Assessment of Population Dynamics of Specific Strains in Groundwater Bioaugmentation Experiments by Two Different Molecular Techniques

A set of microcosm experiments was performed to understand the behaviour of special degraders in bioaugmentation experiments. In the experiments the following chlorobenzene degraders were used: the genetically modified Pseudomonas putida F1CC, and the two wild-type strains Pseudomonas putida GJ31 and Pseudomonas aeruginosa RHO1. These strains were used at an initial cell density of 10⁵ cells mL^–1 groundwater which had been spiked with 1,2-dichlorobenzene (1,2-DCB), 1,4-dichlorobenzene (1,4-DCB) and, as main contaminant, chlorobenzene (CB). The population dynamics and behaviour of the three special degraders within the groundwater microcosms were studied by single-strand conformation polymorphism (SSCP) analysis of 16S rDNA fragments amplified from directly extracted community DNA and fluorescent in situ hybridization (FISH) with species-specific probes. RHO1 disappeared after 4 days as detected by FISH in contrast to SSCP-detection where RHO1 could be found during the whole incubation time. Whereas GEM F1CC and wild-type strain GJ31 survived the whole incubation for 20 days. With both methods we were able to detect all strains with high specificity among the indigenous microbial community. The data sets obtained from SSCP analysis and FISH were highly correlated. Specific band intensity within the SSCPfingerprints and the cell counts determined by FISH gave a quantitative overview about the introduced strains.     

Population dynamics during startup of thermophilic anaerobic digesters: The mixing factor

Two thermophilic digesters were inoculated with manure and started-up under mixed and stagnant conditions. The Archaea in the mixed digester (A) were dominated by hydrogenotrophic Methanobateriaceae (61%) with most of the methane being produced via syntrophic pathways. Methanosarcinales (35%) were the only acetoclastic methanogens present. Acetate dissipation seems to depend on balanced hydrogenotrophic-to-acetotrophic abundance, which in turn was statistically correlated to free ammonia levels. Relative abundance of bacterial community was associated with the loading rate. However, in the absence of mixing (digester B), the relationship between microbial composition and operating parameters was not discernible. This was attributed to the development of microenvironments where environmental conditions are significantly different from average measured parameters. The impact of microenvironments was accentuated by the use of a non-acclimated seed that lacks adequate propionate degraders. Failure to disperse the accumulated propionate, and other organics, created high concentration niches where competitive and inhibiting conditions developed and favored undesired genera, such as Halobacteria (65% in B). As a result, digester B experienced higher acid levels and lower allowable loading rate. Mixing was found necessary to dissipate potential inhibitors, and improve stability and loading capacity, particularly when a non-acclimated seed, often lacking balanced thermophilic microflora, is used.