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Browsing by Person "Lilge, Lars"

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Characterization ofantifungal properties of lipopeptide-producing Bacillus velezensis strains and their proteome-based response to the phytopathogens, Diaporthe spp
(2023) Akintayo, Stephen Olusanmi; Hosseini, Behnoush; Vahidinasab, Maliheh; Messmer, Marc; Pfannstiel, Jens; Bertsche, Ute; Hubel, Philipp; Henkel, Marius; Hausmann, Rudolf; Vögele, Ralf; Lilge, Lars
Introduction: B. velezensis strains are of interest in agricultural applications due to their beneficial interactions with plants, notable through their antimicrobial activity. The biocontrol ability of two new lipopeptides-producing B. velezensis strains ES1-02 and EFSO2-04, against fungal phytopathogens of Diaporthe spp., was evaluated and compared with reference strains QST713 and FZB42. All strains were found to be effective against the plant pathogens, with the new strains showing comparable antifungal activity to QST713 and slightly lower activity than FZB42. Methods: Lipopeptides and their isoforms were identified by high-performance thin-layer chromatography (HPTLC) and mass spectrometric measurements. The associated antifungal influences were determined in direct in vitro antagonistic dual culture assays, and the inhibitory growth effects on Diaporthe spp. as representatives of phytopathogenic fungi were determined. The effects on bacterial physiology of selected B. velezensis strains were analyzed by mass spectrometric proteomic analyses using nano-LC-MS/MS. Results and Discussion: Lipopeptide production analysis revealed that all strains produced surfactin, and one lipopeptide of the iturin family, including bacillomycin L by ES1-02 and EFSO2-04, while QST713 and FZB42 produced iturin A and bacillomycin D, respectively. Fengycin production was however only detected in the reference strains. As a result of co-incubation of strain ES1-02 with the antagonistic phytopathogen D. longicolla, an increase in surfactin production of up to 10-fold was observed, making stress induction due to competitors an attractive strategy for surfactin bioproduction. An associated global proteome analysis showed a more detailed overview about the adaptation and response mechanisms of B. velezensis, including an increased abundance of proteins associated with the biosynthesis of antimicrobial compounds. Furthermore, higher abundance was determined for proteins associated with oxidative, nitrosative, and general stress response. In contrast, proteins involved in phosphate uptake, amino acid transport, and translation were decreased in abundance. Altogether, this study provides new insights into the physiological adaptation of lipopeptide-producing B. velezensis strains, which show the potential for use as biocontrol agents with respect to phytopathogenic fungi.
Design and evaluation of a 3D‐printed, lab‐scale perfusion bioreactor for novel biotechnological applications
(2023) Merkel, Manuel; Noll, Philipp; Lilge, Lars; Hausmann, Rudolf; Henkel, Marius
3D‐printing increased in significance for biotechnological research as new applications like lab‐on‐a‐chip systems, cell culture devices or 3D‐printed foods were uncovered. Besides mammalian cell culture, only few of those applications focus on the cultivation of microorganisms and none of these make use of the advantages of perfusion systems. One example for applying 3D‐printing for bioreactor development is the microbial utilization of alternative substrates derived from lignocellulose, where dilute carbon concentrations and harmful substances present a major challenge. Furthermore, quickly manufactured and affordable 3D‐printed bioreactors can accelerate early development phases through parallelization. In this work, a novel perfusion bioreactor system consisting of parts manufactured by fused filament fabrication (FFF) is presented and evaluated. Hydrophilic membranes are used for cell retention to allow the application of dilute substrates. Oxygen supply is provided by membrane diffusion via hydrophobic polytetrafluoroethylene membranes. An exemplary cultivation of Corynebacterium glutamicum ATCC 13032 supports the theoretical design by achieving competitive biomass concentrations of 18.4 g L−1 after 52 h. As a proof‐of‐concept for cultivation of microorganisms in perfusion mode, the described bioreactor system has application potential for bioconversion of multi‐component substrate‐streams in a lignocellulose‐based bioeconomy, for in‐situ product removal or design considerations of future applications for tissue cultures. Furthermore, this work provides a template‐based toolbox with instructions for creating reference systems in different application scenarios or tailor‐made bioreactor systems.
