A scalable bubble-free membrane aerator for biosurfactant production

The bioeconomy is a paramount pillar in the mitigation of greenhouse gas emissions and climate change. Still, the industrialization of bioprocesses is limited by economical and technical obstacles. The synthesis of biosurfactants as advanced substitutes for crude-oil-based surfactants is often restrained by excessive foaming. We present the synergistic combination of simulations and experiments towards a reactor design of a submerged membrane module for the efficient bubble-free aeration of bioreactors. A digital twin of the combined bioreactor and membrane aeration module was created and the membrane arrangement was optimized in computational fluid dynamics studies with respect to fluid mixing. The optimized design was prototyped and tested in whole-cell biocatalysis to produce rhamnolipid biosurfactants from sugars. Without any foam formation, the new design enables a considerable higher space-time yield compared to previous studies with membrane modules. The design approach of this study is of generic nature beyond rhamnolipid production

Metabolic Engineering of Pseudomonas putida KT2440 for enhanced rhamnolipid production

The production of chemicals and fuels is mainly based on fossil resources. The reduced availability of these resources and thus the increasing prices for crude oil as well as the resulting pollution of the environment require alternative strategies to be developed. One approach is the employment of microorganisms for the production of platform molecules using renewable resources as substrate. Biosurfactants, such as rhamnolipids, are an example for such products as they can be naturally produced by microorganisms and are biodegradable in contrast to chemical surfactants. The bio-based production of chemicals has to be efficient and sustainable to become competitive on the market. Several strategies can be applied to increase the efficiency of a microbial cell factory, e.g., streamlining the chassis. Here, we show the heterologous production of rhamnolipids with the non-pathogenic Pseudomonas putida KT2440 with the aim of increasing the yield. P. putida KT2440 is a well-characterized microorganism and its genome is sequenced and well annotated. Thus, the targeted removal of genes is possible and can lead to a reduction of the metabolic burden and by-product formation, which can result in a higher yield. Furthermore, the efficient supply of precursors is an important factor for optimized production processes. Rhamnolipids are amphiphilic molecules containing rhamnose and ß-hydroxy fatty acids. These precursors are synthesized by two pathways, the fatty acid de novo synthesis and the rhamnose pathway. We performed gene deletions to avoid the synthesis of by-products, like pyoverdine, exopolysaccharides, and large surface proteins and energy consuming devices as the flagellum. Most of the genome-reduced mutants reached a higher yield compared to the strain with wildtype background. With the best chassis, the yield could be increased by 35%. Furthermore, we conducted the overexpression of genes for precursor supply, either plasmid-based or genomically integrated. In this regard, the genes for the phosphoglucomutase, the complete rhamnose-synthesis pathway operon, and different enzymes in the pathway for acetyl-CoA synthesis were targeted. Various combinations were tested, and the highest yield reached was 51% higher compared to the initial rhamnolipid producer. Finally, a genome-reduced mutant was equipped with the overexpression modules and the rhamnolipid titer was increased from approximately 590 mg/L for the wildtype background to 960 mg/L, which represents a 63% increase. In conclusion, we were able to enhance the yield of rhamnolipids per glucose using metabolic engineering

Deep genome editing of Pseudomonas putida for rhamnolipid production using non-conventional substrates

Biosurfactants, such as rhamnolipids, are valuable products with many potential applications. Their amphiphilic structure allows their use in different fields. The overall aim of this thesis was to produce rhamnolipids from renewable substrates using the nonpathogenic Pseudomonas putida KT2440, which would contribute to a sustainable bioeconomy. Various substrates were implemented, which allowed new insights into the respective metabolism. In order to reduce the costs for rhamnolipid production, lignocellulose-derived substrates, such as xylose and galacturonic acid, can be used. While P. putida can natively utilize galacturonic acid, the use of xylose does not lead to growth. Three bacterial xylose utilization pathways were introduced and the engineered strains were characterized regarding their growth and production. While the implementation of the oxidative xylose pathways resulted in higher biomass production and rhamnolipid titers, the product yield was higher for the Isomerase pathway caused by the different stoichiometries of the pathways. Rhamnolipid production from galacturonic acid was immediately efficient, however the production rate was improved by laboratory evolution. Ethanol is another sustainable carbon source as it can be produced out of lignocellulosic biomass by microbes. Laboratory evolution was applied to obtain a strain with a high growth rate on ethanol at elevated concentrations. Genome re-sequencing and gene expression studies revealed a rerouting of the metabolism for carbon-efficient ethanol utilization. The resulting biosurfactant producer was used in a fed-batch process, where the water-soluble ethanol served as carbon source and defoamer. This unique setup enabled a biosurfactant production of over 5 g/L in one day with a great space time yield of 0.23 g/L/h. In addition, P. putida KT2440 was modified to generate a genome-reduced chassis by targeted deletion of dispensable elements or processes. Several deletions led to a 66% improved rhamnolipid production. As precursor supply seemed to be a bottleneck, the overexpression of the phosphoglucomutase and rhamnose-operon was performed. The combination of a reduced genome and the overexpression further increased the rhamnolipid production up to 94%. In conclusion, this thesis clearly illustrates that rhamnolipid production from renewable substrates is feasible. Further, the application of P. putida KT2440 as microbial cell factory inindustrial processes is demonstrated. A combination of the improved chassis and the use of any renewable lignocellulose-derived substrate will truly contribute to establish a competitive production process

