Unravelling the hydrolytic activity of sludge degrading aquatic worms

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The overall objective of this thesis was to investigate ways to improve the extent and rate of waste activated sludge (WAS) hydrolysis by researching the WAS degrading activities and mechanisms of the aquatic worm Tubifex tubifex (T. tubifex) as a starting point. The WAS degrading aquatic worms were taken as a model “biochemical reactor” of which its conversion processes still need to be unravelled. Because the worms are known for their excellent performance in WAS-solids reduction, i.e., up to 45% volatile solids (VS) reduction in 4 – 5 days, the focus was on worm-based enzymatic processes for improving WAS hydrolysis.

Generally, T. tubifex predation shows significantly higher WAS conversion rates compared to anaerobic and aerobic digestion processes. However, information on the effect of WAS predation on the overall WAS biodegradability was lacking. Hereto, experiments were conducted to assess the ultimate WAS biodegradability potential, after which results were used as a reference to compare the biodegradability potential of different combinations of worm predation and anaerobic digestion. Interestingly, worm predation combinations showed superior solids removal rates and superior overall conversion rates, compared to solely conventional anaerobic digestion. However, the overall WAS biodegradability potential was similar in both experimental set-ups, reaching 58% and 49% removal for chemical oxygen demand (COD) and VS respectively.

The improved WAS conversion rates during worm predation were related to the efficient removal of protein-like and, to a smaller extent, polysaccharide-like substances from the sludge matrix. Additionally, alginate-like exopolysaccharides (ALE), were partly consumed during worm treatment of
WAS. The removal of protein, polysaccharide and ALE-like substances resulted in the disintegration of sludge flocs and the release of fulvic and humic substances as well as the cations Mg2+, Al3+ and Fe3+ from the sludge matrix. The cations and the humic and fulvic substances have a known structural function in the extracellular polymeric substances (EPS) of sludge flocs and are therefore, most
likely tightly associated with the removed protein-like fraction.

Corroborating with the removal of a protein-like fraction, an increased protease activity was observed in the predated WAS. The improved protease activity was likely related to T. tubifex based enzymes and/or the excretion of intestinal proteolytic bacteria. More specifically, a maximum of 73% of the proteolytic activity, related to the conversion of the model substrate casein, was due to the activity of the worms, while the remaining activity could be linked to the intestinal proteolytic bacteria.

The synergy between bacteria and worms was further investigated using microbial community analysis. We showed that the worm faeces produced through WAS predation shared more similarities in microbial structure with predated protein rich substrates as compared to the WAS itself. The microbial change towards a microbiome, which was apparently related to protein degradation, was probably due to favourable conditions in the worm gut that facilitated a protein-degrading microbial community. It was further found that the genera Burkholderiales, Chryseobacterium and Flavobacterium were
associated with predation by T. tubifex and are likely related to protein degradation.

Overall, the research demonstrated that the key aspects of efficient WAS hydrolysis are related to the removal and conversion of protein- and alginate-like substances as well as elevated protease activity. The type of proteases and possibly other mechanisms such as the lytic capabilities of the aquatic worms
are yet to be investigated.