Individual methanogenic granules are whole-ecosystem replicates with reproducible responses to environmental cues

Background In this study, individual methanogenic (anaerobic), granular biofilms were used as true community replicates to assess whole-microbial-community responses to environmental cues. The aggregates were sourced from a lab-scale, engineered, biological wastewater treatment system, were size-separated, and the largest granules were individually subjected to controlled environmental cues in micro-batch reactors (μBRs). Results Individual granules were identical with respect to the structure of the active community based on cDNA analysis. Additionally, it was observed that the active microbial community of individual granules, at the depth of 16S rRNA gene sequencing, produced reproducible responses to environmental changes in pH, temperature, substrate, and trace-metal supplementation. We identified resilient and susceptible taxa associated with each environmental condition tested, as well as selected specialists, whose niche preferences span the entire trophic chain required for the complete anaerobic degradation of organic matter. Conclusions We found that single anaerobic granules can be considered highly-replicated whole-ecosystems with potential usefulness for the field of microbial ecology. Additionally, they act as the smallest whole-community unit within the meta-community of an engineered bioreactor. When subjected to various environmental cues, anaerobic granules responded reproducibly allowing for rare or unique opportunities for high-throughput studies testing whole-community responses to a wide range of environmental conditions

First evidence for temperature influencing the enrichment, assembly and activity of polyhydroxyalkanoate-synthesizing mixed microbial communities

Polyhydroxyalkanoates (PHA) are popular biopolymers due to their potential use as biodegradable thermoplastics. In this study, three aerobic sequencing batch reactors were operated identically except for their temperatures, which were set at 15 °C, 35 °C, and 48 °C. The reactors were subjected to a feast–famine feeding regime, where carbon sources are supplied intermittently, to enrich PHA-accumulating microbial consortia. The biomass was sampled for 16S rRNA gene amplicon sequencing of both DNA (during the enrichment phase) and cDNA (during the enrichment and accumulation phases). All temperatures yielded highly enriched PHA-accumulating consortia. Thermophilic communities were significantly less diverse than those at low or mesophilic temperatures. In particular, Thauera was highly adaptable, abundant, and active at all temperatures. Low temperatures resulted in reduced PHA production rates and yields. Analysis of the microbial community revealed a collapse of community diversity during low-temperature PHA accumulation, suggesting that the substrate dosing strategy was unsuccessful at low temperatures. This points to future possibilities for optimizing low-temperature PHA accumulation

