Evaluation of an external foam column for in situ product removal in aerated surfactin production processes.

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.

Expression of degQ gene and its effect on lipopeptide production as well as formation of secretory proteases in Bacillus subtilis strains.

Bacillus subtilis is described as a promising production strain for lipopeptides. In the case of B. subtilis strains JABs24 and DSM10T , surfactin and plipastatin are produced. Lipopeptide formation is controlled, among others, by the DegU response regulator. The activating phospho-transfer by the DegS sensor kinase is stimulated by the pleiotropic regulator DegQ, resulting in enhanced DegU activation. In B. subtilis 168, a point mutation in the degQ promoter region leads to a reduction in gene expression. Corresponding reporter strains showed a 14-fold reduced expression. This effect on degQ expression and the associated impact on lipopeptide formation was examined for B. subtilis JABs24, a lipopeptide-producing derivative of strain 168, and B. subtilis wild-type strain DSM10T , which has a native degQ expression. Based on the stimulatory effects of the DegU regulator on secretory protease formation, the impact of degQ expression on extracellular protease activity was additionally investigated. To follow the impact of degQ, a deletion mutant was constructed for DSM10T , while a natively expressed degQ version was integrated into strain JABs24. This allowed strain-specific quantification of the stimulatory effect of degQ expression on plipastatin and the negative effect on surfactin production in strains JABs24 and DSM10T . While an unaffected degQ expression reduced surfactin production in JABs24 by about 25%, a sixfold increase in plipastatin was observed. In contrast, degQ deletion in DSM10T increased surfactin titer by threefold but decreased plipastatin production by fivefold. In addition, although significant differences in extracellular protease activity were detected, no decrease in plipastatin and surfactin produced during cultivation was observed.

Modeling the time course of ComX: towards molecular process control for Bacillus wild-type cultivations.

Wild-type cultivations are of invaluable relevance for industrial biotechnology when it comes to the agricultural or food sector. Here, genetic engineering is hardly applicable due to legal barriers and consumer's demand for GMO-free products. An important pillar for wild-type cultivations displays the genus Bacillus. One of the challenges for Bacillus cultivations is the global ComX-dependent quorum sensing system. Here, molecular process control can serve as a tool to optimize the production process without genetic engineering. To realize this approach, quantitative knowledge of the mechanism is essential, which, however, is often available only to a limited extent. The presented work provides a case study based on the production of cyclic lipopeptide surfactin, whose expression is in dependence of ComX, using natural producer B. subtilis DSM 10 T. First, a surfactin reference process with 40 g/L of glucose was performed as batch fermentation in a pilot scale bioreactor system to gain novel insights into kinetic behavior of ComX in relation to surfactin production. Interestingly, the specific surfactin productivity did not increase linearly with ComX activity. The data were then used to derive a mathematic model for the time course of ComX in dependence of existing biomass, biomass growth as well as a putative ComX-specific protease. The newly adapted model was validated and transferred to other batch fermentations, employing 20 and 60 g/L glucose. The applied approach can serve as a model system for molecular process control strategies, which can thus be extended to other quorum sensing dependent wild-type cultivations.

Bacillus subtilis High Cell Density Fermentation Using a Sporulation-Deficient Strain for the Production of Surfactin.

Bacillus subtilis 3NA is a strain capable of reaching high cell densities. A surfactin producing sfp+ variant of this strain, named JABs32, was utilized in fed-batch cultivation processes. Both a glucose and an ammonia solution were fed to set a steady growth rate µ of 0.1 h-1. In this process, a cell dry weight of up to 88 g L-1 was reached after 38 h of cultivation, and surfactin titers of up to 26.5 g L-1 were detected in this high cell density fermentation process, achieving a YP/X value of 0.23 g g-1 as well as a qP/X of 0.007 g g-1 h-1. In sum, a 21-fold increase in surfactin titer was obtained compared with cultivations in shake flasks. In contrast to fed-batch operations using Bacillus subtilis JABs24, an sfp+ variant derived from B. subtilis 168, JABs32, reached an up to fourfold increase in surfactin titers using the same fed-batch protocol. Additionally, a two-stage feed process was established utilizing strain JABs32. Using an optimized mineral salt medium in this high cell density fermentation approach, after 31 h of cultivation, surfactin titers of 23.7 g L-1 were reached with a biomass concentration of 41.3 g L-1, thus achieving an enhanced YP/X value of 0.57 g g-1 as well as a qP/X of 0.018 g g-1 h-1. The mutation of spo0A locus and an elongation of AbrB in the strain utilized in combination with a high cell density fed-batch process represents a promising new route for future enhancements on surfactin production. KEY POINTS: • Utilization of a sporulation deficient strain for fed-batch operations • High cell density process with Bacillus subtilis for lipopeptide production was established • High titer surfactin production capabilities confirm highly promising future platform strain.

