Background: The production of surfactin, an extracellular accumulating lipopeptide produced by various Bacillus species, is a well-known representative of microbial biosurfactant. However, only limited information is available on the correlation between the growth rate of the production strain, such as B. subtilis BMV9, and surfactin production. To understand the correlation between biomass formation over time and surfactin production, the availability of glucose as carbon source was considered as main point. In fed-batch bioreactor processes, the B. subtilis BMV9 was used, a strain well-suited for high cell density fermentation. By adjusting the exponential feeding rates, the growth rate of the surfactin-producing strain, was controlled. Results: Using different growth rates in the range of 0.075 and 0.4 h^-1, highest surfactin titres of 36 g/L were reached at 0.25 h^-1 with production yields Y_P/S of 0.21 g/g and Y_P/X of 0.7 g/g, while growth rates lower than 0.2 h^-1 resulted in insufficient and slowed biomass formation as well as surfactin production (Y_P/S of 0.11 g/g and Y_P/X of 0.47 g/g for 0.075 h^-1). In contrast, feeding rates higher than 0.25 h^-1 led to a stimulation of overflow metabolism, resulting in increased acetate formation of up to 3 g/L and an accumulation of glucose due to insufficient conversion, leading to production yields Y_P/S of 0.15 g/g and Y_P/X of 0.46 g/g for 0.4 h^-1. Conclusions: Overall, the parameter of adjusting exponential feeding rates have an important impact on the B. subtilis productivity in terms of surfactin production in fed-batch bioreactor processes. A growth rate of 0.25 h^-1 allowed the highest surfactin production yield, while the total conversion of substrate to biomass remained constant at the different growth rates.

In a scenario of increasing alarm about food waste due to rapid urbanization, population growth and lifestyle changes, this study aims to explore the valorization of waste from the retail sector as potential substrates for the biotechnological production of biosurfactants. With a perspective of increasingly contributing to the realization of the circular bioeconomy, a vegetable juice, derived from unsold fruits and vegetables, as a carbon source was used to produce lipopeptides such as surfactin and fengycin. The results from the shake flask cultivations revealed that different concentrations of vegetable juice could effectively serve as carbon sources and that the fed-batch bioreactor cultivation strategy allowed the yields of lipopeptides to be significantly increased. In particular, the product/substrate yield of 0.09 g/g for surfactin and 0.85 mg/g for fengycin was obtained with maximum concentrations of 2.77 g/L and 27.53 mg/L after 16 h, respectively. To conclude, this study provides the successful fed-batch cultivation of B. subtilis using waste product as the carbon source to produce secondary metabolites. Therefore, the consumption of agricultural product wastes might be a promising source for producing valuable metabolites which have promising application potential to be used in several fields of biological controls of fungal diseases.

Background: In biotechnology, B. subtilis is established for heterologous protein production. In addition, the species provides a variety of bioactive metabolites, including the non-ribosomally produced surfactin lipopeptide. However, to control the formation of the target product-forming enzyme, different expression systems could be introduced, including the principle of genetic code expansion by the incorporation of externally supplied non-canonical amino acids. Methods: Integration of an amber stop codon into the srfA operon and additional chromosomal integration of an aminoacyl-tRNA synthetase/tRNA mutant pair from Methanococcus jannaschii enabled site-directed incorporation of the non-canonical amino acid O-methyl-L-tyrosine (OMeY). In different fed-batch bioreactor approaches, OMeY-associated surfactin production was quantified by high-performance thin-layer chromatography (HPTLC). Physiological adaptations of the B. subtilis production strain were analyzed by mass spectrometric proteomics. Results: Using a surfactin-forming B. subtilis production strain, which enables high cell density fermentation processes, the principle of genetic code expansion was introduced. Accordingly, the biosynthesis of the surfactin-forming non-ribosomal peptide synthetase (NRPS) was linked to the addition of the non-canonical amino acid OMeY. In OMeY-associated fed-batch bioreactor fermentation processes, a maximum surfactin titre of 10.8 g/L was achieved. In addition, the effect of surfactin induction was investigated by mass spectrometric proteome analyses. Among other things, adaptations in the B. subtilis motility towards a more sessile state and increased abundances of surfactin precursor-producing enzymes were detected. Conclusions: The principle of genetic code expansion enabled a precise control of the surfactin bioproduction as a representative of bioactive secondary metabolites in B. subtilis. This allowed the establishment of inducer-associated regulation at the post-transcriptional level with simultaneous use of the native promoter system. In this way, inductor-dependent control of the production of the target metabolite-forming enzyme could be achieved.

