The next frontier of the anaerobic digestion microbiome : from ecology to process control

The anaerobic digestion process has been one of the key processes for renewable energy recovery from organic waste streams for over a century. The anaerobic digestion microbiome is, through the continuous development of novel techniques, evolving from a black box to a well-defined consortium, but we are not there yet. In this perspective, I provide my view on the current status and challenges of the anaerobic digestion microbiome, as well as the opportunities and solutions to exploit it. I consider identification and fingerprinting of the anaerobic digestion microbiome as complementary tools to monitor the anaerobic digestion microbiome. However, data availability, method-inherent biases and correct taxa identification hamper the accuracy and reproducibility of anaerobic digestion microbiome characterization. Standardisation of microbiome research in anaerobic digestion and other engineered systems will be essential in the coming decades, for which I proposed some targeted solutions. These will bring anaerobic digestion from a single-purpose energy-recovery technology to a versatile process for integrated resource recovery. It is my opinion that the exploitation of the microbiome will be a driver of innovation, and that it has a key role to play in the bio-based economy of the decades to come. (C) 2020 The Author(s). Published by Elsevier B.V. on behalf of Chinese Society for Environmental Sciences, Harbin Institute of Technology, Chinese Research Academy of Environmental Sciences

Methanosaeta vs. Methanosarcina in anaerobic digestion: the quest for enhanced biogas production

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’

Terminal restriction fragment length polymorphism is an “old school” reliable technique for swift microbial community screening in anaerobic digestion

The microbial community in anaerobic digestion has been analysed through microbial fingerprinting techniques, such as terminal restriction fragment length polymorphism (TRFLP), for decades. In the last decade, high-throughput 16S rRNA gene amplicon sequencing has replaced these techniques, but the time-consuming and complex nature of high-throughput techniques is a potential bottleneck for full-scale anaerobic digestion application, when monitoring community dynamics. Here, the bacterial and archaeal TRFLP profiles were compared with 16S rRNA gene amplicon profiles (Illumina platform) of 25 full-scale anaerobic digestion plants. The α-diversity analysis revealed a higher richness based on Illumina data, compared with the TRFLP data. This coincided with a clear difference in community organisation, Pareto distribution, and co-occurrence network statistics, i.e., betweenness centrality and normalised degree. The β-diversity analysis showed a similar clustering profile for the Illumina, bacterial TRFLP and archaeal TRFLP data, based on different distance measures and independent of phylogenetic identification, with pH and temperature as the two key operational parameters determining microbial community composition. The combined knowledge of temporal dynamics and projected clustering in the β-diversity profile, based on the TRFLP data, distinctly showed that TRFLP is a reliable technique for swift microbial community dynamics screening in full-scale anaerobic digestion plants

The active microbial community more accurately reflects the anaerobic digestion process: 16S rRNA (gene) sequencing as a predictive tool

Background: Amplicon sequencing methods targeting the 16S rRNA gene have been used extensively to investigate microbial community composition and dynamics in anaerobic digestion. These methods successfully characterize amplicons but do not distinguish micro-organisms that are actually responsible for the process. In this research, the archaeal and bacterial community of 48 full-scale anaerobic digestion plants were evaluated on DNA (total community) and RNA (active community) level via 16S rRNA (gene) amplicon sequencing. Results: A significantly higher diversity on DNA compared with the RNA level was observed for archaea, but not for bacteria. Beta diversity analysis showed a significant difference in community composition between the DNA and RNA of both bacteria and archaea. This related with 25.5 and 42.3% of total OTUs for bacteria and archaea, respectively, that showed a significant difference in their DNA and RNA profiles. Similar operational parameters affected the bacterial and archaeal community, yet the differentiating effect between DNA and RNA was much stronger for archaea. Co-occurrence networks and functional prediction profiling confirmed the clear differentiation between DNA and RNA profiles. Conclusions: In conclusion, a clear difference in active (RNA) and total (DNA) community profiles was observed, implying the need for a combined approach to estimate community stability in anaerobic digestion

Enhancement of biogas potential of primary sludge by co-digestion with cow manure and brewery sludge

