Anaerobic Digestion Microbial Community Research Articles

Optimizing the microbial community composition in anaerobic digesters to improve biogas yields from food waste

Methods: Food waste collected from local restaurants and households in New York city will be used in this study. The food waste will be characterized to analyze its composition. Batch anaerobic digesters will be inoculated with microbial communities from existing food waste digesters. DNA analysis using 16S rRNA gene sequencing will be done to show the initial microbial consortia. Various perturbations will be applied to the digesters including changes in temperature, pH, feeding rates, mixing intensities to select for microbial populations yielding higher biogas production. Biogas production will be monitored over time. DNA analysis will be repeated after perturbation to analyze changes in the microbial community structure. Results: Preliminary results show Food waste characterized was found to contain 35% proteins, 45% carbohydrates and 20% fats. The initial microbial consortia in the digesters was dominated by bacteria from the genera Clostridium, Bacteroides and Methanothermobacter. Increasing feeding rates led to a 40% increase in biogas yields. Microbial analysis indicated a shift in populations with Clostridium becoming the most abundant genus. Decreasing pH from 7 to 6.5 further enhanced biogas by 25% with Clostridium and Syntrophomonas becoming dominant. Discussion: The study demonstrates that optimizing operational conditions can effectively manipulate the microbial communities in anaerobic digesters processing food waste. By applying selective pressures, populations better suited to degrade the local food waste and produce higher methane yields can be enriched. Further experiments will aim to construct stable, optimized consortia through controlled perturbation for robust and efficient food waste digestion. The approach holds promise for improving biogas production from food waste globally

Effect of hydrothermal-acid pretreatment on methane yield and microbial community in anaerobic digestion of rice straw

Hydrothermal pretreatment has been proposed to enhance straw methane yield during anaerobic digestion recently. However, the combined effect of hydrothermal and organic acid pretreatment (HTOAP) needs further investigation. This study identified optimal pretreatment at 120 °C with 3 % acetic acid for 24 h by orthogonal design method. The HTOAP increased the reducing sugar content by destroying the lignocellulosic structure. A 79 % increment of methane production after HTOAP was observed compared to the untreated group. Microbial analysis showed that HTOAP enriched the relative abundance of lignocellulose-degraders, such as W5053, Thermanaerovibrio, Caldicoprobacter, as well as the syntrophic acetate oxidizing bacteria Syntrophaceticus. Moreover, Methanobacterium conducted hydrogenotrophic methanogenesis dominantly. Furthermore, the potential function analysis showed that HTOAP stimulated the expression of key enzymes in the hydrogenotrophic pathway, including carbon-monoxide dehydrogenase (EC 1.2.7.4) and coenzyme F420 hydrogenase (EC 1.12.98.1). This investigation illustrated the potential of HTOAP of rice straw to facilitate methane production.

Adaptation of Anaerobic Digestion Microbial Communities to High Ammonium Levels: Insights from Strain-Resolved Metagenomics.

Ammonia release from proteinaceous feedstocks represents the main inhibitor of the anaerobic digestion (AD) process, which can result in a decreased biomethane yield or even complete failure of the process. The present study focused on the adaptation of mesophilic AD communities to a stepwise increase in the concentration of ammonium chloride in synthetic medium with casein used as the carbon source. An adaptation process occurring over more than 20 months allowed batch reactors to reach up to 20 g of NH4+ N/L without collapsing in acidification nor ceasing methane production. To decipher the microbial dynamics occurring during the adaptation and determine the genes mostly exposed to selective pressure, a combination of biochemical and metagenomics analyses was performed, reconstructing the strains of key species and tracking them over time. Subsequently, the adaptive metabolic mechanisms were delineated by following the single nucleotide variants (SNVs) characterizing the strains and prioritizing the associated genes according to their function. An in-depth exploration of the archaeon Methanoculleus bourgensis vb3066 and the putative syntrophic acetate-oxidizing bacteria Acetomicrobium sp. ma133 identified positively selected SNVs on genes involved in stress adaptation. The intraspecies diversity with multiple coexisting strains in a temporal succession pattern allows us to detect the presence of an additional level of diversity within the microbial community beyond the species level.