Evaluation of an external foam column for in situ product removal in aerated surfactin production processes
(2023) Treinen, Chantal; Claassen, Linda; Hoffmann, Mareen; Lilge, Lars; Henkel, Marius; Hausmann, Rudolf
In Bacillus fermentation processes, severe foam formation may occur in aerated bioreactor systems caused by surface-active lipopeptides. Although they represent interesting compounds for industrial biotechnology, their property of foaming excessively during aeration may pose challenges for bioproduction. One option to turn this obstacle into an advantage is to apply foam fractionation and thus realize in situ product removal as an initial downstream step. Here we present and evaluate a method for integrated foam fractionation. A special feature of this setup is the external foam column that operates separately in terms of, e.g., aeration rates from the bioreactor system and allows recycling of cells and media. This provides additional control points in contrast to an internal foam column or a foam trap. To demonstrate the applicability of this method, the foam column was exemplarily operated during an aerated batch process using the surfactin-producing Bacillus subtilis strain JABs24. It was also investigated how the presence of lipopeptides and bacterial cells affected functionality. As expected, the major foam formation resulted in fermentation difficulties during aerated processes, partially resulting in reactor overflow. However, an overall robust performance of the foam fractionation could be demonstrated. A maximum surfactin concentration of 7.7 g/L in the foamate and enrichments of up to 4 were achieved. It was further observed that high lipopeptide enrichments were associated with low sampling flow rates of the foamate. This relation could be influenced by changing the operating parameters of the foam column. With the methodology presented here, an enrichment of biosurfactants with simultaneous retention of the production cells was possible. Since both process aeration and foam fractionation can be individually controlled and designed, this method offers the prospect of being transferred beyond aerated batch processes.
Evaluation of an oxygen‐dependent self‐inducible surfactin synthesis in B. subtilis by substitution of native promoter PsrfA by anaerobically active PnarG and PnasD
(2021) Hoffmann, Mareen; Braig, Alina; Fernandez Cano Luna, Diana Stephanie; Rief, Katharina; Becker, Philipp; Treinen, Chantal; Klausmann, Peter; Morabbi Heravi, Kambiz; Henkel, Marius; Lilge, Lars; Hausmann, Rudolf
A novel approach targeting self-inducible surfactin synthesis under oxygen-limited conditions is presented. Because both the nitrate (NarGHI) and nitrite (NasDE) reductase are highly expressed during anaerobic growth of B. subtilis, the native promoter PsrfA of the surfactin operon in strain B. subtilis JABs24 was replaced by promoters PnarG and PnasD to induce surfactin synthesis anaerobically. Shake flask cultivations with varying oxygen availabilities indicated no significant differences in native PsrfA expression. As hypothesized, activity of PnarG and PnasD increased with lower oxygen levels and surfactin was not produced by PsrfA::PnarG as well as PsrfA::PnasD mutant strains under conditions with highest oxygen availability. PnarG showed expressions similar to PsrfA at lowest oxygen availability, while maximum value of PnasD was more than 5.5-fold higher. Although the promoter exchange PsrfA::PnarG resulted in a decreased surfactin titer at lowest oxygen availability, the strain carrying PsrfA::PnasD reached a 1.4-fold increased surfactin concentration with 696 mg/L and revealed an exceptional high overall YP/X of 1.007 g/g. This value also surpassed the YP/X of the reference strain JABs24 at highest and moderate oxygen availability. Bioreactor cultivations illustrated that significant cell lysis occurred when the process of “anaerobization” was performed too fast. However, processes with a constantly low agitation and aeration rate showed promising potential for process improvement, especially by employing the strain carrying PsrfA::PnasD promoter exchange. Additionally, replacement of other native promoters by nitrite reductase promoter PnasD represents a promising tool for anaerobic-inducible bioprocesses in Bacillus.