A scalable bubble‐free membrane aerator for biosurfactant production

The bioeconomy is a paramount pillar in the mitigation of greenhouse gas emissions and climate change. Still, the industrialization of bioprocesses is limited by economical and technical obstacles. The synthesis of biosurfactants as advanced substitutes for crude-oil-based surfactants is often restrained by excessive foaming. We present the synergistic combination of simulations and experiments towards a reactor design of a submerged membrane module for the efficient bubble-free aeration of bioreactors. A digital twin of the combined bioreactor and membrane aeration module was created and the membrane arrangement was optimized in computational fluid dynamics studies with respect to fluid mixing. The optimized design was prototyped and tested in whole-cell biocatalysis to produce rhamnolipid biosurfactants from sugars. Without any foam formation, the new design enables a considerable higher space-time yield compared to previous studies with membrane modules. The design approach of this study is of generic nature beyond rhamnolipid production

A Straightforward Assay for Screening and Quantification of Biosurfactants in Microbial Culture Supernatants

A large variety of microorganisms produces biosurfactants with the potential for a number of diverse industrial applications. To identify suitable wild-type or engineered production strains, efficient screening methods are needed, allowing for rapid and reliable quantification of biosurfactants in multiple cultures, preferably at high throughput. To this end, we have established a novel and sensitive assay for the quantification of biosurfactants based on the dye Victoria Pure Blue BO (VPBO). The assay allows the colorimetric assessment of biosurfactants directly in culture supernatants and does not require extraction or concentration procedures. Working ranges were determined for precise quantification of different rhamnolipid biosurfactants; titers in culture supernatants of recombinant Pseudomonas putida KT2440 calculated by this assay were confirmed to be the same ranges detected by independent high-performance liquid chromatography (HPLC)-charged aerosol detector (CAD) analyses. The assay was successfully applied for detection of chemically different anionic or non-ionic biosurfactants including mono- and di-rhamnolipids (glycolipids), mannosylerythritol lipids (MELs, glycolipids), 3-(3-hydroxyalkanoyloxy) alkanoic acids (fatty acid conjugates), serrawettin W1 (lipopeptide), and N-acyltyrosine (lipoamino acid). In summary, the VPBO assay offers a broad range of applications including the comparative evaluation of different cultivation conditions and high-throughput screening of biosurfactant-producing microbial strains

Integrated strain- and process design enable production of 220 g L−1 itaconic acid with Ustilago maydis

BackgroundItaconic acid is an unsaturated, dicarboxylic acid which finds a wide range of applications in the polymer industry and as a building block for fuels, solvents and pharmaceuticals. Currently, Aspergillus terreus is used for industrial production, with titers above 100 g L−1 depending on the conditions. Besides A. terreus, Ustilago maydis is also a promising itaconic acid production host due to its yeast-like morphology. Recent strain engineering efforts significantly increased the yield, titer and rate of production.ResultsIn this study, itaconate production by U. maydis was further increased by integrated strain- and process engineering. Next-generation itaconate hyper-producing strains were generated using CRISPR/Cas9 and FLP/FRT genome editing tools for gene deletion, promoter replacement, and overexpression of genes. The handling and morphology of this engineered strain were improved by deletion of fuz7, which is part of a regulatory cascade that governs morphology and pathogenicity. These strain modifications enabled the development of an efficient fermentation process with in situ product crystallization with CaCO3. This integrated approach resulted in a maximum itaconate titer of 220 g L−1, with a total acid titer of 248 g L−1, which is a significant improvement compared to best published itaconate titers reached with U. maydis and with A. terreus.ConclusionIn this study, itaconic acid production could be enhanced significantly by morphological- and metabolic engineering in combination with process development, yielding the highest titer reported with any microorganism