Propidium monoazide - polymerase chain reaction reveals viable microbial community shifts in anaerobic membrane bioreactors treating domestic sewage at low temperature

An anaerobic membrane bioreactor (AnMBR) treated domestic sewage at 15 °C under different hydraulic retention time (HRT) conditions (6, 12, 16, and 24 h). Propidium monoazide (PMA)-PCR excluded microorganisms without intact cell membranes, focusing on the viable microbial community in anaerobic digestion. The results showed that the 6-hour HRT had poor treatment performance: low chemical oxygen demand removal efficiency (below 80%) and high mean trans-membrane pressure and flux (15 kPa and 9.4 L/(m2 h)). Comparatively, PMA-PCR combined with next-generation sequencing improved the identification of microbial changes compared to conventional 16S rRNA gene sequencing. HRT influenced microorganisms in the hydrolysis and acid-production stages, including carbohydrate-degrading bacteria such as Bifidobacterium and Prevotella 1. Remarkably, a comparison with an AnMBR at 25 °C showed Proteobacteria to be the main cause of membrane fouling in the low-temperature AnMBR, with most operational taxonomic units negatively correlated with HRT and solids retention time.

Ultrasonication-enhanced biogas production in anaerobic digestion of waste active sludge: A pilot scale investigation

In this study, long-term pilot experiments were conducted to investigate the effects of ultrasonication (US) pretreatment on anaerobic digestion (AD) performance. Life cycling assessment (LCA) was used to assess energy efficiency and environmental impacts. With the response to 90-s US at 0.8 W/mL, the soluble COD concentrations increased by 359.0%, whereas the sludge particle size decreased by 56.7%. The positive effects of US increased biogas production by 15.0% and 25.6% at raw sludge VS/TS averages of 0.60 and 0.43, respectively. LCA results indicated that energy neutralization for US-AD was feasible, with a total energy balance of 461.3 kW·h/t DS because of increased energy recovery. The use of US pretreatment allowed for a more diverse microbial community in AD, thereby improving resilience to environmental fluctuations. Unfortunately, US-AD exhibited weak efficiency in inactivating pathogens and antibiotic resistance genes (ARGs), and US could hardly improve the AD control of pathogens carrying ARGs.

Influence of organic load on biogas production and response of microbial community in anaerobic digestion of food waste

Organic load (OL) is one important parameter influencing performance of anaerobic digestion, yet it is unclear how it affects the biogas composition and microbial community. This work investigated the influence of OL on biogas production from food waste (FW) and the response of microbial community. Results showed that the main biogas component was methane at low OLs (<10 g VS/L) while it turned into hydrogen at high OLs (>10 g VS/L). The optimum methane and hydrogen yields were 184.4 and 61.3 mL/g VS, corresponding to the OLs of 4 and 20 g VS/L, respectively. Analysis of microbial community indicated that high OLs leaded to the decrease of hydrolysis bacteria and methanogen while it helped to increase the relative abundance of hydrogen-producing bacteria. This study cast an insight that it is essential to control the OL and reinforce the hydrogenogen to obtain high output of hydrogen energy.

Effect of magnetite addition on transcriptional profiles of syntrophic Bacteria and Archaea during anaerobic digestion of propionate in wastewater sludge.

Anaerobic digestion (AD) is an important technology for the effective conversion of waste and wastewater to methane. Here, syntrophic bacteria transfer molecular hydrogen (H2 ), formate, or directly supply electrons (direct interspecies electron transfer, DIET) to the methanogens. Evidence is accumulating that the methanation of short-chain fatty acids can be enhanced by the addition of conductive material to the anaerobic digester, which has often been attributed to the stimulation of DIET. Since little is known about the transcriptional response of a complex AD microbial community to the addition of conductive material, we added magnetite to propionate-fed laboratory-scale reactors that were inoculated with wastewater sludge. Compared to the control reactors, the magnetite-amended reactors showed improved methanation of propionate. A genome-centric metatranscriptomics approach identified the active SCFA-oxidizing bacteria that affiliated with Firmicutes, Desulfobacterota and Cloacimonadota. The transcriptional profiles revealed that the syntrophic bacteria transferred acetate, H2 and formate to acetoclastic and hydrogenotrophic methanogens, whereas transcription of potential determinants for DIET such as conductive pili and outer-membrane cytochromes did not significantly change with magnetite addition. Overall, changes in the transcriptional profiles of syntrophic Bacteria and Archaea in propionate-fed lab-scale reactors amended with magnetite refute a major role of DIET in the studied system.

Roadmap from Microbial Communities to Individuality Modeling for Anaerobic Digestion of Sewage Sludge.

