Dominant Denaturing Gradient Gel Electrophoresis Bands Research Articles

Spatial heterogeneity in a deep artificial lake plankton community revealed by PCR-DGGE fingerprinting

To explore the spatial heterogeneity of plankton communities in a deep artificial lake (Songhua Lake, China), samples were collected at seven sites. Samples were investigated by denaturing gradient gel electrophoresis (DGGE) analysis of the PCR-amplified 16S and 18S rRNA genes and specific bands were sequenced. Cluster analysis of the DGGE profiles revealed that all of the samples grouped into two distinct clusters, in accordance with sampling site; while in each cluster, the divergence of sub-clusters correlated with sampling depth. Sequence analysis of selected dominant DGGE bands revealed that most sequenced phylotypes (84%) exhibited ≥97% similarity to the closest sequences in GenBank, and were affiliated with ten common freshwater plankton phyla (Proteobacteria, Actinobacteria, Bacteroidetes, Cyanobacteria, Bacillariophyta, Pyrrophyta, Cryptophyta, Ciliophora, Stramenopiles, and Rotifera). Several of these groups are also found worldwide, indicating the cosmopolitan distribution of the phylotypes. The relationships between DGGE patterns and environmental factors were analyzed by redundancy analysis (RDA). The results suggested that, total nitrogen, nitrate, nitrite, ammonia, and CODMn concentrations, and water temperature were strongly correlated with the variation in plankton composition.

English

The rotating biological contactor (RBC) process was frequently used for the biological wastewater treatment in order to remove pollutants and to improve the water qualitybefore discharge to the environment. The presence of bacteria species in the secondary treated wastewater indicates the necessity of a tertiary treatment process [ultraviolet (UV)-C radiation disinfection] to reduce the number of living organisms in the water. Denaturing gradient gel electrophoresis (DGGE) method using 16S rDNA was commonly used for a direct comparison of structural diversity among different microbial communities. In the present study, community in treated and untreated wastewater from RBC treatment plantwas investigated using DGGE coupled with sequence analysis of 16S rRNA gene fragments from bands of interest. The analysis of the DGGE profiles and the sequence of the dominant DGGE bands showed a variability of the bacterial community with season. DGGE patterns of samples collected in summer were more complex than those collected in winter. In addition, the investigation of the effect of increasing UV253.7 germicidal doses on the bacterial community, in secondary treated wastewater effluent, revealed variabilityin bacterial tolerance to UV253.7 radiation. This variability is inter-specific and is dependent on the UV-C dose used and the bacterial specie irradiated. Consequently, this study demonstrated that DGGE method coupled with sequencing provides precise information on RBC and UV-C wastewater treatment process.   Key words: Wastewater, biological treatment, bacterial communities, polymerase chain reaction - denaturing gradient gel electrophoresis (PCR-DGGE), 16S rDNA, ultraviolet (UV)-C radiation disinfection.

Dynamics of indigenous bacterial communities associated with crude oil degradation in soil microcosms during nutrient-enhanced bioremediation

Bacterial population dynamics were examined during bioremediation of an African soil contaminated with Arabian light crude oil and nutrient enrichment (biostimulation). Polymerase chain reaction followed by denaturing gradient gel electrophoresis (DGGE) were used to generate bacterial community fingerprints of the different treatments employing the 16S ribosomal ribonucleic acid (rRNA) gene as molecular marker. The DGGE patterns of the nutrient-amended soils indicated the presence of distinguishable bands corresponding to the oil-contaminated-nutrient-enriched soils, which were not present in the oil-contaminated and pristine control soils. Further characterization of the dominant DGGE bands after excision, reamplification and sequencing revealed that Corynebacterium spp., Dietzia spp., Rhodococcus erythropolis sp., Nocardioides sp., Low G+C (guanine plus cytosine) Gram positive bacterial clones and several uncultured bacterial clones were the dominant bacterial groups after biostimulation. Prominent Corynebacterium sp. IC10 sequence was detected across all nutrient-amended soils but not in oil-contaminated control soil. Total heterotrophic and hydrocarbon utilizing bacterial counts increased significantly in the nutrient-amended soils 2 weeks post contamination whereas oil-contaminated and pristine control soils remained fairly stable throughout the experimental period. Gas chromatographic analysis of residual hydrocarbons in biostimulated soils showed marked attenuation of contaminants starting from the second to the sixth week after contamination whereas no significant reduction in hydrocarbon peaks were seen in the oil-contaminated control soil throughout the 6-week experimental period. Results obtained indicated that nutrient amendment of oil-contaminated soil selected and enriched the bacterial communities mainly of the Actinobacteria phylogenetic group capable of surviving in toxic contamination with concomitant biodegradation of the hydrocarbons. The present study therefore demonstrated that the soil investigated harbours hydrocarbon-degrading bacterial populations which can be biostimulated to achieve effective bioremediation of oil-contaminated soil.

