PCR detection, denaturing gradient gel electrophoresis (DGGE) fingerprinting and identification of the microbial consortium in different types of UASB granules
Thesis (PhD (Food Science))--University of Stellenbosch, 2006.
High-rate anaerobic bioreactors are used for the treatment of various wastewaters, of which the upflow anaerobic sludge blanket (UASB) bioreactor has the widest application, especially in the food and beverage industries. In an UASB bioreactor sludge develops in a particular granular or flocculent form and the success of the anaerobic process relies on the formation of active and settable granules. These granules are formed by self-aggregation of bacteria that can be divided into different trophic groups that are responsible for the metabolic breakdown of organic substrates. The successful performance of a bioreactor is influenced by the composition of the substrate which subsequently may have an impact on the microbial consortium present in the UASB granules. In order to determine if a change in the structure of the non-methanogenic microbial community takes place, UASB brewery granules were subjected to the sudden addition of different carbon sources at different concentrations. A shift in the microbial community did occur when the granules were subjected to lactate medium (5 g.l-1). No changes in the microbial community were observed when the granules were stressed with glucose medium as carbon source, regardless of an increase in the glucose concentration. In order to better understand the effect that different wastewaters may have on the microbial consortium present in different UASB granules, the polymerase chain reaction (PCR) based denaturing gradient gel electrophoresis (DGGE) technique and sequence analysis were used to fingerprint and identify the Bacteria and Archaea present in either, winery, brewery, distillery or peach-lye canning UASB granules. Each granule type showed distinct PCR-based DGGE fingerprints with unique bands, while other bands were found to be present in all the granules regardless of the wastewater being treated. Bacillus, Pseudomonas, Bacteroides, Enterococcus, Alcaligenes, Clostridium, Shewanella, Microbacterium, Leuconostoc, Sulfurospirillum, Acidaminococcus, Vibrio, Aeromonas, Nitrospira, Synergistes, Rhodococcus, Rhodocyclus, Syntrophobacter and uncultured bacteria were identified, representing different acidogenic, acetogenic and homoacetogenic Bacteria.Different methanogenic bacteria such as Methanosaeta, Methanosarcina, Methanobacterium and uncultured bacteria belonging to the group Archaea were also fingerprinted and identified from different UASB granules. In both these studies a DGGE marker was constructed that may be used to assist in the identification of bacteria. The DGGE marker can also be used to monitor the presence of bacteria over a time period during anaerobic digestion. Bioaugmentation or the enrichment of granules results in tailor-made granules that may be used for the treatment of specific wastewaters. One of the most important contributions to the maintenance and enhancement of UASB granule formation is the inclusion of suitable microbes in the granule structure. Enterobacter sakazakii was isolated from raw winery wastewater and was found to produce sufficient amounts of desired fatty acids. This bacteria was, therefore, incorporated into batch cultured granular sludge. In order to identify and monitor the presence of the incorporated E. sakazakii in the tailor-made granules, 16S rRNA gene sequence primers and PCR conditions were developed. The use of molecular techniques such as PCR-based DGGE and sequence analysis proved to be successful methods to fingerprint and identify the microbial consortium present in the different UASB granules.