Enhancing acetate-based biosurfactant production by Pseudomonas putida KT2440 through fermentation optimization

There is an increasing demand for sustainable chemicals, although "green premium" can be rarily achieved. In this context, biosurfactants emerge as promising substitutes for petroleum- or palm-oil-based surfactants due to their potentially lower environmental impact. 3-(3-hydroxyalkanoyloxy)alkanoic acids (HAAs) are biosurfactants composed of dimeric esterified hydroxy fatty acids. They serve as precursor molecules for rhamnolipids. Here, acetate was chosen as potentially CO₂-derived carbon source to replace conventional sugar-based feedstocks for HAA production using the versatile bacterium Pseudomonas putida KT2440 as heterologous production host.

In this study, a fed-batch fermentation for microbial HAA production with acetate as sole carbon source was developed and optimized. First, different pH values were evaluated to determine optimal cultivation conditions, and a pH of 7.8 was identified as the optimal setpoint. To enhance the overall productivity of the fermentation process, different feeding strategies were investigated. Acetate feeding was controlled either by dissolved oxygen (DO)-triggered substrate addition or by pH-coupled feeding using acetic acid as titrant and carbon source. Both feeding strategies resulted in periods of carbon limitation, which promoted HAA degradation. To overcome this challenge, urea was investigated as an alternative nitrogen source in the pH-coupled feeding strategy. Here, the highest reported HAA concentration of 1 g L⁻¹ was achieved, with more than 3.5-fold increases in space-time and product-to-substrate yields compared to the ammonium-based pH-coupled feeding. As a next step, the implementation of CO₂-derived acetate as carbon source was tackled to improve the overall carbon footprint of the bioprocess. To this end, different substrate mixtures obtained from chemocatalytic CO₂ conversion were evaluated for biocompatibility, growth, and HAA production. The results demonstrate the suitability of the CO₂-derived acetate-formate mixture for the bioprocess without purification, achieving growth and HAA production comparable to the reference.

This study demonstrates the successful establishment of a bioprocess for heterologous production of HAAs using acetate as carbon source. Urea as alternative nitrogen source not only enhanced process performance and simplified process control but also improved the overall sustainability of the fermentation process. Finally, the successful valorization of CO₂-derived acetate further enhances its role as a next-generation feedstock in an emerging bioeconomy.