Soil purple phototrophic bacterial diversity under double cropping (rice-wheat) with free-air CO2 enrichment (FACE)

Soil purple phototrophic bacterial (PPB) communities and their responses to elevated atmospheric carbon dioxide (CO 2 ) concentration and nitrogen (N) fertilizer were investigated under a rotation of paddy rice (Oryza sativa L.) and winter wheat (Triticum aestivum L. cv. Yangmai 14) cultivation in a FACE (free-air CO 2 enrichment) system. Community structures and abundances of PPB were determined by denaturing gradient gel electrophoresis (DGGE) and real-time quantitative PCR respectively, targeting the pufM gene, which encodes a protein in the light reaction centre of PPB. Soil PPB communities were more diverse and larger under rice than under wheat cultivation, which may be attributed to the flooding of the paddy field and soil moisture changes. Elevated atmospheric CO 2 concentration significantly increased the abundance and biodiversity of PPB in soils under rice cultivation, while N fertilizer application rate had less effect on the abundance and diversity. Phylogenetic analysis showed that two common dominant DGGE bands belonged to Bradyrhizobium- and Rhodopseudomonas palustris-like PPB in both rice and wheat soils. The results demonstrated a significant shift in soil PPB communities during the rice-wheat rotation, and a strong positive response of PPB communities to an elevated atmospheric CO 2 concentration. Our results also indicated that a diverse and abundant soil PPB community could occur in upland crop fields as well as in aquatic environments and paddy-rice fields. These findings extend our understanding of the ecological significance of PPB in terrestrial soil environments and their responses to future climate change.

Biodegradation of specified risk material and characterization of actinobacterial communities in laboratory-scale composters

As a result of bovine spongiform encephalopathy in Canada, specific tissues at risk of harbouring prions are not allowed to enter the food chain. Composting may be a viable alternative to rendering and land filling for the disposal of specified risk material (SRM). Two types of laboratory-scale composters, actively-heated and ambient systems were constructed to assess the biodegradation of SRM over 30days. A second heating cycle was generated by mixing the compost after 15days. Compared to ambient composters, temperature profiles in actively-heated composters were above 50°C for 5 and 4days longer in the first and second composting cycles, respectively. Degradation of SRM was similar between two composter types during two composting cycles, averaging 52.2% in the first cycle and 43.9% in second cycle. Denaturing gradient gel electrophoresis (DGGE) revealed that changes in the actinobacteria populations in the first composting cycle were of a temporal nature, whereas alterations in populations in the second composting cycle were more related to active heating of compost. Sequencing of the dominant DGGE bands showed the predominance of Corynebacterium, Promicromonospora, Pseudonocardia, and Thermobifida in the first composting cycle and Corynebacterium, Mycobacterium, Nocardia, Saccharomonospora, and Streptomyces in the second composting cycle. Active heating can alter the nature of actinobacteria populations in compost, but does not appear to have a major impact on the extent of degradation of SRM.

