Microbial community analysis in sludge of anaerobic wastewater treatment systems : integrated culture-dependent and culture-independent approaches

The need for clean water is increasing and anaerobic wastewater treatment can be used as a cost-effective solution for purification of organically polluted industrial waste streams. This thesis presents results from microbiological investigations of several full-scale and lab-scale anaerobic wastewater treatments systems. Anaerobic wastewater treatment has gained popularity and is now one of the key technologies in environmental biotechnology. However, knowledge of the microbial community structure – function relationships is limited. A combination of cultivation-dependent and cultivation-independent techniques can be used to improve this knowledge. In this thesis, batch serial dilution incubations from a methanol-fed lab-scale thermophilic (55°C) methanogenic bioreactor indicated that syntrophic interspecies hydrogen transfer-dependent methanol conversion is at least equally important as direct methanogenesis in this lab-scale reactor. A direct methanol-utilizing Methanomethylovorans hollandica-related strain was detected up to a 10⁸-fold dilution, while Thermodesulfovibrio relatives and Methanothermobacter thermoautotrophicus strains were found till 10⁹–fold dilutions in the presence of H₂/CO₂. Microbial diversity was further evaluated in two expanded granular sludge bed reactors fed with increasing oleic acid loading rates. The archaeal community in the reactor inoculated with granular sludge stayed quite stable and active, whereas the relative abundance of Methanosaeta-like organisms gradually decreased in the reactor inoculated with suspended sludge when oleate loads were increased to 8 kg of chemical oxygen demand m^-3 day^-1. Desulfomicrobium and Methanobacterium were found to dominate the start-up of a full-scale synthesis gas fed gas-lift reactor treating metal and sulphate rich wastewater. Most Probable Number (MPN) counts confirmed that heterotrophic sulphate reducing bacteria (SRB) were dominant (10¹¹-10¹²cells/g VSS) compared to homoacetogens (10⁵-10⁶cells/g VSS) and methanogens (10⁸-10⁹cells/g VSS). Methanogens can still persist in sulphate-reducing bioreactors with short sludge retention time, since competition for hydrogen is determined by Monod kinetics and not by hydrogen threshold values. The microbial community in a full-scale upflow anaerobic sludge blanket reactor treating paper mill wastewater operated at 37°C was relatively stable over a period of 3 years as indicated by a high similarity (>75%) of denaturing gradient gel electrophoresis profiles of 16S ribosomal RNA gene fragments. Batch incubations at different temperatures resulted in microbial community changes. While the archaeal community composition differed significantly between incubations at 45 and 55°C, the bacterial composition changed between 37 and 45°C. Overall the bacterial community was dominated by Firmicutes (68% of the clones) and Delta-Proteobacteria (17% of the clones). A sequential degradation of first butyrate and then propionate at 37°C was linked to strong presence of Syntrophomonas sp. and Desulfobulbus propionicus, respectively. MPN series allowed estimating the number of micro-organisms per ml sludge that could use propionate without sulphate (10⁹), propionate and sulphate (10⁵), butyrate without sulphate (10⁸), butyrate with sulphate (10⁵), glucose (10⁹) and H₂/CO₂ (10¹⁰). Archaea were mainly dominated by Methanosaeta, but also Crenarchaeota-relatives were identified. Bacterial clone sequences were related to a variety of different known species, with expected functions in anaerobic digestion like fermentative bacteria, syntrophic short chain fatty acids oxidisers and SRB. However, about 80% of the clones was similar to sequences in the database without close cultured relatives, but many of these appeared to be present in anaerobic environments. It is important to improve knowledge of these unknown micro-organisms and fast accurate monitoring and identification could be instrumental in realising this. Therefore, a pilot macro-array was developed and tested. It appeared that combining probes generated by PCR amplification of the V1 and V6 variable regions of the 16S rRNA gene provided accurate differentiation of closely related organisms. The integrated application of molecular and cultivation dependent analyses of microbiota structure and function of a broad variety of anaerobic wastewater treatment systems described in this thesis has been used to improve insight of the ecophysiology in such reactors. Some general commonalities of anaerobic systems have been found, but also system-specific characteristics. This provides potential identification of general and system-specific indicator populations, allowing improved diagnostics and reactor predictability.