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Anaerobic digestion (AD) for waste, and wastewater, management was identified in the 1970s as a forerunner in the push for sustainability. The development of AD applications resulted in the discove...

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. IMPORTANCE Complex microbial communities in engineered environments can aggregate to form surface-attached biofilms. Others form suspended biofilms, such as methanogenic granules. The formation of dense, methanogenic granules underpins the performance of high-rate, anaerobic bioreactors in industrial wastewater treatment. Granule formation (granulation) has been well studied from a physico-chemical perspective, but the ecological basis is poorly understood. We identified a distinct, flocculent, microbial community, which was present alongside granules, comprising primary consumers likely key in providing simpler substrates to granules. This flocculent community is understudied in anaerobic digestion and may initiate, or perpetuate, granule formation. We propose that it may be possible to influence bioreactor performance (e.g., to regulate volatile fatty acid concentrations) by manipulating this community. The patterns of microbial community diversity and assembly revealed by the study indicate that cycles of granule growth and breakage lead to overall diversification of the bioreactor meta-community, with implications for bioreactor process stability.

Identifying species interactions in a microbial community and how this relates to community function is a key challenge. Towards addressing this challenge, we present here an extensive genome-resolved, longitudinal dataset and associated metadata. We collected weekly samples of microbial communities and recorded operating conditions from industrial methane producing anaerobic digestion reactors for a year. This allowed us to recover 2240 dereplicated metagenome assembled genomes (dMAGs), together with their coverage dynamics and functional annotations from which functional traits were inferred. Of these dMAGs, 1910 were novel species, with 22 representing novel orders and classes. Methanogenic communities are expected to be strongly structured by syntrophic and other associations between the methanogens and syntrophs that produce their substrates. We identified 450 potential syntrophic dMAGs by searching for pairs of methanogenic and non-methanogenic dMAGs that had highly correlated time-series. Genomes of potential syntrophs were enriched for oxidoreductases and sugar transport genes and there was a strong taxonomic signal in their associations with methanogens. Of particular note, we found that Bathyarchaeiea associated specifically with methanogens from the Thermoplasmata, and Thermococci classes. Same syntrophic associations were only rarely observed across multiple reactors, suggesting that syntrophies might be facultative, with particular strains within a species forming syntrophic associations only sometimes and not necessarily always with the same methanogenic partner. The presented results show that longitudinal metagenomics is a highly valuable approach for identifying species and their interactions in microbial communities.One Sentence SummaryLongitudinal study of microbial communities identifies novel species and predicts their interactions and role in community function.

Use of high-throughput sequencing is widespread in efforts to understand the microbial communities in natural and engineered systems. Many built ecosystems, in particular those used for engineered wastewater treatment, have harnessed the metabolic capacity of complex microbial communities for the effective removal and recovery of organic pollutants. Recent efforts to better understand and precisely engineer such systems have increasingly used high-throughput sequencing to map the structure and function of wastewater treatment microbiomes. An enormous amount of data is readily available on online repositories such as the National Center for Biotechnology Information Short Read Archive (NCBI SRA). Here, we describe and provide an optimised meta-analysis workflow to utilise this resource to collate heterogenous studies together for anaerobic digestion research. We analysed 16S rRNA gene Illumina Miseq amplicon sequencing data from 31 anaerobic digestion studies (from high-rate digesters), including >1,300 samples. Additionally, we compare several methodological choices: extraction method, v-region, taxonomical database, and the classifier. We demonstrate that collation of data from multiple v-regions can be achieved by using only the taxa for which sequences are available in the reference databases, without losses in diversity trends. This is made possible by focusing on alternative strategies for taxonomic assignments, namely, bayesian lowest common ancestor (BLCA) algorithm which offers increased resolution to the traditional naïve bayesian classifier (NBC). While we demonstrate this using an anaerobic digestion wastewater treatment dataset, this methodology can be translated to perform meta-analysis on amplicon sequences in any field. These findings not only provide a roadmap for meta-analysis in any field, but additionally provide an opportunity to reuse extensive data resources to ultimately advance knowledge of wastewater treatment systems.ImportanceIn this study, we have combined sequencing data from 31 individual studies with the purpose of identifying a meta-analysis workflow which can accurately collate data derived from sequencing different v-regions with minimal data loss and more accurate diversity patterns. While we have used Anaerobic Digestion (AD) communities for our proof-of-concept, our workflow (Fig 1) can be translated to any Illumina MiSeq meta-analysis study, in any field. Thereby, we provide the foundation for intensive data mining of existing amplicon sequencing resources. Such data-mining can provide a global perspective on complex microbial communities.Graphical Abstract

