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| Home > Publications database > Application of functional gene arrays for monitoring influences of plant/seasons on bacterial functions and community structures in constructed wetland (Bitterfeld, Germany) |
| Home > Publications database > Application of functional gene arrays for monitoring influences of plant/seasons on bacterial functions and community structures in constructed wetland (Bitterfeld, Germany) |
| Dissertation / PhD Thesis/Book | PreJuSER-17113 |
2011
Forschungszentrum Jülich GmbH Zentralbibliothek, Verlag
Jülich
ISBN: 978-3-89336-724
Abstract: With the increasing application of constructed wetlands (CWs) around the world to improve water quality, it becomes more and more important to gain details about microbial ecology in this “black box”. In order to better understand general relationships among bacterial community structures, functions and environmental influencing factors in CWs, soil samples taken from the planted and the unplanted CWs in Bitterfeld (Germany), as well as those taken in warm- and cold-seasons, were compared in this study by using functional gene arrays (the GeoChip). In addition, other assessment methods, such as chemical analyses, fluorescent microscopic enumeration and PCRbased DGGE ($\underline{D}$enaturing $\underline{G}$radient $\underline{G}$el $\underline{E}$lectrophoresis) were also applied to complete the conclusions obtained by using the GeoChip. In general, this study consisted of two parts. The first part focused on evaluations of suitable DNA preparation procedures for functional gene arrays. The whole genome amplification was taken out of the primary plan due to biases in proportions of bacterial populations in DGGE band patterns, which were observed not only among the replications of the same DNA template but also between the products with and without this treatment. While evaluating procedure of DNA purification, the gel filtration column failed to purify DNA extracts of our samples, probably due to high amount of humic substances in the extracts. Among the three DNA extraction kits compared in this study (e.g. Bio101 FastDNA Spin Kit for Soil, UltraClean Soil DNA Isolation Kit and PowerSoil DNA Isolation Kit), extracts of the Bio101 were proved to be more suitable for the DGGE analysis with respect to their purity, yield and representativeness, while those of the PowerSoil showed better results in the microarray analysis. In addition, basing on the DNA extracts revealing different quantity and purity indices (A260/280, A260/230 and A320), influences of these indices on subsequent assessment methods of microbial communities (e.g. PCR-based DGGE and functional gene arrays) were also investigated. It was concluded that the success in PCR performance and consequently also in the DGGE analysis is more affected by the A260/280 and A320 values than by the ratio A260/230, and conditionally by the DNA yield. Moreover, the DGGE band pattern could also be affected by the preferential extraction due to chemical agents applied in the extraction. However, unlike the DGGE analysis, DNA microarrays were more affected by the A260/230 and A320 values. Thus, the primary goal of DNA extraction to obtain DNA extracts with the highest purity is not accurate enough any more in the environmental studies applying various analyzing methods. The performance of DNA extraction should vary with the subsequent [...]
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