Phylogenetic composition of Arctic Ocean archaeal assemblages and comparison with Antarctic assemblages

Archaea assemblages from the Arctic Ocean and Antarctic waters were compared by PCR-denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA genes amplified using the Archaea-specific primers 344f and 517r. Inspection of the DGGE fingerprints of 33 samples from the Arctic Ocean (from SCICEX submarine cruises in 1995, 1996, and 1997) and 7 Antarctic samples from Gerlache Strait and Dallman Bay revealed that the richness of Archaea assemblages was greater in samples from deep water than in those from the upper water column in both polar oceans. DGGE banding patterns suggested that most of the Archaea ribotypes were common to both the Arctic Ocean and the Antarctic Ocean. However, some of the Euryarchaeota ribotypes were unique to each system. Cluster analysis of DGGE fingerprints revealed no seasonal variation but supported depth-related differences in the composition of the Arctic Ocean Archaea assemblage. The phylogenetic composition of the Archaea assemblage was determined by cloning and then sequencing amplicons obtained from the Archaea-specific primers 21f and 958r. Sequences of 198 clones from nine samples covering three seasons and all depths grouped with marine group I Crenarchaeota (111 clones), marine group II Euryarchaeota (86 clones), and group IV Euryarchaeota (1 clone). A sequence obtained only from a DGGE band was similar to those of the marine group III Euryarchaeota:     

Seasonal and Spatial Variability of Bacterial and Archaeal Assemblages in the Coastal Waters near Anvers Island, Antarctica

A previous report of high levels of members of the domain Archaea in Antarctic coastal waters prompted us to investigate the ecology of Antarctic planktonic prokaryotes. rRNA hybridization techniques and denaturing gradient gel electrophoresis (DGGE) analysis of the bacterial V3 region were used to study variation in Antarctic picoplankton assemblages. In Anvers Island nearshore waters during late winter to early spring, the amounts of archaeal rRNA ranged from 17.1 to 3.6% of the total picoplankton rRNA in 1996 and from 16.0 to 1.0% of the total rRNA in 1995. Offshore in the Palmer Basin, the levels of archaeal rRNA throughout the water column were higher (average, 24% of the total rRNA) during the same period in 1996. The archaeal rRNA levels in nearshore waters followed a highly seasonal pattern and markedly decreased during the austral summer at two stations. There was a significant negative correlation between archaeal rRNA levels and phytoplankton levels (as inferred from chlorophyll a concentrations) in nearshore surface waters during the early spring of 1995 and during an 8-month period in 1996 and 1997. In situ hybridization experiments revealed that 5 to 14% of DAPI (4′,6-diamidino-2-phenylindole)-stained cells were archaeal, corresponding to 0.9 × 10⁴ to 2.7 × 10⁴ archaeal cells per ml, in late winter 1996 samples. Analysis of bacterial ribosomal DNA fragments by DGGE revealed that the assemblage composition may reflect changes in water column stability, depth, or season. The data indicate that changes in Antarctic seasons are accompanied by significant shifts in the species composition of bacterioplankton assemblages and by large decreases in the relative proportion of archaeal rRNA in the nearshore water column.     

Phylogenetic Composition of Arctic Ocean Archaeal Assemblages and Comparison with Antarctic Assemblages

Archaea assemblages from the Arctic Ocean and Antarctic waters were compared by PCR-denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA genes amplified using the Archaea-specific primers 344f and 517r. Inspection of the DGGE fingerprints of 33 samples from the Arctic Ocean (from SCICEX submarine cruises in 1995, 1996, and 1997) and 7 Antarctic samples from Gerlache Strait and Dallman Bay revealed that the richness of Archaea assemblages was greater in samples from deep water than in those from the upper water column in both polar oceans. DGGE banding patterns suggested that most of the Archaea ribotypes were common to both the Arctic Ocean and the Antarctic Ocean. However, some of the Euryarchaeota ribotypes were unique to each system. Cluster analysis of DGGE fingerprints revealed no seasonal variation but supported depth-related differences in the composition of the Arctic Ocean Archaea assemblage. The phylogenetic composition of the Archaea assemblage was determined by cloning and then sequencing amplicons obtained from the Archaea-specific primers 21f and 958r. Sequences of 198 clones from nine samples covering three seasons and all depths grouped with marine group I Crenarchaeota (111 clones), marine group II Euryarchaeota (86 clones), and group IV Euryarchaeota (1 clone). A sequence obtained only from a DGGE band was similar to those of the marine group III Euryarchaeota.     

