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Research on the hydrolysis step of the AD became more important with the increased use of recalcitrant waste products such as manure, sewage sludge and agricultural biomass for biogas production. Hydrolysis is often the rate limiting step of the overall AD. Hydrolysis enhancement is one of the required steps to optimise biogas production. Despite the progress to overcome the limitations of hydrolysis, inhibition of hydrolysis is still poorly researched. Humic acid-like molecules (HA) are one of the inhibitors of the anaerobic hydrolysis and their effect on the overall AD process is generally overlooked. In this thesis, the HA inhibition on anaerobic digestion of cellulosic material and mitigation strategies, using cation and enzyme addition, to overcome the inhibition were investigated. In addition, the microbial community dynamics during AD in the presence and absence of HA were examined. In this scope, in Chapter 2, we reviewed the literature and pinpointed the urgent need for comprehensive studies on the role of hydrolytic microorganisms and environmental factors that effects their abundance within biogas plants. Consequently, the hydrolysis mechanism and involved hydrolytic enzymes were discussed. The overall discussion showed that a holistic approach, including microbiological and engineering studies should be chosen to disclose the role of hydrolytic microbes within biogas reactors. In Chapter 3 and, Chapter 4 the effect of HA on anaerobic cellulose hydrolysis and methanogenesis, in batch wise incubations is reported, respectively. Our results showed that pulse addition of 5 g L-1 HA caused a 50 % decrease in hydrolysis rate of anaerobic cellulose degradation (Chapter 3). Moreover, VFA accumulation was observed in the presence of HA during the anaerobic cellulose degradation, which indicated the possible inhibition of HA on methanogenesis. Based on the results of Chapter 3, pure cultures of methanogens and a mixed culture were tested to study the vulnerability of methanogenesis to HA inhibition. Hydrogenotrophic methanogenesis in pure cultures was inhibited by more than 75% in the presence of 1 g L-1 HA whereas, acetoclastic methanogenesis by Methanosaeta concilii was only slightly affected by HA up to 3 g L-1. When methanogenic granular sludge was incubated with HA, the specific methanogenic activity tests showed less inhibition, when compared to the pure cultures of methanogens. HA inhibition was also observed during long-term CSTR operation at an HRT of 20 days, 35°C and a mixture of cellulose and xylan as a subtrate (Chapter 6). 8 g L-1 HA inhibited the hydrolysis efficiency of the cellulose and xylan digestion by 40 % and concomitantly reduced the methane yields. Mitigation of the HA inhibition is required to increase the hydrolysis efficiency and methane yields of cellulosic biomass digestion. Therefore, two different strategies were tested for their potential use as mitigation agents, viz. addition of cations such as, calcium magnesium and iron (Chapter 3 and Chapter 6) and addition of hydrolytic enzymes (Chapter 6). Addition of magnesium, calcium and iron salts mitigated the HA inhibition and hydrolysis efficiencies reached up to 75, 65 and 72%, respectively, compared to the control groups in the batch wise incubations (Chapter 3). However, in long term CSTR operations, calcium addition did not show a positive effect on hydrolysis inhibition. On the other hand, enzyme addition helped to reverse the negative effect of HA. The microbial communities involved in AD were also studied. Chapter 5 and Chapter 6 dealt with microbial community analyses with 16S rRNA next generation sequencing. In Chapter 5, five replicate reactors were monitored during the start-up period. Transient feeding strategy was used to acclimatise anaerobic sludge to efficient cellulose and xylan degradation. During the experiment, Bacteriodales, Clostridiales and Anaerolineales became dominant bacterial populations while, Methanobacteriaceae and Methanospirillaceae were the dominant archaeal populations within the reactors. In Chapter 6, the microbial population dynamics in the presence and absence of HA were monitored. Microbiological analyses showed that the abundance of hydrolytic/fermentative bacterial groups such as Clostridiales, Bacteroidales and Anaerolineales was significantly lowered by the presence of HA. HA also affected the archaeal populations. Mostly hydrogenotrophic methanogens were negatively affected by HA. In conclusion, this thesis confirms that HA inhibit the hydrolysis and methanogenesis within both batch incubations and CSTR systems. Microbial populations were also affected by HA. Therefore, hydrolytic enzyme addition can be an option to mitigate HA inhibition and enhance hydrolysis and methanogenesis during conversion of biomass to biogas.

Anaerobic digestion of cellulose and hemicellulose in the presence of humic acids