Sludge management has gained importance over the years due to high sludge treatment and disposal cost, stringent disposal laws, and a need to move towards sustainable energy production. Anaerobic Digestion (AD) of waste activated sludge (WAS) is a favoured sludge stabilisation technique due to its low energy footprint. Hydrolysis – the first step in AD is often the rate-limiting step and thus pre-treatment such as Thermal Hydrolysis Process (THP) are implemented before AD to improve the biodegradability of WAS and biogas production. However, THP leads to the formation of recalcitrant compounds such as melanoidins which are presumed to have a similar effect as Humic substances on AD. Also, THP leads to the release of metals incorporated in sludge flocs by the degradation of extracellular polymeric substances (EPS) structures. Therefore, this study aimed to understand the effect of melanoidins and metals on the hydrolysis step in AD.
The interaction between melanoidins and cations Fe²⁺, Ca²⁺, Mg²⁺, Cu²⁺, K⁺, and NH₄⁺ was studied using ultra-filtration and ICP-MS. The effect of these melanoidins-metals interactions on enzymatic hydrolysis of cellulose and proteins was investigated using fluorescence essay and Response Surface Methodology (RSM) modeling. The mechanism of inhibition of hydrolysis by melanoidins and metal was investigated using SEC-HPLC.
The results showed that complexation of melanoidins with metals shows the trend - Fe²⁺ > Ca²⁺ > Mg²⁺ > Cu²⁺ > K⁺ ≥ NH₄⁺. The ions with higher charge and ionic radius interacted with melanoidins more effectively. The melanoidins-metal interaction increased with an increase in melanoidins concentration most likely due to more phenolic and carboxylic functional groups available for binding. Fe³⁺ leads to an increase in molecular weight (MW) of melanoidins due to inter-molecular interaction caused by complete charge neutralisation, while no MW changes are observed with Cu²⁺ and Ca²⁺. Individually, melanoidins decreased, Fe³⁺ increased, while Cu²⁺ decreased the rate of hydrolysis of cellulose. In the presence of melanoidins, the inhibiting effect of Cu²⁺ is decreased with increasing melanoidins concentration. Similarly, the positive effect of Fe³⁺ is decreased with increasing melanoidins concentration. Protein hydrolysis was completely inhibited with 3 mM metals concentration. Melanoidins and Fe³⁺ concentrations had no major impact on protein hydrolysis while Cu²⁺ inhibits hydrolysis even at 0.15 mM concentration.
Therefore, it was hypothesised that Cu²⁺ causes enzymatic inhibition, but in the presence of melanoidins, melanoidins-Cu²⁺ complexes formation mitigates the toxic effect of Cu²⁺ on hydrolysis thus improving the rate of hydrolysis. While, Fe³⁺ improved hydrolysis by facilitating substrate-enzyme interaction but in presence of melanoidins, melanoidins-Fe³⁺ complexation leads to the non-availability of Fe³⁺ to facilitate hydrolysis process thus having no major impact on the rate of hydrolysis.