Linking microbial community structure and function during the acidified anaerobic digestion of grass

This research was funded by the Irish Higher Education Authority Program for Research in Third Level Institutions Cycle 5: – PRTLI-5 ESI Ph.D. ENS Program. This work was also supported by the Wellcome Trust (grant number 094476/Z/10/Z for the TripleTOF 5600 mass spectrometer at the University of St Andrews), NERC (grant number NE/L011956/1), and a Royal Irish Academy Mobility Grant.Harvesting valuable bioproducts from various renewable feedstocks is necessary for the critical development of a sustainable bioeconomy. Anaerobic digestion is a well-established technology for the conversion of wastewater and solid feedstocks to energy with the additional potential for production of process intermediates of high market values (e.g. carboxylates). In recent years, first-generation biofuels typically derived from food crops have been widely utilised as a renewable source of energy. The environmental and socioeconomic limitations of such strategy, however, have led to the development of second-generation biofuels utilising, amongst other feedstocks, lignocellulosic biomass. In this context, the anaerobic digestion of perennial grass holds great promise for the conversion of sustainable renewable feedstock to energy and other process intermediates. The advancement of this technology however, and its implementation for industrial applications, relies on a greater understanding of the microbiome underpinning the process. To this end, microbial communities recovered from replicated anaerobic bioreactors digesting grass were analysed. The bioreactors leachates were not buffered and acidic pH (between 5.5 and 6.3) prevailed at the time of sampling as a result of microbial activities. Community composition and transcriptionally active taxa were examined using 16S rRNA sequencing and microbial functions were investigated using metaproteomics. Bioreactor fraction, i.e. grass or leachate, was found to be the main discriminator of community analysis across the three molecular level of investigation (DNA, RNA and proteins). Six taxa, namely Bacteroidia, Betaproteobacteria, Clostridia, Gammaproteobacteria, Methanomicrobia and Negativicutes accounted for the large majority of the three datasets. The initial stages of grass hydrolysis were carried out by Bacteroidia, Gammaproteobacteria and Negativicutes in the grass biofilms, in addition to Clostridia in the bioreactor leachates. Numerous glycolytic enzymes and carbohydrate transporters were detected throughout the bioreactors in addition to proteins involved in butanol and lactate production. Finally, evidence of the prevalence of stressful conditions within the bioreactors and particularly impacting Clostridia was observed in the metaproteomes. Taken together, this study highlights the functional importance of Clostridia during the anaerobic digestion of grass and thus research avenues allowing members of this taxon to thrive should be explored.Publisher PDFPeer reviewe

Beyond basic diversity estimates – analytical tools for mechanistic interpretations of amplicon sequencing data

Understanding microbial ecology through amplifying short read regions, typically 16S rRNA for prokaryotic species or 18S rRNA for eukaryotic species, remains a popular, economical choice. These methods provide relative abundances of key microbial taxa, which, depending on the experimental design, can be used to infer mechanistic ecological underpinnings. In this review, we discuss recent advancements in in situ analytical tools that have the power to elucidate ecological phenomena, unveil the metabolic potential of microbial communities, identify complex multidimensional interactions between species, and compare stability and complexity under different conditions. Additionally, we highlight methods that incorporate various modalities and additional information, which in combination with abundance data, can help us understand how microbial communities respond to change in a typical ecosystem. Whilst the field of microbial informatics continues to progress substantially, our emphasis is on popular methods that are applicable to a broad range of study designs. The application of these methods can increase our mechanistic understanding of the ongoing dynamics of complex microbial communities

Growth and break-up of methanogenic granules suggests mechanisms for biofilm and community development

