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Abstract The complex regulatory network in Bacillus, known as quorum sensing, offers many opportunities to modify bacterial gene expression and hence to control bioprocesses. One target regulated by this mechanism is the activity of the P srfA promoter, which is engaged in the formation of lipopeptide surfactin. It was hypothesised that deletion of rapC, rapF and rapH, encoding for prominent Rap-phosphatases known to affect P srfA activity, would enhance surfactin production. Therefore, these genes were deleted in a sfp + derivative of B. subtilis 168 with subsequent evaluation of quantitative data. Up to the maximum product formation of the reference strain B. subtilis KM1016 after 16 h of cultivation, the titers of the rap deletion mutants did not exceed the reference. However, an increase in both product yield per biomass Y P/X and specific surfactin productivity q surfactin was observed, without any considerable effect on the ComX activity. By extending the cultivation time, a 2.7-fold increase in surfactin titer was observed after 24 h for strain CT10 (ΔrapC) and a 2.5-fold increase for CT11 (ΔrapF) compared to the reference strain KM1016. In addition, Y P/X was again increased for strains CT10 and CT11, with values of 1.33 g/g and 1.13 g/g, respectively. Interestingly, the effect on surfactin titer in strain CT12 (ΔrapH) was not as distinct, although it achieved the highest promoter activity (P srfA -lacZ). The data presented support the possibility of involving the quorum sensing system of Bacillus in bioprocess control as shown here on the example of lipopeptide production.

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.

Abstract 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.

Abstract A key challenge to advance the efficiency of bioprocesses is the uncoupling of biomass from product formation, as biomass represents a by-product that is in most cases difficult to recycle efficiently. Using the example of rhamnolipid biosurfactants, a temperature-sensitive heterologous production system under translation control of a fourU RNA thermometer from Salmonella was established to allow separating phases of preferred growth from product formation. Rhamnolipids as bulk chemicals represent a model system for future processes of industrial biotechnology and are therefore tied to the efficiency requirements in competition with the chemical industry. Experimental data confirms function of the RNA thermometer and suggests a major effect of temperature on specific rhamnolipid production rates with an increase of the average production rate by a factor of 11 between 25 and 38 °C, while the major part of this increase is attributable to the regulatory effect of the RNA thermometer rather than an unspecific overall increase in bacterial metabolism. The production capacity of the developed temperature sensitive-system was evaluated in a simple batch process driven by a temperature switch. Product formation was evaluated by efficiency parameters and yields, confirming increased product formation rates and product-per-biomass yields compared to a high titer heterologous rhamnolipid production process from literature.

Abstract 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 P srfA of the surfactin operon in strain B. subtilis JABs24 was replaced by promoters P narG and P nasD to induce surfactin synthesis anaerobically. Shake flask cultivations with varying oxygen availabilities indicated no significant differences in native P srfA expression. As hypothesized, activity of P narG and P nasD increased with lower oxygen levels and surfactin was not produced by P srfA ::P narG as well as P srfA ::P nasD mutant strains under conditions with highest oxygen availability. P narG showed expressions similar to P srfA at lowest oxygen availability, while maximum value of P nasD was more than 5.5-fold higher. Although the promoter exchange P srfA ::P narG resulted in a decreased surfactin titer at lowest oxygen availability, the strain carrying P srfA ::P nasD reached a 1.4-fold increased surfactin concentration with 696 mg/L and revealed an exceptional high overall Y P/X of 1.007 g/g. This value also surpassed the Y P/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 P srfA ::P nasD promoter exchange. Additionally, replacement of other native promoters by nitrite reductase promoter P nasD represents a promising tool for anaerobic-inducible bioprocesses in Bacillus.

Abstract 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.

Abstract The microbial production of rhamnolipids has been in the focus of research for the last decades. Today, mainly heterologous production systems are targeted due to the advantage of non-pathogenic hosts as well as uncoupling from complex quorum sensing regulatory networks compared to their natural producer Pseudomonas aeruginosa. In the recent past, the presence and function of a ROSE-like RNA-thermometer located in the 5′UTR of the rhamnosyltransferase genes rhlAB has been reported in wild type P. aeruginosa. In this study, the temperature-induced regulation of this native RNA-thermometer for heterologous rhamnolipid production was evaluated and its potential application for process control is discussed. For this purpose, the non-pathogenic production host P. putida KT2440 containing the rhlAB genes with the native P. aeruginosa 5′-UTR region was used. The system was evaluated and characterized regarding the effect of temperature on growth and product formation, as represented by efficiency parameters and yields. Experimental data suggests a major effect of temperature on specific rhamnolipid production rates. With maximum values of 0.23 g/(g h) at 37 °C, this constitutes a more than 60% increase compared to the production rate of 0.14 g/(g h) at the growth optimum of 30 °C. Interestingly however, control experiments unveiled that besides the regulatory effect of the RNA-thermometer, multiple metabolic effects may contribute equally to the observed increase in production rate. As such, this work constitutes an important step towards the utilization of temperature-based process designs and enables the possibility for novel approaches for process control.