The production of renewable energy from organic waste streams is one of the most important aspects in the concept of sustainable development. Anaerobic digestion can be considered one of the main techniques to treat organic waste streams, allowing both waste stabilization and renewable energy production in the form of biogas. Its widespread application on full-scale relates to the fact that anaerobic digestion has, apart from biogas production and organic waste stabilization, several other advantages over alternative biological processes, e.g. a low cell yield, a high organic loading rate, limited nutrient demands, and low costs for operation and maintenance of the reactor system. The methanogenic archaea are responsible for the final and critical step of anaerobic digestion, as they produce valuable methane. One of the major drawbacks of anaerobic digestion is, however, the sensitivity of the methanogenic community to different environmental factors or stressors.
At this point, our knowledge of the microbial community taking care of the different stages in anaerobic digestion is still limited and, therefore, anaerobic digestion can still be considered a ‘black box’. Indeed, our knowledge of the bacterial community is restricted to the attribution of the first three steps in anaerobic digestion, i.e. hydrolysis, acidogenesis and acetogenesis. Although several key populations have already been identified, the exact contribution of the different bacterial phyla remains, however, to be elucidated. Methanogenesis, the last step, is carried out by archaea. The methanogenic community can be divided into two different groups, related to their main methanogenic pathway, i.e. hydrogenotrophic and acetoclastic methanogens. Thus far, only two genera, Methanosaeta and Methanosarcina, are reported to be able to carry out acetoclastic methanogenesis. Due to a distinct difference in physiology, morphology and metabolic potential, these two genera are expected to occupy different niches in anaerobic digestion. However, up until now, little is known about the specific contribution of both genera to methanogenesis in anaerobic digestion.
The main objective of this research was to unravel the ‘black box’ of anaerobic digestion to allow better and more solid process engineering. Several strategies were applied to improve biogas production and process stability, by (in)directly influencing the microbial community. A main focus was placed on the methanogenic community, as methanogenesis can be considered the weak link in the chain, because of the sensitivity of the methanogenic community to different environmental factors. However, to reach stable methane production, a close interaction between the bacterial and methanogenic community is required, hence, the bacterial community was also examined in terms of composition and organization.
In Chapter 2, A-sludge originating from the A-stage of the ‘Adsorptions-Belebungsverfahren’, was co-digested with kitchen waste to increase biogas production. This Fe-rich A-sludge appeared to be a suitable co-substrate for kitchen waste, as methane production rate values of 1.15 ± 0.22 and 1.12 ± 0.28 L L-1 d-1 were obtained during mesophilic and thermophilic co-digestion, respectively, of a feed-mixture consisting of 15% kitchen waste and 85% A-sludge. Mono-digestion of kitchen waste resulted in process failure. The thermophilic process led to higher residual volatile fatty acid concentrations, up to 2070 mg COD L-1, hence, the mesophilic process can be considered the most ‘stable’.
The optimal combination of A-sludge and kitchen waste served as a basis for the co-digestion of A-sludge with kitchen waste or molasses at mesophilic conditions in Chapter 3. In this chapter the objective was to evaluate the exact stabilizing mechanism of A-sludge as co-substrate in anaerobic digestion. Co-digestion of kitchen waste and molasses with A-sludge resulted in stable methane production, as values up to 1.53 L L-1 d-1 for kitchen waste and 1.01 L L-1 d-1 for molasses were obtained. The stabilizing effect of A-sludge in anaerobic digestion could not be attributed to bioaugmentation, despite its indigenous methanogenic activity, and therefore was dominated by nutrient addition. Methanosaetaceae maintained high copy numbers, between 109 and 1010 copies g-1 sludge, as long as optimal conditions were maintained, irrespective of the selected (co-)substrates. However, an increase in volatile fatty acids and a decrease in pH resulted in a decreased abundance of Methanosaetaceae.
In Chapter 4, a different feeding pattern was applied to obtain a higher degree of functional stability by (in)directly changing the evenness, dynamics and richness of the bacterial community. A short-term stress test revealed that pulse feeding leads to a higher tolerance of the digester to an organic shock load of 8 g COD L-1 and total ammonia levels up to 8000 mg N L-1. The bacterial community showed a high degree of dynamics over time, yet the methanogenic community remained constant. These results suggest that the regular application of a limited pulse of organic material and/or a variation in the substrate composition might promote higher functional stability in anaerobic digestion.