Methanogenic sludge granules are densely packed, small, spherical biofilms found in anaerobic digesters used to treat industrial wastewaters, where they underpin efficient organic waste conversion and biogas production. Each granule theoretically houses representative microorganisms from all of the trophic groups implicated in the successive and interdependent reactions of the anaerobic digestion (AD) process. Information on exactly how methanogenic granules develop, and their eventual fate will be important for precision management of environmental biotechnologies. Granules from a full-scale bioreactor were size-separated into small (0.6–1 mm), medium (1– 1.4 mm), and large (1.4–1.8 mm) size fractions. Twelve laboratory-scale bioreactors were operated using either small, medium, or large granules, or unfractionated sludge. After >50 days of operation, the granule size distribution in each of the small, medium, and large bioreactor sets had diversified beyond—to both bigger and smaller than—the size fraction used for inoculation. Interestingly, extra-small (XS; <0.6 mm) granules were observed, and retained in all of the bioreactors, suggesting the continuous nature of granulation, and/or the breakage of larger granules into XS bits. Moreover, evidence suggested that even granules with small diameters could break. “New” granules from each emerging size were analyzed by studying community structure based on high-throughput 16S rRNA gene sequencing. Methanobacterium, Aminobacterium, Propionibacteriaceae, and Desulfovibrio represented the majority of the community in new granules. H2-using, and not acetoclastic, methanogens appeared more important, and were associated with abundant syntrophic bacteria. Multivariate integration (MINT) analyses identified distinct discriminant taxa responsible for shaping the microbial communities in different-sized granules

De novo growth of methanogenic granules indicates a biofilm life-cycle with complex ecology

Methanogenic sludge granules are densely packed, small (diameter, approx. 0.5-2.0 mm) spherical biofilms found in anaerobic digesters used to treat industrial wastewaters, where they underpin efficient organic waste conversion and biogas production. A single digester contains millions of individual granules, each of which is a highly-organised biofilm comprised of a complex consortium of likely billions of cells from across thousands of species – but not all granules are identical. Whilst each granule theoretically houses representative microorganisms from all of the trophic groups implicated in the successive and interdependent reactions of the anaerobic digestion process, parallel granules function side-by-side in digesters to provide a ‘meta-organism’ of sorts. Granules from a full-scale bioreactor were size-separated into small, medium and large granules. Laboratory-scale bioreactors were operated using only small (0.6–1 mm), medium (1–1.4 mm) or large (1.4–1.8 mm) granules, or unfractionated (naturally distributed) sludge. After >50 days of operation, the granule size distribution in each of the small, medium and large bioreactor types had diversified beyond – to both bigger and smaller than – the size fraction used for inoculation. ‘New’ granules were analysed by studying community structure based on high-throughput 16S rRNA gene sequencing. Methanobacterium, Aminobacterium, Propionibacteriaceae and Desulfovibrio represented the majority of the community in new granules. H2-using, and not acetoclastic, methanogens appeared more important, and were associated with abundant syntrophic bacteria. Multivariate integration analyses identified distinct discriminant taxa responsible for shaping the microbial communities in different-sized granules, and along with alpha diversity data, indicated a possible biofilm life cycle. Importance: Methanogenic granules are spherical biofilms found in the built environment, where despite their importance for anaerobic digestion of wastewater in bioreactors, little is understood about the fate of granules across their entire life. Information on exactly how, and at what rates, methanogenic granules develop will be important for more precise and innovative management of environmental biotechnologies. Microbial aggregates also spark interest as subjects in which to study fundamental concepts from microbial ecology, including immigration and species sorting affecting the assembly of microbial communities. This experiment is the first, of which we are aware, to compartmentalise methanogenic granules into discrete, size-resolved fractions, which were then used to separately start up bioreactors to investigate the granule life cycle. The evidence, and extent, of de novo granule growth, and the identification of key microorganisms shaping new granules at different life-cycle stages, is important for environmental engineering and microbial ecology

Anaerobic microbial hydrolysis and fermentation of food waste for volatile fatty acid production

