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Water affects the susceptibility of stone to alteration by facilitating physical, chemical and biological weathering. Stone properties determine water transport and retention, but it is also expected that biofilms and extracellular polymeric substances could alter the water-stone relationship. A lot of research on this subject has been carried out on soils, but the effect on stones is understudied. For this reason, three sedimentary building stones, Ernzen, Euville and Savonnieres, each with a different pore size distribution, were biofouled with cyanobacteria. Their relationship with the stone material was investigated by optical and electron microscopy, and the effect of cyanobacterial biofilms on water transport and retention was studied. The results showed that the cyanobacteria primarily colonize the building stones on the outer surface and have a limited effect on the water transport properties. They slightly reduced the capillary coefficient and drying rate of the stones, but enhanced the water content in the stone and increased water vapour sorption. They induced (near) hydrophobic conditions, but had no measurable effect on the gas permeability and water vapour diffusion. Moreover, swelling and shrinkage of the biofilms were observed, which could potentially induce physical weathering. It is expected that these changes could influence other forms of weathering, such as freeze-thaw weathering and salt weathering.

N,N-Dimethylformamide (DMF) is an organic solvent produced in large quantities worldwide. It is considered as a hazardous air pollutant and its emission should be controlled. However, only a limited number of studies have been performed on the removal of gaseous DMF by biological technologies. In this paper, we evaluate the removal of DMF under mesophilic and thermophilic conditions in a lab-scale biofilter for 472 days. The results show that, at ambient temperature, the biofilter achieved an average removal efficiency (RE) of 99.7 ± 0.3 % at Inlet Loads (ILs) up to 297 ± 52 g DFM m−3 h−1 (Empty Bed Residence Time (EBRTs) of 10.7 s). However, a decrease in EBRT (6.4 s) led to an unstable outlet concentration and, thus, to a drop in the biofilter performance (average RE: 90 ± 9 %). Moreover, an increase in temperature up to 65 °C led to a gradual decrease in RE (till 91 ± 7 %). Microbial analysis indicates that once the microorganisms encountered DMF, Rhizobiaceae dominated followed by Alcaligenaceae. Afterwards, a strong decrease in Rhizobiaceae was observed at every increase in temperature, and at 65 °C, the taxa were more heterogeneous. Overall, our experimental results indicate that biofiltration is a promising technique to remove DMF from waste gas streams.

Natural stones are essential for our built heritage. They are considered durable and permanent. However, they deteriorate through physical, chemical and biological action with water as the principal facilitator. Within a stone, water transport and retention depend on the stone's characteristics. It is expected that biofilms and extracellular polymeric substances change these properties and thus the water-stone relationship. To study the effect of the biofilms, three porous natural building stones: Ernzen, Euville and Savonnières, were biofouled with cyanobacteria Phormidium autumnale. The amount of biofilm was estimated by spectrophotometry, characterized by optical microscopy, and (environmental) scanning electron microscopy. Water transport and retention for biofouled and untreated samples were determined following European standards. Microscopy showed that the biofilms primarily covered the stone's outer surface. They experienced swelling and shrinkage dependent on the subjected relative humidity conditions, which could facilitate physical weathering. The biofilms slightly affected the water transport properties and induced a (near) hydrophobic surface. They reduced the drying rate and capillary coefficient. Moreover, biofouled samples absorbed more water vapor, had a higher water content, while no direct effect was measured on the water vapor diffusion and gas permeability. These changes could affect future weathering processes, including freeze-thaw and salt weathering.

Commonwealth war cemeteries commemorate the fallen of both world wars. Every casualty is remembered with a memorial or on a headstone. However, the headstones need to be maintained extensively, as microorganisms easily colonise them, affecting legibility and the stone substrate in the longer term. In the past, pesticides and other chemicals were popular to clean headstones, but due to raised environmental concerns, new treatment strategies are necessary. Within conservation science, enzymes have emerged as a popular tool for restoration. However, studies related to the use of enzymes for stone conservation are limited. Within this preliminary study, we applied commercially available enzyme-based treatments on biofouled natural building stones in the laboratory and in situ. Photography and spectrophotometry were used to monitor the effect of the treatment. The application of enzymes resulted in rapid disintegration of biological pigments, whereas visual improvement occurred more gradually. The successful application of enzymes suggests their potential to replace pesticides as the principal cleaning agent for headstones and natural building stones in a more general fashion.

