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Towards a sustainable bioeconomy

Research • Educate • Connect
Towards a sustainable bioeconomy
FocusLab Bio²

Integration of next generation biosurfactant production into biorefinery processes

The FocusLab Bio² aimed at the development of a competitive biorefinery process for the production of bio-based surfactants.  Rhamnolipids (RLs) and mannosylerythritol lipids (MELs) were produced by recombinant strains of Pseudomonas putida and Ustilago maydis, respectively. The project combined strain optimization with process development and in situ product recovery approaches to account for the complex process requirements. A socioeconomic evaluation via life cycle assessment guided the process design up to the evaluation in technical scale. By opening up new raw material sources, increasing yields and providing new analytical techniques, the project helped to enhance biosurfactant competitiveness on the market.

Completely bio-based surfactants („biosurfactants“) represent a sustainable alternative to surfactants produced from crude oil. (Bio)surfactants reduce the surface tension of liquids or the interfacial tension of dispersions (Figure 1). This enables a wide range of applications. Consequently, surfactants are used as detergents in cleaning agents, as foaming agents in firefighting, and as emulsifiers in the food industry. Biosurfactants are of microbial origin and can be produced from renewable raw materials.

Fig. 1: The functional principle of surfactants

Production costs for biosurfactants are higher compared to surfactants produced from crude oil. This is caused by the use of expensive raw materials and by low yields and production efficiencies of the microorganisms. Furthermore, the use of sugars or oils as raw materials competes with food production and has to be evaluated critically. Additionally, process control in classical bioreactors is hampered by the biosurfactant’s inherent property of foam formation. Although foaming can be reduced by adding antifoaming agents, this impedes the purification process.

The aim of Bio2 was to address these challenges in a holistic approach along the entire process chain. Therefore, strain and process engineering were considered simultaneously and process stages were interconnected. The work was accompanied by a socio-economic analysis to evaluate and improve the resulting process in terms of ecological, economic and social aspects. Rhamnolipids produced by Pseudomonas putida and mannosylerithrytollipids (MEL) produced by Ustilago maydis were selected as examples of biosurfactants.

Fig. 2: The Bio² process.

The holistic approach could only be implemented in a highly interdisciplinary team. Accordingly, natural scientists and engineers from different disciplines worked closely together within the project.

Growth on renewable resources

For Pseudomonas putida, the substrate spectrum was extended to xylose and ethanol. Xylose is an important constituent of lignocellulose. Consequently, investigation of rhamnolipid production for example from beech wood hydrolysate will be possible. This way, a direct link to the FocusLab AP³ was created. Expansion of the substrate spectrum towards ethanol opens up further options to use alternative carbon sources.

For Ustilago maydis, metabolization of polygalacturonic acid was achieved by the overexpression of heterologous polygalacturonases. This leads the way to using substrates containing pectin, such as sugar beet pulp (SBP). SBP is a by-product of sugar beet processing. Also, it was evaluated if the biomass remaining after the production process can be used as fermentation substrate to utilize remaining waste streams.

Strain development to increase product quantities

For P. putida, numerous promoter systems for the expression of the rhamnolipid biosynthesis genes were adapted. These increase the genetic stability and allow the optimization of the production, for example by adjusting the induction timepoint. Additionally, the genome was reduced to reduce energy-intensive applications such as the formation of flagella. A specially developed high-throughput assay in microtiter plate format considerably simplifies the comparative analysis of glycolipid producers.

When using renewable raw materials, the nitrogen content cannot be controlled at will. To reduce the nitrogen dependency of the MEL production in U. maydis, a 2A peptide strategy for the artificial regulation of the biosynthesis steps was established. Furthermore, important steps for the production of pure MEL-D production could be achieved by eliminating side products such as ustilaginic acid and itaconic acid.

Holistic process development

A new concept for aerobic cultivation - membrane gassing - was developed within the project and a patent application was filed. The integration of the corresponding modules into existing bioreactors enables almost bubble-free aeration of the bioreactor and nearly completely avoids foam formation in the process. The interconnection of the membrane module with a filtration membrane for cell retention also provides the basis for in-situ product separation.

