Genome-informed bioaugmentation with Bacillus subtilis TLO3 in pilot-scale sewage sludge composting improves...

Sewage sludge composting often suffers from incomplete stabilization, nutrient loss, and pollutant risks that limit safe reuse. This study introduces a genome-informed, multi-stage bioaugmentation strategy using Bacillus subtilis TLO3, integrating genomic functional annotation with phase-specific inoculation to enhance composting efficiency and safety. Here, we evaluated bioaugmentation with Bacillus subtilis TLO3 through pilot-scale trials (∼150 kg piles) combined with genome- informed functional analysis. Three treatments were compared: regular multi-stage inoculation, single late inoculation, and a non-inoculated control. Regular bioaugmentation sustained thermophilic conditions (>55 °C) for >14 days and accelerated organic matter transformation, achieving 48.9 % reduction in total organic carbon and 68.7 % increase in total Kjeldahl nitrogen by day 42. Importantly, by the end of composting (Day 42), diazotrophic populations were strongly enriched (4.7 × 10⁷ CFU/g, ∼8-fold higher than the control), directly supporting enhanced nitrogen retention. Environmental safety improved, with germination indices reaching 135.15 % (Lactuca sativa) and 131 % (Raphanus sativus), and total metal concentrations substantially reduced (Cd -92 %, Pb -48.5 %, Cu -47.3 %), all within EU 2019/1009 limits. Genome annotation revealed a rich repertoire of carbohydrate-active enzymes, nitrogen assimilation pathways, siderophore biosynthesis, and stress-resilience genes, supporting enhanced decomposition, nutrient conservation, and heavy metal detoxification. Motility and antimicrobial biosynthetic clusters further explain the strain's persistence and ecological competitiveness in dynamic composting environments. These results demonstrate that genome-informed bioaugmentation can accelerate compost stabilization, mitigate pollutant risks, and enable regulation-compliant recycling of sewage sludge into safe organic fertilizers, advancing circular bioeconomy goals.