Landfill, incineration, compositing and anaerobic digestion (AD) are the principal food-waste (FW) treatment methods used in the European Union. Because of the EU landfill directive and waste-management policies on organic wastes, however, the landfill approach is no longer a sustainable strategy. The incineration of FW is generally perceived to be energy demanding and inappropriate because of the high water content (>70%) of FW. Composting and AD both fit well in the “3R” waste management hierarchy and are therefore the most appropriate strategies for FW treatment. AD is more attractive than composting, however, due to its ability to stabilise FW and to generate valuable end-products such as organic acids, biogas and fertilisers. The use of FW as sustainable feedstock for the production of these valuable products through AD processes would contribute to reduce the green house gas emission and could enable to meet the EU 2020 renewable energy target; it could also enable an increase in chemical supply. However when dealing with mixed feedstock such as FW, AD process for methane production as the sole beneficiary product is usually less attractive. An alternative approach to the anaerobic digestion of this type of biomass is to aim for production of organic acids which have higher added value than methane. The sustainability of this approach depends on the extent of FW stabilisation, however, as well as on the yields, rates and profiles of the organic acids that are generated. Although FW is generally regarded as being readily biodegradable because of its high volatile solid fraction (90% of total solids), its hydrolysis is still perceived as a rate-limiting step. The enhancement of the hydrolysis step during anaerobic digestion could improve the rate and yield of organic acid accumulation and shorten the solid retention time required for biodegradation. Furthermore, there is a need to uncover the microbial groups involved in the hydrolytic-acidogenic stage as this could help in selecting the best operational parameters for their growth, which in turn could improve the rate of the processes. The objective of this thesis was to investigate and optimise the accumulation of organic acids from restaurant food waste (RFW) AD. The first phase of this study (Chapter 2) was conducted to evaluate and optimise the biodegradation efficiency of RFW using biomethane potential assay; the effect of the FW composition (fat, protein, hemicellulose and cellulose) on biodegradation rates was assessed. In addition, a bioaugmentation strategy was used to enhance the hydrolysis efficiency of the RFW components. The RFW biodegradation efficiency was enhanced by 10 to 15% using the bioaugmentation approach, which consisted of supplementing the primary inoculum with enriched culture developed on pure substrates. The hydrolysis rate constant for the different fractions of the RFW indicated that hemicellulose fraction was easily hydrolysable, while fat was the most recalcitrant. Hemicellulose and cellulose were the two fractions of the RFW enhanced as the result of enriched cultures supplementation. Bacteroides graminisolvens and species affiliated with Porphyromonadaceae were identified as potential cellulose and hemicellulose hydrolysers (respectively) using 16S rRNA profiling. The data obtained suggested a fourteen-day solid retention time for maximum biodegradation of RFW and the possibility of shortening this time through a bioaugmentation strategy. In the second phase of this study (Chapter 3), three leach-bed reactors fed with RFW and initially inoculated with granular sludge were operated at 37oC in a semi-continuous mode. Based on the results obtained in Chapter 2, the solid retention time of fourteen days was applied; a ratio of 1:4 (inoculum:RFW) was chosen to favour the rapid accumulation of organic acids inside the reactors. Stable bioprocess performance was demonstrated, with volatile solid (VS) efficiency above 60%. The hydrolysis of the components of the RFW was believed to be efficient over the initial two days of the incubation, as indicated by the maximum soluble chemical oxygen demand (sCOD) accumulation over the same period. Leachate analysis revealed the accumulation of up to 49 g l-1 of volatile fatty acids (VFAs), of which circa 35% was butyric acid and 25% acetic acid. Microbial communities identified from 16S rRNA-based Illumina sequencing analysis of leach-bed reactors identified Enterococcus as potential hydrolysers. The important fermentative groups (identified as Lactobacillus, Clostridium and Bifidobacterium) were likely responsible for the production of lactic acid, butyric acid and acetic acid, respectively. The results gathered in this second phase suggested that it is feasible to biodegrade the RFW over short periods (two days) while at the same time accumulating organic acids. In the final phase of this study (Chapter 4), process optimisation strategies were investigated in terms of promoting VFAs accumulation; the feasibility to selectively produce caproic and butyric acid from RFW was also assessed. Based on the data generated in Chapter 3, which showed that maximum hydrolysis efficiency was achieved in two days, the solid retention time (SRT) in the leach-bed reactors was reduced from fourteen- to seven days (Chapter 4). Increasing the recirculation regime (frequency) from once to three times per day and reducing the starting liquor VFAs’ concentration from 15 to 6 g COD l-1 resulted in a 55% improvement of VFAs production. With these parameters, VS removal efficiency of over 70% was achieved; caproic acid at the concentration of 21.86 g COD l-1 was the highest VFA produced in the leach-bed reactors. The composition of VFAs was influenced by hydrolysis rate, pH, loading rate and the depletion rate of short chain volatile fatty acids (SCVFAs). The selective production of caproic acid from RFW leachate at the rate of 3 g l-1 d-1 was demonstrated in this study by using hydrogen or the combination of hydrogen and ethanol supplementation. Butyric acid accumulation was observed in the presence of ethanol. Microbial community analysis based on the 16S rRNA sequencing suggested the implication of Clostridium and Peptoniphilus in the generation of butyric acid, while Lactobacillus reuteri could play a role in the accumulation of caproic acid. This study has set some basis for the selective production of caproic and butyric acids from FW. This thesis demonstrates the feasibility of biodegrading RFW while promoting the accumulation of valuable organic acids (mainly VFAs) using leach-bed reactors. The combination of bioprocess monitoring with molecular analyses provided several valuable insights into the complex hydrolytic-acidogenic microbial communities which underpin these processes.2018-09-3

