Publications by authors named "Till Tiso"

31 Publications

A scalable bubble-free membrane aerator for biosurfactant production.

Biotechnol Bioeng 2021 May 17. Epub 2021 May 17.

Chemical Process Engineering (AVT.CVT), RWTH Aachen University, Aachen, Germany.

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.
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http://dx.doi.org/10.1002/bit.27822DOI Listing
May 2021

Towards bio-upcycling of polyethylene terephthalate.

Metab Eng 2021 07 16;66:167-178. Epub 2021 Apr 16.

iAMB - Institute of Applied Microbiology. ABBt - Aachen Biology and Biotechnology, RWTH Aachen University, Worringerweg 1, D-52074, Aachen, Germany. Electronic address:

Over 359 million tons of plastics were produced worldwide in 2018, with significant growth expected in the near future, resulting in the global challenge of end-of-life management. The recent identification of enzymes that degrade plastics previously considered non-biodegradable opens up opportunities to steer the plastic recycling industry into the realm of biotechnology. Here, the sequential conversion of post-consumer polyethylene terephthalate (PET) into two types of bioplastics is presented: a medium chain-length polyhydroxyalkanoate (PHA) and a novel bio-based poly(amide urethane) (bio-PU). PET films are hydrolyzed by a thermostable polyester hydrolase yielding highly pure terephthalate and ethylene glycol. The obtained hydrolysate is used directly as a feedstock for a terephthalate-degrading Pseudomonas umsongensis GO16, also evolved to efficiently metabolize ethylene glycol, to produce PHA. The strain is further modified to secrete hydroxyalkanoyloxy-alkanoates (HAAs), which are used as monomers for the chemo-catalytic synthesis of bio-PU. In short, a novel value-chain for PET upcycling is shown that circumvents the costly purification of PET monomers, adding technological flexibility to the global challenge of end-of-life management of plastics.
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http://dx.doi.org/10.1016/j.ymben.2021.03.011DOI Listing
July 2021

Impact of the number of rhamnose moieties of rhamnolipids on the structure, lateral organization and morphology of model biomembranes.

Soft Matter 2021 Mar 23;17(11):3191-3206. Epub 2021 Feb 23.

Physical Chemistry I - Biophysical Chemistry, Faculty of Chemistry and Chemical Biology, TU Dortmund University, Otto-Hahn Street 4a, 44227 Dortmund, Germany.

Various studies have described remarkable biological activities and surface-active properties of rhamnolipids, leading to their proposed use in a wide range of industrial applications. Here, we report on a study of the effects of monorhamnolipid RhaCC and dirhamnolipid RhaRhaCC incorporation into model membranes of varying complexity, including bacterial and heterogeneous model biomembranes. For comparison, we studied the effect of HAA (CC, lacking a sugar headgroup) partitioning into these membrane systems. AFM, confocal fluorescence microscopy, DSC, and Laurdan fluorescence spectroscopy were employed to yield insights into the rhamnolipid-induced morphological changes of lipid vesicles as well as modifications of the lipid order and lateral membrane organization of the model biomembranes upon partitioning of the different rhamnolipids. The partitioning of the three rhamnolipids into phospholipid bilayers changes the phase behavior, fluidity, lateral lipid organization and morphology of the phospholipid membranes dramatically, to what extent, depends on the headgroup structure of the rhamnolipid, which affects its packing and hydrogen bonding capacity. The incorporation into giant unilamellar vesicles (GUVs) of a heterogeneous anionic raft membrane system revealed budding of domains and fission of daughter vesicles and small aggregates for all three rhamnolipids, with major destabilization of the lipid vesicles upon insertion of RhaCC, and also formation of huge GUVs upon the incorporation of RhaRhaCC. Finally, we discuss the results with regard to the role these biosurfactants play in biology and their possible impact on applications, ranging from agricultural to pharmaceutical industries.
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http://dx.doi.org/10.1039/d0sm01934hDOI Listing
March 2021

Upcycling of hydrolyzed PET by microbial conversion to a fatty acid derivative.

Methods Enzymol 2021 23;648:391-421. Epub 2021 Jan 23.

iAMB-Institute of Applied Microbiology, ABBt-Aachen Biology and Biotechnology, RWTH Aachen University, Aachen, Germany. Electronic address:

The enzymatic degradation of polyethylene terephthalate (PET) results in a hydrolysate consisting almost exclusively of its two monomers, ethylene glycol and terephthalate. To biologically valorize the PET hydrolysate, microbial upcycling into high-value products is proposed. Fatty acid derivatives hydroxyalkanoyloxy alkanoates (HAAs) represent such valuable target molecules. HAAs exhibit surface-active properties and can be exploited in the catalytical conversion to drop-in biofuels as well as in the polymerization to bio-based poly(amide urethane). This chapter presents the genetic engineering methods of pseudomonads for the metabolization of PET monomers and the biosynthesis of HAAs with detailed protocols concerning product purification.
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http://dx.doi.org/10.1016/bs.mie.2020.12.025DOI Listing
June 2021

Uncoupling Foam Fractionation and Foam Adsorption for Enhanced Biosurfactant Synthesis and Recovery.

Microorganisms 2020 Dec 18;8(12). Epub 2020 Dec 18.

iAMB-Institute of Applied Microbiology, ABBt-Aachen Biology and Biotechnology, RWTH Aachen University, 52074 Aachen, Germany.