Cultivation-Independent Analysis of the Development of the Lactobacillus spp. Community in the Intestinal Tract of Newborn Piglets

Abstract Molecular diversity and development of the Lactobacillus community in the intestinal tract, as influenced by age and intestinal compartment, were studied in one litter of 12 conventionally raised piglets. Piglets were euthanized at each week (3 animals per time). Digesta and tissue samples from stomach, duodenum, jejunum, ileum, caecum, colon, and rectum were collected and analysed by using 16S ribosomal RNA-based methods. DGGE (denaturing gradient gel electrophoresis) profiles revealed that the Lactobacillus communities throughout the GI tract from duodenum to rectum showed good stability at same age. This indicates that fecal Lactobacillus communities can effectively represent the intestinal community. Two dominant bands were found in tissue samples of the small intestine, suggesting that the lactobacilli can adhere to the small intestinal wall. The Lactobacillus communities in different GI tract compartments developed over time. A successional change of Lactobacillus communities was observed from birth, through creep feeding to one week after weaning, showing a trend from simple to complex and back to simple. Furthermore, a clone library of Lactobacillus spp. 16S rRNA gene sequences were generated from jejunal and colonic chymes. Six dominant DGGE bands generated from jejunal chymes were matched with sequences that show 94-98% similarity to the bands derived from L. reuteri, L. delbrueckii, and L. crispatus. Seven dominant DGGE bands generated from colon chymes were matched with sequences that show 88-99% similarity to those derived from L. reuteri, L. delbrueckii, L. amylovorus/L. sobrius, and L. acidophilus. Amplicons related to L. reuteri were found in all DGGE fingerprints from jejunal digesta of age of weeks 1, 3, and 4. Amplicons related to L. amylovorus/L. sobrius were present in all DGGE fingerprints from colonic digesta of age of week 1, 3, and 4. Amplicons related to L. delbrueckii were found before weaning, L. crispatus after creep feeding before weaning and L. acidophilus after weaning. This indicates that L. reuteri and L. amylovorus/L. sobrius probably belong to the permanent composition, while L. delbruckii, L. acidophilus, and L. crispatus probably belong to the temporal groups of Lactobacillus communities in the GI tract of piglets.

Community Structure of Bacteria Associated with Microcystis Colonies from Cyanobacterial Blooms

ABSTRACT Thirty Microcystis colonies, belonging to three species (Microcystis aeruginosa, Microcystis flos-aquae, and Microcystis wesenbergii) were isolated from Lake Taihu and Lake Chaohu in China. Bacterial communities associated with the single Microcystis colonies were investigated using a seminested polymerase chain reaction-amplification along with denaturing gradient gel electrophoresis (DGGE). The bacterial communities of most colonies of the same Microcystis species in each lake tended to be similar. This indicates that species-specific associations may exist between colony-associated bacteria and Microcystis spp. Sequence analysis of dominant DGGE bands revealed that the dominant bacterial groups associated with Microcystis colonies were Alphaproteobacteria, Betaproteobacteria, Garnmaproteobacteria, Bacteroidetes, Firmicutes, Deinococcus-Thermus, and Gemmatimonadetes. Phylotypes related to Pseudomonas sp., Serratia sp., and Flexibacteraceae were dominant in colonies of M. wesenbergii, M flos-aquae, and M aeruginosa, respectively.

Bacterial and archaeal assemblages in sediments of a large shallow freshwater lake, Lake Taihu, as revealed by denaturing gradient gel electrophoresis

To explore the association of microbial community structure with the development of eutrophication in a large shallow freshwater lake, Lake Taihu. The bacterial and archaeal assemblages in sediments of different lake areas were analysed using denaturing gradient gel electrophoresis (DGGE) of amplified 16S rDNA fragments. The bacterial DGGE profiles showed that eutrophied sites, grass-bottom areas and relatively clean sites with a eutrophic (albeit dredged) site are three respective clusters. Fifty-one dominant bacterial DGGE bands were detected and 92 corresponding clones were sequenced, most of which were affiliated with bacterial phylotypes commonly found in freshwater ecosystems. Actinobacteria were detected in the centre of the lake and not at eutrophied sites whereas the opposite was found with respect to Verrucomicrobiales. Twenty-five dominant archaeal DGGE bands were detected and 31 corresponding clones were sequenced, most of which were affiliated with freshwater archaeal phylotypes. The bacterial community structures in the sediments of different areas with similar water quality and situation tend to be similar in Taihu Lake. This study may expand our knowledge on the relationship between the overall microbial assemblages and the development of eutrophication in the shallow freshwater lake.