Spatiotemporal variations in an assemblage of closely related planktonic aerobic methanotrophs

1. The assemblage of aerobic methane‐oxidising bacteria (MOB) was investigated in different seasons in the water column of a stratified freshwater lake. Species composition was analysed by performing denaturing gradient gel electrophoresis (DGGE) of the 16S rRNA genes and cloning analysis of the pmoA gene, which encodes the α‐subunit of particulate methane monooxygenase. The relative abundance of MOB to total bacteria was deduced from the copy number of the pmoA gene and 16S rRNA gene using real‐time polymerase chain reaction. 2. The profiles of the DGGE banding patterns changed with water depth, and these changes correlated with oxygen concentration and water temperature. The sequences of the DGGE bands obtained were all associated with the genus Methylobacter. During the analysis of pmoA gene, all clones sequenced were that of the Methylobacter/Methylosarcina group. The relative abundances of pmoA gene peaked around the oxycline, and small peaks of pmoA gene were also observed near the surface when peaks of methane were observed at the corresponding depth. 3. Profiles of the DGGE banding patterns suggested that ecophysiological characteristics differ among members of the genus Methylobacter; this indicates the importance of investigating the MOB assemblage at the species level or lower. Planktonic MOB seemed to be abundant around oxycline.     

Dominant bacterioplankton populations in the meromictic Lake Suigetsu as determined by denaturing gradient gel electrophoresis of 16S rRNA gene fragments

Lake Suigetsu is a typical meromictic lake in Japan characterized by a permanent chemocline at a depth of between 3 and 8 m separating the oxic freshwater mixolimnion from anoxic saline sulfidogenic monimolimnion. Dominant bacterioplankton populations in Lake Suigetsu were investigated using PCR-denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene fragments. The bacterial population was vertically stratified, and temporal shifts in the microbial communities were observed in both the oxic and anoxic layers of Lake Suigetsu during the sampling period. Several dominant DGGE bands were excised and sequenced. In the chemocline, green sulfur bacteria phylogenetically related to the genera Prosthecochloris, Pelodyctyon, and Chlorobium within the phylum Chlorobi were dominant; the colorless sulfur bacteria closely related to the genus Thiomicrospira were detected. These sulfur bacterial groups appear to be important in the biogeochemical cycling of sulfur and/or carbon in Lake Suigetsu. Bacterial sequences affiliated with the Bacteroidetes phylum were frequent among the dominant fragments in the DGGE profiles throughout the water column. Populations possessing a fermentative metabolism exist in Bacteroidetes, suggesting they may contribute to the degradation of organic matter in the anoxic environment of Lake Suigetsu.     

Diel and seasonal vertical distribution of meiobenthic copepods in muddy sediments of a eutrophic lagoon (fish ponds of Arcachon Bay)

The vertical distribution of meiobenthic copepods was investigated within muddy sediments of a eutrophic lagoon (fish ponds of Arcachon Bay, France). The aim of the study was to determine if in muddy sediments, as previously established in sandy sediments, meiobenthic copepods migrate vertically according to the seasons or diel periods. Two experimental approaches were used, viz: a three-season comparison was made of the diel vertical distribution of the harpacticoid Canuella perplexa T. & A. Scott (1893) and secondly the depth distribution of a meiobenthic copepod assemblage was followed for a 24 h period, in shallow water subtidal locations. The harpacticoid C. perplexa vertically migrated through the top three centimeters of the sediment, showing diel and seasonal variations in depth distribution. The differential vertical distributions shown by the dominant meiobenthic populations suggest that emergence into the water column may mainly concern surface dwelling copepods. The physical and biological factors affecting seasonal and diel changes in the copepod assemblage of the fish ponds are discussed.     