The production of biosurfactants is often hampered by excessive foaming in the bioreactor, impacting system scale-up and downstream processing. Foam fractionation was proposed to tackle this challenge by combining in situ product removal with a pre-purification step. In previous studies, foam fractionation was coupled to bioreactor operation, hence it was operated at suboptimal parameters. Here, we use an external fractionation column to decouple biosurfactant production from foam fractionation, enabling continuous surfactant separation, which is especially suited for system scale-up. As a subsequent product recovery step, continuous foam adsorption was integrated into the process. The configuration is evaluated for rhamnolipid (RL) or 3-(3-hydroxyalkanoyloxy)alkanoic acid (HAA, i.e., RL precursor) production by recombinant non-pathogenic KT2440. Surfactant concentrations of 7.5 g/L and 2.0 g/L were obtained in the fractionated foam. 4.7 g RLs and 2.8 g HAAs could be separated in the 2-stage recovery process within 36 h from a 2 L culture volume. With a culture volume scale-up to 9 L, 16 g RLs were adsorbed, and the space-time yield (STY) increased by 31% to 0.21 gRL/L·h. We demonstrate a well-performing process design for biosurfactant production and recovery as a contribution to a vital bioeconomy.
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http://dx.doi.org/10.3390/microorganisms8122029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7766737PMC
December 2020

Coupling an Electroactive KT2440 with Bioelectrochemical Rhamnolipid Production.

Microorganisms 2020 Dec 10;8(12). Epub 2020 Dec 10.

Leibniz Institute for Natural Product Research and Infection Biology-Hans Knöll Institute, 07745 Jena, Germany.

Sufficient supply of oxygen is a major bottleneck in industrial biotechnological synthesis. One example is the heterologous production of rhamnolipids using KT2440. Typically, the synthesis is accompanied by strong foam formation in the reactor vessel hampering the process. It is caused by the extensive bubbling needed to sustain the high respirative oxygen demand in the presence of the produced surfactants. One way to reduce the oxygen requirement is to enable the cells to use the anode of a bioelectrochemical system (BES) as an alternative sink for their metabolically derived electrons. We here used a KT2440 strain that interacts with the anode using mediated extracellular electron transfer via intrinsically produced phenazines, to perform heterologous rhamnolipid production under oxygen limitation. The strain RL-PCA successfully produced 30.4 ± 4.7 mg/L mono-rhamnolipids together with 11.2 ± 0.8 mg/L of phenazine-1-carboxylic acid (PCA) in 500-mL benchtop BES reactors and 30.5 ± 0.5 mg/L rhamnolipids accompanied by 25.7 ± 8.0 mg/L PCA in electrode containing standard 1-L bioreactors. Hence, this study marks a first proof of concept to produce glycolipid surfactants in oxygen-limited BES with an industrially relevant strain.
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http://dx.doi.org/10.3390/microorganisms8121959DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7763313PMC
December 2020

Genetic Cell-Surface Modification for Optimized Foam Fractionation.

Front Bioeng Biotechnol 2020 29;8:572892. Epub 2020 Oct 29.

iAMB - Institute of Applied Microbiology, ABBt - Aachen Biology and Biotechnology, RWTH, Aachen University, Aachen, Germany.

Rhamnolipids are among the glycolipids that have been investigated intensively in the last decades, mostly produced by the facultative pathogen 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 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 ( = 0.17 g/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 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 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.
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http://dx.doi.org/10.3389/fbioe.2020.572892DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7658403PMC
October 2020

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

Front Bioeng Biotechnol 2020 7;8:596414. Epub 2020 Oct 7.

iAMB - Institute of Applied Microbiology, ABBt - Aachen Biology and Biotechnology, RWTH Aachen University, Aachen, Germany.

[This corrects the article DOI: 10.3389/fbioe.2020.00899.].
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http://dx.doi.org/10.3389/fbioe.2020.596414DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7576688PMC
October 2020

Hyphenation of supercritical fluid chromatography with different detection methods for identification and quantification of liamocin biosurfactants.

J Chromatogr A 2020 Sep 24;1631:461584. Epub 2020 Sep 24.

Institute of Inorganic and Analytical Chemistry, University of Münster, D-48149 Münster, Germany. Electronic address:

Liamocins are a class of biosurfactants with growing interest. However, methods for identification and quantification of liamocins on the molecular level are lagging behind. Therefore, we developed a chromatographic separation based on supercritical fluid chromatography (SFC) for liamocins and structurally related exophilins. The different congeners could be separated on a charge modulated hydroxyethyl amide functionalized silica-based column. Coupling to high-resolution mass spectrometry (MS) revealed four exophilin species and four liamocin species with mannitol and arabitol as polyol head group in a sample of the yeast-like fungus Aureobasidium pullulans (A. pullulans). In contrast to a recently published reversed phase high-performance liquid chromatography (HPLC) method, the different subclasses (exophilins, mannitol liamocins and arabitol liamocins) were additionally separated by means of SFC. The structures were confirmed by their accurate masses and tandem mass spectrometry (MS/MS). A complementary quantification method was developed using SFC coupled to charged-aerosol detection (CAD) to overcome the disadvantages of quantification by means of MS without authentic standards. A flow compensation by varying the make-up flow was used to obtain a constant composition of the mobile phase during detection and to ensure a stable detector response. The concentrations of the individual liamocin species were determined using an external calibration with n-octyl-β-d-glycopyranoside. The total amount of these concentrations agrees with the dry weight of an aliquot of the heavy oil. The developed SFC-based method has the advantage of shorter analysis time and superior selectivity compared to the previously published LC separation. In brief, the here presented SFC hyphenations enable comprehensive analysis of liamocin biosurfactants providing identification and absolute quantification of individual congeners.
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http://dx.doi.org/10.1016/j.chroma.2020.461584DOI Listing
September 2020

Integration of Genetic and Process Engineering for Optimized Rhamnolipid Production Using .