Exploring the diversity of bacterial communities in sediments of urban mangrove forests

Municipal sewage, urban runoff and accidental oil spills are common sources of pollutants in urban mangrove forests and may have drastic effects on the microbial communities inhabiting the sediment. However, studies on microbial communities in the sediment of urban mangroves are largely lacking. In this study, we explored the diversity of bacterial communities in the sediment of three urban mangroves located in Guanabara Bay (Rio de Janeiro, Brazil). Analysis of sediment samples by means of denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene fragments suggested that the overall bacterial diversity was not significantly affected by the different levels of hydrocarbon pollution at each sampling site. However, DGGE and sequence analyses provided evidences that each mangrove sediment displayed a specific structure bacterial community. Although primer sets for Pseudomonas, alphaproteobacterial and actinobacterial groups also amplified ribotypes belonging to taxa not intended to be enriched, sequence analyses of dominant DGGE bands revealed ribotypes related to Alteromonadales, Burkholderiales, Pseudomonadales, Rhodobacterales and Rhodocyclales. Members of these groups were often shown to be involved in aerobic or anaerobic degradation of hydrocarbon pollutants. Many of these sequences were only detected in the sampling sites with high levels of anthropogenic inputs of hydrocarbons. Many dominant DGGE ribotypes showed low levels of sequence identity to known sequences, indicating a large untapped bacterial diversity in mangrove ecosystems.

Removal of volatile fatty acids (VFA) by microbial fuel cell with aluminum electrode and microbial community identification with 16S rRNA sequence

Removal of volatile fatty acids in anaerobic digestion of organic wastes can accelerate eventual decomposition of organic wastes to CO2 and H2O using a recovery of electric energy by a microbial fuel cell. The fuel cell anode chamber was a 10 cm (I.D.)×20 cm long cylindrical Plexiglass having an ion ceramic cylinder separator (I.D.10 mm, O.D.12 mm, 0.3 μm average pore size). The aluminum foil cathode (12 cm2 surface area) was located inside the ceramic cylinder. Between the two cylinders, 1 liter of activated carbon particles was packed as anode electrode having a void fraction of 0.4. This fuel cell was connected to a 5 liter bioreactor (working volume 1.5 liter), and the bioreactor was run in batch mode by re-circulating a synthetic wastewater of 5 g/L glucose. Maximum TVFA (total volatile fatty acids) and SCOD (soluble chemical oxygen demand) removal rate were 3.79 g/L·day, 5.88 g/L·day, respectively. TVFA removal efficiency (92.7%) and SCOD removal efficiency (94.7%) under maximum current density operation were higher than the operation with maximum power density. In acid fermentation, butyric acid concentration was highest because Clostridium butyricum was a dominant microbial communitiy in the inoculum. The microbial cells collected from the anode bio-film samples were affiliated with Bacillus cereus based on the nucleotide sequences of dominant DGGE (denaturing gradient gel electrophoresis) bands.

Resilience of the rhizosphere Pseudomonas and ammonia‐oxidizing bacterial populations during phytoextraction of heavy metal polluted soil with poplar

We assessed the effects of phytoextraction on the dynamics of Pseudomonas spp. and ammonia-oxidizing bacterial populations in a heavy metal (HM) polluted soil. Hybrid poplars were grown in two-compartment root containers with a medium history (> 4 years) of HM pollution for 13 weeks. Bulk and poplar rhizosphere soils were analysed by denaturing gradient gel electrophoresis (DGGE) of Pseudomonas (sensu stricto) 16S rRNA and amoA gene fragments. DGGE patterns revealed that Pseudomonas and amoA-containing populations in the contaminated soils were markedly different from those in the uncontaminated soils. Pseudomonas and amoA profiles appeared to be stable over time in the bulk soils. In contrast, contaminated rhizosphere soils revealed a clear shift of populations with removal of HM becoming similar or at least shifted to the populations of the uncontaminated soils. The effect of phytoextraction was, however, not evident in the bulk samples, which still contained large amounts of HM. Cloning and sequencing of dominant DGGE bands revealed that Pseudomonas were phylogenetically related to the Pseudomonas fluorescens cluster and the amoA sequences to Nitrosospira spp. At the last sampling, major prominent band sequences from contaminated rhizosphere soils were identical to sequences obtained from uncontaminated rhizosphere soils, indicating that the populations were dominated by the same phylotypes. This study suggests that two taxonomically different populations are able to recover after the relief of HM stress by phytoextraction practices, whereas bulk microbial activities still remained depressed.