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.     

Microheterogeneity in 16S ribosomal DNA-defined bacterial populations from a stratified planktonic environment is related to temporal changes and to ecological adaptations

Temporal changes of the bacterioplankton from a meromictic lake (Lake Vilar, Banyoles, Spain) were analyzed with four culture-independent techniques: epifluorescence microscopy, PCR-denaturing gradient gel electrophoresis (DGGE) fingerprinting, fluorescence in situ whole-cell hybridization and flow cytometry sorting. Microscopically, blooms of one cyanobacterium (Synechococcus sp.-like), one green sulfur bacterium (Chlorobium phaeobacteroides-like), and one purple sulfur bacterium (Thiocystis minor-like) were observed at different depths and times. DGGE retrieved these populations and, additionally, populations related to the Cytophaga-Flavobacterium-Bacteroides phylum as predominant community members. The analyses of partial 16S ribosomal DNA sequences from the DGGE fingerprints (550 bp analyzed) revealed higher genetic diversity than expected from microscopic observation for most of these groups. Thus, the sequences of two Synechococcus spp. (both had a similarity of 97% to Synechococcus sp. strain PCC6307 in 16S rRNA), two Thiocystis spp. (similarities to Thiocystis minor of 93 and 94%, respectively), and three Cytophaga spp. (similarities to Cytophaga fermentans of 88 and 89% and to Cytophaga sp. of 93%, respectively) were obtained. The two populations of Synechococcus exhibited different pigment compositions and temporal distributions and their 16S rRNA sequences were 97.3% similar. The two Thiocystis populations differed neither in pigment composition nor in morphology, but their 16S rRNA sequences were only 92.3% similar and they also showed different distributions over time. Finally, two of the Cytophaga spp. showed 96.2% similarity between the 16S rRNA sequences, but one of them was found to be mostly attached to particles and only in winter. Thus, the identity of the main populations changed over time, but the function of the microbial guilds was maintained. Our data showed that temporal shifts in the identity of the predominant population is a new explanation for the environmental 16S rRNA microdiversity retrieved from microbial assemblages and support the hypothesis that clusters of closely related 16S rRNA environmental sequences may actually represent numerous closely related, yet ecologically distinct, populations.     

Distribution of sulfate-reducing bacteria in a stratified fjord (Mariager Fjord, Denmark) as evaluated by most-probable-number counts and denaturing gradient gel electrophoresis of PCR-amplified ribosomal DNA fragments.

The sulfate-reducing bacterial populations of a stratified marine water column, Mariager Fjord, Denmark, were investigated by molecular and culture-dependent approaches in parallel. Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA and DNA encoding rRNA (rDNA) isolated from the water column indicated specific bacterial populations in different water column layers and revealed a highly differentiated pattern of rRNA- and rDNA-derived PCR amplificates, probably reflecting active and resting bacterial populations. Hybridization of DGGE patterns with rRNA probes indicated the increased presence and activity (by at least 1 order of magnitude) of sulfate-reducing bacteria within and below the chemocline. Parallel to this molecular approach, an approach involving most-probable-number (MPN) counts was used, and it found a similar distribution of cultivable sulfate-reducing bacteria in the water column of Mariager Fjord, Approximately 25 cells and 250 cells per ml above and below the chemocline, respectively, were found. Desulfovibrio- and Desulfobulbus-related strains occurred in the oxic zone. DGGE bands from MPN cultures were sequenced and compared with those obtained from nucleic acids extracted from water column samples. The MPN isolates were phylogenetically affiliated with sulfate-reducing delta subdivision proteobacteria (members of the genera Desulfovibrio, Desulfobulbus, and Desulfobacter), whereas the molecular isolates constituted an independent lineage of the delta subdivision proteobacteria. DGGE of PCR-amplified nucleic acids with general eubacterial PCR primers conceptually revealed the general bacterial population, whereas the use of culture media allowed cultivable sulfate-reducing bacteria to be selected. A parallel study of Mariager Fjord biogeochemistry, bacterial activity, and bacterial counts complementing this investigation has been presented elsewhere (N.B. Ramsing, H. Fossing, T. G. Ferdelman, F. Andersen, and B. Thamdrup, Appl. Environ.     