Front Bioeng Biotechnol 2020 20;8:976. Epub 2020 Aug 20.

iAMB - Institute of Applied Microbiology, ABBt - Aachen Biology and Biotechnology, RWTH Aachen University, Aachen, Germany.

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.
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http://dx.doi.org/10.3389/fbioe.2020.00976DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7468518PMC
August 2020

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

Front Bioeng Biotechnol 2020 20;8:958. Epub 2020 Aug 20.

Forschungszentrum Jülich GmbH, Institute of Bio- and Geosciences IBG 1: Biotechnology, Jülich, Germany.

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 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 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.
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http://dx.doi.org/10.3389/fbioe.2020.00958DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7468441PMC
August 2020

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

Front Bioeng Biotechnol 2020 7;8:899. Epub 2020 Aug 7.

iAMB - Institute of Applied Microbiology, ABBt - Aachen Biology and Biotechnology, RWTH Aachen University, Aachen, Germany.

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 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 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. 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.
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http://dx.doi.org/10.3389/fbioe.2020.00899DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7427536PMC
August 2020

Comprehensive liamocin biosurfactants analysis by reversed phase liquid chromatography coupled to mass spectrometric and charged-aerosol detection.

J Chromatogr A 2020 Sep 15;1627:461404. Epub 2020 Jul 15.

Institute of Inorganic and Analytical Chemistry, University of Münster, D-48149 Münster, Germany. Electronic address:

Liamocin biosurfactants and structurally related exophilins secreted by the Aureobasidium pullulans (A. pullulans) strain NRRL62031 were firstly analyzed by hyphenation of high-performance liquid chromatography (HPLC) with high-resolution mass spectrometry (HRMS). Ten different analytes were detected and identified by their accurate masses and divided into subclasses according to their different head groups: three liamocins with arabitol as head group, three mannitol liamocins, and four exophilins. A baseline separation of congeners within the subclasses was achieved by reversed phase HPLC on a C18 stationary phase, whereas an overlap of subclasses occurred. The structures were simultaneously confirmed by online tandem mass spectrometry (MS/MS) experiments in positive and negative ionization mode. The assigned polyol head groups and thus the feasibility of this method were confirmed by gas chromatography (GC)-MS data obtained after hydrolysis and derivatization of the liamocins. Based on the varying structural characteristics of liamocins, e.g. the polyol head group (or even none for exophilins) and the degree of acetylation, different detector response in LC-MS was expected, impairing relative quantification of congeners. Therefore, a complementary quantification method was developed using HPLC coupled to charged-aerosol detection (CAD), which allows the determination of the amount of the individual liamocin species without authentic liamocin standards. Hence, the here presented hyphenated techniques facilitate comprehensive analysis of liamocin biosurfactants.
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http://dx.doi.org/10.1016/j.chroma.2020.461404DOI Listing
September 2020

Interaction of rhamnolipids with model biomembranes of varying complexity.

Biochim Biophys Acta Biomembr 2020 11 1;1862(11):183431. Epub 2020 Aug 1.

Physical Chemistry I - Biophysical Chemistry, Faculty of Chemistry and Chemical Biology, TU Dortmund University, Otto-Hahn Street 4a, 44227 Dortmund, Germany. Electronic address:

Rhamnolipids represent a large class of biologically produced surface-active compounds, which participate in various essential cellular functions. While many studies have reported on the antibacterial and antifungal effects of rhamnolipids, only a few tried to describe the molecular mechanisms underlying these effects. Here, we first review the literature on rhamnolipid-phospholipid interactions and then add own results on a prominent monorhamnolipid congener, RhaCC. By focusing on the interactions between the rhamnolipid and lipid model membranes of different complexity, up to heterogeneous raft-like model biomembranes, we gained new insights into changes of the lateral membrane organization and morphological changes of membrane vesicles induced by partitioning of the rhamnolipid. To this end, AFM, confocal fluorescence microscopy, and Laurdan fluorescence spectroscopy analyses were employed. In summary, we provide a concise description of the physio-chemical effects rhamnolipids impose on lipid membranes, which help us to understand their physiological role.
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http://dx.doi.org/10.1016/j.bbamem.2020.183431DOI Listing
November 2020

Double bond localization in unsaturated rhamnolipid precursors 3-(3-hydroxyalkanoyloxy)alkanoic acids by liquid chromatography-mass spectrometry applying online Paternò-Büchi reaction.

Anal Bioanal Chem 2020 Sep 5;412(23):5601-5613. Epub 2020 Jul 5.

Institute of Inorganic and Analytical Chemistry, University of Münster, Corrensstraße 30, 48149, Münster, Germany.

