Background: Methane (CH4) is an attractive carbon feedstock for sustainable biomanufacturing, but poor CH4 solubility and gas-liquid mass-transfer limitations continue to constrain methanotroph-based CH4 valorization. Here, we evaluated biocompatible bionanofluids as additive-based process intensification tools and examined whether two operationally distinct bionanofluid application strategies produce distinct time-dependent physiological responses beyond simple growth enhancement.
Results: Two bionanofluids, chitosan/oleamide (CS/OA) nanoparticles and tannic acid–Fe3+ complex-coated cellulose nanocrystals (TA-Fe3+CNCs), were evaluated in 5-L cultures of Methylosinus trichosporium OB3b using two different application approaches: CS/OA seed adaptation followed by cultivation in conventional medium, and TA-Fe3+CNCs supplementation in the main culture. In both approaches, time-resolved profiling of gas uptake, nitrogen species, extracellular metabolites, and poly(3-hydroxybutyrate) (PHB) showed higher CH4 and O2 utilization, with 1.5–1.6- and 2.0–2.1-fold increases in specific CH4 and O2 uptake rates, respectively, and faster growth, with 1.3–1.5-fold higher µmax, than in the conventional control. These changes were accompanied by consistently lower PHB content, faster nitrate depletion, higher ammonium accumulation, and distinct extracellular metabolite patterns. Notably, CS/OA seed adaptation was associated with transient succinate accumulation during exponential growth, whereas TA-Fe3+CNCs supplementation led to faster methanol accumulation without succinate release. These observations are consistent with a process-level response in which higher CH4/O2 uptake and faster growth coincided with lower PHB content under the tested conditions. As an initial proof-of-concept experiment, a consecutive strategy combining CS/OA seed adaptation with TA-Fe3+CNCs supplementation in Methylomonas sp. DH-1 was associated with greater transient succinate accumulation and a higher peak succinate titer than under the conventional condition.
Conclusions: Comparative time-resolved profiling of two different bionanofluid approaches indicated that bionanofluid application was associated with higher CH4/O2 uptake, faster growth, lower PHB accumulation, and distinct time-dependent metabolite patterns under the tested conditions. These findings support the use of biocompatible bionanofluids as practical media additives that can be applied without major bioreactor redesign and can motivate future component-controlled testing of sequential strategies for growth-linked CH4 bioconversion.