Biological models describing anaerobic digestion (AD) of sewage sludge have been widely applied to test various control and operation strategies. Anaerobic digestion model 1 (ADM1) provides a generic platform that includes the main processes of AD, excluding homoacetogenesis and the microbial structure. Homoacetogenic bacteria have been identified as important competitors for hydrogen consumption and acetate production. Although recent advances in meta-omics techniques have improved our characterization of AD microbial communities, conventional models treat functional groups as homogeneous and overlook the physiology and behavior of microbial individuality, limiting insights into mechanisms governing process performance. A novel microbial individuality model (MIM) that integrates kinetics, energetics, and agent-based modeling to describe a microbiome's behavior as heterogenic populations, including homoacetogenesis, was developed. The MIM was validated with two datasets from previous studies through daily biogas production, methane content, compound concentrations, and microbial relative abundance changes. The MIM identified the emergence of Methanosaeta at low concentrations of acetate. Moreover, this simulation supports experimental studies confirming that the overlooked homoacetogenesis is an important hydrogen sink in AD. Validated MIMs are expected to provide insights into syntrophic and competitive interactions among microbiomes in AD systems while testing different operational parameters in a virtual environment. The MIM offers a methodological framework to other biological treatment systems and their microbial community dynamics.

Effect of Hydrogen Peroxide on Hydrogen Production from Melon Fruit (Cucumis melo L.) Waste by Anaerobic Digestion Microbial Community

Biohydrogen (H2) production has the potential to provide clean, environmentally friendly, and cost-effective energy sources. The effect of increasing oxidative stress on biohydrogen production by acid-treated anaerobic digestion microbial communities was studied. The use of varying amounts of hydrogen peroxide (H2O2; 0.1, 0.2, and 0.4 mM) for enhancing hydrogen production from melon fruit waste was investigated. It was found that H2O2 amendment to the H2-producing mixed culture increased hydrogen production. Treatment with 0.4 mM H2O2 increased cumulative H2 output by 7.7% (954.6 mL/L), whereas treatment with 0.1 mM H2O2 enhanced H2 yield by 23.8% (228.2 mL/gVS) compared to the untreated control. All treatments showed a high H2 production rate when the pH was 4.5 – 7.0. H2O2-treated samples exhibited greater resilience to pH reduction and maintained their H2 production rate as the system became more acidic during H2 fermentation. The application of H2O2 affected the volatile fatty acid (VFA) profile during biohydrogen fermentation, with an increase in acetic and propionic acid and a reduction in formic acid concentration. The H2O2 treatment positively affects H2 production and is proposed as an alternative way of improving H2 fermentation.

Physicochemical Parameters and Microbial Community in Anaerobic Digestion of Organic Wastes

The demand for an alternative source of energy and challenge of increase in wastes pollution initiates the need for renewable energy and management of waste using anaerobic digestion (AD). Anaerobic digestion is an effective and efficient method of waste treatment and energy generation. The study focused on investigating the physicochemical parameters and microbial community in anaerobic digestion of organic wastes and was conducted using chicken wastes and food wastes as organic substrate under semi-continuous conditions at hydraulic retention time (HRT) of forty-two (42) days in fifteen liter (15L) fabricated digesters labeled D1, D2 and D3 at 37OC. The pH, volatile fatty acid (VFA), moisture content (MC), total ammonia, total solid, volatile solid, alkalinity was assessed before and after digestion while the microbial community diversity was analyzed using 16S rRNA amplicon-based nextgeneration sequencing (NGS). The results indicated a pH value of 6.65 ± 0.12, 7.27 ± 0.13, 6.43 ± 0.27, volatile fatty acid of 72.17 ± 1.42, 58.35 ± 2.58, 40.56 ± 0.38 and moisture content of 98.9 ± 2.65, 92.3 ± 1.81, 96.4 ± 3.60 at day 42 for D1 (Chicken waste and food wastes), D2 (Chicken wastes+), D3 (control) respectively. A collective biogas yield of 686±17.00 kpa for D1, 700±11.00kpa for D2 and 521±21.00 kpa for D3 were recorded. The characterization of biogas analyzed with nondispersive infrared (NDIR) gas analyzer (gas board 3100p) revealed a percentage methane content of 46.11±1.11, 52.4±1.05, 50.31±1.33 for D1, D2 and D3 respectively. The microbial community identified phylum Bacteroidetes, Firmicutes, proteobacteria, Tenericutes, Verrucomicrobia, Actinobacteria, Euryarchaeota among others. The study shows that physicochemical properties and microbial community diversity are useful tools to indicate digester performance and also to enhance anaerobic digestion process.