Lipids are biomolecules with a broad variety of chemical structures, which renders them essential not only for various biological functions but also interestingly for biotechnological applications. Rhamnolipids are microbial glycolipids with surface-active properties and are widely used biosurfactants. They are composed of one or two L-rhamnoses and up to three hydroxy fatty acids. Their biosynthetic precursors are 3-hydroxy(alkanoyloxy)alkanoic acids (HAAs). The latter are also present in cell supernatants as complex mixtures and are extensively studied for their potential to replace synthetically derived surfactants. The carbon chain lengths of HAAs determine their physical properties, such as their abilities to foam and emulsify, and their critical micelle concentration. Despite growing biotechnological interest, methods for structural elucidation are limited and often rely on hydrolysis and analysis of free hydroxy fatty acids losing the connectivity information. Therefore, a high-performance liquid chromatography-mass spectrometry method was developed for comprehensive structural characterization of intact HAAs. Information is provided on chain length and number of double bonds in each hydroxy fatty acid and their linkage by tandem mass spectrometry (MS/MS). Post-column photochemical derivatization by online Paternὸ-Büchi reaction and MS/MS fragmentation experiments generated diagnostic fragments allowing structural characterization down to the double bond position level. Furthermore, the presented experiments demonstrate a powerful approach for structure elucidation of complex lipids by tailored fragmentation.
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http://dx.doi.org/10.1007/s00216-020-02776-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7413879PMC
September 2020

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

Front Bioeng Biotechnol 2019 17;7:480. Epub 2020 Jan 17.

iAMB - Institute of Applied Microbiology, ABBt - Aachen Biology and Biotechnology, RWTH Aachen University, Aachen, Germany.

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 and . For isolating the Dahms pathway in 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. After adaptive laboratory evolution the lag phase was reduced significantly. The highest titers of 720 mg L mono-rhamnolipids and 30 mg L 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.
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http://dx.doi.org/10.3389/fbioe.2019.00480DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6978631PMC
January 2020

Exploiting the Natural Diversity of RhlA Acyltransferases for the Synthesis of the Rhamnolipid Precursor 3-(3-Hydroxyalkanoyloxy)Alkanoic Acid.

Appl Environ Microbiol 2020 03 2;86(6). Epub 2020 Mar 2.

RWTH Aachen University, iAMB (Institute of Applied Microbiology, ABBt), Aachen Biology and Biotechnology, Aachen, Germany

While rhamnolipids of the type are commercially available, the natural diversity of rhamnolipids and their origin have barely been investigated. Here, we collected known and identified new genes encoding the acyltransferase responsible for the synthesis of the lipophilic rhamnolipid precursor 3-(3-hydroxyalkanoyloxy)alkanoic acid (HAA). Generally, all homologs were found in and A likely horizontal gene transfer event into is the only identified exception. The phylogeny of the RhlA homologs from and species is consistent with the organism phylogeny, and genes involved in rhamnolipid synthesis are located in operons. In contrast, RhlA homologs from the do not follow the organisms' phylogeny but form their own branch. Furthermore, in many and from the , an isolated homolog can be found in the genome. The RhlAs from PA01, LMG 05825, LMG 20103, PG1, LMG 19182, sp. strain R57-5, Ech586, and PRI-2C were expressed in and tested for HAA production. Indeed, except for the RhlA, HAAs were produced with the engineered strains. A detailed analysis of the produced HAA congeners by high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) highlights the congener specificity of the RhlA proteins. The congener length varies from 4 to 18 carbon atoms, with the main congeners consisting of different combinations of saturated or monounsaturated C, C, and C fatty acids. The results are discussed in the context of the phylogeny of this unusual enzymatic activity. The RhlA specificity explains the observed differences in 3-(3-hydroxyalkanoyloxy)alkanoic acid (HAA) congeners. Whole-cell catalysts can now be designed for the synthesis of different congener mixtures of HAAs and rhamnolipids, thereby contributing to the envisaged synthesis of designer HAAs.
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http://dx.doi.org/10.1128/AEM.02317-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7054101PMC
March 2020

Complete Genome Sequence and Annotation of the Paracoccus pantotrophus Type Strain DSM 2944.

Microbiol Resour Announc 2020 Jan 2;9(1). Epub 2020 Jan 2.

Institute of Applied Microbiology, RWTH Aachen University, Aachen, Germany

spp. are metabolically versatile alphaproteobacteria able to perform heterotrophic and chemoautotrophic growth. This study describes the whole-genome sequence of the type strain DSM 2944 (ATCC 35512, LMD 82.5, GB17). The genome sequence revealed the presence of a complete gene cluster related to polyhydroxyalkanoate metabolism.
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http://dx.doi.org/10.1128/MRA.01290-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6940293PMC
January 2020

Biotechnological upcycling of plastic waste and other non-conventional feedstocks in a circular economy.

Curr Opin Biotechnol 2020 04 24;62:212-219. Epub 2019 Dec 24.

BEACON SFI Bioeconomy Research Centre and School of Biomolecular and Biomedical Science, University College Dublin, Belfield, Dublin 4, Ireland.

The envisaged circular economy requires absolute carbon efficiency and in the long run abstinence from fossil feedstocks, and integration of industrial production with end-of-life waste management. Non-conventional feedstocks arising from industrial production and societal consumption such as CO and plastic waste may soon enable manufacture of multiple products from simple bulk chemicals to pharmaceuticals using biotechnology. The change to these feedstocks could be faster than expected by many, especially if the true cost, including the carbon footprint of products, is considered. The efficiency of biotechnological processes can be improved through metabolic engineering, which can help fulfill the promises of the Paris agreement.
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http://dx.doi.org/10.1016/j.copbio.2019.11.011DOI Listing
April 2020

A pH shift induces high-titer liamocin production in Aureobasidium pullulans.

