Abstract
Rhamnolipids are biosurfactants produced by microorganisms with the potential to replace synthetic compounds with petrochemical origin. To promote industrial use of rhamnolipids, recombinant rhamnolipid production from sugars needs to be intensified. Since this remains challenging, the aim of the presented research is to utilize a multidisciplinary approach to take a step toward developing a sustainable rhamnolipid production process. Here, we developed expression cassettes for stable integration of the rhamnolipid biosynthesis genes into the genome outperformed plasmid-based expression systems. Furthermore, the genetic stability of the production strain was improved by using an inducible promoter. To enhance rhamnolipid synthesis, energy- and/or carbon-consuming traits were removed: mutants negative for the synthesis of the flagellar machinery or the storage polymer PHA showed increased production by 50%. Variation of time of induction resulted in an 18% increase in titers. A scale-up from shake flasks was carried out using a 1-L bioreactor. By recycling of the foam, biomass loss could be minimized and a rhamnolipid titer of up to 1.5 g/L was achieved without using mechanical foam destroyers or antifoaming agents. Subsequent liquid–liquid extraction was optimized by using a suitable minimal medium during fermentation to reduce undesired interphase formation. A technical-scale production process was designed and evaluated by a life-cycle assessment (LCA). Different process chains and their specific environmental impact were examined. It was found that next to biomass supply, the fermentation had the biggest environmental impact. The present work underlines the need for multidisciplinary approaches to address the challenges associated with achieving sustainable production of microbial secondary metabolites. The results are discussed in the context of the challenges of microbial biosurfactant production using hydrophilic substrates on an industrial scale.
Highlights
Rhamnolipids are versatile anionic glycolipid biosurfactants produced by different bacteria (Germer et al, 2020)
To address the challenges associated with rhamnolipid production on all levels, strain engineering, process development, and environmental impact were simultaneously considered
Phase 1: We established a strategy for rhamnolipid production in a non-pathogenic strain and a suitable medium sustaining growth and high production
Summary
Rhamnolipids are versatile anionic glycolipid biosurfactants produced by different bacteria (Germer et al, 2020). The three surface active compounds HAAs and mono- and dirhamnolipids show differences in physiochemical parameters like the critical micelle concentration (Tiso et al, 2017b). The biosynthesis of rhamnolipids starts with the formation of HAA from two β-hydroxy fatty acids by the enzyme RhlA (Déziel et al, 2003). The substrate spectra of different RhlA enzymes are responsible for the chain-length spectrum of the final biosurfactant (Dulcey et al, 2019; Germer et al, 2020). Rhamnose units are attached to the HAA by activity of RhlB and RhlC, respectively, to form mono- and dirhamnolipids (Ochsner et al, 1994; Rahim et al, 2001). The mono-rhamnolipid biosynthesis encoding genes rhlA and rhlB are joint to one transcriptional unit in all producer strains known so far (Tiso et al, 2017a). The precursors (hydroxy fatty acids and rhamnose as activated conjugates) are provided by the cell’s central metabolism (Tiso et al, 2016)
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