Wastewater from the food and agro-industry is filled with organic contaminants. If these substances are discharged into surface water, they promote the growth of unwanted microorganisms. To prevent this, contaminants are removed from the water through wastewater treatment. This is done using anaerobic digestion with microorganisms. Various types of microorganisms convert the organic compounds into methane gas, recovering some of the energy. The final step in anaerobic digestion, the conversion to methane, is the limiting factor in the process. A high concentration of methane-producing archaea is desired for rapid methane production.

Growing microorganisms in granules enables them to remain longer in the reactor, leading to an increased biomass concentration. The granules consist of multiple layers, each layer containing organisms that perform specific steps in the conversion to methane. These granules are a specific type of biofilm, made up of microorganisms embedded in a self-produced extracellular matrix. This matrix is composed of extracellular polymeric substances (EPS), which are produced and secreted by the microorganisms in the biofilm. EPS are a complex combination of proteins, polysaccharides, and lipids. Besides these basic polymers, combinations such as glycoproteins and lipopolysaccharides are also produced by the microorganisms. Charged polymers can form a polymer network with oppositely charged polymers or ions, contributing to the strength of the granular sludge. It is, therefore, no surprise that negatively charged particles, or acidic polymers, are often found in biofilms. However, how specific components in the EPS composition affect the structure and physical properties of granular sludge has been unclear until now.

The aim of this thesis is to study the EPS composition of anaerobic granular sludge, focusing on three main aspects: the identification of specific polymers, visualization of these polymers in the extracellular matrix, and identification of EPS synthesis pathways. While this thesis primarily characterizes the EPS composition of anaerobic granular sludge, the findings and methods are applicable to biofilms in general. By gaining a better understanding of the role of specific EPS components, we can better control biofilm processes....