Microbial hotspots in anchialine blue holes: initial discoveries from the Bahamas

Inland blue holes of the Bahamas are anchialine ecosystems with distinct fresh and salt water layers, and anoxic or microoxic conditions at depth. Scientific cave diving and geomicrobiology exploration of blue holes are providing a first glimpse of the geochemistry and microbial life in these vertically stratified karst features. We hypothesized that two geographically adjacent, sunlit blue holes on Abaco Island would have comparable biogeochemistry and microbial life. Water samples were analyzed using in situ multiparameter dataloggers and field and laboratory tests, and diver-retrieved microbial samples were analyzed using nucleic acid analysis. Microbial 16S rRNA genes were dominated by members of the anoxygenic phototroph clade Chlorobi, with smaller numbers of Deltaproteobacteria, in both blue holes. However, spatial distributions of microbial biomass and species present within these major clades were significantly different. We also found that differences in the intensity of solar insolation, terrestrial and marine inputs, water residence time, depth to the halo/chemocline, and cave passage geometry strongly influence geochemical changes with depth. The biogeochemical diversity of inland blue holes in the Bahamas make them valuable as natural laboratories, repositories of microbial diversity, and analogs for stratified and sulfidic oceans present early in Earth’s history.     

Distribution of bacterioplankton in meromictic Lake Saelenvannet, as determined by denaturing gradient gel electrophoresis of PCR-amplified gene fragments coding for 16S rRNA.

The community structure of bacterioplankton in meromictic Lake Saelenvannet was examined by PCR amplification of the V3 region of 16S rRNA from microbial communities recovered from various depths in the water column. Two different primer sets were used, one for amplification of DNA from the domain Bacteria and another specific for DNA from the domain Archaea. Amplified DNA fragments were resolved by denaturing gradient gel electrophoresis (DGGE), and the resulting profiles were reproducible and specific for the communities from different depths. Bacterial diversity estimated from the number and intensity of specific fragments in DGGE profiles decreased with depth. The reverse was true for the Archaea, with the diversity increasing with depth. Hybridization of DGGE profiles with oligonucleotide probes specific for phylogenetic groups of microorganisms showed the presence of both sulfate-reducing bacteria and methanogens throughout the water column, but they appeared to be most abundant below the chemocline. Several dominant fragments in the DGGE profiles were excised and sequenced. Among the dominant populations were representatives related to Chlorobium phaeovibrioides, chloroplasts from eukaryotic algae, and unidentified Archaea.     

Microheterogeneity in 16S Ribosomal DNA-Defined Bacterial Populations from a Stratified Planktonic Environment Is Related to Temporal Changes and to Ecological Adaptations

Temporal changes of the bacterioplankton from a meromictic lake (Lake Vilar, Banyoles, Spain) were analyzed with four culture-independent techniques: epifluorescence microscopy, PCR-denaturing gradient gel electrophoresis (DGGE) fingerprinting, fluorescence in situ whole-cell hybridization and flow cytometry sorting. Microscopically, blooms of one cyanobacterium (Synechococcus sp.-like), one green sulfur bacterium (Chlorobium phaeobacteroides-like), and one purple sulfur bacterium (Thiocystis minor-like) were observed at different depths and times. DGGE retrieved these populations and, additionally, populations related to the Cytophaga-Flavobacterium-Bacteroides phylum as predominant community members. The analyses of partial 16S ribosomal DNA sequences from the DGGE fingerprints (550 bp analyzed) revealed higher genetic diversity than expected from microscopic observation for most of these groups. Thus, the sequences of two Synechococcus spp. (both had a similarity of 97% to Synechococcus sp. strain PCC6307 in 16S rRNA), two Thiocystis spp. (similarities to Thiocystis minor of 93 and 94%, respectively), and three Cytophaga spp. (similarities to Cytophaga fermentans of 88 and 89% and to Cytophaga sp. of 93%, respectively) were obtained. The two populations of Synechococcus exhibited different pigment compositions and temporal distributions and their 16S rRNA sequences were 97.3% similar. The two Thiocystis populations differed neither in pigment composition nor in morphology, but their 16S rRNA sequences were only 92.3% similar and they also showed different distributions over time. Finally, two of the Cytophaga spp. showed 96.2% similarity between the 16S rRNA sequences, but one of them was found to be mostly attached to particles and only in winter. Thus, the identity of the main populations changed over time, but the function of the microbial guilds was maintained. Our data showed that temporal shifts in the identity of the predominant population is a new explanation for the environmental 16S rRNA microdiversity retrieved from microbial assemblages and support the hypothesis that clusters of closely related 16S rRNA environmental sequences may actually represent numerous closely related, yet ecologically distinct, populations.     