In Chapter 2-4, the anaerobic sludge originating from the same sludge digester was used as inoculum. The contribution of the inoculum to stable methane production and stress tolerance was investigated in Chapter 5. A different response in terms of start-up efficiency and ammonium tolerance was observed between the different inocula. Methanosaeta was the dominant acetoclastic methanogen, yet Methanosarcina increased in abundance at elevated ammonium concentrations. A shift from a Firmicutes to a Proteobacteria dominated bacterial community was observed in failing digesters. Methane production was strongly positively correlated with Methanosaetaceae, but with several bacterial populations as well. Overall, these results indicated the importance of inoculum selection to ensure stable operation and stress tolerance in anaerobic digestion.
In several studies, the positive effect of a bioelectrochemical system on biogas production in anaerobic digestion is described, however, the main mechanism behind this remained unsolicited, and primary controls were not executed. In Chapter 6, the stabilizing ability of a bioelectrochemical system for molasses digestion was evaluated in a 154 days experiment. A high abundance of Methanosaeta was detected on the electrodes, however, irrespective of the applied cell potential. This study demonstrated that, in addition to other studies reporting only an increase in methane production, a bioelectrochemical system can also remediate anaerobic digestion systems that exhibited process failure. However, the lack of difference between current driven and open circuit systems indicates that the key impact is through biomass retention, especially Methanosaetaceae, rather than electrochemical interaction with the electrodes.
Anaerobic membrane bioreactors with different fouling prevention strategies, i.e. biogas recirculation or membrane vibration, were applied to increase the retention of slow growing methanogens in Chapter 7. Biogas recirculation was the best mechanism to avoid membrane fouling, while the trans membrane pressures in the vibrating membrane bioreactor increased over time, due to cake layer formation. Stable methane production, up to 2.05 L L-1 d-1 and a concomitant COD removal of 94.4%, were obtained, only when diluted molasses were used, since concentrated molasses resulted in process failure. Real-time PCR results revealed a clear dominance of Methanosaetaceae over Methanosarcinaceae as the main acetoclastic methanogens in both anaerobic membrane bioreactor systems.
In Chapter 8, an extensive evaluation of 38 samples from 29 full-scale anaerobic digestion plants was carried out to relate operational parameters to microbial community composition and organization. The bacterial community was dominated by representatives of the Firmicutes, Bacteroidetes and Proteobacteria, covering 86.1 ± 10.7% of the total bacterial community. Acetoclastic methanogenesis was dominated by Methanosaetaceae, yet, only Methanobacteriales significantly positively correlated to biogas production. Three potential clusters, that could be considered as ‘AD-types’, were identified. These so-called ‘AD-types’ were determined by total ammonia concentration, free ammonia concentration and temperature, and characterized by an increased abundance of the Bacteroidales, Clostridiales and Lactobacillales, respectively. The identification of these three potential AD-types may serve as a basis for directly engineering the microbial community in anaerobic digestion. However, further research will be required to validated the actual existence of these three clusters in AD.
This research demonstrated the potential of several operational and technological strategies to improve biogas production and process stability in anaerobic digestion. Stable anaerobic digestion hosts a static methanogenic community, as long as evolving operational parameters or substrate composition do not influence the optimal conditions for methanogenesis, and an ever dynamic bacterial community. Methanosaetaceae are the uncontested dominant methanogens in anaerobic digestion, irrespective of the substrate, operational conditions or reactor configuration. However, increasing ammonium, salt and volatile fatty acid concentrations cause a shift from acetoclastic methanogenesis by Methanosaetaceae to hydrogenotrophic methanogenesis. Comparison of the lab-scale reactor results with full-scale plant microbial community analysis results showed a high similarity on bacterial level. However, at ‘deteriorating’ conditions at lab-scale a transition to a Methanosarcinaceae dominated methanogenesis was observed, while this shift could not be observed in full-scale plants. Hence, instead of Methanosarcinaceae, the Methanobacteriales are to be considered as the main drivers of so-called high-rate anaerobic digestion. The identification of the three AD-types can serve as a basis for unravelling the anaerobic digestion microbiome. Further in-depth research, however, will be required to determine the exact role of the core micro-organisms in each cluster to allow microbial community based engineering of anaerobic digestion ecosystems. The application of RNA, protein and metabolite based methods will be essential to estimate the effective metabolic activity of the microbial community in anaerobic digestion, thus, allowing more in-depth process control and further unravelling of the anaerobic digestion ‘black box’.