA (involved in desulfonation) indicated that there were clear potato rhizosphere effects on the abundance of this gene. Interestingly, both cultivar type and plant growth stage affected the community under some circumstances.

Seasonal modulation of bacterioplankton community at a temperate eutrophic shallow lake

At the present time, there is still a lack of information about environmental parameters modulating variations on bacterial diversity in temperate lakes, particularly from Portugal. Fermentelos Lake (Central Portugal) is a shallow water body that sustains an important wetland area. The strong nutrient inputs from agriculture and industrial runoffs have led to its current eutrophic status. The present work aimed to understand which factors modulate the seasonal bacterioplankton diversity at this lake using 16S rRNA PCR-denaturing gradient gel electrophoresis (DGGE) and multivariate analysis. Environmental data demonstrated eutrophic features throughout all samples with nitrate concentrations reaching 12.0 mg N (NO3−) l−1 in March 2006, while the highest conductivity (609 μS cm−1), soluble reactive phosphorus (0.37 mg l−1), total suspended solids (87.2 mg l−1) and chlorophyll a (286.6 μg l−1) levels were recorded in August 2007. Over the past two decades there was a general increase in nitrate, pH and conductivity levels at this lake, suggesting the eutrophication process is still in progress. Multivariate analysis showed that summer versus winter DGGE patterns could be established for bacterial assemblages and were mainly defined by water temperature and chlorophyll a. Actinobacteria were dominant throughout the study period although a general preference for higher temperature, pH, total suspended solids, conductivity, soluble reactive phosphorus (SRP) and chlorophyll a levels was observed. The highest concentrations of nitrogen sources were related to Bacteroidetes and phototrophic eukaryote (cryptophycean) dominance. The expansion of Betaproteobacteria, Alphaproteobacteria and Cyanobacteria phylotypes was generally associated to high temperature, pH, conductivity and SRP values.

The bacterial community of tomato rhizosphere is modified by inoculation with arbuscular mycorrhizal fungi but unaffected by soil enrichment with mycorrhizal root exudates or inoculation with Phytophthora nicotianae

Arbuscular mycorrhizal (AM) fungi have been shown to induce the biocontrol of soilborne diseases, to change the composition of root exudates and to modify the bacterial community structure of the rhizosphere, leading to the formation of the mycorrhizosphere. Tomato plants were grown in a compartmentalized soil system and were either submitted to direct mycorrhizal colonization or to enrichment of the soil with exudates collected from mycorrhizal tomato plants, with the corresponding negative controls. Three weeks after planting, the plants were inoculated or not with the soilborne pathogen Phytophthora nicotianae growing through a membrane from an adjacent infected compartment. At harvest, a PCR-Denaturing gradient gel electrophoresis analysis of 16S rRNA gene fragments amplified from the total DNA extracted from each plant rhizosphere was performed. Root colonization with the AM fungi Glomus intraradices or Glomus mosseae induced significant changes in the bacterial community structure of tomato rhizosphere, compared to non-mycorrhizal plants, while enrichment with root exudates collected from mycorrhizal or non-mycorrhizal plants had no effect. Our results support that the effect of AM fungi on rhizosphere bacteria would not be mediated by compounds present in root exudates of mycorrhizal plants but rather by physical or chemical factors associated with the mycelium, volatiles and/or root surface bound substrates. Moreover, infection of mycorrhizal or non-mycorrhizal plants with P. nicotianae did not significantly affect the bacterial community structure suggesting that rhizosphere bacteria would be less sensitive to the pathogen invasion than to mycorrhizal colonization. Of 96 unique sequences detected in the tomato rhizosphere, eight were specific to mycorrhizal fungi, including two Pseudomonas, a Bacillus simplex, an Herbaspirilium and an Acidobacterium. One Verrucomicrobium was common to rhizospheres of mycorrhizal plants and of plants watered with mycorrhizal root exudates.

Characterization of microbial communities in the aqueous phase of a constructed model wetland treating 1,2-dichloroethene-contaminated groundwater

The dynamics and composition of microbial communities in the aqueous phase of a model wetland supplied with cis- and trans-1,2-dichloroethenes (DCE)-contaminated groundwater was characterized. PCR-denaturing gradient gel electrophoresis analysis of water samples obtained from different parts of the wetland revealed that changes of the bacterial community structure coincided with a succession of the hydrochemical conditions in the wetland, from oxic towards anoxic conditions. During this transition phase, the appearance of vinyl chloride and ethene correlated with the presence of putative dechlorinating bacteria (Dehalococcoides spp., Geobacter spp. and Dehalobacter spp.). Additionally, a shift of the DCE isotopic composition indicated the progressive prevalence of reductive dechlorination in the wetland. Although the DCE degradation processes varied over time, biodegradation activity was maintained in the wetland system. 16S rRNA gene libraries revealed that Proteobacteria accounted for >50% of 16S rRNA genes clone libraries, whereas approximately 17% of the sequences from the wetland were related to sulphate reducers. Based on a multiple-method approach, this study illustrates the linkage between microbial community dynamics and composition, changes of hydrochemical conditions and processes of DCE degradation in a wetland system.

Bacterial dynamics during the spontaneous fermentation of cassava dough in gari production

The microbial dynamics and diversity during solid substrate fermentation of cassava, a case study of gari production in West Africa, was investigated. The 16S rDNA gene sequence analysis of the PCR-Denaturing Gradient Gel Electrophoresis (PCR–DGGE) analysis of microbial community DNA and Pulsed Field Gel Electrophoresis (PFGE) of selected isolates as well as culturing techniques using different selective media were used to monitor the bacterial dynamics during cassava fermentation. The V3 variable region of the 16S gene was analyzed and the closest relatives of Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus pentosus, Lactobacillus acidophilus and Lactobacillus casei were identified by sequencing of the DGGE band amplimers. The DGGE amplimers also revealed the succession and dynamics of LAB; there was a progressive increase in their population proportional to the fermentation period. The analysis of the PFGE band patterns showed that five diverse species of LAB were involved in the fermentation. The representative isolate of each of the PFGE clusters was phenotypicaly identified as L. plantarum, L. fermentum and Leuconostoc mesenteroides by the API 50 CHL sugar fermentation profile. These combinations of parameters identified heterofermentative LAB as bacteria that initiated the fermentation, reduced the pH below four and increased the acidity of the fermentation mash. Information such as this is relevant for the development of starter cultures and predictability of the process for traditional fermented foods and to aid their intermediate and large scale production.