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. 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). 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.

Bacillus strains can produce various lipopeptides, known for their antifungal properties. This makes them attractive metabolites for applications in agriculture. Therefore, identification of productive wild-type strains is essential for the development of biopesticides. Bacillus velezensis FZB42 is a well-established strain for biocontrol of plant pathogens in agriculture. Here, we characterized an alternative strain, B. velezensis UTB96, that can produce higher amounts of all three major lipopeptide families, namely surfactin, fengycin, and iturin. UTB96 produces iturin A. Furthermore, UTB96 showed superior antifungal activity towards the soybean fungal pathogen Diaporthe longicolla compared to FZB42. Moreover, the additional provision of different amino acids for lipopeptide production in UTB96 was investigated. Lysine and alanine had stimulatory effects on the production of all three lipopeptide families, while supplementation of leucine, valine and isoleucine decreased the lipopeptide bioproduction. Using a 45-litre bioreactor system for upscaling in batch culture, lipopeptide titers of about 140 mg/L surfactin, 620 mg/L iturin A, and 45 mg/L fengycin were achieved. In conclusion, it becomes clear that B. velezensis UTB96 is a promising strain for further research application in the field of agricultural biological controls of fungal diseases.

Background: Plipastatin is a potentBacillusantimicrobial lipopeptide with the prospect to replace conventional antifungal chemicals for controlling plant pathogens. However, the application of this lipopeptide has so far been investigated in a few cases, principally because of the yield in low concentration and unknown regulation of biosynthesis pathways.B. subtilissynthesizes plipastatin by a non-ribosomal peptide synthetase encoded by theppsABCDEoperon. In this study,B. subtilis3NA (a non-sporulation strain) was engineered to gain more insights about plipastatin mono-production.Results: The 4-phosphopantetheinyl transferase Sfp posttranslationally converts non-ribosomal peptide synthetases from inactive apoforms into their active holoforms. In case of 3NA strain,sfpgene is inactive. Accordingly, the first step was an integration of a repairedsfpversion in 3NA to construct strain BMV9. Subsequently, plipastatin production was doubled after integration of a fully expresseddegQversion fromB. subtilisDSM10^Tstrain (strain BMV10), ensuring stimulation of DegU-P regulatory pathway that positively controls theppsABSDEoperon. Moreover, markerless substitution of the comparably weak native plipastatin promoter (P_pps) against the strong constitutive promoter P_vegled to approximately fivefold enhancement of plipastatin production in BMV11 compared to BMV9. Intriguingly, combination of both repaireddegQexpression and promoter exchange (P_pps::P_veg) did not increase the plipastatin yield. Afterwards, deletion of surfactin (srfAA-AD) operon by the retaining the regulatorycomSwhich is located withinsrfABand is involved in natural competence development, resulted in the loss of plipastatin production in BMV9 and significantly decreased the plipastatin production of BMV11. We also observed that supplementation of ornithine as a precursor for plipastatin formation caused higher production of plipastatin in mono-producer strains, albeit with a modified pattern of plipastatin composition.Conclusions: This study provides evidence thatdegQstimulates the native plipastatin production. Moreover, a full plipastatin production requires surfactin synthetase or some of its components. Furthermore, as another conclusion of this study, results point towards ornithine provision being an indispensable constituent for a plipastatin mono-producerB. subtilisstrain. Therefore, targeting the ornithine metabolic flux might be a promising strategy to further investigate and enhance plipastatin production byB. subtilisplipastatin mono-producer strains.

Temperature is an important parameter in bioprocesses, influencing the structure and functionality of almost every biomolecule, as well as affecting metabolic reaction rates. In industrial biotechnology, the temperature is usually tightly controlled at an optimum value. Smart variation of the temperature to optimize the performance of a bioprocess brings about multiple complex and interconnected metabolic changes and is so far only rarely applied. Mathematical descriptions and models facilitate a reduction in complexity, as well as an understanding, of these interconnections. Starting in the 19th century with the “primal” temperature model of Svante Arrhenius, a variety of models have evolved over time to describe growth and enzymatic reaction rates as functions of temperature. Data-driven empirical approaches, as well as complex mechanistic models based on thermodynamic knowledge of biomolecular behavior at different temperatures, have been developed. Even though underlying biological mechanisms and mathematical models have been well-described, temperature as a control variable is only scarcely applied in bioprocess engineering, and as a conclusion, an exploitation strategy merging both in context has not yet been established. In this review, the most important models for physiological, biochemical, and physical properties governed by temperature are presented and discussed, along with application perspectives. As such, this review provides a toolset for future exploitation perspectives of temperature in bioprocess engineering.

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 σ^B and σ^W 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.

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 producerPseudomonas aeruginosa. In the recent past, the presence and function of a ROSE-like RNA-thermometer located in the 5′UTR of the rhamnosyltransferase genesrhlABhas been reported in wild typeP. 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 hostP. putidaKT2440 containing therhlABgenes with the nativeP. aeruginosa5′-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.