Deeper Insights into the Effects of Microwave Hydrothermal Pretreatment on Methane Production and Microbial Community in Anaerobic Digestion

Deeper Insights into the Effects of Microwave Hydrothermal Pretreatment on Methane Production and Microbial Community in Anaerobic Digestion

Monitoring of seven industrial anaerobic digesters supplied with biochar

BackgroundRecent research articles indicate that direct interspecies electron transfer (DIET) is an alternative metabolic route for methanogenic archaea that improves microbial methane productivity. It has been shown that multiple conductive materials such as biochar can be supplemented to anaerobic digesters to increase the rate of DIET. However, the industrial applicability, as well as the impact of such supplements on taxonomic profiles, has not been sufficiently assessed to date.ResultsSeven industrial biogas plants were upgraded with a shock charge of 1.8 kg biochar per ton of reactor content and then 1.8 kg per ton were added to the substrate for one year. A joint analysis for all seven systems showed a decreasing trend for the concentration of acetic acid (p < 0.0001), propionic acid (p < 0.0001) and butyric acid (p = 0.0022), which was significant in all cases. Quantification of the cofactor F420 using fluorescence microscopy showed a reduction in methanogenic archaea by up to a power of ten. Methanogenic archaea could grow within the biochar, even if the number of cells was 4 times less than in the surrounding sludge. 16S-rRNA gene amplicon sequencing showed a higher microbial diversity in the biochar particles than in the sludge, as well as an accumulation of secondary fermenters and halotolerant bacteria. Taxonomic profiles indicate microbial electroactivity, and show the frequent occurrence of Methanoculleus, which has not been described in this context before.ConclusionsOur results shed light on the interplay between biochar particles and microbial communities in anaerobic digesters. Both the microbial diversity and the absolute frequency of the microorganisms involved were significantly changed between sludge samples and biochar particles. This is particularly important against the background of microbial process monitoring. In addition, it could be shown that biochar is suitable for reducing the content of inhibitory, volatile acids on an industrial scale.

Metagenomic insights into the effect of thermal hydrolysis pre-treatment on microbial community of an anaerobic digestion system

Thermal hydrolysis process (THP) is an effective pre-treatment method to reduce solids volume and improve biogas production during anaerobic digestion (AD) via increasing the biodegradability of waste activated sludge (WAS). However, the effects of THP pre-treated sludge on microbial diversity, interspecies interactions, and metabolism in AD systems remain largely unknown. We therefore setup and operated an anaerobic digester during a long-term period to shed light on the effect of THP pre-treatment on AD microbial ecology in comparison to conventional AD via Illumina based 16S rRNA gene amplicon sequencing and genome-centric metagenomics analysis. Results showed THP sludge significantly reduced the microbial diversity, shaped the microbial community structure, and resulted in more intense microbial interactions. Compared to WAS as the feed sludge, THP sludge shaped the core functional groups, but functional redundancy ensured the system's stability. The metabolic interactions between methanogens and syntrophic bacteria as well as the specific metabolic pathways were further elucidated. Hydrogenotrophic methanogens, Methanospirillum sp. and Methanolinea sp., were the primary contributors for methane production when treating THP and WAS, respectively, which also have potential for acetate oxidation to methane. Collectively, this study provides in-depth information on the interspecies interactions to better understand how THP pre-treatment influences AD microbial community.

Effects of different pretreatment methods on biogas production and microbial community in anaerobic digestion of wheat straw.

The pretreatment of wheat straw has been recognized to be an essential step prior to anaerobic digestion, owing to the high abundance of lignocellulosic materials. In order to choose economical and effective techniques for the disposal of wheat straw, effects of five pretreatment methods including acid, alkali, co-pretreatment of acid and alkali, CaO2, and liquid digestate of municipal sewage sludge on anaerobic digestion of wheat straw were investigated by analyzing biogas production and organic matter degradation in the study. The results showed that among these pretreatment methods, the methane yield was highest in the liquid digestate pretreated-wheat straw with 112.6mL gTS-1, followed by the acid, alkali, and CaO2 pretreatments, and the lowest was observed in the co-pretreatment of acid and alkali. Illumina MiSeq sequencing of the microbial communities in the anaerobic digesters revealed that the genera Ruminiclostridium including Ruminiclostridium and Ruminiclostridium 1, Hydrogenispora, and Capriciproducens were the main hydrolytic bacteria, acidogenic bacteria, and acetogenic bacteria, respectively, in the anaerobic digesters. Capriciproducens and Hydrogenispora dominated in the first and the later stages, respectively, in the anaerobic digesters, which could work as indicators of the anaerobic co-digestion stage of sludge and wheat straw. The total solid and SO42--S contents of the solid digestate and the NH4+-N concentration of the liquid digestate had a significant influence on the microbial community in the digesters. These findings indicated that liquid digestate pretreatment was a potential option to improve the anaerobic digestion of wheat straw, due to the low cost without additional chemical agents.