Reproducible, high-yielding, biological caproate production from food waste using a single-phase anaerobic reactor system

Background: Nowadays, the vast majority of chemicals are either synthesised from fossil fuels or are extracted from agricultural commodities. However, these production approaches are not environmentally and economically sustainable, as they result in the emission of greenhouse gases and they may also compete with food production. Because of the global agreement to reduce greenhouse gas emissions, there is an urgent interest in developing alternative sustainable sources of chemicals. In recent years, organic waste streams have been investigated as attractive and sustainable feedstock alternatives. In particular, attention has recently focused on the production of caproate from mixed culture fermentation of low-grade organic residues. The current approaches for caproate synthesis from organic waste are not economically attractive, as they involve the use of two-stage anaerobic digestion systems and the supplementation of external electron donors, both of which increase its production costs. This study investigates the feasibility of producing caproate from food waste (FW) without the supplementation of external electron donors using a single-phase reactor system.& para;& para;Results: Replicate leach-bed reactors were operated on a semi-continuous mode at organic loading of 80 g VS FW l(-1) and at solid retention times of 14 and 7 days. Fermentation, rather than hydrolysis, was the limiting step for caproate production. A higher caproate production yield 21.86 +/- 0.57 g COD l(-1) was achieved by diluting the inoculating leachate at the beginning of each run and by applying a leachate recirculation regime. The mixed culture batch fermentation of the FW leachate was able to generate 23 g caproate COD l(-1) (10 g caproate l(-1)), at a maximum rate of 3 g caproate l(-1) day(-1) under high H-2 pressure. Lactate served as the electron donor and carbon source for the synthesis of caproate. Microbial community analysis suggested that neither Clostridium kluyveri nor Megasphaera elsdenii, which are well-characterised caproate producers in bioreactors systems, were strongly implicated in the synthesis of caproate, but that rather Clostridium sp. with 99% similarity to Ruminococcaceae bacterium CPB6 and Clostridium sp. MT1 likely played key roles in the synthesis of caproate. This finding indicates that the microbial community capable of caproate synthesis could be diverse and may therefore help in maintaining a stable and robust process.& para;& para;Conclusions: These results indicate that future, full-scale, high-rate caproate production from carbohydrate-rich wastes, associated with biogas recovery, could be envisaged

Current Trends in Biological Valorization of Waste-Derived Biomass: The Critical Role of VFAs to Fuel A Biorefinery