Surfactin shows relatively low antimicrobial activity against Bacillus subtilis and other bacterial model organisms in the absence of synergistic metabolites
(2022) Lilge, Lars; Ersig, Nadine; Hubel, Philipp; Aschern, Moritz; Pillai, Evelina; Klausmann, Peter; Pfannstiel, Jens; Henkel, Marius; Morabbi Heravi, Kambiz; Hausmann, Rudolf
Surfactin is described as a powerful biosurfactant and is natively produced by Bacillus subtilis in notable quantities. Among other industrially relevant characteristics, antimicrobial properties have been attributed to surfactin-producing Bacillus isolates. To investigate this property, stress approaches were carried out with biotechnologically established strains of Corynebacterium glutamicum, Bacillus subtilis, Escherichia coli and Pseudomonas putida with the highest possible amounts of surfactin. Contrary to the popular opinion, the highest growth-reducing effects were detectable in B. subtilis and E. coli after surfactin treatment of 100 g/L with 35 and 33%, respectively, while P. putida showed no growth-specific response. In contrast, other antimicrobial biosurfactants, like rhamnolipids and sophorolipids, showed significantly stronger effects on bacterial growth. Since the addition of high amounts of surfactin in defined mineral salt medium reduced the cell growth of B. subtilis by about 40%, the initial stress response at the protein level was analyzed by mass spectrometry, showing induction of stress proteins under control of alternative sigma factors σB and σW as well as the activation of LiaRS two-component system. Overall, although surfactin is associated with antimicrobial properties, relatively low growth-reducing effects could be demonstrated after the surfactin addition, challenging the general claim of the antimicrobial properties of surfactin.
Toward effects of hydrophobicity on biosurfactant production by Bacillus subtilis isolates from crude-oil-exposed environments
(2024) Hashemi, Seyedeh Zahra; Fooladi, Jamshid; Vahidinasab, Maliheh; Hubel, Philipp; Pfannstiel, Jens; Pillai, Evelina; Hrenn, Holger; Hausmann, Rudolf; Lilge, Lars
Background: Due to their structural features, biosurfactants reveal promising physicochemical properties, making them interesting for various applications in different fields, such as the food, cosmetics, agriculture, and bioremediation sectors. In particular, the bioproduction of surfactin, one of the most potent microbially synthesized biosurfactant molecules, is of great interest. However, since the wild-type productivities are comparably low, stimulatory environmental conditions have to be identified for improved bioproduction This study aims to find a correlation between the hydrophobicity and production of the biosurfactant surfactin by B. subtilis isolates from crude-oil-contaminated soil and water. Methods: The surfactin production yield was characterized in adapted batch cultivations using high-performance thin-layer liquid chromatography (HPTLC). Defined hydrophobic environmental conditions were achieved by supplementation with hexadecane or polystyrene beads, and the effects on biosurfactant production were measured. Adaptations at the protein level were analyzed using mass spectrometry measurements. Results: The correlation between hydrophobicity and surfactin production was characterized using Bacillus subtilis strains ZH1 and P7 isolated from crude-oil-contaminated soil and water. Since these isolates show the biodegradation of crude oil and hexadecane as hydrophobic substrates, respectively, a first-time approach, using polystyrene beads, was applied to provide a hydrophobic environment. Interestingly, contrary to popular opinion, reduced biosurfactant production was determined. Using mass spectrometric approaches, the physiological effects of co-cultivation and the cellular response at the protein level were investigated, resulting in altered quantities of stress proteins and proteins involved in the carbon metabolism counter to polystyrene beads. Conclusions: Contrary to common opinion, increasing hydrophobicity does not have a stimulating effect, and even reduces the effect on the bioproduction of surfactin as the main biosurfactant using selected B. subtilis strains.

Browsing by Person "Lilge, Lars"

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