Litter Breakdown and Microbial Succession on Two Submerged Leaf Species in a Small Forested Stream

Microbial succession during leaf breakdown was investigated in a small forested stream in west-central Georgia, USA, using multiple culture-independent techniques. Red maple (Acer rubrum) and water oak (Quercus nigra) leaf litter were incubated in situ for 128 days, and litter breakdown was quantified by ash-free dry mass (AFDM) method and microbial assemblage composition using phospholipid fatty acid analysis (PLFA), ribosomal intergenic spacer analysis (RISA), denaturing gradient gel electrophoresis (DGGE), and bar-coded next-generation sequencing of 16S rRNA gene amplicons. Leaf breakdown was faster for red maple than water oak. PLFA revealed a significant time effect on microbial lipid profiles for both leaf species. Microbial assemblages on maple contained a higher relative abundance of bacterial lipids than oak, and oak microbial assemblages contained higher relative abundance of fungal lipids than maple. RISA showed that incubation time was more important in structuring bacterial assemblages than leaf physicochemistry. DGGE profiles revealed high variability in bacterial assemblages over time, and sequencing of DGGE-resolved amplicons indicated several taxa present on degrading litter. Next-generation sequencing revealed temporal shifts in dominant taxa within the phylum Proteobacteria, whereas γ-Proteobacteria dominated pre-immersion and α- and β-Proteobacteria dominated after 1 month of instream incubation; the latter groups contain taxa that are predicted to be capable of using organic material to fuel further breakdown. Our results suggest that incubation time is more important than leaf species physicochemistry in influencing leaf litter microbial assemblage composition, and indicate the need for investigation into seasonal and temporal dynamics of leaf litter microbial assemblage succession.     

Spatial and Temporal Analysis of the Microbial Community in Slow Sand Filters Used for Treating Horticultural Irrigation Water

An experimental slow sand filter (SSF) was constructed to study the spatial and temporal structure of a bacterial community suppressive to an oomycete plant pathogen, Phytophthora cryptogea. Passage of water through the mature sand column resulted in complete removal of zoospores of the plant pathogen. To monitor global changes in the microbial community, bacterial and fungal numbers were estimated on selective media, direct viable counts of fungal spores were made, and the ATP content was measured. PCR amplification of 16S rRNA genes and denaturing gradient gel electrophoresis (DGGE) were used to study the dynamics of the bacterial community in detail. The top layer (1 cm) of the SSF column was dominated by a variable and active microbial population, whereas the middle (50 cm) and bottom (80 cm) layers were dominated by less active and diverse bacterial populations. The major changes in the microbial populations occurred during the first week of filter operation, and these populations then remained to the end of the study. Spatial and temporal nonlinear mapping of the DGGE bands provided a useful visual representation of the similarities between SSF samples. According to the DGGE profile, less than 2% of the dominating bands present in the SSF column were represented in the culturable population. Sequence analysis of DGGE bands from all depths of the SSF column indicated that a range of bacteria were present, with 16S rRNA gene sequences similar to groups such as Bacillus megaterium, Cytophaga, Desulfovibrio, Legionella, Rhodococcus rhodochrous, Sphingomonas, and an uncharacterized environmental clone. This study describes the characterization of the performance, and microbial composition, of SSFs used for the treatment of water for use in the horticultural industry. Utilization of naturally suppressive population of microorganisms either directly or by manipulation of the environment in an SSF may provide a more reproducible control method for the future.     