Surfactin from Bacillus subtilis displays promising characteristics as O/W-emulsifier for food formulations.

BACKGROUND: Biosurfactants are surface-active molecules produced by different microorganisms and display a promising alternative to synthetically derived food emulsifiers. One of these biosurfactants, synthesized by Bacillus subtilis, is the lipopeptide surfactin, which composes a linear fatty acid and cyclic peptide moiety. This study explores the interfacial and emulsion forming properties of surfactin to further characterize its suitability as an O/W emulsifier in food formulations. RESULTS: Surfactin revealed a high interfacial activity with a reduction of interfacial tension of 83.26 % to 4.21 ± 0.11 mN/m. O/W emulsions (coil = 10 % w/w) were prepared by high-pressure homogenization, which yielded volume-based mean particle sizes below 1 µm already at low emulsifier concentrations of 0.01 % (w/w). Environmental stress experiments revealed that emulsions were stable between pH 6 to pH 9. Furthermore, neither phase separation nor extensive emulsion instability was observed with NaCl addition up to 0.5 M. However, CaCl2 addition (> 3 mM) destabilized surfactin mediated emulsions. Finally, the main emulsion forming and stabilization effect of surfactin was related to its high interfacial activity and the high degree of electrostatic repulsion between the oil droplets (i.e. zeta-potential of up to -100 mV). CONCLUSION: In comparison to other natural and synthetic emulsifiers, the results showed that surfactin is a strong candidate to form and stabilize O/W emulsions under the reported conditions.

Evaluation of an oxygen-dependent self-inducible surfactin synthesis in B. subtilis by substitution of native promoter PsrfA by anaerobically active PnarG and PnasD.

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.

Towards the Anaerobic Production of Surfactin Using Bacillus subtilis.

The anaerobic growth of B. subtilis to synthesize surfactin poses an alternative strategy to conventional aerobic cultivations. In general, the strong foam formation observed during aerobic processes represents a major obstacle. Anaerobic processes have, amongst others, the distinct advantage that the total bioreactor volume can be exploited as foaming does not occur. Recent studies also reported on promising product per biomass yields. However, anaerobic growth in comparison to aerobic processes has several disadvantages. For example, the overall titers are comparably low and cultivations are time-consuming due to low growth rates. B. subtilis JABs24, a derivate of strain 168 with the ability to synthesize surfactin, was used as model strain in this study. Ammonium and nitrite were hypothesized to negatively influence anaerobic growth. Ammonium with initial concentrations up to 0.2 mol/L was shown to have no significant impact on growth, but increasing concentrations resulted in decreased surfactin titers and reduced nitrate reductase expression. Anaerobic cultivations spiked with increasing nitrite concentrations resulted in prolonged lag-phases. Indeed, growth rates were in a similar range after the lag-phase indicating that nitrite has a neglectable effect on the observed decreasing growth rates. In bioreactor cultivations, the specific growth rate decreased with increasing glucose concentrations during the time course of both batch and fed-batch processes to less than 0.05 1/h. In addition, surfactin titers, overall Y P/X and Y P/S were 53%, ∼42%, and ∼57% lower than in serum flask with 0.190 g/L, 0.344 g/g and 0.015 g/g. The Y X/S, on the contrary, was 30% lower in the serum flask with 0.044 g/g. The productivities q were similar with ∼0.005 g/(g⋅h). However, acetate strongly accumulated during cultivation and was posed as further metabolite that might negatively influence anaerobic growth. Acetate added to anaerobic cultivations in a range from 0 g/L up to 10 g/L resulted in a reduced maximum and overall growth rate µ by 44% and 30%, respectively. To conclude, acetate was identified as a promising target for future process enhancement and strain engineering. Though, the current study demonstrates that the anaerobic cultivation to synthesize surfactin represents a reasonable perspective and feasible alternative to conventional processes.