Biological wastewater conversion processes collectively constitute one of the single biggest worldwide applications of microbial communities. There is an obvious requirement, therefore, to study the microbial systems central to the success of such technologies. Methanogenic granules, in particular, are architecturally fascinating biofilms that facilitate highly organized cooperation within the metabolic network of the anaerobic digestion (AD) process and, thus, are especially intriguing model systems for microbial ecology. This study, in a way not previously reported, provoked syntrophic and methanogenic activity and the structure of the microbial community, using specific substrates targeting the key trophic groups in AD. Unexpectedly, granule size more strongly than substrate shaped the active portion of the microbial community. Importantly, the findings suggest the size, or age, of granules inherently shapes the active microbiome linked to a life cycle. This provides exciting insights into the function of, and the potential for additional modeling of biofilm development in, methanogenic granules., 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. IMPORTANCE Biological wastewater conversion processes collectively constitute one of the single biggest worldwide applications of microbial communities. There is an obvious requirement, therefore, to study the microbial systems central to the success of such technologies. Methanogenic granules, in particular, are architecturally fascinating biofilms that facilitate highly organized cooperation within the metabolic network of the anaerobic digestion (AD) process and, thus, are especially intriguing model systems for microbial ecology. This study, in a way not previously reported, provoked syntrophic and methanogenic activity and the structure of the microbial community, using specific substrates targeting the key trophic groups in AD. Unexpectedly, granule size more strongly than substrate shaped the active portion of the microbial community. Importantly, the findings suggest the size, or age, of granules inherently shapes the active microbiome linked to a life cycle. This provides exciting insights into the function of, and the potential for additional modeling of biofilm development in, methanogenic granules.

Background In this study, individual anaerobic granular biofilms were used as true community replicates to assess whole-microbial-community responses to environmental cues. The aggregates originated from three different biomass sources, i.e. three different engineered biological wastewater treatment systems, were each size-separated into three fractions – small, medium and large – and characterised according to organic matter concentrations and rates of methanogenic activity. Differences in the microbial community structure of each size fraction from each source were determined using 16S rRNA gene sequencing. Subsequently, single granules from the large size fraction of one of the sources were separately subjected controlled environmental cues in novel micro batch reactors (mBRs). Results Organic content, methanogenic activity, and microbial community were significantly different between the three size fractions, with diversity trajectories replicated across the three sludge sources – indicating a potential development model as granules age. Individual large granules from one of these sources were statistically identical with respect to the structure of the active community based on cDNA analysis. It was observed that the active microbial community of individual granules, at the depth of 16S rRNA sequencing, produced reproducible responses to environmental conditions. While each condition resulted in the up-regulation of particular OTUs and clades, the core microbiome, consisting of many fermentative bacteria along with methanogenic archaea, namely, Methanosarcina and Methanobacterium , persisted. Conclusions At this level, single anaerobic granules can be considered highly-replicated whole-ecosystems, opening the door to high-throughput studies in Microbial Ecology.

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.