Seasonal Population Changes in the Restriction Fragment Length Polymorphism (RFLP) Patterns from PCR-Amplified 16S rRNA Genes of Predominant Ribotypes in Microbial Mat Samples from the Ebro Delta (Spain)

The stratified benthic microbial communities, which developed as a consequence of the physico-chemical gradients and the physiology of the inhabiting microorganisms, from Ebro Delta microbial mats were analyzed. 16S rRNA eubacterial and archaeal genes were amplified by polymerase chain reaction (PCR). PCR products were separately digested with three different restriction enzymes (AluI, HinfI, and RsaI) and later separated by horizontal electrophoresis to determine whether changes of predominant ribotypes are occurring over a period of a year. Comparison of total restriction patterns was performed by scoring similarities by using the Jaccard coefficient and then building a multidimensional scaling (MDS) map from the resulting similarities matrix. The three enzymes gave a consistent result, a seasonal distribution instead of a spatial and/or physiological one. Received: 25 April 2002 / Accepted: 31 May 2002     

Spatial and temporal analysis of the microbial community in slow sand filters used for treating horticultural irrigation water

An experimental slow sand filter (SSF) was constructed to study the spatial and temporal structure of a bacterial community suppressive to an oomycete plant pathogen, Phytophthora cryptogea. Passage of water through the mature sand column resulted in complete removal of zoospores of the plant pathogen. To monitor global changes in the microbial community, bacterial and fungal numbers were estimated on selective media, direct viable counts of fungal spores were made, and the ATP content was measured. PCR amplification of 16S rRNA genes and denaturing gradient gel electrophoresis (DGGE) were used to study the dynamics of the bacterial community in detail. The top layer (1 cm) of the SSF column was dominated by a variable and active microbial population, whereas the middle (50 cm) and bottom (80 cm) layers were dominated by less active and diverse bacterial populations. The major changes in the microbial populations occurred during the first week of filter operation, and these populations then remained to the end of the study. Spatial and temporal nonlinear mapping of the DGGE bands provided a useful visual representation of the similarities between SSF samples. According to the DGGE profile, less than 2% of the dominating bands present in the SSF column were represented in the culturable population. Sequence analysis of DGGE bands from all depths of the SSF column indicated that a range of bacteria were present, with 16S rRNA gene sequences similar to groups such as Bacillus megaterium, Cytophaga, Desulfovibrio, Legionella, Rhodococcus rhodochrous, Sphingomonas, and an uncharacterized environmental clone. This study describes the characterization of the performance, and microbial composition, of SSFs used for the treatment of water for use in the horticultural industry. Utilization of naturally suppressive population of microorganisms either directly or by manipulation of the environment in an SSF may provide a more reproducible control method for the future.     

Comparative studies to assess bacterial communities on the clover phylloplane using MLST, DGGE and T-RFLP

Denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism (T-RFLP) were used to characterise the changes that occurred in Bacillus cereus group strains present in the phylloplane of clover Trifolium hybridum over 4 months. These strains had previously been analysed by multiple locus sequence typing (MLST). DGGE displayed many equally intense bands which indicated many equally abundant ribotypes. The bacterial community composition was variable and the leaves sampled as little as a week apart were found to have some temporal variability, indicating that diverse phylloplane bacterial communities follow sequential patterns from time to time. The B. cereus group community clearly clustered into early, mid and late branches, possibly due to multiple successional sequences occurring during growing seasons. The functionally and phylogenetically diverse microbial communities appeared to exhibit predictable successional patterns over shorter time scales. DGGE analysis with the molecular marker rpoB gave better resolution than 16S rRNA amplicons. There were no strong similarities between the dendrograms produced by DGGE, MLST and T-RFLP and the clustering produced by the automated T-RFLP method was variable even between the three restriction enzymes used. The DGGE–MLST method emerged as a superior method to T-RFLP–MLST for rapid typing of bacterial communities.     

