Isabel Bator

RWTH Aachen University

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Lars M. Blank

RWTH Aachen University

Till Tiso

RWTH Aachen University

Tobias Karmainski

RWTH Aachen University

Michael Feldbrügge

Heinrich Heine University Düsseldorf

Nina Ihling

RWTH Aachen University

Stephan Thies

Forschungszentrum Jülich

Sonja Kubicki

Heinrich Heine University Düsseldorf

Jochen Büchs

RWTH Aachen University

Anita Loeschcke

Heinrich Heine University Düsseldorf

Karl‐Erich Jaeger

Hamburg University of Technology

Cooperative Institutions

RWTH Aachen University

Forschungszentrum Jülich

Heinrich Heine University Düsseldorf

FH Aachen

Cluster of Excellence on Plant Sciences

DWI – Leibniz Institute for Interactive Materials

Stadtwerke Jülich (Germany)

Norwegian Institute of Bioeconomy Research

Software (Spain)

Max Planck Society

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A scalable bubble‐free membrane aerator for biosurfactant production

Biotechnology and Bioengineering (2021)

Patrick Bongartz Isabel Bator Kristina Baitalow Robert Keller Till Tiso

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.

Rhamnolipid

Membrane reactor

10.1002/bit.27822

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Citations (16)

Corrigendum: Killing Two Birds With One Stone – Strain Engineering Facilitates the Development of a Unique Rhamnolipid Production Process

Frontiers in Bioengineering and Biotechnology (2020)

Isabel Bator Tobias Karmainski Till Tiso Lars M. Blank

[This corrects the article DOI: 10.3389/fbioe.2020.00899.].

Rhamnolipid

Strain (injury)

10.3389/fbioe.2020.596414

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Citations (16)

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

Biotechnology for Biofuels (2019)

Hamed Hosseinpour Tehrani Johanna Becker Isabel Bator Katharina Maria Saur Svenja Meyer

Abstract Background Itaconic 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. Results In 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 CaCO 3 . 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 . Conclusion In 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.

Itaconic acid

Aspergillus terreus

Ustilago

Strain (injury)

Metabolic Engineering

10.1186/s13068-019-1605-6

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Citations (73)

Genetic Cell-Surface Modification for Optimized Foam Fractionation

Frontiers in Bioengineering and Biotechnology (2020)

Christian C. Blesken Isabel Bator Christian Eberlein Hermann J. Heipieper Till Tiso

Rhamnolipids are among the glycolipids that have been investigated intensively in the last decades, mostly produced by the facultative pathogen Pseudomonas aeruginosa using plant oils as carbon source and antifoam agent. Simplification of downstream processing is envisaged using hydrophilic carbon sources, such as glucose, employing recombinant non-pathogenic Pseudomonas putida KT2440 for rhamnolipid or 3-(3-hydroxyalkanoyloxy)alkanoic acid (HAA, i.e., rhamnolipid precursors) production. However, during scale-up of the cultivation from shake flask to bioreactor, excessive foam formation hinders the use of standard fermentation protocols. In this study, the foam was guided from the reactor to a foam fractionation column to separate biosurfactants from medium and bacterial cells. Applying this integrated unit operation, the space-time yield (STY) for rhamnolipid synthesis could be increased by a factor of 2.8 (STY = 0.17 gRL/L·h) compared to the production in shake flasks. The accumulation of bacteria at the gas-liquid interface of the foam resulted in removal of whole-cell biocatalyst from the reactor with the strong consequence of reduced rhamnolipid production. To diminish the accumulation of bacteria at the gas-liquid interface, we deleted genes encoding cell-surface structures, focusing on hydrophobic proteins present on P. putida KT2440. Strains lacking, e.g., the flagellum, fimbriae, exopolysaccharides, and specific surface proteins, were tested for cell surface hydrophobicity and foam adsorption. Without flagellum or the large adhesion protein F (LapF), foam enrichment of these modified P. putida KT2440 was reduced by 23 and 51%, respectively. In a bioreactor cultivation of the non-motile strain with integrated rhamnolipid production genes, biomass enrichment in the foam was reduced by 46% compared to the reference strain. The intensification of rhamnolipid production from hydrophilic carbon sources presented here is an example for integrated strain and process engineering. This approach will become routine in the development of whole-cell catalysts for the envisaged bioeconomy. The results are discussed in the context of the importance of interacting strain and process engineering early in the development of bioprocesses.

Foam fractionation

Surface Modification

Foam cell

10.3389/fbioe.2020.572892

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Citations (26)

Killing Two Birds With One Stone – Strain Engineering Facilitates the Development of a Unique Rhamnolipid Production Process

Frontiers in Bioengineering and Biotechnology (2020)