As the global demand for water increases, so does the quantity of wastewater requiring treatment. Due to a relatively low carbon footprint, compared with conventional wastewater treatment technologies, anaerobic digestion (AD) was identified in the 1970s as a forerunner in the push for sustainability, when interest in sustainable technologies and renewable energy sources was first sparked. AD technology development ultimately resulted in the discovery of the ‘anaerobic granule’. It is a spontaneously-forming bio-aggregate of microbial cells capable of digesting pollutants and producing methane-rich biogas as a renewable source of bioenergy. The high settling velocity of such granules meant that AD systems could be operated as high-rate treatment processes, because the active, relatively-slow-growing, pollutant-removing biomass would be retained inside, and not washed out of, even bioreactors operated at extremely high volumetric loading rates. In the intervening years the emergence of the anaerobic ammonium oxidising (anammox) granule, aerobic granule, hydrogenic granule, oxygenic photogranule, and many other functionally-specialised granules, has opened new opportunities in wastewater treatment biotechnology. Whilst environmental engineering based around wastewater treatment is still a growing field of research and implementation, the granule (in all forms) is starting to catch the attention of microbial ecologists. It is a self-immobilised biofilm, with many of the properties of ‘conventional’ biofilms formed in Nature. However, as a single entity, a granule represents an entire community of microorganisms, competing or functioning cooperatively or in syntrophy. Together, inside a bioreactor, granules perform side-by-side arguably representing a meta-organism. Granules are gaining traction as the perfect samples for high-throughput studies on fundamental ecological concepts. Granular biofilms can be used to test hypotheses around drivers of diversity, community assembly, biofilm formation and maturation, community expansion and succession, community stress response, among others. This review outlines the history of three of the most influential types of granules: the anaerobic (methanogenic), aerobic and anammox granule. The main biochemical processes found in each type; their primary characteristics; and the typical makeup of the microbial community underpinning the processes are compared. Finally, the adoption of granules as the perfect ‘playground’ for experiments in microbial ecology is reviewed.

Up-flow anaerobic bioreactors are widely applied for high-rate digestion of industrial wastewaters and rely on formation, and retention, of methanogenic granules, comprising of dense, fast-settling, microbial aggregates (approx. 0.5–4.0 mm in diameter). Granule formation (granulation) mechanisms have been reasonably well hypothesized and documented. However, this study used laboratory-scale bioreactors, inoculated with size-separated granular sludge to follow new granule formation, maturation, disintegration and re-formation. Temporal size profiles, volatile solids content, settling velocity, and ultrastructure of granules were determined from each of four bioreactors inoculated only with small granules, four with only large granules, and four with a full complement of naturally-size-distributed granules. Constrained granule size profiles shifted toward the natural distribution, which was associated with maximal bioreactor performance. Distinct morphological features characterized different granule sizes and biofilm development stages, including ‘young’, ‘juvenile’, ‘mature’ and ‘old’. The findings offer opportunities toward optimizing management of high-rate, anaerobic digesters by shedding light on the rates of granule growth, the role of flocculent sludge in granulation and how shifting size distributions should be considered when setting upflow velocities.

Low temperature anaerobic digestion remains in its infancy, despite increasing interest for the treatment of complex wastewaters. In this study, the feasibility of low-temperature anaerobic treatment of dairy wastewater was assessed during a 443-day laboratory-scale bioreactor trial. The bioreactors were operated in triplicate at organic loading rates of 7.5–9 kgCODm−3d−1 throughout five operational phases. The structure of the microbial community was analysed using quantitative real-time PCR and amplicon sequencing of 16S rRNA genes from DNA and rRNA. The results indicated that low-temperature treatment of dairy wastewater is feasible at 15 °C, but that reactor configuration remains extremely important. The upflow anaerobic sludge bed (UASB) configuration out-performed the expanded granular sludge bed (EGSB)-based configurations. Decreased temperatures resulted in significant reductions in microbiome diversity. Methanosaeta was identified as a dominant genus throughout the trial, while Lactococcus was identified as an important bacterial genus at low-temperatures. However, the relative abundance of Lactococcus was significantly influenced by reactor configuration.

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. Electronic supplementary material The online version of this article (10.1186/s13068-018-1101-4) contains supplementary material, which is available to authorized users.

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,PropionibacteriaceaeandDesulfovibriorepresented 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.ImportanceMethanogenic 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, ofde novogranule growth, and the identification of key microorganisms shaping new granules at different life-cycle stages, is important for environmental engineering and microbial ecology.

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.