Illumina sequencing of 16S rRNA genes reveals a unique microbial community in three anaerobic sludge digesters of Dubai.

Understanding the microbial communities in anaerobic digesters, especially bacteria and archaea, is key to its better operation and regulation. Microbial communities in the anaerobic digesters of the Gulf region where climatic conditions and other factors may impact the incoming feed are not documented. Therefore, Archaeal and Bacterial communities of three full-scale anaerobic digesters, namely AD1, AD3, and AD5 of the Jebel Ali Sewage water Treatment Plant (JASTP) were analyzed by Illumina sequencing of 16S rRNA genes. Among bacteria, the most abundant genus was fermentative bacteria Acetobacteroides (Blvii28). Other predominant bacterial genera in the digesters included thermophilic bacteria (Fervidobacterium and Coprothermobacter) and halophilic bacteria like Haloterrigena and Sediminibacter. This can be correlated with the climatic condition in Dubai, where the bacteria in the incoming feed may be thermophilic or halophilic as much of the water used in the country is desalinated seawater. The predominant Archaea include mainly the members of the phyla Euryarchaeota and Crenarchaeota belonging to the genus Methanocorpusculum, Metallosphaera, Methanocella, and Methanococcus. The highest population of Methanocorpusculum (more than 50% of total Archaea), and other hydrogenotrophic archaea, is in agreement with the high population of bacterial genera Acetobacteroides (Blvii28) and Fervidobacterium, capable of fermenting organic substrates into acetate and H2. Coprothermobacter, which is known to improve protein degradation by establishing syntrophy with hydrogenotrophic archaea, is also one of the digesters' dominant genera. The results suggest that the microbial community in three full-scale anaerobic digesters is different. To best of our knowledge this is the first detailed report from the UAE.

Anaerobic digestion of microalgal biomass for bioenergy production, removal of nutrients and microcystin: current status

Using renewable microalgal biomass as active feedstocks for biofuels and bioproducts is explored to substitute petroleum-based fuels and chemicals. In the last few years, the importance of microalgae biomass has been realized as a renewable feedstock due to several positive attributes associated with it. Biorefinery via anaerobic digestion (AD) of microalgal biomass is a promising and sustainable method to produce value-added chemicals, edible products and biofuels. Microalgal biomass pretreatment is a significant process to enhance methane production by AD. Findings on the AD microbial community's variety and organization can give novel in turn on digester steadiness and presentation. This review presents a vital study of the existing facts on the AD microbial community and AD production. Co-digestion of microalgal biomass with different co-substrates was used in AD to enhance biogas production, and the process was economically viable with improved biodegradability. Microcystins, which are produced by toxic cyanobacterial blooms, create a severe hazard to environmental health. Anaerobic biodegradation is an effective method to degrade the microcystins and convert into nontoxic products. However, for the cost-effective conversion of biomass to energy and other beneficial byproducts, additional highly developed research is still required for large-scale AD of microalgal biomass.

Comparison of the microbial communities in anaerobic digesters treating high alkalinity synthetic wastewater at atmospheric and high-pressure (11 bar)

High-pressure anaerobic digestion is an appealing concept since it can upgrade biogas directly within the reactor. However, the decline of pH caused by the dissolution of CO2 is the main barrier that prevents a good operating high-pressure anaerobic digestion process. Therefore, in this study, a high-pressure anaerobic digestion was studied to treat high alkalinity synthetic wastewater, which could not be treated in a normal-pressure anaerobic digester. In the high-pressure reactor, the pH value was 7.5 ~ 7.8, and the CH4 content reached 88% at 11 bar. Unlike its normal-pressure counterpart (2285 mg/L acetic acid), the high-pressure reactor ran steadily (without volatile fatty acids inhibition). Furthermore, the microbial community changed in the high-pressure reactor. Specifically, key microbial guilds (Syntrophus (11.2%), Methanosaeta concilii (50.9%), and Methanobrevibacter (26.8%)) were dominant in the high-pressure reactor at 11 bar, indicating their fundamental roles under high-pressure treating high alkalinity synthetic wastewater.