Appl Microbiol Biotechnol 2019 Jun 25;103(12):4741-4752. Epub 2019 Apr 25.

iAMB-Institute of Applied Microbiology, ABBt-Aachen Biology and Biotechnology, RWTH Aachen University, Worringerweg 1, D-52074, Aachen, Germany.

Liamocins are biosurfactants produced by the fungus Aureobasidium pullulans. A. pullulans belongs to the black yeasts and is known for its ability to produce pullulan and melanin. However, the production of liamocins has not been investigated intensively. Initially, HPLC methods for the quantification of liamocin and the identification of liamocin congeners were established. Eleven congeners could be detected, differing in the polyol head groups arabitol or mannitol. In addition, headless molecules, so-called exophilins, were also identified. The HPLC method reported here allows quick and reliable quantification of all identified congeners, an often-overlooked prerequisite for the investigation of valuable product formation. Liamocin synthesis was optimized during cultivation in lab-scale fermenters. While the pH can be kept constant, the best strategy for liamocin synthesis consists of a growth phase at neutral pH and a subsequent production phase induced by a manual pH shift to pH 3.5. Finally, combining increased nitrogen availability with a pulsed fed-batch fermentation, cell growth, and liamocin titers could be enhanced. Here, the maximal titers of above 10 g/L that were reached are the highest reported to date for liamocin synthesis using A. pullulans in lab-scale fermenters.
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http://dx.doi.org/10.1007/s00253-019-09677-3DOI Listing
June 2019

Mass spectrometric characterization of siderophores produced by Pseudomonas taiwanensis VLB120 assisted by stable isotope labeling of nitrogen source.

Biometals 2018 10 28;31(5):785-795. Epub 2018 Jun 28.

Institute of Inorganic and Analytical Chemistry, University of Münster, Corrensstraße 30, 48149, Münster, Germany.

The structures of three previously unknown siderophores produced by the fluorescent, biotechnologically relevant Pseudomonas taiwanensis (P. taiwanensis) VLB120 bacteria were elucidated by means of hydrophilic interaction liquid chromatography (HILIC) hyphenated to high-resolution tandem mass spectrometry (HRMS/MS). They could be verified as iron(III)- and aluminum(III) complexes as well as the protonated molecules of the siderophores formed by in-source fragmentation. The siderophores were separated according to their different acyl side chains and additionally according their central ions. One of the siderophores was identified as pyoverdine with a malic acid (hydroxy succinic acid) amide side chain and a peptide moiety consisting of Orn-Asp-OHAsn-Thr-AcOHOrn-Ser-cOHOrn. The other analytes were assigned to an azotobactin with the identical peptide chain linked to the characteristic chromophoric unit and a pyoverdine with a variation in the amino acid sequence. Proline is directly linked to the pyoverdine chromophore instead of ornithine. Acidic and enzymatic hydrolyses were carried out to analyze the individual amino acids. Beside OHAsn, each amino acid of the peptide part was identified by HILIC-HRMS and comparison to authentic standards. Additionally, N-labeled cellular supernatants were analyzed by means of HRMS/MS. The results of the MS/MS experiments complemented by accurate mass data facilitated elucidation of the structures studied in this work and provided further confirmation of the three recently described pyoverdines of P. taiwanensis VLB120 (Baune et al. in Biometals 30:589-597, 2017. https://doi.org/10.1007/s10534-017-0029-7 ).
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http://dx.doi.org/10.1007/s10534-018-0122-6DOI Listing
October 2018

Heterologous production of long-chain rhamnolipids from Burkholderia glumae in Pseudomonas putida-a step forward to tailor-made rhamnolipids.

Appl Microbiol Biotechnol 2018 Feb 20;102(3):1229-1239. Epub 2017 Dec 20.

Ulm Center for Peptide Pharmaceuticals (U-PEP), Ulm University, Albert-Einstein-Allee 11, 89081, Ulm, Germany.

Rhamnolipids are biosurfactants consisting of rhamnose (Rha) molecules linked through a β-glycosidic bond to 3-hydroxyfatty acids with various chain lengths, and they have an enormous potential for various industrial applications. The best known native rhamnolipid producer is the human pathogen Pseudomonas aeruginosa, which produces short-chain rhamnolipids mainly consisting of a Rha-Rha-C-C congener. Bacteria from the genus Burkholderia are also able to produce rhamnolipids, which are characterized by their long-chain 3-hydroxyfatty acids with a predominant Rha-Rha-C-C congener. These long-chain rhamnolipids offer different physicochemical properties compared to their counterparts from P. aeruginosa making them very interesting to establish novel potential applications. However, widespread applications of rhamnolipids are still hampered by the pathogenicity of producer strains and-even more important-by the complexity of regulatory networks controlling rhamnolipid production, e.g., the so-called quorum sensing system. To overcome encountered challenges of the wild type, the responsible genes for rhamnolipid biosynthesis in Burkholderia glumae were heterologously expressed in the non-pathogenic Pseudomonas putida KT2440. Our results show that long-chain rhamnolipids from Burkholderia spec. can be produced in P. putida. Surprisingly, the heterologous expression of the genes rhlA and rhlB encoding an acyl- and a rhamnosyltransferase, respectively, resulted in the synthesis of two different mono-rhamnolipid species containing one or two 3-hydroxyfatty acid chains in equal amounts. Furthermore, mixed biosynthetic rhlAB operons with combined genes from different organisms were created to determine whether RhlA or RhlB is responsible to define the fatty acid chain lengths in rhamnolipids.
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http://dx.doi.org/10.1007/s00253-017-8702-xDOI Listing
February 2018

Designer rhamnolipids by reduction of congener diversity: production and characterization.