Rapid contamination of drinking water in distribution and storage systems can occur due to pressure drop, backflow, cross-connections, accidents, and bio-terrorism. Small volumes of a concentrated contaminant (e.g., wastewater) can contaminate large volumes of water in a very short time with potentially severe negative health impacts. The technical limitations of conventional, cultivation-based microbial detection methods neither allow for timely detection of such contaminations, nor for the real-time monitoring of subsequent emergency remediation measures (e.g., shock-chlorination). Here we applied a newly developed continuous, ultra high-frequency flow cytometry approach to track a rapid pollution event and subsequent disinfection of drinking water in an 80-min laboratory scale simulation. We quantified total (TCC) and intact (ICC) cell concentrations as well as flow cytometric fingerprints in parallel in real-time with two different staining methods. The ingress of wastewater was detectable almost immediately (i.e., after 0.6% volume change), significantly changing TCC, ICC, and the flow cytometric fingerprint. Shock chlorination was rapid and detected in real time, causing membrane damage in the vast majority of bacteria (i.e., drop of ICC from more than 380 cells μl-1 to less than 30 cells μl-1 within 4 min). Both of these effects as well as the final wash-in of fresh tap water followed calculated predictions well. Detailed and highly quantitative tracking of microbial dynamics at very short time scales and for different characteristics (e.g., concentration, membrane integrity) is feasible. This opens up multiple possibilities for targeted investigation of a myriad of bacterial short-term dynamics (e.g., disinfection, growth, detachment, operational changes) both in laboratory-scale research and full-scale system investigations in practice., Frontiers in Microbiology, 8, ISSN:1664-302X

Plant-based sediment microbial fuel cells (PMFCs) couple the oxidation of root exudates in living rice plants to current production. We analysed the composition of the microbial community on anodes from PMFC with natural rice field soil as substratum for rice by analysing 16S rRNA as an indicator of microbial activity and diversity. Terminal restriction fragment length polymorphism (TRFLP) analysis indicated that the active bacterial community on anodes from PMFCs differed strongly compared with controls. Moreover, clones related to Deltaproteobacteria and Chloroflexi were highly abundant (49% and 21%, respectively) on PMFCs anodes. Geobacter (19%), Anaeromyxobacter (15%) and Anaerolineae (17%) populations were predominant on anodes with natural rice field soil and differed strongly from those previously detected with potting soil. In open circuit (OC) control PMFCs, not allowing electron transfer, Deltaproteobacteria (33%), Betaproteobacteria (20%), Chloroflexi (12%), Alphaproteobacteria (10%) and Firmicutes (10%) were detected. The presence of an electron accepting anode also had a strong influence on methanogenic archaea. Hydrogenotrophic methanogens were more active on PMFC (21%) than on OC controls (10%), whereas acetoclastic Methanosaetaceae were more active on OC controls (31%) compared with PMFCs (9%). In conclusion, electron accepting anodes and rice root exudates selected for distinct potential anode-reducing microbial populations in rice soil inoculated PMFC.

The region of Flanders in Belgium is, due to its high population density, intensive industry and livestock production, a nutrient-rich region. This results in important anthropogenic emissions to the environment, but also a large potential for the recovery and reuse of nitrogen (N ) and phosphorus (P) from waste streams. In this study, a substance flow analysis study for N and P is presented, in which the anthropogenic fluxes, stocks and hot spots of these two nutrients are quantified throughout the Flemish economy and environment. The environmental impact of the different economic sectors is addressed through the determination of the N and P footprint. The importance of food production in the nutrient cycle is thereby demonstrated through the large contribution of agriculture to the nutrient footprint (49% of N and 36% of P). Further focus is placed on the nutrient use efficiencies across the different sectors of the food supply nexus to target key nutrient losses and inefficiencies. This leads to an overall fertilizer-to-consumer efficiency of 14% for N and P, with the main nutrient losses originating from livestock production and food processing. At the end of the production and consumption chain, important nutrient quantities are embedded in concentrated waste streams such as excess manure, food processing waste streams and activated sludge. This demonstrates the large potential for nutrient recovery as a tool to improve nutrient use efficiencies and reduce the dependency of inorganic fertilizers. Several nutrient recovery strategies, both physicochemical and microbial, were evaluated for their economic feasibility and their impact on the primary energy demand of the total food supply chain.

