a Extracellular processes in wastewater treatment

Abstract

PhD ThesisOne of the limitations of low-temperature anaerobic treatment of domestic wastewater is poor lipid degradation. Even when psychrophiles are used as an inoculum, the lipids degrade relatively less than carbohydrates and proteins. The first step towards the rational engineering of lipolysis in any system is to identify the lipolytic bacteria. In this study the combination of metagenomics and metaproteomics is used to screen for potential and actual lipolytic bacteria and their extracellular lipases in anaerobic membrane bioreactors treating domestic wastewater at 4℃ and 15℃. The reactors were inoculated by psychrophilic biomass collected from the sediment and soils of Lake Geneva, Switzerland (annual temperature range -11 – 21 °C) and Svalbard, Norway (annual temperature range - 16 – 6 °C), respectively. The feed of the reactors was primary influent collected from an activated sludge plant. The bacterial psychrophilic community and their lipases at 4℃ and 15℃ were compared. Of the 40 recovered putative lipolytic metagenome-assembled genomes (MAGs), only three (Chlorobium, Desulfobacter, and Mycolicibacterium) were common and abundant (relative abundance ≥ 1%) in all reactors. Notably, some MAGs that represented aerobic autotrophs (Nitrosomonas) contained lipases. Therefore, the lipases found may not always be associated with exogenous lipid degradation and may have other roles such as polyhydroxyalkanoates accumulation/degradation and interference with the outer membranes of other bacteria. Different protein classification tools were used for the putative lipase sequences identified by metagenomics to verify if they have potential lipolytic activity. None of the current tools, including InterProScan, could precisely assign lipolytic activity to these sequences. Enrichment of public databases by lipase sequences that have been experimentally tested can alleviate this problem. Metaproteomics did not provide sufficient proteome coverage for relatively lower abundant proteins such as lipases. The expression of fadL genes (long-chain fatty acid transporters) was confirmed for four genera (Dechloromonas, Azoarcus, Aeromonas and Sulfurimonas), but none of them was recovered as putative lipolytic MAGs. Metaproteomics also confirmed the presence of 15 relatively abundant (≥1%) genera in all reactors, of which at least 6 can potentially accumulate lipid/polyhydroxyalkanoates. For most putative lipolytic MAGs, there was no statistically significant correlation between the read abundance and ii reactor conditions such as temperature, phase (biofilm and bulk liquid), and feed type (treated by ultraviolet light or not). Reactor temperature had no statistical correlation with the length of the lipases either. Results obtained by metagenomics and metaproteomics did not confirm each other and further work is required to identify the true lipid degraders in these systems