Scaling glucose-based rhamnolipid production with engineered Pseudomonas putida: Environmental and economic challenges from plasmid-based pilot test to antibiotic-free industry simulation

Background Rhamnolipids are biodegradable biosurfactants with applications in cosmetics, agriculture, and environmental remediation. However, industrial production faces environmental and economic challenges due to resource-intensive fermentation, aeration, and downstream processing. Additionally, non-pathogenic hosts and antibiotic-free operation are required, which typically result in lower titers. This study evaluates the environmental and economic sustainability of rhamnolipid production using engineered Pseudomonas putida KT2440 on glucose as the sole carbon source. Methods Experimental data from 30 L and 150 L pilot fermentations using a plasmid-based expression system with antibiotics were used to develop process simulations representing industrial-scale production (5,000 kg per batch) with genome-integrated, antibiotic-free strains. Life Cycle Assessment (LCA) and Life Cycle Costing (LCC) methodologies were integrated to quantify environmental burdens and economic performance. Life Cycle Inventory data captured resource inputs, emissions, and cost elements for the glucose-based fermentation process. Results Pilot fermentations achieved rhamnolipid titers of 1.9–2.21 g/L with yields of 0.01–0.11 g/g glucose. At industrial scale, the process exhibited a global warming potential of 109.30 kg CO 2 -eq per kg product, with fermentation (50.70 kg CO 2 -eq) and media preparation (44.96 kg CO 2 -eq) as primary contributors. Water consumption reached 185.28 m 3 water deprivation per kg product. Glucose consumption (61.80 kg/kg product) dominated costs at €33.25/kg product, representing 95% of media preparation expenses. Downstream purification contributed 12.30 kg CO2-eq and required 137.34 kg water per kg product. Scale-up improved economic viability through economies of scale, though environmental impacts increased without parallel efficiency improvements. Conclusions This integrated LCA-LCC framework identifies glucose consumption and water-intensive processing as critical sustainability bottlenecks. Future strain engineering must balance antibiotic-free operation with productivity improvements to achieve economically viable and environmentally sustainable industrial-scale rhamnolipid production.