Microb Cell Fact 2017 Dec 14;16(1):225. Epub 2017 Dec 14.

iAMB-Institute of Applied Microbiology, ABBt-Aachen Biology and Biotechnology, RWTH Aachen University, Worringerweg 1, 52074, Aachen, Germany.

Background: Rhamnolipids are biosurfactants featuring surface-active properties that render them suitable for a broad range of industrial applications. These properties include their emulsification and foaming capacity, critical micelle concentration, and ability to lower surface tension. Further, aspects like biocompatibility and environmental friendliness are becoming increasingly important. Rhamnolipids are mainly produced by pathogenic bacteria like Pseudomonas aeruginosa. We previously designed and constructed a recombinant Pseudomonas putida KT2440, which synthesizes rhamnolipids by decoupling production from host-intrinsic regulations and cell growth.

Results: Here, the molecular structure of the rhamnolipids, i.e., different congeners produced by engineered P. putida are reported. Natural rhamnolipid producers can synthesize mono- and di-rhamnolipids, containing one or two rhamnose molecules, respectively. Of each type of rhamnolipid four main congeners are produced, deviating in the chain lengths of the β-hydroxy-fatty acids. The resulting eight main rhamnolipid congeners with variable numbers of hydrophobic/hydrophilic residues and their mixtures feature different physico-chemical properties that might lead to diverse applications. We engineered a microbial cell factory to specifically produce three different biosurfactant mixtures: a mixture of di- and mono-rhamnolipids, mono-rhamnolipids only, and hydroxyalkanoyloxy alkanoates, the precursors of rhamnolipid synthesis, consisting only of β-hydroxy-fatty acids. To support the possibility of second generation biosurfactant production with our engineered microbial cell factory, we demonstrate rhamnolipid production from sustainable carbon sources, including glycerol and xylose. A simple purification procedure resulted in biosurfactants with purities of up to 90%. Finally, through determination of properties specific for surface active compounds, we were able to show that the different mixtures indeed feature different physico-chemical characteristics.

Conclusions: The approach demonstrated here is a first step towards the production of designer biosurfactants, tailor-made for specific applications by purposely adjusting the congener composition of the mixtures. Not only were we able to genetically engineer our cell factory to produce specific biosurfactant mixtures, but we also showed that the products are suited for different applications. These designer biosurfactants can be produced as part of a biorefinery from second generation carbon sources such as xylose.
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http://dx.doi.org/10.1186/s12934-017-0838-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5729600PMC
December 2017

Anionic Extraction for Efficient Recovery of Biobased 2,3-Butanediol-A Platform for Bulk and Fine Chemicals.

ChemSusChem 2017 08 2;10(16):3252-3259. Epub 2017 Aug 2.

Chair of Heterogeneous Catalysis and Chemical Technology, RWTH Aachen University, Worringerweg 2, 52074, Aachen, Germany.

2,3-Butanediol (BDO) presents a promising platform molecule for the synthesis of basic and fine chemicals. Biotechnological production of BDO from renewable resources with living microbes enables high concentrations in the fermentation broth. The recovery of high-boiling BDO from an aqueous fermentation broth presents a subsequent challenge. A method is proposed for BDO isolation based on reversible complexation with phenylboronate in an anionic complex. BDO can be recovered by back-extraction into an acidic solution. The composition of the extracted species was determined by NMR spectroscopy, MS, and GC-MS methods. The conditions of extraction and back-extraction were optimized by using commercial BDO and finally applied to different fermentation broths. Up to 72-93 % BDO can be extracted and up to 80-90 % can be back-extracted under the optimized conditions. Purified bio-BDO was used in the presence of sulfuric acid for the synthesis of methyl ethyl ketone, an established organic solvent and discussed tailor-made biofuel.
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http://dx.doi.org/10.1002/cssc.201700899DOI Listing
August 2017

Novel insights into biosynthesis and uptake of rhamnolipids and their precursors.

Appl Microbiol Biotechnol 2017 Apr 17;101(7):2865-2878. Epub 2016 Dec 17.

Ulm Center for Peptide Pharmaceuticals (U-PEP), Ulm University, Albert-Einstein-Allee 11, 89081, Ulm, Germany.