Molecular characterization of methane-oxidizing bacteria associated with rice straw decomposition in a rice field

Methane-oxidizing bacteria (MOB) are crucial to the reduction of CH4 emitted to the atmosphere. However, it is unclear how MOB in rice straw are affected by straw decomposition processes. In a Japanese rice field, a year-round experiment was set up to study the effects of agricultural practice (rice cultivation/winter fallow), straw parts (leaf sheath/blade) and the site of straw placement (plow layer/soil surface) on MOB communities in rice straw using denaturing gradient gel electrophoresis (DGGE) and DNA sequencing analyses of key MOB functional genes (pmoA and amoA). Thirty-eight different DGGE bands were observed over the entire investigation period. Principal component analysis of DGGE pattern suggested that agricultural practice is the key factor regulating the MOB communities. Sequencing of dominant DGGE bands showed that: (1) during the rice cultivation period, methanotrophs (particularly type I methanotrophs) dominated the MOB community, (2) during the winter fallow season both type I and type II methanotrophs were dominant in sheath segments placed both on the soil surface and in the plow layer, whereas ammonia oxidizers seemed to dominate blade segments placed in the plow layer. Alignment of diagnostic amino acid sequences of MOB suggested the presence of novel ammonia oxidizers in rice straw in rice fields.

Isolation of Methylophaga spp. from Marine Dimethylsulfide-Degrading Enrichment Cultures and Identification of Polypeptides Induced during Growth on Dimethylsulfide

Dimethylsulfide (DMS)-degrading enrichment cultures were established from samples of coastal seawater, nonaxenic Emiliania huxleyi cultures, and mixed marine methyl halide-degrading enrichment cultures. Bacterial populations from a broad phylogenetic range were identified in the mixed DMS-degrading enrichment cultures by denaturing gradient gel electrophoresis (DGGE). Sequences of dominant DGGE bands were similar to those of members of the genera Methylophaga and Alcanivorax. Several closely related Methylophaga strains were obtained that were able to grow on DMS as the carbon and energy source. Roseobacter-related populations were detected in some of the enrichment cultures; however, none of the Roseobacter group isolates that were tested were able to grow on DMS. Oxidation of DMS by Methylophaga sp. strain DMS010 was not affected by addition of the inhibitor chloroform or methyl tert-butyl ether, suggesting that DMS metabolism may occur by a route different from those described for Thiobacillus species and other unidentified marine isolates. Addition of DMS and methanethiol to whole-cell suspensions of strain DMS010 induced oxygen uptake when strain DMS010 was grown on DMS but not in cells grown on methanol. The apparent K(m)s of strain DMS010 for DMS and for methanethiol were 2.1 and 4.6 microM, respectively, when grown on DMS. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the biomass of strain DMS010 and analysis of peptide bands by mass spectrometry techniques and N-terminal sequencing provided the first insight into the identity of polypeptides induced during growth on DMS. These included XoxF, a homolog of the large subunit of methanol dehydrogenase for which a biological role has not been identified previously.

Analysis of bacterial communities on aging flue-cured tobacco leaves by 16S rDNA PCR–DGGE technology

Many microorganisms, growing on aging flue-cured tobacco leaves, play a part in its fermentation process. These microflora were identified and described by culture-dependent methods earlier. In this study we report the identity of the microflora growing on the tobacco leaf surface by employing culture-independent methods. We have amplified microbial 16S rDNA sequences directly from the leaf surface and used denaturing gradient gel electrophoresis (DGGE) to identify bacterial community on the tobacco leaves. Our culture-independent methods for the study of microbial community on tobacco leaves showed that microbial community structures on leaves of variety Zhongyan 100, NC89 and Zhongyan 101 were similar between 0 and 6 months aging, and between 9 and 12 months aging, while the similarity is low between 0 and 6, and between 9 and 12 months aging, respectively. There were certain similarities of bacterial communities (similarity up to 63%) among the three tobacco varieties for 0 to 6 months aging. Five dominant 16S rDNA DGGE bands A, B, C, D and E were isolated, cloned, and sequenced. They were most similar to two cultured microbial species Bacteriovorax sp. EPC3, Bacillus megaterium, and three uncultured microbial species, respectively.

