Diversity converges during community assembly in methanogenic granules, suggesting a biofilm life-cycle

Anaerobic biological decomposition of organic matter is ubiquitous in Nature wherever anaerobic environments prevail, and is catalysed by hydrolytic, fermentative, acetogenic, methanogenic, and various other groups, including syntrophic bacteria. It is also harnessed in innovative ways in engineered systems that may rely on small (0.1-4.0 mm), spherical, anaerobic granules, which we have found to be highly-replicated, whole-ecosystems harbouring the entire community necessary to mineralise complex organics. We hypothesised distinct granule sizes correspond to stages in a biofilm life-cycle, in which small granules are ‘young’ and larger ones are ‘old’. Here, granules were separated into 10 size fractions used for physico-chemical and ecological characterisation. Gradients of volatile solids, density, settleability, biofilm morphology, methanogenic activity, and EPS profiles were observed across size fractions. Sequencing of 16S rRNA genes indicated linear convergence of diversity during community assembly as granules increased in size. A total of 155 discriminant OTUs were identified, and correlated strongly with physico-chemical parameters. Community assembly across sizes was influenced by a niche effect, whereby Euryarchaeota dominated a core microbiome presumably as granules became more anaerobic. The findings indicate opportunities for precision management of environmental biotechnologies, and the potential of aggregates as playgrounds to study assembly and succession in whole microbiomes

Size shapes the active microbiome of the methanogenic granules, corroborating a biofilm life cycle

Methanogenic archaea are key players in cycling organic matter in nature but also in engineered waste treatment systems, where they generate methane, which can be used as a renewable energy source. In such systems in the built environment, complex methanogenic consortia are known to aggregate into highly organized, spherical granular biofilms comprising the interdependent microbial trophic groups mediating the successive stages of the anaerobic digestion (AD) process. This study separated methanogenic granules into a range of discrete size fractions, hypothesizing different biofilm growth stages, and separately supplied each with specific substrates to stimulate the activity of key AD trophic groups, including syntrophic acid oxidizers and methanogens. Rates of specific methanogenic activity were measured, and amplicon sequencing of 16S rRNA gene transcripts was used to resolve phylotranscriptomes across the series of size fractions. Increased rates of methane production were observed in each of the size fractions when hydrogen was supplied as the substrate compared with those of volatile fatty acids (acetate, propionate, and butyrate). This was connected to a shift toward hydrogenotrophic methanogenesis dominated by Methanobacterium and Methanolinea. Interestingly, the specific active microbiomes measured in this way indicated that size was significantly more important than substrate in driving the structure of the active community in granules. Multivariate integration studywise discriminant analysis identified 56 genera shaping changes in the active community across both substrate and size. Half of those were found to be upregulated in the medium-sized granules, which were also the most active and potentially of the most important size, or life stage, for precision management of AD systems

Diversity converges during community assembly in methanogenic granules, suggesting a biofilm life-cycle

Anaerobic biological decomposition of organic matter is ubiquitous in Nature wherever anaerobic environments prevail, and is catalysed by hydrolytic, fermentative, acetogenic, methanogenic, and various other groups, including syntrophic bacteria. It is also harnessed in innovative ways in engineered systems that may rely on small (0.1-4.0 mm), spherical, anaerobic granules, which we have found to be highly-replicated, whole-ecosystems harbouring the entire community necessary to mineralise complex organics. We hypothesised distinct granule sizes correspond to stages in a biofilm life-cycle, in which small granules are ‘young’ and larger ones are ‘old’. Here, granules were separated into 10 size fractions used for physico-chemical and ecological characterisation. Gradients of volatile solids, density, settleability, biofilm morphology, methanogenic activity, and EPS profiles were observed across size fractions. Sequencing of 16S rRNA genes indicated linear convergence of diversity during community assembly as granules increased in size. A total of 155 discriminant OTUs were identified, and correlated strongly with physico-chemical parameters. Community assembly across sizes was influenced by a niche effect, whereby Euryarchaeota dominated a core microbiome presumably as granules became more anaerobic. The findings indicate opportunities for precision management of environmental biotechnologies, and the potential of aggregates as playgrounds to study assembly and succession in whole microbiomes

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

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

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

A distinct, flocculent, acidogenic microbial community accompanies methanogenic granules in anaerobic digesters

The formation of dense, well-settling methanogenic granules is essential for the operation of high-rate, up-flow anaerobic bioreactors used for wastewater treatment. Granule formation (granulation) mechanisms have been previously proposed, but an ecological understanding of granule formation is still lacking. Additionally, much of the current research on granulation only examines the start-up phase of bioreactor operation, rather than monitoring the fate of established granules and how new granules emerge over time. This paper, therefore, attempts to provide an insight into the microbial ecology of granule formation outside the start-up phase of bioreactor operation and develop an ecological granulation model. The microbial communities of granules actively undergoing growth, breakage, and reformation were examined, and an ecological granulation model was proposed. A distinct pregranular microbial community, with a high proportion of acidogenic organisms, such as the Streptococcaceae, was identified and suggested to have a role in initiating granulation by providing simpler substrates for the methanogenic and syntrophic communities which developed during granule growth. After initial granule formation, deterministic influences on microbial community assembly increased with granule size and indicated that microbial community succession was influenced by granule growth, leading to the formation of a stepwise ecological model for granulation

Unifying concepts in methanogenic, aerobic, and anammox sludge granulation

The retention of dense and well-functioning microbial biomass is crucial for effective pollutant removal in several biological wastewater treatment technologies. High solids retention is often achieved through aggregation of microbial communities into dense, spherical aggregates known as granules, which were initially discovered in the 1980s. These granules have since been widely applied in upflow anaerobic digesters for waste-to-energy conversions. Furthermore, granular biomass has been applied in aerobic wastewater treatment and anaerobic ammonium oxidation (anammox) technologies. The mechanisms underpinning the formation of methanogenic, aerobic, and anammox granules are the subject of ongoing research. Although each granule type has been extensively studied in isolation, there has been a lack of comparative studies among these granulation processes. It is likely that there are some unifying concepts that are shared by all three sludge types. Identifying these unifying concepts could allow a unified theory of granulation to be formed. Here, we review the granulation mechanisms of methanogenic, aerobic, and anammox granular sludge, highlighting several common concepts, such as the role of extracellular polymeric substances, cations, and operational parameters like upflow velocity and shear force. We have then identified some unique features of each granule type, such as different internal structures, microbial compositions, and quorum sensing systems. Finally, we propose that future research should prioritize aspects of microbial ecology, such as community assembly or interspecies interactions in individual granules during their formation and growth

First proof of concept for full-scale, direct, low-temperature anaerobic treatment of municipal wastewater.

Municipal wastewater constitutes the largest fraction of wastewater, and yet treatment processes are largely removal-based. High-rate anaerobic digestion (AD) has revolutionised the sustainability of industrial wastewater treatment and could additionally provide an alternative for municipal wastewater. While AD of dilute municipal wastewater is common in tropical regions, the low temperatures of temperate climates has resulted in slow uptake. Here, we demonstrate for the first time, direct, high-rate, low-temperature AD of low-strength municipal wastewater at full-scale. An 88 m3 hybrid reactor was installed at the municipal wastewater treatment plant in Builth Wells, UK and operated for 290 days. Ambient temperatures ranged from 2 to 18 °C, but remained below 15 °C for > 100 days. Influent BOD fluctuated between 2 and 200 mg L-1. However, BOD removal often reached > 85%. 16S rRNA amplicon sequencing of DNA from the biomass revealed a highly adaptable core microbiome. These findings could provide the basis for the next-generation of municipal wastewater treatment