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

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

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

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

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

The life-cycle of methanogenic granular biofilms

Anaerobic granules, sometimes referred to as methanogenic granules, underpin the sustainable treatment of wastewater in high-rate anaerobic bioreactors. They are spontaneously-forming self-immobilised biofilm aggregates that contain the entire microbial community necessary to completely mineralise complex organics into a methane-based biogas. Their discovery in the 1970s completely revolutionised wastewater treatment engineering and prompted the design of numerous new, sustainable, and efficient systems based around anaerobic digestion (AD). Moreover, the success of the anaerobic granule has driven the engineered design of various type of granules, each with unique functions. Indeed, the aerobic granule has played a significant role in upgrading conventional aerobic treatment processes, and the anaerobic ammonium oxidation (anammox) granule has challenged the well-established practices in nutrient removal – providing a sustainable path for nitrogen transformations. The unique properties of granules including their settleability, and their densely-packed, diverse microbial communities have been consistently cited as the primary characteristics influencing their success. However, while granules have had a significant impact on environmental engineering, they are recently catching the interest of microbial ecologists. In no other form in Nature do biofilms exist with such defined boundaries, wide applications, and complex communities, creating whole-ecosystems in a 1-4 mm diameter. The potential of such microbial aggregates for high-throughput investigations into ecological theory is endless. They can be used to pursue the nature of microbial stress responses, community assembly, community expansion and succession, mechanisms around biofilm disintegration, cellular communication and signaling, and so on. The objective of this thesis was to use anaerobic granules as a means to study biofilm growth, life-cycle, function, and microbial diversity. Each experimental chapter herein operates on the hypothesis that size can be used as an indicator of growth-stage, where small granules are ‘young’ and large granules are ‘old’. Each experimental setup involved the separation of anaerobic granules into discrete size fractions, and the comparison of these sizes in terms of structure, function, and microbial diversity. The first experimental chapter (Chapter 2) describes a characterisation-based study in which granules were separated into 10 discrete size fractions. Each size was compared based on several different physico-chemical, and molecular parameters. It was determined that size matters for the structure and function of anaerobic granular biofilms as strong gradients were observed across the granular ultrastructure using scanning electron microscopy (SEM); in the makeup of the loosely-bound extracellular polymeric substances (EPS); in granular density and settleability; and in the rates of methane generation. Additionally, as granule size increased – representing granule growth – the rarefied richness and Shannon entropy of the microbial community decreased linearly, becoming increasingly dominated by a core group of methanogenic archaea. The diversity of these Euryarchaeota, however, was not increasing with increasing granule size. It is therefore likely that a functional group theory explains the pronounced diversity shifts in these communities. Furthermore, it is proposed that these granular biofilms may undergo a predictable life-cycle and growth model, in which it is possible that medium-sized granules may be the most optimal for efficient bioreactor function. In the second experimental chapter (Chapter 3) the growth hypothesis was tested by operating a series of lab-scale bioreactors each inoculated with granules from within a defined size range (small, medium, or large). Following a 51-day trial, the size distribution of the sludge was reexamined and was found to have diversified beyond the size fraction used for inoculation. In particular, a full range of sizes was found in the bioreactors initially containing only large granules indicating the emergence of both ‘de novo’ and bigger granules. Sequencing of 16S rRNA gene amplicons from granules from each of these emerging sizes revealed that Methanobacterium, Aminobacterium, Propionibacteriaceae and Desulfovibrio made up the majority of the microbial community. Based on the relative abundances of microbial taxa, a clear shift to the hydrogenotrophic pathway for methanogenesis was apparent, and the responsive emergence of several syntrophic bacteria was observed. This chapter describes how, over a short trial, granules do ‘grow’ in a progressive manner – from small into medium and, eventually, large. It remains unclear, however, whether or not, or by which mechanisms, this process is cyclical. To help elucidate the nature of the life-cycle, in Chapter 4 a cDNA-based approach was used to analyse the active microbiome of the size fractions. Each fraction was separately supplied with a series of methanogenic substrates: acetate, propionate, butyrate and H2/CO2. The biomass was sampled from incubations during the exponential phase of biogas production, and the cDNA was used for 16S rRNA amplicon sequence analysis. The makeup of the active microbiome, and specifically the beta-diversity analyses, indicated that a granule biofilm life-cycle is likely in such systems; the active community structure of the largest and smallest sizes was more similar than previously observed with DNA-based analyses. Moreover, the active microbiome provided insights into key organisms involved in the degradation of specific substrates. However, size remained a more significant driver than substrate for overall active community structure. In summary, this thesis provides evidence in support of a growth, and life-cycle model, for methanogenic granular biofilms

