Periodic Reporting for period 2 - MIBIREM (MIBIREM – Toolbox for Microbiome based Remediation)
Reporting period: 2024-04-01 to 2025-09-30
MIBIREM's overall objective is to develop tools that enable cleaning of contaminated soil and groundwater by microbiomes. Due to industrial and commercial activities, at many sites toxic chemicals were released into the environment, leading to contaminations of soil and groundwater. Some of these chemicals are very persistent and stay in place for decades, if no remediation measures are taken. In Europe, there are 324,000 severely contaminated sites that require urgent clean-up. In principle, biological, chemical and physical remediation technologies are available. Among these biological methods, like the use of microorganisms, have the advantage of being cost-effective and eco-friendly to remediate sites polluted with organic contaminants: no net water extraction, no extensive on-site treatment plants, no waste and no transport to and treatment by an off-site soil cleaner are required. The key for development and application of microbial remediation technologies is the identification of microbial communities (microbiomes) and strains that are able to use the contaminants as sources of energy and/or nutrients. A full degradation of organic contaminants in the environment requires the interplay of several microbes, similar to digestion of food in the gut. The isolation and characterisation and conservation of such microbiomes that can degrade organic contaminants is key to MIBIREM. For this purpose, three use-cases were defined each containing one group of contaminants: PHC, HCH and Cyanides. These three groups of substances can be found at many contaminated sites across Europe and cause risks for humans and the ecosystem. The toolbox being developed in MIBIREM for these use-cases can be later applied to develop microbiome-based remediation technologies for other contaminants and related applications like wastewater treatment.
In WP1, RNA-extraction was added to the handbook (D1.4). Permits for pilots in Ploufragan, Bitterfeld and Stockach were obtained by site owners. The DMP set up in P1 was thoroughly updated at the end of P2 (D1.3). For WP2, extensive microbiome analyses were conducted across the three use cases (PHC, HCH, and cyanides). Soil and groundwater samples were collected, DNA was extracted, and 16S rRNA gene amplicon and shotgun metagenomic sequencing were performed to characterize microbial communities. Enrichment cultures with high degradation capacities were established for PHC, HCH, and cyanides from diverse contaminated sites. Metagenomic and metatranscriptomic analyses were applied to the most promising microbiomes to reconstruct metagenome-assembled genomes, identify key biodegradation pathways, and elucidate gene expression profiles during contaminant degradation. DNA-SIP experiments using labeled substrates (e.g. 13C-hexadecane) were performed to identify active degraders in situ. Furthermore, an isolation campaign yielded over 4600 bacterial strains across the three use cases. These isolates were taxonomically identified via MALDI-TOF MS and a selection of 321 strains was whole-genome sequenced. Functional gene screening revealed the presence of canonical degradation genes, although for HCH degradation, classical lin genes were often absent or incomplete in isolates, suggesting alternative degradation pathways. This integrated approach has established a solid foundation for developing effective bioaugmentation consortia. In WP3, the experiment on preservation of a PHC-degrading microbiome (set up in P1) was continued. Flow cytometry data was collected from 3 additional time-points, up to 2 years post-preservation. In addition, experiments were set up on the preservation of a cyanide- and HCH-degrading microbiome. Flow cytometry data was collected from two (cyanide) and three (HCH) time-points. Pure cultures were whole genome sequenced: 109 for cyanide, 92 for HCH and 120 for PHC. In WP4, the prediction tool continued its development integrating microbiological, chemical, hydrological and physical data and processes. Work in P2 focused on the design and implementation of software components for the tool. Their application to MIBIREM field sites will follow in P3. Relevant software packages of the prediction tool (mibiscreen, mibitrans, mibiremo) have been implemented with publication as python packages and doi (via Zenodo). In WP5, safety assessments have been developed and conducted for selected consortia. Annotated genomes from isolated single strains provided by WP3 were screened for presence of antimicrobial resistance and virulence genes. Four selected consortia were screened using selective media. Phytotoxicity and ecotoxicity tests have been performed for the HCH and PHC use-cases (cyanide use-case pending). For the HCH and PHC-use case, mesocosm experiments followed by the set-up of large-scale tests were conducted. For the cyanide use-case, large-scale columns tests had been prepared and an up-scaling experiment was conducted. For each use-case, the set-up of the respective pilot test has been engineered. In WP6, a regulatory review on soil bioremediation was conducted, based on practical insights from consultants/contractors, national, EU and international regulations, and interviews with MIBIREM site owners. The review also provided valuable insights for safety assessment in WP5. The MIBIREM-led ALL4BIOREM project cluster continued its activities with two successful stakeholder workshops, the launch of its visual identity and dedicated online presence (LinkedIn). An Innovation Task Force was also launched in MIBIREM, resulting in four preliminary business models for four key focus areas of the MIBIREM toolbox, to be further refined in P3.
Recent analyses revealed the presence of microbial communities capable of degrading HCH despite the apparent absence of canonical lin genes typically associated with the HCH degradation pathway. Across all contaminated sites investigated, several microbiomes exhibited functional HCH transformation activity while harboring genomes that do not encode recognizable components of the established lin degradation machinery. These findings suggest the existence of alternative metabolic routes or yet-uncharacterized enzymatic systems enabling HCH turnover, thereby expanding our current understanding of organochlorine biodegradation. This evidence not only highlights the metabolic plasticity and adaptive potential of environmental microbiomes but also provides a foundation for enriching genomic databases with novel gene candidates and pathway information for HCH bioremediation.