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.