Effects of site and plant species on rhizosphere community structure as revealed by molecular analysis of microbial guilds

The bacterial and fungal rhizosphere communities of strawberry (Fragaria ananassa Duch.) and oilseed rape (Brassica napus L.) were analysed using molecular fingerprints. We aimed to determine to what extent the structure of different microbial groups in the rhizosphere is influenced by plant species and sampling site. Total community DNA was extracted from bulk and rhizosphere soil taken from three sites in Germany in two consecutive years. Bacterial, fungal and group-specific (Alphaproteobacteria, Betaproteobacteria and Actinobacteria) primers were used to PCR-amplify 16S rRNA and 18S rRNA gene fragments from community DNA prior to denaturing gradient gel electrophoresis (DGGE) analysis. Bacterial fingerprints of soil DNA revealed a high number of equally abundant faint bands, while rhizosphere fingerprints displayed a higher proportion of dominant bands and reduced richness, suggesting selection of bacterial populations in this environment. Plant specificity was detected in the rhizosphere by bacterial and group-specific DGGE profiles. Different bulk soil community fingerprints were revealed for each sampling site. The plant species was a determinant factor in shaping similar actinobacterial communities in the strawberry rhizosphere from different sites in both years. Higher heterogeneity of DGGE profiles within soil and rhizosphere replicates was observed for the fungi. Plant-specific composition of fungal communities in the rhizosphere could also be detected, but not in all cases. Cloning and sequencing of 16S rRNA gene fragments obtained from dominant DGGE bands detected in the bacterial profiles of the Rostock site revealed that Streptomyces sp. and Rhizobium sp. were among the dominant ribotypes in the strawberry rhizosphere, while sequences from Arthrobacter sp. corresponded to dominant bands from oilseed rape bacterial fingerprints.

Diversity and Functional Analysis of Bacterial Communities Associated with Natural Hydrocarbon Seeps in Acidic Soils at Rainbow Springs, Yellowstone National Park

In this paper we describe the bacterial communities associated with natural hydrocarbon seeps in nonthermal soils at Rainbow Springs, Yellowstone National Park. Soil chemical analysis revealed high sulfate concentrations and low pH values (pH 2.8 to 3.8), which are characteristic of acid-sulfate geothermal activity. The hydrocarbon composition of the seep soils consisted almost entirely of saturated, acyclic alkanes (e.g., n-alkanes with chain lengths of C15 to C30, as well as branched alkanes, predominately pristane and phytane). Bacterial populations present in the seep soils were phylogenetically characterized by 16S rRNA gene clone library analysis. The majority of the sequences recovered (>75%) were related to sequences of heterotrophic acidophilic bacteria, including Acidisphaera spp. and Acidiphilium spp. of the alpha-Proteobacteria. Clones related to the iron- and sulfur-oxidizing chemolithotroph Acidithiobacillus spp. were also recovered from one of the seep soils. Hydrocarbon-amended soil-sand mixtures were established to examine [14C]hexadecane mineralization and corresponding changes in the bacterial populations using denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene fragments. Approximately 50% of the [14C]hexadecane added was recovered as 14CO2 during an 80-day incubation, and this was accompanied by detection of heterotrophic acidophile-related sequences as dominant DGGE bands. An alkane-degrading isolate was cultivated, whose 16S rRNA gene sequence was identical to the sequence of a dominant DGGE band in the soil-sand mixture, as well as the clone sequence recovered most frequently from the original soil. This and the presence of an alkB gene homolog in this isolate confirmed the alkane degradation capability of one population indigenous to acidic hydrocarbon seep soils.

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.