The human pathogenic bacterium Pseudomonas aeruginosa produces rhamnolipids, glycolipids with functions for bacterial motility, biofilm formation, and uptake of hydrophobic substrates. Rhamnolipids represent a chemically heterogeneous group of secondary metabolites composed of one or two rhamnose molecules linked to one or mostly two 3-hydroxyfatty acids of various chain lengths. The biosynthetic pathway involves rhamnosyltransferase I encoded by the rhlAB operon, which synthesizes 3-(3-hydroxyalkanoyloxy)alkanoic acids (HAAs) followed by their coupling to one rhamnose moiety. The resulting mono-rhamnolipids are converted to di-rhamnolipids in a third reaction catalyzed by the rhamnosyltransferase II RhlC. However, the mechanism behind the biosynthesis of rhamnolipids containing only a single fatty acid is still unknown. To understand the role of proteins involved in rhamnolipid biosynthesis the heterologous expression of rhl-genes in non-pathogenic Pseudomonas putida KT2440 strains was used in this study to circumvent the complex quorum sensing regulation in P. aeruginosa. Our results reveal that RhlA and RhlB are independently involved in rhamnolipid biosynthesis and not in the form of a RhlAB heterodimer complex as it has been previously postulated. Furthermore, we demonstrate that mono-rhamnolipids provided extracellularly as well as HAAs as their precursors are generally taken up into the cell and are subsequently converted to di-rhamnolipids by P. putida and the native host P. aeruginosa. Finally, our results throw light on the biosynthesis of rhamnolipids containing one fatty acid, which occurs by hydrolyzation of typical rhamnolipids containing two fatty acids, valuable for the production of designer rhamnolipids with desired physicochemical properties.
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http://dx.doi.org/10.1007/s00253-016-8041-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5352749PMC
April 2017

Rhamnolipid biosurfactant analysis using online turbulent flow chromatography-liquid chromatography-tandem mass spectrometry.

J Chromatogr A 2016 Sep 21;1465:90-7. Epub 2016 Aug 21.

Institute of Inorganic and Analytical Chemistry, University of Münster, Corrensstr. 30, 48149 Münster, Germany. Electronic address:

Rhamnolipids are biosurfactants produced by a variety of bacterial species that present a promising alternative to surfactants from petrochemical or oleochemical origin. The success of the fermentation is evaluated by subsequent qualitative and quantitative analysis. However, the sample preparation for high numbers of samples is often laborious and inefficient. In this study an online sample preparation is developed for the qualitative and quantitative analysis of rhamnolipids by LC-MS/MS. Online sample preparation is carried out on a TurboFlow Cyclone MAX column using turbulent flow chromatography. Sample preparation prior the analysis is minimized to a dilution and syringe filtration step leading to an instrumental analysis time of 33min. The limit of detection and the limit of quantification were 0.4ng and 0.6ng on column, respectively. Recovery of the main mono- and di-rhamnolipids from a fermentation sample was 102-104%. Additionally, the rhamnolipid biosynthetic precursors 3-hydroxy(alkanoyloxy)alkanoic acids (HAAs) are covered, albeit extraction is not quantitative (85-90%). The analysis of rhamnolipids from four different microbial species was in good agreement with previous reports. The presented method allows rapid and comprehensive analysis of rhamnolipids with minimal sample preparation directly from the fermentation broth. The application of complementary data-dependent MS/MS acquisition enables non-target screening of rhamnolipids.
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http://dx.doi.org/10.1016/j.chroma.2016.08.044DOI Listing
September 2016

Creating metabolic demand as an engineering strategy in - Rhamnolipid synthesis as an example.

Metab Eng Commun 2016 Dec 8;3:234-244. Epub 2016 Aug 8.

iAMB - Institute of Applied Microbiology, ABBt - Aachen Biology and Biotechnology, RWTH Aachen University, Worringerweg 1, D-52074 Aachen, Germany.

Metabolic engineering of microbial cell factories for the production of heterologous secondary metabolites implicitly relies on the intensification of intracellular flux directed toward the product of choice. Apart from reactions following peripheral pathways, enzymes of the central carbon metabolism are usually targeted for the enhancement of precursor supply. In , a Gram-negative soil bacterium, central carbon metabolism, i.e., the reactions required for the synthesis of all 12 biomass precursors, was shown to be regulated at the metabolic level and not at the transcriptional level. The bacterium's central carbon metabolism appears to be driven by demand to react rapidly to ever-changing environmental conditions. In contrast, peripheral pathways that are only required for growth under certain conditions are regulated transcriptionally. In this work, we show that this regulation regime can be exploited for metabolic engineering. We tested this driven-by-demand metabolic engineering strategy using rhamnolipid production as an example. Rhamnolipid synthesis relies on two pathways, i.e., fatty acid synthesis and the rhamnose pathway, providing the required precursors hydroxyalkanoyloxy-alkanoic acid (HAA) and activated (dTDP-)rhamnose, respectively. In contrast to single-pathway molecules, rhamnolipid synthesis causes demand for two central carbon metabolism intermediates, i.e., acetyl-CoA for HAA and glucose-6-phosphate for rhamnose synthesis. Following the above-outlined strategy of driven by demand, a synthetic promoter library was developed to identify the optimal expression of the two essential genes () for rhamnolipid synthesis. The best rhamnolipid-synthesizing strain had a yield of 40% rhamnolipids on sugar [Cmol/Cmol], which is approximately 55% of the theoretical yield. The rate of rhamnolipid synthesis of this strain was also high. Compared to an exponentially growing wild type, the rhamnose pathway increased its flux by 300%, whereas the flux through fatty acid synthesis increased by 50%. We show that the central carbon metabolism of is capable of meeting the metabolic demand generated by engineering transcription in peripheral pathways, thereby enabling a significant rerouting of carbon flux toward the product of interest, in this case, rhamnolipids of industrial interest.
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http://dx.doi.org/10.1016/j.meteno.2016.08.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5678820PMC
December 2016

High performance liquid chromatography-charged aerosol detection applying an inverse gradient for quantification of rhamnolipid biosurfactants.

