Diversity and seasonal changes of uncultured Planctomycetales in river biofilms.

Cell counts of planctomycetes showed that there were high levels of these organisms in the summer and low levels in the winter in biofilms grown in situ in two polluted rivers, the Elbe River and the Spittelwasser River. In this study 16S rRNA-based methods were used to investigate if these changes were correlated with changes in the species composition. Planctomycete-specific clone libraries of the 16S rRNA genes found in both rivers showed that there were seven clusters, which were distantly related to the genera Pirellula, Planctomyces, and Gemmata. The majority of the sequences from the Spittelwasser River were affiliated with a cluster related to Pirellula, while the majority of the clones from the Elbe River fell into three clusters related to Planctomyces and one deeply branching cluster related to Pirellula. Some clusters also contained sequences derived from freshwater environments worldwide, and the similarities to our biofilm clones were as high as 99.8%, indicating the presence of globally distributed freshwater clusters of planctomycetes that have not been cultivated yet. Community fingerprints of planctomycete 16S rRNA genes were generated by temperature gradient gel electrophoresis from Elbe River biofilm samples collected monthly for 1 year. Sixteen bands were identified, and for the most part these bands represented organisms related to the genus Planctomyces. The fingerprints showed that there was strong seasonality of most bands and that there were clear differences in the summer and the winter. Thus, seasonal changes in the abundance of Planctomycetales in river biofilms were coupled to shifts in the community composition.

Diversity and seasonal variability of beta-Proteobacteria in biofilms of polluted rivers: analysis by temperature gradient gel electrophoresis and cloning.

The beta-subgroup of the Proteobacteria has been shown to be important in aquatic habitats and was investigated in depth here by molecular 16S rRNA techniques in biofilms of the Elbe River and its polluted tributary, the Spittelwasser River. The bacterial 16S rRNA genes were cloned from each site, screened for beta-proteobacterial clones and sequenced. River biofilm clones from both rivers grouped into 9 clusters (RBFs). RBFs 1, 2, and 3 fell into the recently described betaI cluster of cosmopolitan freshwater bacteria, where they represented new species related to Rhodoferax, Aquaspirillum, and Hydrogenophaga: RBFs 4 to 7 affiliated with Aquabacterium commune, Ideonella dechloratans, and Sphaerotilus natans, respectively. The two remaining RBFs were uncultivated clusters, one of them being distantly related to Gallionella ferruginea. Seasonal changes in the relative intensity of the beta-proteobacterial 16S rRNA genes of biofilms harvested monthly for 1 year were determined by specific amplification and separation by temperature gradient gel electrophoresis (TGGE). Bands were identified by comparison of clones to community fingerprints by TGGE. Eight of 13 identified bands were shared by both habitats but showed different relative abundance and seasonal variability in the two rivers, probably caused by differences in temperature and pollutants. The data indicate new not-yet-cultivated clusters of river biofilm organisms, some of them probably distributed globally. They confirm the importance of certain known freshwater genera in river biofilms. The high phylogenetic resolution obtained by clone library analysis combined with the high temporal resolution obtained by TGGE suggest that the observed microdiversity in the river biofilm clone libraries might be caused by phylogenetically closely related microbial populations which are adapted to ecological parameters.

Biofilm community structure in polluted rivers: abundance of dominant phylogenetic groups over a complete annual cycle.

The seasonal dynamics of river biofilm communities in two German rivers, the Elbe and one of its tributaries, the Spittelwasser, were investigated for the first time by using fluorescence in situ hybridization and a standardized biofilm sampling procedure. We show the importance of members of the beta subclass of the class Proteobacteria, which formed the largest single group in the massively polluted Spittelwasser at all times. Clear seasonal peaks of abundance were observed for the planctomycetes and the Cytophaga-Flavobacterium cluster.

Microcosm enrichment of biphenyl-degrading microbial communities from soils and sediments.

A microcosm enrichment approach was employed to isolate bacteria which are representative of long-term biphenyl-adapted microbial communities. Growth of microorganisms was stimulated by incubating soil and sediment samples from polluted and nonpolluted sites with biphenyl crystals. After 6 months, stable population densities between 8 x 10(9) and 2 x 10(11) CFU/ml were established in the microcosms, and a large percentage of the organisms were able to grow on biphenyl-containing minimal medium plates. A total of 177 biphenyl-degrading strains were subsequently isolated and characterized by their ability to grow on biphenyl in liquid culture and to accumulate a yellow meta cleavage product when they were sprayed with dihydroxybiphenyl. Isolates were identified by using a polyphasic approach, including fatty acid methyl ester (FAME) analysis, 16S rRNA gene sequence comparison, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell proteins, and genomic fingerprinting based on sequence variability in the 16S-23S ribosomal DNA intergenic spacer region. In all of the microcosms, isolates identified as Rhodococcus opacus dominated the cultivable microbial community, comprising a cluster of 137 isolates with very similar FAME profiles (Euclidean distances, <10) and identical 16S rRNA gene sequences. The R. opacus isolates from the different microcosms studied could not be distinguished from each other by any of the fingerprint methods used. In addition, three other FAME clusters were found in one or two of the microcosms analyzed; these clusters could be assigned to Alcaligenes sp., Terrabacter sp., and Bacillus thuringiensis on the basis of their FAME profiles and/or comparisons of the 16S rRNA gene sequences of representatives. Thus, the microcosm enrichments were strongly dominated by gram-positive bacteria, especially the species R. opacus, independent of the pollution history of the original sample. R. opacus, therefore, is a promising candidate for development of effective long-term inocula for polychlorinated biphenyl bioremediation.

Metal selectivity of in situ microcolonies in biofilms of the Elbe river.

The ultrastructure of natural complex biofilm communities of the Elbe river grown in situ on microscopic glass coverslips was studied by using transmission electron microscopy and energy-dispersive x-ray (EDX) analysis. Characteristic microcolonies which measured between 3.3 and 9.3 microm in diameter were frequently observed. They had an outer envelope and harbored 6 to 30 cells. The cells formed short rods measuring 1.09 +/- 0.28 microm (n = 10) in length and 0.55 + 0.07 microm (n = 21) in width. They were surrounded by a thick layer of electron-transparent, nonosmicated matter, 120 to 300 nm thick. Individual cells exhibited a unique ultrastructural trait, namely, a concentric membrane stack which completely surrounded the cytoplasm. It consisted of three membrane doublets, which showed an overall thickness of 57 to 66 nm. The center-to-center spacing between two membrane doublets was 22.2 +/- 1.0 nm (n = 12). The bacterial cell wall seemed to be of the gram-negative type. The fact that upon shrinkage hexagonal clefts appeared proved the cells to be tightly packed, and septum formation by binary fissions was observed. All of these morphological details indicate that the cells within these microcolonies were actively growing and did not represent spore-like states. EDX analysis showed that only the electron-dense surface deposit of the microcolonies contained Mn and Fe in significant amounts, while these two elements were absent from the intercellular space and the cytoplasm of the microorganisms. In contrast, aluminum ions were able to penetrate the outer envelope of the microcolonies and were detected in the intercellular space. They were, however, completely absent from the microbial cytoplasm, indicating a filter cascade with respect to aluminum. From the ultrastructural data together with the deposition of iron and manganese on the microcolony surface, it appears that these organisms may belong to the genus Siderocapsa or Nitrosomonas. They do not precisely match any of the described species and may therefore represent a new species.