Distribution of eukaryotic picoplankton assemblages across hydrographic fronts in the Southern Ocean, studied by denaturing gradient gel electrophoresis

We used a molecular fingerprinting technique to analyze the distribution and composition of eukaryotic picoplankton along latitudinal transects in the Southern Ocean. First, primers specific for eukaryotic 18S rDNA were used in a polymerase chain reaction (PCR) with environmental DNA. The amplification products were subjected to denaturing gradient gel electrophoresis (DGGE). Transect DOVETAIL (44°W) went from the ice edge (at 60°S) across the Weddell‐Scotia confluence and north to 58°S; it was sampled in January 1998 (summer). Transect DHARMA (between 53°W and 58°W) went from the ice edge in the Weddell Sea (63°S) across the Drake Passage to the South American continental platform (55°S); it was sampled in December 1998 (late spring). DGGE band patterns were used to build dendrograms combining samples from each cruise. Samples were grouped in several distinct clusters that were generally consistent with the hydrography of the area. In DOVETAIL, the upper water column was stratified and the DGGE band patterns varied with depth. In DHARMA the upper mixed layer showed the same composition of the eukaryotic picoplankton at all depths. The most dominant DGGE bands were excised and sequenced. Some were closely related to well‐known components of the plankton such as prasinophytes, prymnesiophytes, dinoflagellates, and diatoms. A significant number of sequences were related to previously unknown phylogenetic groups, including novel stramenopiles and alveolates or to poorly known groups such as cercomonads. This fingerprinting technique is useful for a rapid evaluation of the spatial distribution of picoeukaryotic assemblages in the oceans.

Dynamics of fungal communities in bulk and maize rhizosphere soil in the tropics.

The fungal population dynamics in soil and in the rhizospheres of two maize cultivars grown in tropical soils were studied by a cultivation-independent analysis of directly extracted DNA to provide baseline data. Soil and rhizosphere samples were taken from six plots 20, 40, and 90 days after planting in two consecutive years. A 1.65-kb fragment of the 18S ribosomal DNA (rDNA) amplified from the total community DNA was analyzed by denaturing gradient gel electrophoresis (DGGE) and by cloning and sequencing. A rhizosphere effect was observed for fungal populations at all stages of plant development. In addition, pronounced changes in the composition of fungal communities during plant growth development were found by DGGE. Similar types of fingerprints were observed in two consecutive growth periods. No major differences were detected in the fungal patterns of the two cultivars. Direct cloning of 18S rDNA fragments amplified from soil or rhizosphere DNA resulted in 75 clones matching 12 dominant DGGE bands. The clones were characterized by their HinfI restriction patterns, and 39 different clones representing each group of restriction patterns were sequenced. The cloning and sequencing approach provided information on the phylogeny of dominant amplifiable fungal populations and allowed us to determine a number of fungal phylotypes that contribute to each of the dominant DGGE bands. Based on the sequence similarity of the 18S rDNA fragment with existing fungal isolates in the database, it was shown that the rhizospheres of young maize plants seemed to select the Ascomycetes order Pleosporales, while different members of the Ascomycetes and basidiomycetic yeast were detected in the rhizospheres of senescent maize plants.

Effect of fermentable carbohydrates on piglet faecal bacterial communities as revealed by denaturing gradient gel electrophoresis analysis of 16S ribosomal DNA

The effect of fermentable carbohydrates (sugar beet pulp and fructooligosaccharides) on the faecal bacterial communities of weaning piglets was analysed using 16S rDNA-based approaches. Amplicons of the V6-V8 variable regions of bacterial 16S rDNA were analysed by denaturing gradient gel electrophoresis (DGGE), cloning and sequencing. Differences in piglet faecal bacterial community structure were determined based on the Dice coefficients for pairwise comparison of the DGGE fingerprints and revealed significant changes in the faecal microbiota immediately after weaning. Piglets fed with fermentable carbohydrates showed a higher bacterial diversity and a more rapid stabilisation of the bacterial community compared with that of the animals fed with the control diet. Thirteen dominant DGGE bands were matched with sequences that showed 91-97% similarity to those derived from the Clostridium coccoides group and the Clostridium leptum subgroup. Amplicons related to Ruminococcus-like species were found in all DGGE fingerprints derived from pigs on the diet containing sugar beet pulp and fructooligosaccharides, but not in pigs on the control diet. These results indicate that these bacteria may play a role in the utilisation of dietary fibres.