Jo De Vrieze

Introduction: Microbial protein (MP) is a promising alternative protein source that can be produced using methane as a substrate derived from undervalued resources. Methylococcus capsulatus is one of the most prominent microbial species employed for industrial production, while other potentially suitable methanotrophs remain underexplored. Methods: Here, we selected the alternative methanotrophs Methylosinus sp. and Methylomonas methanica with high growth rates and potential for halotolerance and compared them to M. capsulatus . Results and Discussion: Methylosinus sp. and Methylomonas methanica achieved up to 1.87-fold higher maximum specific growth rates and 2.52-fold higher biomass concentrations, compared to M. capsulatus . When the pH was allowed to acidify below optimum for growth, the protein content of M. methanica (45% ± 5% CDW) was 1.55 times compared to pH-controlled conditions. Cultivation at an increased salt content (20 g NaCl L ⁻¹ ) boosted the protein content of M. methanica to 81% ± 7% CDW, showing its applicability for MP production using saline water. Co-culturing M. methanica with the methylotrophic yeast Komagataella phaffii led to a 1.27-fold higher essential amino acid content, which indicates an improvement in protein quality, while the use of synthetic biogas increased the protein yields up to 1.5-fold, compared to purified methane. In summary, M. methanica co-cultured with selected methylotrophs has the potential to enable more sustainable MP production under saline conditions using biogas, as an alternative to natural gas-based production with M. capsulatus . These strategic upstream choices could bring us closer to a future-proof protein production system.

Anaerobic digestate is increasingly recognized as a secondary resource for nutrient recovery, yet this depends on nutrient speciation and the physicochemical and microbial characteristics governing nutrient availability. Comparative information on how digestion systems influence phosphorous partitioning, ionic chemistry, volatile fatty acids (VFA) profiles and microbial characteristics under full-scale operating conditions remains limited. In this study, we investigated 41 digestate samples collected from three full-scale mesophilic anaerobic digestion systems in Belgium representing sewage sludge digestion (n=12), wet co-digestion of food and plant residues (n=25), and dry high-solids co-digestion of source-separated municipal biowaste (n=4) to evaluate how system-dependent characteristics influence phosphorous occurrence and nutrient recovery potential. Multivariate analyses demonstrated that process type was the primary determinant of digestate characteristics, producing distinct physicochemical and microbial signatures. Although sewage sludge digestates contained the highest total phosphorous (TP) levels (4266±1210 mg/L), dry digestion exhibited a significantly greater dissolved orthophosphate fraction (median PO₄³⁻/TP=0.37±0.16 versus 0.09±0.06 in sludge digestates; p<0.05), demonstrating that phosphorous partitioning provides a more meaningful indicator of recovery potential than TP concentration alone. Distinct ionic environments (K+ concentration and conductivity, in particular) further differentiate the digestion systems, accompanied by process-specific microbial physiological states identified through flow cytometry. Microbial biomass ranged from 5.6×10⁹ to 5.0×10¹⁰ cells/mL, while methanogen abundance was inversely associated with the fraction of damaged cells across the combined dataset (p<0.001). Overall, integrating phosphorous partitioning, ionic composition and microbial physiological state provide a more comprehensive framework for process-specific digestate characterization and for selecting effective phosphorous recovery strategies.

Microbial chain elongation enables the conversion of organic waste into higher-value products and is therefore a promising process for circular biomanufacturing. However, the microbial interactions governing chain elongation communities remain poorly understood. While quorum sensing has been extensively studied in the context of pathogens and model organisms, research on the perception of quorum-sensing molecules by non-model organisms and their effects within microbial consortia has remained limited. Here, Lactiplantibacillus plantarum and Megasphaera elsdenii were selected as representatives of two key functional guilds in chain elongation communities, namely lactic acid bacteria and chain-elongating bacteria. The effects of different exogenous quorum sensing molecules were evaluated in pure cultures and co-cultures using microtiter plates and serum bottles. Both organisms exhibited distinct molecule-dependent responses for both growth and biofilm formation. Moreover, the response of M. elsdenii was highly dependent on the supplied substrate. Despite changes in growth and/or biofilm formation, product yield and product spectra remained largely unaffected. Importantly, responses observed in pure cultures did not predict co-culture behavior, and no clear response to the tested molecules was detected in the co-culture grown in serum bottles. These findings demonstrate that responses to quorum sensing molecules are strongly dependent on the signal, substrate, microbial context, and cultivation conditions. These results highlight the limited predictive power of pure-culture assays for microbial communication in interacting communities and emphasize the importance of studying signal perception under process-relevant cultivation conditions.