The looming climate and energy crises, exacerbated by increased waste generation, are driving research and development of sustainable resource management systems. Research suggests that organic materials, such as food waste, grass, and manure, have potential for biotransformation into a range of products, including: high-value volatile fatty acids (VFAs); various carboxylic acids; bioenergy; and bioplastics. Valorizing these organic residues would additionally reduce the increasing burden on waste management systems. Here, we review the valorization potential of various sustainably sourced feedstocks, particularly food wastes and agricultural and animal residues. Such feedstocks are often micro-organism-rich and well-suited to mixed culture fermentations. Additionally, we touch on the technologies, mainly biological systems including anaerobic digestion, that are being developed for this purpose. In particular, we provide a synthesis of VFA recovery techniques, which remain a significant technological barrier. Furthermore, we highlight a range of challenges and opportunities which will continue to drive research and discovery within the field. Analysis of the literature reveals growing interest in the development of a circular bioeconomy, built upon a biorefinery framework, which utilizes biogenic VFAs for chemical, material, and energy applications

Current Trends in Biological Valorization of Waste-Derived Biomass: The Critical Role of VFAs to Fuel A Biorefinery

The looming climate and energy crises, exacerbated by increased waste generation, are driving research and development of sustainable resource management systems. Research suggests that organic materials, such as food waste, grass, and manure, have potential for biotransformation into a range of products, including: high-value volatile fatty acids (VFAs); various carboxylic acids; bioenergy; and bioplastics. Valorizing these organic residues would additionally reduce the increasing burden on waste management systems. Here, we review the valorization potential of various sustainably sourced feedstocks, particularly food wastes and agricultural and animal residues. Such feedstocks are often micro-organism-rich and well-suited to mixed culture fermentations. Additionally, we touch on the technologies, mainly biological systems including anaerobic digestion, that are being developed for this purpose. In particular, we provide a synthesis of VFA recovery techniques, which remain a significant technological barrier. Furthermore, we highlight a range of challenges and opportunities which will continue to drive research and discovery within the field. Analysis of the literature reveals growing interest in the development of a circular bioeconomy, built upon a biorefinery framework, which utilizes biogenic VFAs for chemical, material, and energy applications

Reproducible, high-yielding, biological caproate production from food waste using a single-phase anaerobic reactor system

Abstract Background Nowadays, the vast majority of chemicals are either synthesised from fossil fuels or are extracted from agricultural commodities. However, these production approaches are not environmentally and economically sustainable, as they result in the emission of greenhouse gases and they may also compete with food production. Because of the global agreement to reduce greenhouse gas emissions, there is an urgent interest in developing alternative sustainable sources of chemicals. In recent years, organic waste streams have been investigated as attractive and sustainable feedstock alternatives. In particular, attention has recently focused on the production of caproate from mixed culture fermentation of low-grade organic residues. The current approaches for caproate synthesis from organic waste are not economically attractive, as they involve the use of two-stage anaerobic digestion systems and the supplementation of external electron donors, both of which increase its production costs. This study investigates the feasibility of producing caproate from food waste (FW) without the supplementation of external electron donors using a single-phase reactor system. Results Replicate leach-bed reactors were operated on a semi-continuous mode at organic loading of 80 g VS FW l−1 and at solid retention times of 14 and 7 days. Fermentation, rather than hydrolysis, was the limiting step for caproate production. A higher caproate production yield 21.86 ± 0.57 g COD l−1 was achieved by diluting the inoculating leachate at the beginning of each run and by applying a leachate recirculation regime. The mixed culture batch fermentation of the FW leachate was able to generate 23 g caproate COD l−1 (10 g caproate l−1), at a maximum rate of 3 g caproate l−1 day−1 under high H2 pressure. Lactate served as the electron donor and carbon source for the synthesis of caproate. Microbial community analysis suggested that neither Clostridium kluyveri nor Megasphaera elsdenii, which are well-characterised caproate producers in bioreactors systems, were strongly implicated in the synthesis of caproate, but that rather Clostridium sp. with 99% similarity to Ruminococcaceae bacterium CPB6 and Clostridium sp. MT1 likely played key roles in the synthesis of caproate. This finding indicates that the microbial community capable of caproate synthesis could be diverse and may therefore help in maintaining a stable and robust process. Conclusions These results indicate that future, full-scale, high-rate caproate production from carbohydrate-rich wastes, associated with biogas recovery, could be envisaged