The region of Flanders in Belgium has due to its high population density, intensive livestock production and industry a nutrient surplus available in waste streams. It therefore possesses a large potential towards the recovery and reuse of nitrogen and phosphorus. In this study, a substance flow analysis study for nitrogen and phosphorus is presented, in which the fluxes, stocks and hot spots of both nutrients are quantified throughout the economy. The predominant nutrient fluxes are found within the food supply chain, which connects industry, agriculture and consumption. This food supply chain revealed a fertilizer-to-consumer efficiency of 14% for N as well as for P. At the end of the production and consumption chain, a total of 17 kg N cap-1 yr-1 and 8.3 kg P cap-1 yr-1 of nutrients ends up in waste streams. Only a fraction of these nutrients (17% of N and 12% of P) is currently recycled. Several nutrient recovery strategies, both for chemicals and single cell protein production, were therefore evaluated for their economic feasibility and their impact on the energy demand of the food supply chain.

The occurrence of a range of recalcitrant organic micropollutants in our aquatic environment has led to the development of various tertiary wastewater treatment methods. In this study, biogenic manganese oxides (Bio-MnOx), biogenic silver nanoparticles (Bio-Ag0) and ionic silver were used for the oxidative removal of the frequently encountered drug diclofenac and its dechlorinated form, 2-anilinophenylacetate (APA). Diclofenac was rapidly degraded during ongoing manganese oxidation by Pseudomonas putida MnB6. Furthermore, whereas preoxidized Bio-MnOx, Bio-Ag0 and Ag+ separately did not show any removal capacity for diclofenac, an enhanced removal occurred when Bio-MnOx and silver species were combined. Similar results were obtained for APA. Finally, a slow removal of diclofenac but more rapid APA degradation was observed when silver was added to manganese-free P. putida biomass. Combining these results, three mechanisms of diclofenac and APA removal could be distinguished: (i) a co-metabolic removal during active Mn2+ oxidation by P. putida; (ii) a synergistic interaction between preoxidized Bio-MnOx and silver species; and (iii) a (bio)chemical process by biomass enriched with silver catalysts. This paper demonstrates the use of P. putida for water treatment purposes and is the first report of the application of silver combined with biogenic manganese for the removal of organic water contaminants.

Anaerobic digestion is an environmental key technology in the future bio-based economy. To achieve functional stability, a minimal microbial community diversity is required. This microbial community should also have a certain “elasticity”, i.e. the ability to rapidly adapt to sub-optimal conditions or stress. In this study it was evaluated whether a higher degree of functional stability could be achieved by changing the feeding pattern, which can change the evenness, dynamics and richness of the bacterial community. The first reactor (CSTRstable) was fed on daily basis, whereas the second reactor (CSTRdynamic) was fed every two days. Average biogas production was 0.30L CH4 L-1 d-1 in both reactors, although daily variation was up to four times higher in the CSTRdynamic compared to the CSTRstable during the first 50 days. Bacterial analysis revealed that this CSTRdynamic had a two times higher degree of bacterial community dynamics. The CSTRdynamic also appeared to be more tolerant to an organic shock load of 8 g COD L-1 and ammonium levels up to 8000 mg TAN L-1. 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.

Microbial metabolism in a mixed culture anode of microbial fuel cell fed with mixed sources of organic carbon largely resembles the metabolism found in anaerobic environments (digesters, sediments) where CH4 is the end product instead of electrical current. It is hypothesized that current generation is a viable competitor to steer or control CH4 emissions from (cultivated) wetlands. It has already been shown that a plant-MFC is capable of efficiently converting organic carbon derived from rhizodeposition into an electrical current. To understand the interaction between methanogenic metabolism and current generation in waterlogged sediments, several microcosm studies have been carried out.

While precious metals are available to a very limited extent, there is an increasing demand to use them as catalyst. This is also true for palladium (Pd) catalysts and their sustainable recycling and production are required. Since Pd catalysts exist nowadays mostly under the form of nanoparticles, these particles need to be produced in an environment-friendly way. Biological synthesis of Pd nanoparticles (bio-Pd) is an innovative method for both metal recovery and nanocatalyst synthesis. This review will discuss the different bio-Pd precipitating microorganisms, the applications of the catalyst (both for environmental purposes and in organic chemistry) and the state of the art of the reactors based on the bio-Pd concept. In addition, some main challenges are discussed, which need to be overcome in order to create a sustainable nanocatalyst. Finally, some outlooks for bio-Pd in environmental technology are presented.

Numerous membrane fouling studies have been conducted to predict and prevent membrane fouling. It was only recently that a new parameter, TEP, was introduced in this research. The deposition of TEP on reverse osmosis (RO) membranes has already been imaged, correlations between ultrafiltration (UF) fouling and TEP concentrations have been reported. Furthermore, TEP deposition takes place in an early stage of aquatic biofilm formation, making TEP one of the accused in search for biofilm initiation factors. After literature reporting about TEP in marine, surface and wastewater, this is the very first research focusing on TEP through in drinking water. Every single treatment step in three completely different drinking water production plants was scored on TEP removal. It could be concluded that TEP concentrations were very dependent of the raw water source but in none of the installations, TEP was able to reach the final drinking water in significant concentrations. The combination of coagulation and sand filtration proved efficient in strongly reducing TEP levels, while the combination of UF and RO could provide a total TEP removal.