A Rapid, Sensitive, Low-Cost Assay for Detecting Hydrogenotrophic Methanogens in Anaerobic Digesters Using Loop-Mediated Isothermal Amplification.

Understanding how the presence, absence, and abundance of different microbial genera supply specific metabolic functions for anaerobic digestion (AD) and how these impact on gas production is critical for a long-term understanding and optimization of the AD process. The strictly anaerobic methanogenic archaea are essential for methane production within AD microbial communities. Methanogens are a phylogenetically diverse group that can be classified into three metabolically distinct lineages based on the substrates they use to produce methane. While process optimization based on physicochemical parameters is well established in AD, measurements that could allow manipulation of the underlying microbial community are seldom used as they tend to be non-specific, expensive, or time-consuming, or a combination of all three. Loop-mediated isothermal amplification (LAMP) assays combine a simple, rapid, low-cost detection technique with high sensitivity and specificity. Here, we describe the optimization of LAMP assays for the detection of four different genera of hydrogenotrophic methanogens: Methanoculleus, Methanothermobacter, Methanococcus, and Methanobrevibacter spp. By targeting archaeal elongation factor 2 (aEF2), these LAMP assays provide a rapid, low-cost, presence/absence indication of hydrogenotrophic methanogens that could be used as a real-time measure of process conditions. The assays were shown to be sensitive to 1 pg of DNA from most tested methanogen species, providing a route to a quantitative measure through simple serial dilution of samples. The LAMP assays described here offer a simple, fast, and affordable method for the specific detection of four different genera of hydrogenotrophic methanogens. Our results indicate that this approach could be developed into a quantitative measure that could provide rapid, low-cost insight into the functioning and optimization of AD and related systems.

Microbial communities in anaerobic digesters change over time and sampling depth.

Anaerobic digestion (AD) is a process resulting from the anaerobic metabolism of specific microorganisms that produce an eco-friendly type of energy and a stabilized soil fertilizer. We described the microbial communities and their changes in three depths of BioKöhler® biodigester, fed with cattle manure for 18days, under anaerobic incubation at the psychrophilic temperature range (~ 20°C). During the experiment, the maximum methane content in the raw biogas was 79.9%. Non-metric multidimensional scaling (MDS) showed significant differences among microbial communities in the bottom, medium, and upper depths. Considering all the periods of incubation, the microbial communities changed until the eighth day, and they remained stable from eighth to seventeenthdays. Bacteroidetes, Firmicutes, and Synergistetes were the most abundant phyla in samples, representing approximately 41% of the total OTUs. The relative abundance of the phyla Euryarchaeota, Actinobacteria, Firmicutes, and Verrucomicrobia changed from bottom to medium sampling points. Moreover, Crenarchaeota differed in frequencies from medium to upper, and Acidobacteria from bottom to upper samples. Lentisphaerae, Chloroflexi, and LD1 were different solely at the bottom, whereas OP9 and Tenericutes only in the medium. Psychrophilic AD performed in this work removed pathogens like Salmonella and Escherichia, as observed at the digestate analyzed. This type of treatment of raw manure besides producing eco-friendly energy efficiently also generates a stabilized and safe biomass that can be used as fertilizer in soils.

Superior resolution characterisation of microbial diversity in anaerobic digesters using full-length 16S rRNA gene amplicon sequencing

In the past decade, the characterisation of the microbial community in anaerobic digestion was primarily done by using high-throughput short-read amplicon sequencing. However, the short-read approach has inherent primer bias and low phylogenetic resolution. Our previous study using Illumina MiSeq suggested that the heterogeneity of AD microbiome was operation-driven. To advance our knowledge towards the complexity of the AD microbiome, we performed full-length 16S rRNA gene amplicon sequencing using PacBio Sequel for a more accurate phylogenetic identification. To this end, purified DNA samples from 19 global anaerobic digesters were sequenced. Sixteen methanogenic archaea were identified at the species level. Among them, Methanosarcina horonobensis and Methanosarcina flavescens had significant presence under specific operating conditions. Methanothrix concilii presented in all digesters sequenced. Unexpectedly, over 90% of the Smithella detected were closely related to alkane-degrading Smithella strains D17 and M82, not Smithella propionica. Using LEfSe and network analysis, the interspecies relationship between the fermentative and syntrophic bacteria was addressed. Comparison of the short- and long-read sequencing results were performed and discussed. From sample preparation to data analysis, this work characterised the digester microbiomes in a superior resolution.