Anaerobic digestion (AD) has long been used to treat different types of organic wastes especially in the developed world. However, organic wastes are still more often considered as a waste instead of a resource in the developing world, which contributes to environmental pollution arising from their disposal. This study has been conducted at Bugolobi Sewage Treatment Plant (BSTP), where two organic wastes, cow manure and brewery sludge were co-digested with primary sludge in different proportions. This study was done in lab-scale reactors at mesophilic temperature and sludge retention time of 20 d. The main objective was to evaluate the biodegradability of primary sludge generated at BSTP, Kampala, Uganda and enhance its ability of biogas production. When the brewery sludge was added to primary STP sludge at all proportions, the biogas production rate increased by a factor of 3. This was significantly (p<0.001) higher than observed gas yield (337 +/- 18 mL/(L.d)) in the control treatment containing (only STP sludge). Co-digesting STP sludge with cow manure did not show different results compared to the control treatment. In conclusion, Bugolobi STP sludge is poorly anaerobically degradable with low biogas production but co-digestion with brewery sludge enhanced the biogas production rate, while co-digestion with cow manure was not beneficial

Electrochemical technology enables nutrient recovery and ammonia toxicity control in anaerobic digestion

The aim of this study was to investigate the impact of an electrochemical system (ES) on the performance of an anaerobic digester during both low and high ammonium (NH4+) loading rates. For this, a Test (with ES) and Control (without ES) setup was used. Ammonia (NH3), in equilibrium with NH4+, is a toxic compound to the methanogenic community, limits the substrate loading rate and endangers process stability. We hypothesized that, through coupling of an ES to a digester, NH3 toxicity can be controlled with simultaneous recovery of this nutrient. The ES always had a temporary negative effect when switched on. However, during periods of high ammonium loading rates the CH4 production of the Test reactor was at maximum a factor 4.5 higher compared to the Control reactor, which could be explained through a combination of NH4+ extraction and electrochemical pH control. A nitrogen flux of 47 g N m-2 membrane d-1 could be obtained in the Test reactor, resulting in a current and removal efficiency of 38±5% and 28±2%, respectively. For this, an electrochemical power input 17±2 kWh kg-1 N was necessary. In addition, anodic oxidation of sulphide resulted in a significantly lower H2S emission

Integrating anaerobic digestion and slow pyrolysis improves the product portfolio of a cocoa waste biorefinery

The integration of conversion processes with anaerobic digestion is key to increase value from agricultural waste, like cocoa pod husks, generated in developing countries. The production of one metric ton of cocoa beans generates some 15 metric tonnes of organic waste that is today underutilized. This waste can be converted into added value products by anaerobic digestion, converting part of the cocoa pods to biogas while releasing nutrients, and pyrolysis. Here, we compared different scenarios for anaerobic digestion/slow pyrolysis integration in terms of product portfolio (i.e., biogas, pyrolysis liquids, biochar and pyrolysis gases), energy balance and potential for chemicals production. Slow pyrolysis was performed at 350 degrees C and 500 degrees C on raw cocoa pod husks, as well as on digestates obtained from mono-digestion of cocoa pod husks and co-digestion with cow manure. Anaerobic digestion resulted in 20 to 25 wt% of biogas for mono and co-digestion, respectively. Direct pyrolysis of cocoa pod husks mainly resulted in biochar with a maximum yield of 48 wt%. Anaerobic digestion induced compositional changes in the resulting biochar, pyrolysis liquids and evolved gases after pyrolysis. Pyrolysis of mono-digestatee.g., resulted in a more energy-dense organic phase, rich in valuable phenolics while poorer in light oxygenates that hold a modest value. Our comparison shows that co-digestion/slow pyrolysis at 500 degrees C and mono-digestion/slow pyrolysis at 350 degrees C both present high-potential biorefinery schemes. They can be self-sustaining in terms of energy, while resulting in high quality biochar for nutrient recycling and/or energy recovery, and/or phenolics-rich pyrolysis liquids for further upgrading into biorefinery intermediates

Inoculum selection influences the biochemical methane potential of agro-industrial substrates

Obtaining a reliable estimation of the methane potential of organic waste streams in anaerobic digestion, for which a biochemical methane potential (BMP) test is often used, is of high importance. Standardization of this BMP test is required to ensure inter-laboratory repeatability and accuracy of the BMP results. Therefore, guidelines were set out; yet, these do not provide sufficient information concerning origin of and the microbial community in the test inoculum. Here, the specific contribution of the methanogenic community on the BMP test results was evaluated. The biomethane potential of four different substrates (molasses, bio-refinery waste, liquid manure and high-rate activated sludge) was determined by means of four different inocula from full-scale anaerobic digestion plants. A significant effect of the selected inoculum on the BMP result was observed for two out of four substrates. This inoculum effect could be attributed to the abundance of methanogens and a potential inhibiting effect in the inoculum itself, demonstrating the importance of inoculum selection for BMP testing. We recommend the application of granular sludge as an inoculum, because of its higher methanogenic abundance and activity, and protection from bulk solutions, compared with other inocula