The ANAEROBic treasure trunk

ANAEROB aims to generate a platform that enables linking microbial ecology to biotechnology. This platform should be broadly applicable across conditions and applications. Anaerobic processes are performed by a well-structured microbial community and have great potential for upcycling organic wastes and industrial/agricultural residual resources to achieve circular bioeconomy. Upcycling residual resources into materials, biochemicals and energy is a promising route towards sustainable production. Pure culture fermentations are not appropriate when the substrate is too complex, as with waste, and therefore mixed culture microbial consortia are required. Currently, inocula for biological processes utilizing waste as substrates are random self-established cultures. Comprehensive knowledge about the microbial interactions of the anaerobic microbiome is needed for valorization and remediation of biowastes. The overall aim of ANAEROB is to understand how to create/assemble/produce “designer microbial consortia” for specific bioengineering processes based on genetic information of anaerobic microorganisms. The aim will be achieved by: 1. Elucidating the syntrophic mutualistic symbioses of the anaerobic microbiome, and clarifying the role of specific compounds exchanged by microbes; 2. Developing models for prediction of desired pathways; 3. Developing methods for isolation of new unculturable anaerobes; 4. Developing a novel method for designing and establishing microbial “cocktails” for specific functions; and 5. Validating the concept of creating specific anaerobic consortia for upcycling gas streams of carbon-intensive industries. ANAEROB combines engineering and microbiology to improve understanding and exploitation of the anaerobic microbial “treasure”. The project’s successful completion will have major benefits in industrial sectors and environmental applications.

During the January 2024-June 2026 period, the following work was performed with several major achievements:
Continuous stirred tank reactor and batch experiments demonstrated how conductive materials facilitate direct interspecies electron transfer (DIET) and gave new insights into their role in microbial interactions (Objective 1). Under sulfate-rich conditions, magnetite addition stabilized anaerobic digestion performance by mitigating sulfate-reducing bacteria–induced inhibition of methanogenesis. The work further revealed interaction patterns suggesting cooperative relationships between hydrogenotrophic methanogens and sulfate-reducing bacteria under specific environmental conditions.
The role of specific compounds and mechanisms exchanged among syntrophic partners was further clarified through electrochemical and genome-centric analyses (Objectives 1 and 2). Cyclic voltammetry revealed distinct redox activities consistent with DIET. Complementary metagenomic analyses identified microbial populations enriched in pathways related to carbohydrate and amino acid metabolism, cofactor biosynthesis, and electron transport.
Significant progress was achieved in translating genome-centric observations into predictive models of microbial interactions (Objective 2). Genome-centric metagenomics was integrated with genome-scale metabolic models (GEMs) to reconstruct community metabolic networks and investigate interspecies metabolite exchange. The modeling framework identified key metabolites mediating microbial interactions and provided mechanistic insight into anaerobic interactions.
A novel flow-chamber platform was developed to cultivate anaerobic autotrophic gas-fermenting consortia such as those involved in syngas biomethanation, recreating the mass-transfer-limited conditions of biofilm-based reactors like trickle bed reactors (Objectives 1 and 4). The system enabled visualization of biofilm spatial organization, revealing that CO consumers and methanogens were spatially intermixed, highlighting the importance of syntrophic interactions in biofilm organization.
A new approach based on microbial auxotrophies was explored to identify and enrich bacteria beneficial to Methanosarcina species (Objective 1 and 3). Initial results demonstrated the value of predicted auxotrophies for identifying microbial interdependencies. Supplementation of specific auxotrophies enriched selected functional groups, promoting methanogen growth and methane production. To support microbial cocktail design (Objectives 1 and 4), community-level metabolic dependencies were investigated through genome-centric analyses of syngas-biomethanation communities. Key syngas-biomethanating microorganisms lacked the capacity to synthesize specific amino acids and vitamins, whereas low-abundance community members possessed the corresponding biosynthetic pathways.
In parallel, significant progress was made toward the development of novel methodologies for isolating previously unculturable anaerobic microorganisms (Objective 3). A high-throughput isolation strategy was established by combining fluorescence in situ hybridization transcript-annealing molecular beacons (FISH-TAMB) with single-cell dispensing under strictly anoxic conditions. This approach enabled targeted isolation of methanogens and potential syntrophic partners from complex communities and showed how medium and carbon-source regimes steer microbial interactions.
Insights from isolation and community-level studies were then applied to the rational design and establishment of functional microbial cocktails (Objective 4). A combined bioaugmentation strategy using Methanosarcina barkeri DSM800 in conjunction with magnetite demonstrated synergistic enhancement of methane production. Furthermore, the isolation and characterization of Methanosarcina barkeri DTU3 revealed its capacity to accumulate metals and minerals, suggesting an intrinsic strategy to support DIET and reinforcing the concept of designing consortia based on electron-transfer competence.

ANAEROB employed a multidisciplinary strategy integrating experimental microbiology, microbial ecology, electrochemistry, bioinformatics, and systems-level modeling to advance the understanding and exploitation of anaerobic microbial systems. The resulting developments extend beyond the state of the art in microbial interaction analysis, microbiome engineering, and anaerobic biotechnology.
FISH-TAMB combined with single-cell dispensing represents a breakthrough in anaerobe isolation, overcoming long-standing cultivation barriers and enabling systematic exploration of microbial interactions under defined substrate regimes. The integration of electrochemical diagnostics into anaerobic microbiome analysis to detect potential DIET was a novelty. This technology brings a functional, non-destructive tool for probing microbial electron transfer activity in complex anaerobic communities, linking redox behavior to reactor performance.
The demonstration and validation of conductive material-assisted anaerobic digestion represented a major advance for improving process stability and methane production. The mechanism of magnetite was dual, reducing H2S concentration by precipitation while simultaneously promoting collaborative coexistence of methanogens, sulfate-reducing bacteria, and acetogens. These findings challenge the traditional perception of sulfate-reducing bacteria and methanogens as predominantly competing microbial groups and instead suggest that conductive materials can promote stable cooperative networks under sulfate stress. This represents a scalable technological solution for anaerobic digestion facing inhibitory industrial conditions.
The integration of genome-centric metagenomics with GEMs represented an important advance beyond the state of the art. Community metabolic models enabled validation of proposed microbial interaction networks, prediction of metabolite exchange, and mechanistic interpretation of ecological interactions. This provides a framework for better understanding microbial interactions and designing consortia with desired functionalities.
The development of a flow-chamber cultivation platform capable of reproducing the mass-transfer-limited conditions of biofilm-based reactors represented a further advance beyond the state of the art. The platform revealed that syngas-biomethanation biofilms are more strongly shaped by syntrophic interactions than previously assumed. CO-utilizing microorganisms and methanogens were spatially intermixed throughout the biofilm rather than stratified.
Genome-centric metagenomics further demonstrated that key interactions within syngas-biomethanation communities extend beyond carbon and electron exchange. Low-abundance community members supplied essential amino acids and vitamins to dominant functional populations, revealing previously overlooked metabolic dependencies between functional and supporting community members, relevant for designing bottom-up stable engineered microbial consortia.

Periodic Reporting for period 1 - ANAEROB (The ANAEROBic treasure trunk)

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