Comparing activated sludge fungal community population diversity using denaturing gradient gel electrophoresis and terminal restriction fragment length polymorphism

We compared the relative values of denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism (T-RFLP) for profiling fungal communities in wastewater treatment plants using both ITS and 18S rRNA gene fragments as phylogenetic markers. A similar number of fungal ribotypes was obtained with both methods for the same treatment plant when the ITS primer set was used, while a greater number of ribotypes was obtained with T-RFLP compared to DGGE with the 18S rRNA primer set. Non-metric multi-dimensional scaling of presence/absence data and analysis of similarity showed that both methods could distinguish between the different plant communities at a statistically significant level (p < 0.05), regardless of which phylogenetic marker was used. The data suggest that both methods can be used preferably together to profile activated sludge fungal communities. A comparison of profiles generated with both these phylogenetic markers based on the number of ribotypes/bands, suggests that the 18S rRNA region is more discriminatory than the ITS region. Detected differences in fungal community compositions between plants probably reflect differences in their influent compositions and operational parameters.     

Seasonal microbial processes in a high-latitude fjord (Kongsfjorden, Svalbard): I. Heterotrophic bacteria, picoplankton and nanoflagellates

Temporal dynamics of the microbial food web in the Barents Sea and adjacent water masses in the European Arctic are to a large extent unknown. Seasonal variation in stocks and production rates of heterotrophic bacteria and phototrophic and heterotrophic picoplankton and nanoflagellates was investigated in the upper 50 m of the high-latitude Kongsfjorden, Svalbard, during six field campaigns between March and December 2006. Heterotrophic bacteria, picoplankton and nanoflagellates contributed to ecosystem structure and function in all seasons. Activity within the microbial food web peaked during spring bloom in April, parallel to low abundances of mesozooplankton. In the nutrient-limited post-bloom scenario, an efficient microbial loop, fuelled by dissolved organic carbon from abundant mesozooplankton feeding on phytoplankton and protozooplankton, facilitated maximum integrated primary production rates. A tight microbial food web consisting of heterotrophic bacteria and phototrophic and heterotrophic picoplankton and nanoflagellates was found in the stratified water masses encountered in July and September. Microbial stocks and rates were low but persistent under winter conditions. Seasonal comparisons of microbial biomass and production revealed that structure and function of the microbial food web were fundamentally different during the spring bloom when compared with other seasons. While the microbial food web was in a regenerative mode most of the time, during the spring bloom, a microbial transfer mode represented a trophic link for organic carbon in time and space. The microbial food web’s ability to fill different functional roles in periods dominated by new and regenerated production may enhance the ecological flexibility of pelagic ecosystems in the present era of climate change.     

Methanogenic profiles by denaturing gradient gel electrophoresis using order-specific primers in anaerobic sludge digestion

In the present study, the diversity of methanogenic populations was monitored for 25 days, together with the process data for an anaerobic batch reactor treating waste-activated sludge. To understand this microbial diversity and dynamics, 16S rRNA-gene-targeted denaturing gradient gel electrophoresis (DGGE) fingerprinting was conducted at two different taxonomic levels: the domain and order levels. The DGGE profiles of the domain Archaea and the three orders Methanosarcinales, Methanomicrobiales, and Methanobacteriales were comparatively analyzed after each DGGE band was sequenced to enable identification. The DGGE profiles of the three orders showed methanogens belonging to each order that were not detected in the DGGE profile of the Archaea. This discrepancy may have resulted from PCR bias or differences in the abundances of the three microbial orders in the anaerobic bioreactor. In conclusion, to fully understand the detailed methanogenic diversity and dynamics in an anaerobic bioreactor, it is necessary to conduct DGGE analysis with 16S rRNA gene primers that target lower taxonomic groups.