Anaerobic Municipal Wastewater Treatment at Low Temperature: Novel High-throughput Methods for Hydrolysis Monitoring and Response to Temperature Shocks in Differently Inoculated Reactors

Abstract

This dissertation focuses on municipal wastewater, which is produced in everyday household activities. Currently, this wastewater is treated by conventional aerated activated sludge systems. However, these systems are not sustainable since they require substantial amounts of energy for aeration, convert wastewater organic matter only to CO2 and produce large amounts of excess sludge. Anaerobic municipal wastewater treatment (AnMWWT) would tackle all the aforementioned issues, but is challenging at the low wastewater temperatures typical for temperate climate regions, e.g., between 10 °C and 20 °C in Belgium. The main objective of this PhD research was to increase the future applicability of anaerobic municipal wastewater treatment in temperate climate zones. We aimed to achieve this with in-depth experiments organized around three topics. First, we developed a protein hydrolysis assay to quantify the rate at which proteins are converted to peptides and amino acids. A high-throughput assay with a BODIPY FL casein model protein in a 96 well microplate format was developed. This assay provides quantitative results at a high measurement frequency and has a sound theoretical basis. Second, we set out to develop a lipid hydrolysis assay to quantify the rate at which lipids are converted to long-chain fatty acids. A vegetable oil - Rhodamine B lipid emulsion approach in a 96 well microplate format was evaluated with a commercial lipid hydrolysis enzyme, Amano lipase. Third, we aimed to obtain integrated physico-chemical and microbiological knowledge of the changes that occur in anaerobic municipal wastewater treatment when the water temperature drops from 30 °C to 15 °C. In addition, we set out to evaluate the potential of alternative reactor inocula and biofilm-based biomass retention for AnMWWT at a low operational temperature. A lab-scale reactor experiment was conducted over a period of 684 days with a synthetic municipal wastewater fed at a constant rate to the reactor. The operational temperature was varied throughout the experiment from 30 °C to 20 °C, then to 15 °C and again to 30 °C. Polyethylene carriers were added to all reactors to induce biofilm-based biomass retention. The performance and microbial community composition were compared for a traditional sludge digester inoculum (three replicate reactors), a cow rumen inoculum (three replicate reactors) and a sheep rumen inoculum (one reactor). The digester sludge and rumen-inoculated reactors were heavily affected by the stepwise temperature shocks from 30 °C to 20 °C and from 20 °C to 15 °C. A fast increase in the organic acid concentrations with a concomitant drop in biogas production and pH points out inhibition of methanogenesis but not hydrolysis and acidogenesis at low temperature. A steady state with a partial recovery of the initial biogas production rate could be obtained at 20 °C, but not at 15 °C. An adequate reactor performance was obtained in the reactors inoculated with sheep or cow rumen content, but they did not perform better than the digester sludge inoculated reactors. Biomass retention by biofilm formation was intended but was not obtained in this reactor experiment. The combination of turbulence due to continuous mixing and a high hydraulic retention time prevented colonization of the polyethylene carrier surface. The lack of biomass retention most probably affected slow-growing species such as methanogenic Archaea and their syntrophic partners, fatty-acid oxidizing bacteria.status: publishe