For purification of the glycolipids from the fermentation broth, different process alternatives were evaluated, which achieve high product purity combined with high recovery and thus low product losses. Pre-selected separation operations were examined experimentally and model-based screening experiments were performed. Afterwards, purification processes were established by interconnecting the examined separation operations. The corresponding process chains were evaluated for different substrates from a socio-economic point of view. The accompanying evaluation of various environmental indicators as part of a life cycle assessment guided the process optimization.

The holistic process approach was successfully put into practice during the project. This is expressed, among other things, in a joint publication on the project's rhamnolipid part.

Fig. 3: Networking of different disciplines for the production of rhamnolipids with Pseudomonas putida, This Figure was without changes taken from: Tiso T, Ihling N, Kubicki S, Biselli A, Schonhoff A, Bator I, Thies S, Karmainski T, Kruth S, Willenbrink A-L, Loeschcke A, Zapp P, Jupke A, Jaeger K-E, Büchs J and Blank LM (2020) Integration of Genetic and Process Engineering for Optimized Rhamnolipid Production Using Pseudomonas putida. Front. Bioeng. Biotechnol. 8:976. doi: 10.3389/fbioe.2020.00976. The original publication was published in https://www.frontiersin.org/articles/10.3389/fbioe.2020.00976/full#h5 under a Creative Commons CC BY 4.0 license (https://creativecommons.org/licenses/by/4.0/).

Project leader

Dr. Nina Ihling
AVT - Aachener Verfahrenstechnik
RWTH Aachen University
Forckenbeckstraße 51
52074 Aachen, Germany
E-mail: Nina.Ihling@avt.rwth-aachen.de
Phone: +49 241 80-47868
Fax: +49 241 80-22570

 

Funding Period

01.05.2017 – 31.12.2020

 

Funding

The total budget of Bio² was 2,289,709 €. Bio² is part of the NRW-Strategieprojekt BioSC and thus funded by the Ministry of Culture and Science of the German State of North Rhine-Westphalia.

 

Publications

Blesken, C. et al. (2020): Genetic cell-surface modification for optimized foam fractionation. Frontiers in Bioengineering and Biotechnology 8

Bator, I. et al. (2020): Killing Two Birds With One Stone – Strain Engineering Facilitates the Development of a Unique Rhamnolipid Production Process. Frontiers in Bioengineering and Biotechnology 8:899.

Kubicki, S. et al. (2020): A Straightforward Assay for Screening and Quantification of Biosurfactants in Microbial Culture Supernatants. Frontiers in Bioengineering and Biotechnology 8:958.

Tiso, T. et al. (2020): Integration of Genetic and Process Engineering for Optimized Rhamnolipid Production Using Pseudomonas putida. Frontiers in Bioengineering and Biotechnology 8: 976.

Müntjes, K., et al. (2020): Establishing Polycistronic gene expression in the model microorganism Ustilago maydis. Frontiers in Microbiology 11:1384

Biselli, A. et al. (2020): Development, evaluation, and optimisation of downstream process concepts for rhamnolipids and 3-(3-hydroxyalkanoyloxy)alkanoic acids. Separation and Purification Technology, 117031.

Bator, I., et al. (2020): Comparison of three xylose pathways in Pseudomonas putida KT2440 for the synthesis of valuable products. Front. Bioeng. Biotechnol. 7: 1-18.

Stoffels, P., et al. (2020): Complementing the intrinsic repertoire of Ustilago maydis for degradation of the pectin backbone polygalacturonic acid. Journal of Biotechnology 307:148-163.

Domröse, A., et al. (2019) Pseudomonas putida rDNA is a favored site for the expression of biosynthetic genes. Sci Rep 9, 7028 DOI: 10.1038/s41598-019-43405-1

Otto, M., et al. (2019). Targeting 16S rDNA for Stable Recombinant Gene Expression in Pseudomonas. ACS Synthetic Biology 8(8): 1901-1912

Bollinger, A., et al. (2018). The biotechnological potential of marine bacteria in the novel lineage of Pseudomonas pertucinogena. Microbial Biotechnology 13:19-31.

Hage-Hülsmann, J., et al. (2018). Natural biocide cocktails: Combinatorial antibiotic effects of prodigiosin and biosurfactants. PLoS One 13(7): e0200940