Browsing by Person "Hoffmann, Mareen"

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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.
Exploiting novel strategies for the production of surfactin in Bacillus subtilis cultures
(2021) Hoffmann, Mareen; Hausmann, Rudolf
Biosurfactants are synthesized by various microorganisms. These surface-active molecules are a promising alternative to petrochemically and oleochemically produced surfactants. Advantageously, biosurfactants are reported to be better biodegradable and less toxic. The cyclic lipopeptide surfactin synthesized by Bacillus subtilis displays one interesting biosurfactant. Many studies report on the outstanding physico-chemical characteristics and add on benefits such as antimicrobial properties. Hence, surfactin has the potential to be used in a variety of industrial sectors. Nevertheless, processes ensuring both robustness and high titers are rare, especially as conventional aerobic bioreactor cultivations share one major disadvantage, namely excessive foaming. To approach industrial processes, different methods are applied, which can be categorized in three practices. These are (1) media and process parameter optimization, (2) strain engineering, and (3) investigating novel process strategies. For the latter category, the anaerobic growth by nitrate respiration poses an interesting foam-free alternative. In this sense, the anaerobic cultivation of B. subtilis to produce surfactin coupled with the three afore mentioned practices was addressed in this thesis targeting at a foam-free surfactin production process. In the 1st publication, the genome reduced strain B. subtilis IIG-Bs20-5-1, a derivative of the laboratory strain 168 able to synthesize surfactin, was evaluated with respect to its suitability as surfactin producer at various temperatures under both aerobic and anaerobic conditions. It was hypothesized that a deletion of 10% of the genome, e.g., non-essential genes synthesizing prophages or the antibiotic bacilysin, saves metabolic resources and hence results in increased surfactin titers. Strains B. subtilis JABs24, a 168 derivative able to synthesize surfactin but without genome reduction, and the surfactin producer B. subtilis DSM 10T served for comparison. Although strain IIG-Bs20-5-1 was superior regarding specific growth rate µ and biomass yield YX/S, the strain was inferior with respect to surfactin titers, product related yields YP/S and YP/X, and specific productivity q. Indeed, compared to others in literature described strains, B. subtilis JABs24 was emphasized as promising target strain for further process development, reaching high surfactin titers of 1147 mg/L aerobically and 296 mg/L anaerobically as well as exceptionally high product yields YP/X under anaerobic conditions. Accordingly, iterative process optimization was hypothesized to improve anaerobically achieved surfactin titers. However, several aspects to consider of anaerobic growth of B. subtilis by nitrate respiration were described in the 2nd publication. Amongst others, increasing ammonium concentrations, resulting from nitrate reduction to ammonium via nitrite, were shown to have no impact on growth of strain JABs24, but surfactin titers and expression of nitrate reductase promoter PnarG were reduced. Nitrite was shown to peak within the first hours of cultivation and concentrations up to 10 mmol/L resulted in prolonged lag-phases. Moreover, acetate accumulated drastically during the time course of cultivation independent of glucose availability, thus decreasing the glucose flux into biomass. Acetate additionally influenced both specific growth rate µ and PnarG expression negatively. Concluding, the general feasibility of anaerobic fed-batch cultivations to synthesize surfactin was shown, but several aspects must be addressed in future works to make this strategy an equated process with aerobic cultivations. In the 3rd publication, a self-inducible surfactin synthesis process was presented where expression of the surfactin operon in B. subtilis JABs24 was induced under oxygen limited conditions. The native promoter of the srfA operon PsrfA was replaced by anaerobically inducible nitrate reductase promoter PnarG and nitrite reductase promoter PnasD. Shake flask cultivations with varying oxygen availabilities demonstrated that both PnarG and PnasD can serve as auto-inducible promoters. At high oxygen availability, surfactin was not produced in the promoter exchange strains. At lowest oxygen availability, the strain carrying PnarG reached lower surfactin titers than the native JABs24 strain, although expression levels of PnarG and PsrfA were similar. However, strain B. subtilis MG14 with PsrfA::PnasD reached 1.4-fold higher surfactin titers with 696 mg/L and an exceptionally high YP/X of 1.007 g/g with overall lower foam levels. Though, bioreactor cultivations have illustrated that the anaerobic induction must be performed slowly as to avoid cell lysis, resulting in so-defined aerobic-anaerobic switch processes. With further appropriate process optimization, a simple and robust surfactin production process with highly reduced or even no foam formation can be achieved employing strain B. subtilis MG14.