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

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

A high-throughput assay to quantify protein hydrolysis in aerobic and anaerobic wastewater treatment processes

Proteins, an important fraction of the organic matter in wastewater, typically enter a treatment facility as high molecular weight components. These components are degraded by extracellular protein hydrolytic enzymes, denoted as proteases. Adequate protein hydrolysis monitoring is crucial, since protein hydrolysis is often a rate-limiting step in wastewater treatment. However, current monitoring tools lack a high sample throughput and reliable quantification. Here, we present an improved assay for high-throughput protein hydrolysis rate measurements in wastewater treatment applications. A BODIPY FL casein model substrate was implemented in a microplate format for continuous fluorescent quantification. Case studies on a conventional and a high-rate aerobic municipal wastewater treatment plant and a lab-scale, two-stage, anaerobic reactor provided proof-of-concept. The assay presented in this study can help to obtain monitoring-based process insights, which will in turn allow improving biological performance of wastewater treatment installations in the future. KEY POINTS: • Protein hydrolysis is a crucial step in biological wastewater treatment. • Quantification of the protein hydrolysis rate enables in-depth process knowledge. • BODIPY FL casein is a suitable model substrate for a protein hydrolysis assay. • High sample throughput was obtained with fluorescent hydrolysis quantification. Graphical abstract.status: publishe

Microbial community management to optimize anaerobic treatment of domestic wastewater in temperate climates

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Improved protein hydrolysis monitoring tool elucidates the first step in biological wastewater treatment

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A new protein hydrolysis monitoring tool to gain more insight in the initial steps of anaerobic digestion

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Protocol to evaluate and correlate membrane performance and mixed-liquor characteristics of full-scale and pilot-scale AnMBRs

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Cofactor F430 in AnMBRs: a potential biomarker for methanogenic activity?

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Cofactor F430 content as a potential biomarker for methanogenic activity: application to an anaerobic membrane bioreactor system

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Which inoculum do we need for low-temperature, anaerobic treatment of municipal wastewater?

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Cofactor F430 as a biomarker for methanogenic activity: application to an anaerobic bioreactor system

Over the last decades, anaerobic bioreactor technology proved to be a competitive technology for purifying wastewater while producing biogas. Methanogens perform the crucial final step in methane production, and monitoring their activity is of paramount importance for system understanding and management. Cofactor F430 is an essential prosthetic group of the methyl-coenzyme M reductase (MCR) enzyme catalysing this final step. This research investigates whether the quantification of cofactor F430 in bioreactor systems is a viable intermediate-complexity monitoring tool in comparison to the conventional biogas and volatile fatty acid (VFA) concentration follow-up and molecular genetic techniques targeting the mcrA gene encoding the MCR protein or its transcripts. Cofactor F430 was quantified in a lab-scale anaerobic membrane bioreactor (AnMBR) using liquid chromatography. The system was subjected to two organic loading rate shocks, and the F430 content of the sludge was followed up alongside mcrA gene copy and transcript numbers and classical performance monitoring tools. The research showed for the first time the combined mcrA gene transcript and F430 content dynamics in an anaerobic bioreactor system and reveals their significant positive correlation with in situ methane production rate. The main difference between the two monitoring methods relates to the cofactor's slower degradation kinetics. The work introduces the use of cofactor F430 as a biomarker for methanogenic activity and, hence, as a monitoring tool that can be quantified within half a working day, yielding information directly related to in situ methanogenic activity in methanogenic reactors.status: publishe