Conventional wastewater treatment with activated sludge has a large carbon footprint, high aeration rates are combined with CO2 emission from bacteria. By using micro-algal bacterial flocs (MaB-flocs), CO2 could be captured within the biomass and oxygen could be produced in situ. In order to maximize this photosynthetic aeration and CO2 mitigation, we investigated whether inorganic carbon could alter the algae/bacteria ratio while keeping a good removal performance and settleability of the MaB-flocs. Therefore, three illuminated sequencing batch reactors with MaB-flocs were fed with synthetic wastewater enriched with 84.2, 42.1 and 0 mg L-1 C-KHCO3 supplemented with 0, 42.1, 84.2 mg L-1 C-sucrose, respectively, representing the inorganic carbon source compared to an organic carbon source. Bicarbonate significantly increased the chlorophyll a concentration of the MaB-flocs, but only poor settling flocs could be obtained causing a high turbidity of the effluent. Moreover, significant lower nitrogen removal efficiencies were measured feeding bicarbonate compared to sucrose and the pH increased (9.5). Sucrose benefited a good reactor performance and showed a good settleability of MaB-flocs. Despite the lower chlorophyll a concentration of the biomass and the lower in situ oxygen concentration, average soluble COD removal efficiencies of 95 % were achieved with sucrose. Furthermore sucrose was successful in containing the optimal pH at 7. This study shows the importance of the carbon source for a good reactor performance. As a consequence, the inorganic/organic carbon ratio of the wastewater should be taken into account when algal bacterial reactors are used for wastewater treatment.

Natural and managed freshwater ecosystems are hotspots of biodiversity, biogeochemical cycling, and provide numerous societal services (e.g., drinking water production, wastewater treatment). The microbial communities that inhabit these systems form complex assemblages of interacting microbial taxa, for which the community structure and function are not yet fully understood. This research presents new insights into the microbial community ecology of both natural and managed freshwater systems by developing and validating new technologies to study them and by investigating the genomic adaptations to their freshwater habitat. A first line of hypotheses focused on the development and application of novel flow cytometry tools to monitor the structure of aquatic microbial communities in a fast and non-invasive manner. These tools were then applied to both natural (Lake Michigan) and managed (cooling and drinking water) freshwater ecosystems. In parallel, a second line of hypotheses delved into the genomic properties that some of the abundant populations in these communities have evolved to their freshwater environment.

