The objective of this study was to investigate the effect of waste activated sludge (WAS) predation by the aquatic worm Tubifex tubifex (T. tubifex) on the overall biodegradability of WAS. The initial WAS biodegradability potential was determined in 80 days sequential batch-fed anaerobic and aerobic treatment combinations. These treatment combinations were used as a reference for comparison with the effect of 5-day predation and 40-day anaerobic treatment combinations. Predation and the subsequent anaerobic digestion of the predated solids shows superior solids removal and superior overall conversion rates compared to solely conventional anaerobic digestion. Strikingly, the predation and anaerobic treatment combinations reached the same chemical oxygen demand (COD) and volatile solids (VS) reduction as the reference processes, i.e. 58% and 49% for COD and VS, respectively. Our results show that predation and anaerobic treatment combinations increase solids removal rates, but do not alter the overall biodegradability potential of WAS.@en

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. @en

Worm predation (WP) by Tubifex tubifex was investigated using waste activated sludge (WAS) as the substrate. In order to better understand the sludge degradation mechanisms during WP, the activity of five common hydrolytic enzymes was determined and compared among the initial feed activated sludge, endogenous respirated sludge and worm predated sludge. The results showed that the enzymatic activity decreased upon aerobic (worm) treatment of WAS and that this activity was predominantly associated with the removed solids fraction of the sludge. Interestingly, the protease activity showed a smaller decrease in activity when the worms were present. Flow cell cytometry revealed the release of intestinal bacteria from the worms, which are presumed to be largely responsible for the observed protease activity. Additionally, experiments in which T. tubifex were treated with antibiotics showed that the worms are responsible for a maximum of 73% of the observed proteolytic activity. The remaining 27% is attributed to the intestinal bacteria that exhibit a synergistic relationship with T. tubifex towards protein hydrolysis.@en

Sludge predation by aquatic worms results in an increased sludge reduction rate, which is mainly due to the specific removal of a protein fraction from the sludge. As microorganisms play an essential role in sludge hydrolysis a better understanding of the microbial community involved in the worm predation process will provide more insight into the relations between the aquatic worms, their associated microbiome and the efficient sludge reduction. In this study, the microbial community associated with predation by the Tubifex tubifex was investigated. The microbial diversity in the samples of the worm faeces (WF), predated activated sludge and protein-rich substrates were compared. The results indicated that predation on sludge resulted in a microbial change from Actinobacteria (44%) in the sludge, to Proteobacteria (64%) and Bacteriodites (36%) in the WF. Interestingly, the faecal microbial community was more related to the community in (predated) protein-rich substrates than to the community in predated or endogenously respirated activated sludge samples. This similar microbial community could be due to microbial utilisation of protein hydrolysis products. Alternatively, conditions in the worm gut could facilitate a protein hydrolysing community which assists in protein hydrolysis. The genera Burkholderiales, Chryseobacterium and Flavobacterium were found to be associated with predation by T. tubifex.@en

Worm predation (WP) on activated sludge leads to increased sludge degradation rates, irrespective of the type of worm used or reactor conditions employed. However, the cause of the increased sludge degradation rates remains unknown. This paper presents a comparative analysis of the physical and biochemical aspects of predated sludge, providing insight into the hydrolytic mechanisms underlying WP. To this end, the sessile worm Tubifex tubifex was used as a model oligochaete and was batch cultivated in an 18-L airlift reactor. Predation on activated sludge showed an average reduction rate of 12 ± 3.8%/d versus 2 ± 1.3%/d for endogenous respirated sludge. Sludge predation resulted in an increased release of inorganic nitrogen, phosphate and soluble chemical oxygen demand (sCOD). The sCOD consisted mainly of polysaccharides; however, fluorescence excitation emission matrix spectroscopy analysis also revealed the presence of Tryptophan-protein-like substances. Results suggest that the released polysaccharides contain a protein-like element. Additionally, soluble iron increased slightly in concentration after WP. The extent of hydrolysis seemed to reach an average plateau of about 40% volatile solids (VS) reduction after 4 days, which is substantially higher than the 29% VS reduction for endogenous decay of activated sludge after 30 days. Furthermore, T. tubifex predominantly consumed the protein fraction of the extracellular polymeric substances. Results suggest that that the worms specifically target a fraction of the sludge that is predominantly biodegradable under aerobic conditions, albeit at significantly higher degradation rates when compared to the endogenous decay of waste activated sludge.@en