The life-cycle of methanogenic granular biofilms

Anaerobic granules, sometimes referred to as methanogenic granules, underpin the sustainable treatment of wastewater in high-rate anaerobic bioreactors. They are spontaneously-forming self-immobilised biofilm aggregates that contain the entire microbial community necessary to completely mineralise complex organics into a methane-based biogas. Their discovery in the 1970s completely revolutionised wastewater treatment engineering and prompted the design of numerous new, sustainable, and efficient systems based around anaerobic digestion (AD). Moreover, the success of the anaerobic granule has driven the engineered design of various type of granules, each with unique functions. Indeed, the aerobic granule has played a significant role in upgrading conventional aerobic treatment processes, and the anaerobic ammonium oxidation (anammox) granule has challenged the well-established practices in nutrient removal – providing a sustainable path for nitrogen transformations. The unique properties of granules including their settleability, and their densely-packed, diverse microbial communities have been consistently cited as the primary characteristics influencing their success. However, while granules have had a significant impact on environmental engineering, they are recently catching the interest of microbial ecologists. In no other form in Nature do biofilms exist with such defined boundaries, wide applications, and complex communities, creating whole-ecosystems in a 1-4 mm diameter. The potential of such microbial aggregates for high-throughput investigations into ecological theory is endless. They can be used to pursue the nature of microbial stress responses, community assembly, community expansion and succession, mechanisms around biofilm disintegration, cellular communication and signaling, and so on. The objective of this thesis was to use anaerobic granules as a means to study biofilm growth, life-cycle, function, and microbial diversity. Each experimental chapter herein operates on the hypothesis that size can be used as an indicator of growth-stage, where small granules are ‘young’ and large granules are ‘old’. Each experimental setup involved the separation of anaerobic granules into discrete size fractions, and the comparison of these sizes in terms of structure, function, and microbial diversity. The first experimental chapter (Chapter 2) describes a characterisation-based study in which granules were separated into 10 discrete size fractions. Each size was compared based on several different physico-chemical, and molecular parameters. It was determined that size matters for the structure and function of anaerobic granular biofilms as strong gradients were observed across the granular ultrastructure using scanning electron microscopy (SEM); in the makeup of the loosely-bound extracellular polymeric substances (EPS); in granular density and settleability; and in the rates of methane generation. Additionally, as granule size increased – representing granule growth – the rarefied richness and Shannon entropy of the microbial community decreased linearly, becoming increasingly dominated by a core group of methanogenic archaea. The diversity of these Euryarchaeota, however, was not increasing with increasing granule size. It is therefore likely that a functional group theory explains the pronounced diversity shifts in these communities. Furthermore, it is proposed that these granular biofilms may undergo a predictable life-cycle and growth model, in which it is possible that medium-sized granules may be the most optimal for efficient bioreactor function. In the second experimental chapter (Chapter 3) the growth hypothesis was tested by operating a series of lab-scale bioreactors each inoculated with granules from within a defined size range (small, medium, or large). Following a 51-day trial, the size distribution of the sludge was reexamined and was found to have diversified beyond the size fraction used for inoculation. In particular, a full range of sizes was found in the bioreactors initially containing only large granules indicating the emergence of both ‘de novo’ and bigger granules. Sequencing of 16S rRNA gene amplicons from granules from each of these emerging sizes revealed that Methanobacterium, Aminobacterium, Propionibacteriaceae and Desulfovibrio made up the majority of the microbial community. Based on the relative abundances of microbial taxa, a clear shift to the hydrogenotrophic pathway for methanogenesis was apparent, and the responsive emergence of several syntrophic bacteria was observed. This chapter describes how, over a short trial, granules do ‘grow’ in a progressive manner – from small into medium and, eventually, large. It remains unclear, however, whether or not, or by which mechanisms, this process is cyclical. To help elucidate the nature of the life-cycle, in Chapter 4 a cDNA-based approach was used to analyse the active microbiome of the size fractions. Each fraction was separately supplied with a series of methanogenic substrates: acetate, propionate, butyrate and H2/CO2. The biomass was sampled from incubations during the exponential phase of biogas production, and the cDNA was used for 16S rRNA amplicon sequence analysis. The makeup of the active microbiome, and specifically the beta-diversity analyses, indicated that a granule biofilm life-cycle is likely in such systems; the active community structure of the largest and smallest sizes was more similar than previously observed with DNA-based analyses. Moreover, the active microbiome provided insights into key organisms involved in the degradation of specific substrates. However, size remained a more significant driver than substrate for overall active community structure. In summary, this thesis provides evidence in support of a growth, and life-cycle model, for methanogenic granular biofilms