Methane is the most abundant organic greenhouse gas in our atmosphere, and has a strong infrared absorbance, being 25 to 30 times more effective than carbon dioxide on a 100 years scale. Methane therefore plays an important role in the climate warming regulation. Methanotrophs are microorganisms that can consume methane and utilize it as their sole source of carbon and energy. These organisms are the most important biological sink of methane. Their importance is especially demonstrated in marine ecosystems. While the oceans have the potential to produce enormous quantities of methane, a series of very effective microbiological oxidation processes results in the ocean being one of the smallest net global methane sources. Hence, it is of vital importance to understand who is there and what factors may positively or negatively impact the methane-oxidizing activity of these organisms. Despite the importance of the aerobic methane oxidizing bacteria (MOB) in the marine ecosystems currently only a limited amount of ex situ cultures is available. Nevertheless, they are indispensable to link physiology to genomic features and expand our knowledge about the specific habitat preferences of marine MOB. This study focused on aerobic marine MOBs and aimed at designing a large-scale enrichment and isolation strategy to retrieve a maximal MOB diversity from marine sediments. First, the effect of adhesion material and headspace composition on the methane oxidation activity in marine sediment enrichments were investigated. The addition of sterilized natural sediment as well as acid-washed silicium dioxide significantly positively influenced methane oxidation. The exact mechanism of this positive effect needs further investigation but might be the facilitation of methane, carbon dioxide and oxygen gradients in addition to adhesion. Use of adhesion material might thus facilitate the cultivation and subsequent enrichment of members of this functional guild. Next, using these insights, a large scale isolation strategy was performed from sediment collected at six different stations in the North Sea along a transect from estuary to open sea. An initial enrichment step with serial subcultivations was followed by miniaturized extinction culturing mimicking a range of nitrogen and oxygen microniches. A clear decreasing trend of cultivability and detectability was observed along the investigated transect. Furthermore widely applied pmoA primers failed to amplify biomarkers in a large number of active methanotrophic cultures, suggesting enormous underestimation of methanotrophs in situ in PCR-based molecular surveys. Unfortunately, despite the numerous attempts we were not able to obtain axenic methanotrophic cultures, most likely due to tight mutualistic interactions with heterotrophic bacteria.Shot gun sequencing of four methane-oxidizing enrichment cultures revealed the presence of a novel gammaproteobacterial MOB belonging to the deep-sea cluster 2 in two cultures and a novel alphaproteobacterial MOB belonging to the recently described methylotrophic genus Methyloceanibacter in the other two cultures. Methyloceanibacter methanicus represented the first MOB found in an exclusively methylotrophic genus, the first marine type II MOB and only the third taxon in which solely sMMO was resoponsible for methane oxidation. A targeted isolation using methanol as carbon source led to a axenic culture of the MOB, in addition to three closely related novel strict methylotrophic species, M. superfactum, M. stevinii, M. marginalis. Together with the previously described M. caenitepidi, these species exemplify an extreme niche differentiation, with a wide ecotypic variation related to growth kinetics on methanol, and preferences for nitrogen, pH, temperature and salt. Furthermore, the most striking difference of the deep-sea cluster 2 representatives demonstrated a striking difference with other gammaproteobacterial MOB in its lack of a calcium dependent methanol dehydrogenase encoded in the genome. The genome solely contained the genes for xoxF5 for the lanthanide-containing methanol dehydrogenase.Lastly, a preservation protocol was optimized for the long term storage of marine bacteria in order to successfully store the enrichments and axenic cultures obtained throughout this dissertation. Fastidious nitrite-oxidizing bacteria were used as model organisms. They demonstrated that optimal preservation conditions were strain-dependent whereby marine strains, appeared to be more sensitive to freezing than non-marine strains. Nevertheless, a general cryopreservation protocol using 10% dimethyl sulfoxide as cryoprotective agent with or without ten-fold diluted trypticase soy broth and trehalose as a preservation medium allowed successful preservation of all tested strains. Applying the same protocol on whole marine sediment samples allowed successful storage of different key players in the carbon and nitrogen cycle.In conclusion marine MOBs are notoriously difficult to cultivate and isolate. Despite numerous attempts, I was only able obtain one axenic culture, in addition to over 200 enrichment cultures, from the oxygenated zones of North Sea sediments. The availability of a successful preservation protocol allows the storage of this axenic culture, ensuring that novel diversity does not get lost, but also further guaranties archiving of the obtained enrichment cultures and environmental samples. As such these are available for future isolation when novel insights in marine MOB and isolation strategies/techniques become available.

Resource-efficient nutrient management is key to secure food production in the context of a growing global population, rising resource scarcity and increasing environmental pressure. The recycling of nitrogen and phosphorus from wastewaters is seen as a necessary element of sustainable resource management. The aim of this PhD thesis was to investigate and connect different aspects related to biological nutrient recycling: nutrient quantification, recovery and valorization. A detailed quantification of nutrient flows, emissions and waste streams in Flanders is provided which functions as a decision support tool for improved nutrient management. Afterwards a strategy for the biological stabilization and upgrading of concentrated nutrient streams was developed. Additionally, two novel microbial processes for nutrient upgrading were explored, based on nitrate-accumulation. To conclude, the valorization of recycled nutrients as a high-value microalgae fertilizer was investigated. This interdisciplinary approach provides strategies to facilitate the transition towards circular nutrient management.

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’.

A Microbial Fuel Cell (MFC) is a technology which enables the production of electrical current and power by bacteria from the breakdown of organic matter. This system can be applied in waterlogged soils and sediments but here organic carbon supply is limited. Adding a plant to the soil or sediment enables a continuous supply of organic matter in the soil due the photosynthesis and rhizodeposition processes of the plant. In this work, different components and designs of the plant-MFC were investigated in order to optimize electrical power output, find new niches for application or new products other than electrical power. In a first instance the optimal distribution of anode material in the soil was determined. These results were used for the second project which was aimed at studying the influence of a plant-MFC on greenhouse gas emissions from waterlogged environments. A third line of work entailed the concentration of soluble organic material from wastewater by means of a constructed wetland. The effluent of the wetland was used in a MFC to produce an electrical current. The electrical current was not used as electrical power, instead hydrogen peroxide was produced in the cathode for disinfection of the wetland effluent. A fourth line focused entirely on the cathode, where process operation and electrode design was studied for CO2 fixation to acetate by means of a pure culture of Acetobacterium woodii.