Isabel Bator Tobias Karmainski Till Tiso Lars M. Blank

High-titer biosurfactant production in aerated fermenters using hydrophilic substrates is often hampered by excessive foaming. Ethanol has been shown to efficiently destabilize foam of rhamnolipids, a popular group of biosurfactants. To exploit this feature, we used ethanol as carbon source and defoamer, without introducing novel challenges for rhamnolipid purification. In detail, we engineered the non-pathogenic Pseudomonas putida KT2440 for heterologous rhamnolipid production from ethanol. To obtain a strain with high growth rate on ethanol as sole carbon source at elevated ethanol concentrations, adaptive laboratory evolution (ALE) was performed. Genome re-sequencing allowed to allocate the phenotypic changes to emerged mutations. Several genes were affected and differentially expressed including alcohol and aldehyde dehydrogenases, potentially contributing to the increased growth rate on ethanol of 0.51 h-1 after ALE. Further, mutations in genes were found, which possibly led to increased ethanol tolerance. The engineered rhamnolipid producer was used in a fed-batch fermentation with automated ethanol addition over 23 h, which resulted in a 3-(3-hydroxyalkanoyloxy)alkanoates and mono-rhamnolipids concentration of about 5 g L-1. The ethanol concomitantly served as carbon source and defoamer with the advantage of increased rhamnolipid and biomass production. In summary, we present a unique combination of strain and process engineering that facilitated the development of a stable fed-batch fermentation for rhamnolipid production, circumventing mechanical or chemical foam disruption.

Rhamnolipid

Defoamer

Pseudomonas putida

Strain (injury)

Carbon source

10.3389/fbioe.2020.00899

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Citations (36)

Comparison of Three Xylose Pathways in Pseudomonas putida KT2440 for the Synthesis of Valuable Products

Frontiers in Bioengineering and Biotechnology (2020)

Isabel Bator Andreas Wittgens Frank Rosenau Till Tiso Lars M. Blank

Pseudomonas putida KT2440 is a well-established chassis in industrial biotechnology. To increase the substrate spectrum, we implemented three alternative xylose utilization pathways, namely the Isomerase, Weimberg, and Dahms pathways. The synthetic operons contain genes from Escherichia coli and Pseudomonas taiwanensis. For isolating the Dahms pathway in P. putida KT2440 two genes (PP_2836 and PP_4283), encoding an endogenous enzyme of the Weimberg pathway and a regulator for glycolaldehyde degradation, were deleted. Before and after adaptive laboratory evolution, these strains were characterized in terms of growth and synthesis of mono-rhamnolipids and pyocyanin. The engineered strain using the Weimberg pathway reached the highest maximal growth rate of 0.30 h-1. After adaptive laboratory evolution the lag phase was reduced significantly. The highest titers of 720 mg L-1 mono-rhamnolipids and 30 mg L-1 pyocyanin were reached by the evolved strain using the Weimberg or an engineered strain using the Isomerase pathway, respectively. The different stoichiometries of the three xylose utilization pathways may allow engineering of tailored chassis for valuable bioproduct synthesis.

Pseudomonas putida

Pyocyanin

Strain (injury)

10.3389/fbioe.2019.00480

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Citations (90)

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

Frontiers in Bioengineering and Biotechnology (2020)

Sonja Kubicki Isabel Bator S. Jankowski Kerstin Schipper Till Tiso

A large variety of microorganisms produce biosurfactants with the potential for a number of diverse industrial applications. To identify suitable wildtype 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 accordingly were confirmed by independent HPLC-CAD analyses. The assay was successfully applied for detection of chemically different an- 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.

Pseudomonas putida

Lipopeptide

Rhamnolipid

10.3389/fbioe.2020.00958

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Citations (24)

A plea for the integration of Green Toxicology in sustainable bioeconomy strategies – Biosurfactants and microgel-based pesticide release systems as examples

Journal of Hazardous Materials (2021)

Sarah Johann Fabian G. Weichert Lukas Schröer Lucas Stratemann Christoph Kämpfer

10.1016/j.jhazmat.2021.127800

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Citations (9)

Integration of Genetic and Process Engineering for Optimized Rhamnolipid Production Using Pseudomonas putida

Frontiers in Bioengineering and Biotechnology (2020)

Till Tiso Nina Ihling Sonja Kubicki Andreas Biselli Andreas Schonhoff

Rhamnolipids are biosurfactants produced by microorganisms with the potential to replace synthetic compounds with petrochemical origin. Possible industrial applications range from pharmaceuticals to bioremediation agents. As the challenges to intensify recombinant rhamnolipid production from sugars are manifold, multidisciplinary approaches are required. Previously, P. putida KT2440 was developed as heterologous platform strain for rhamnolipid synthesis. Based on this, a multidisciplinary approach towards developing a sustainable rhamnolipid production process is presented. This includes strain development and physiological characterization, improvement of cultivation conditions, and downstream processing as well as life cycle assessment (LCA). Newly developed expression cassettes for stable integration of the rhamnolipid biosynthesis genes into the genome outperformed plasmid-based expression systems. Furthermore, 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 %. 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 anti foaming agents. The usage of a suitable minimal medium reduced undesired interphase formation in the liquid-liquid extraction step. To assess the relevant system variants by their environmental impacts, a technical scale production process was designed (150 L fermentation volume) and evaluated performing LCA. The 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 results are discussed in the context of the challenges of microbial biosurfactant production using hydrophilic substrates on an industrial scale, and how a multidisciplinary approach can be guided by early process evaluations.

Rhamnolipid

Pseudomonas putida

Surfactin

Downstream processing

10.3389/fbioe.2020.00976

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Citations (72)