Anaerobic Conversion of Proteins in Aerobic Granular Sludge

Abstract

In an aerobic granular sludge (AGS) reactor treating urban wastewater, nutrient removal depends on the availability of carbon source. Domestic wastewater consists of 40-60% of slowly biodegradable complex substrates, out of which proteins form a major fraction. Despite this, little is known about the mechanisms of protein degradation in AGS. This research assessed the anaerobic availability of protein substrates for enhanced biological phosphorous removal (EBPR) in an aerobic granular sludge reactor. Proteins have to be first hydrolyzed before being assimilated by the bacteria, and nutrient removal is often limited by the rate of hydrolysis. Therefore, a major part of this thesis attempted to look into the mechanisms of proteolysis in AGS. Next, the utilization of amino acids - the hydrolysis product of proteins - by PAO was explored, based on critical evaluation of available literature. Firstly, it is proposed that the important aspect likely to govern the hydrolysis of proteins in AGS is the substrate-granule interaction, taking into account the diffusion limitation of particulate substrates within the granules and the probable presence of hydrolytic enzymes on the granular surface. Further, it is seen that the amino acids may be utilized by the polyphosphate accumulating organisms (PAOs) either directly or after the anaerobic degradation of amino acids to simple VFAs (volatile fatty acids), which are then taken up by the PAOs. The anaerobic degradation or fermentation of amino acids may occur via two well-known pathways- Stickland pathway and the non-Stickland pathway. Non-Stickland reaction requires syntrophy with hydrogen consuming bacteria whose presence in AGS is questionable. The bacteria responsible for Stickland reaction are obligate anaerobes belonging to the genus Clostridium which has not been found in aerobic granular sludge. Thus, it seems more likely that the amino acids are directly taken up by the PAOs in an AGS reactor. However, the direct uptake of amino acids by the PAOs has been reported only for eleven amino acids in total. More research on the likely fate of the remaining amino acids is recommended, considering that very little is known about the fate of amino acids in AGS. Few laboratory experiments were also conducted to study the effect of substrate size and granule size on the rate of hydrolysis of proteins. From preliminary experiments, it was seen that aerobic granular sludge exhibited significant anaerobic phosphate-release activity when casein (protein) was the only available carbon source. In the lab experiment carried out with different sizes of protein substrates, the observed anaerobic phosphate-release activity was used to obtain the rate of hydrolysis of different sizes of casein. Based on the hydrolysis rate obtained, it is seen that in an AGS reactor with a typical sludge concentration of 8g/l, up to 90% of the proteins present in domestic wastewater influent could be potentially taken up by the PAOs, provided that the proteins are completely dissolved. It was also seen that 60-80% of the particulate casein COD (>0.45um) was hydrolyzed within 24 hours of the assay. In another lab experiment, fluorescent protease assay was performed to assess the effect of granule size on the rate of hydrolysis. A significant decrease (by at least 2 times) in the specific rate of protein hydrolysis was observed when the aerobic granule size was increased from 1-2mm to 3.15-4mm. This may be significant especially when the stratification of granules and plug-flow feeding in an AGS reactor is taken into account. Further research is recommended to analyze the relative effect of substrate size and granule size on the rate of hydrolysis of proteins in AGS.