J Chromatogr A 2016 Jul 25;1455:125-132. Epub 2016 May 25.

Institute of Inorganic and Analytical Chemistry, University of Münster, Corrensstr. 30, 48149 Münster, Germany. Electronic address:

A method using high performance liquid chromatography coupled to charged-aerosol detection (HPLC-CAD) was developed for the quantification of rhamnolipid biosurfactants. Qualitative sample composition was determined by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The relative quantification of different derivatives of rhamnolipids including di-rhamnolipids, mono-rhamnolipids, and their precursors 3-(3-hydroxyalkanoyloxy)alkanoic acids (HAAs) differed for two compared LC-MS instruments and revealed instrument dependent responses. Our here reported HPLC-CAD method provides uniform response. An inverse gradient was applied for the absolute quantification of rhamnolipid congeners to account for the detector's dependency on the solvent composition. The CAD produces a uniform response not only for the analytes but also for structurally different (nonvolatile) compounds. It was demonstrated that n-dodecyl-β-d-maltoside or deoxycholic acid can be used as alternative standards. The method of HPLC-ultra violet (UV) detection after a derivatization of rhamnolipids and HAAs to their corresponding phenacyl esters confirmed the obtained results but required additional, laborious sample preparation steps. Sensitivity determined as limit of detection and limit of quantification for four mono-rhamnolipids was in the range of 0.3-1.0 and 1.2-2.0μg/mL, respectively, for HPLC-CAD and 0.4 and 1.5μg/mL, respectively, for HPLC-UV. Linearity for HPLC-CAD was at least 0.996 (R(2)) in the calibrated range of about 1-200μg/mL. Hence, the here presented HPLC-CAD method allows absolute quantification of rhamnolipids and derivatives.
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http://dx.doi.org/10.1016/j.chroma.2016.05.079DOI Listing
July 2016

Characterization of rhamnolipids by liquid chromatography/mass spectrometry after solid-phase extraction.

Anal Bioanal Chem 2016 Apr 15;408(10):2505-14. Epub 2016 Feb 15.

Institute of Inorganic and Analytical Chemistry, University of Münster, Corrensstr. 30, 48149, Münster, Germany.

Rhamnolipids are surface-active agents with a broad application potential that are produced in complex mixtures by bacteria of the genus Pseudomonas. Analysis from fermentation broth is often characterized by laborious sample preparation and requires hyphenated analytical techniques like liquid chromatography coupled to mass spectrometry (LC-MS) to obtain detailed information about sample composition. In this study, an analytical procedure based on chromatographic method development and characterization of rhamnolipid sample material by LC-MS as well as a comparison of two sample preparation methods, i.e., liquid-liquid extraction and solid-phase extraction, is presented. Efficient separation was achieved under reversed-phase conditions using a mixed propylphenyl and octadecylsilyl-modified silica gel stationary phase. LC-MS/MS analysis of a supernatant from Pseudomonas putida strain KT2440 pVLT33_rhlABC grown on glucose as sole carbon source and purified by solid-phase extraction revealed a total of 20 congeners of di-rhamnolipids, mono-rhamnolipids, and their biosynthetic precursors 3-(3-hydroxyalkanoyloxy)alkanoic acids (HAAs) with different carbon chain lengths from C8 to C14, including three rhamnolipids with uncommon C9 and C11 fatty acid residues. LC-MS and the orcinol assay were used to evaluate the developed solid-phase extraction method in comparison with the established liquid-liquid extraction. Solid-phase extraction exhibited higher yields and reproducibility as well as lower experimental effort.
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http://dx.doi.org/10.1007/s00216-016-9353-yDOI Listing
April 2016

Mechanism-specific and whole-organism ecotoxicity of mono-rhamnolipids.

Sci Total Environ 2016 Apr 20;548-549:155-163. Epub 2016 Jan 20.

Department of Ecosystem Analysis, Inst. for Environmental Research (Biology V), Worringerweg 1, 52074 Aachen, Germany. Electronic address:

Biosurfactants like rhamnolipids are promising alternatives to chemical surfactants in a range of applications. A wider use requires an analysis of their environmental fate and their ecotoxicological potential. In the present study mono-rhamnolipids produced by a recombinant Pseudomonas putida strain were analyzed using the Green Toxicology concept for acute and mechanism-specific toxicity in an ecotoxicological test battery. Acute toxicity tests with the invertebrate Daphnia magna and with zebrafish embryos (Danio rerio) were performed. In addition, microbial and fungicidal effectiveness was investigated. Mutagenicity of the sample was tested by means of the Ames fluctuation assay. A selected mono-rhamnolipid was used for model simulations regarding mutagenicity and estrogenic activity. Our results indicate that mono-rhamnolipids cause acute toxicity to daphnids and zebrafish embryos comparable to or even lower than chemical surfactants. Rhamnolipids showed very low toxicity to the germination of Aspergillus niger spores and the growth of Candida albicans. No frameshift mutation or base substitutions were observed using the Ames fluctuation assay with the two tester strains TA98 and TA100. This result was confirmed by model simulations. Likewise it was computed that rhamnolipids have no estrogenic potential. In conclusion, mono-rhamnolipids are an environmental friendly alternative to chemical surfactants as the ecotoxicological potential is low.
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http://dx.doi.org/10.1016/j.scitotenv.2016.01.066DOI Listing
April 2016
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