Microbial biosurfactants have become increasingly attractive as promising ingredients for environmentally friendly products. The reasons for this are their generally good performance and biodegradability, low toxicity, production from renewable raw materials, and benefits for the environment perceived by consumers. In this study, we investigated the chemical structure and properties of a novel glycolipid from a new biosurfactant-producing strain, Rouxiella badensis DSM 100043^T. Bioreactor cultivation was performed at 30 °C and pH 7.0 for 28 h using 15 g/L glycerol as a carbon source. The glycolipid was successfully purified from the ethyl acetate extract of the supernatant using medium pressure liquid chromatography (MPLC). The structure of the glycolipid was determined by one- and two-dimensional (¹H and ¹³C) nuclear magnetic resonance (NMR) and confirmed by liquid chromatography electrospray ionization mass spectrometry (LC-ESI/MS). NMR analysis revealed the hydrophilic moiety as a glucose molecule and the hydrophobic moieties as 3-hydroxy-5-dodecenoic acid and 3-hydroxydecanoic acid, which are linked with the glucose by ester bonds at the C2 and C3 positions. Surface tension measurement with tensiometry indicated that the glucose–lipid could reduce the surface tension of water from 72.05 mN/m to 24.59 mN/m at 25 °C with a very low critical micelle concentration (CMC) of 5.69 mg/L. Moreover, the glucose–lipid demonstrated very good stability in maintaining emulsification activity at pH 2–8, a temperature of up to 100 °C, and a NaCl concentration of up to 15%. These results show that R. badensis DSM 100043^T produced a novel glycolipid biosurfactant with excellent surface-active properties, making it promising for further research or industrial applications.

Bacillus subtilis is one of the most important production organisms in industrial biotechnology. However, there is still limited knowledge about the kinetics of fed-batch processes in bioreactors, as well as a lack of biological performance indicators, such as production yields, particularly regarding their variation over time. Understanding these kinetics and changes is crucial for optimizing the productivity in fed-batch processes. Fed-batch bioreactor cultures of Bacillus subtilis BMV9 in high cell density processes for surfactin production have been characterized with a kinetic model composed of first-order ordinary differential equations, describing the time course of biomass, substrate, surfactin and acetate. This model contributes to understanding critical restrictions and the knowledge gained was used to design and implement a model-based process. The model integrates biomass growth based on Monod kinetics, substrate consumption, surfactin synthesis and formation of the by-product acetate. After the model was parameterized for B. subtilis BMV9 using 12 different fed-batch bioreactor experiments, the kinetic model was able to accurately describe biomass accumulation, substrate consumption, product formation rates and, to some extent, the overflow metabolism involving acetate. Based on this, the kinetic model was used for a process design, in which the batch was omitted, which led to a product titre of 46.33 g/L and a space–time-yield of 2.11 g/(L*h) was achieved. The kinetic model developed in this study enables the description of the time course of biomass growth, substrate consumption and product formation and thus significantly improves process understanding. The computation of process parameters, which are not analytically accessible at any time, could be realized. A sensitivity analysis identified the maximum specific growth rate, substrate-related maintenance and the maximum acetate formation rate as key parameters influencing model outputs.

The biosynthesis of surfactin, a potent lipopeptide biosurfactant produced by Bacillus subtilis , imposes a significant metabolic burden on the host organism. Understanding the metabolic costs associated with surfactin production, specifically the ATP demand and precursor diversion resulting from the expression of the large srfAA-AD operon ( srfA operon), is crucial for optimizing production strains. In this study, the metabolic burden and growth impacts associated with surfactin biosynthesis in B. subtilis were quantitatively evaluated by comparing a reference surfactin-producing strain (BMV9) with two mutant strains: BMV12, which lacks the srfA operon, and BMV33, which retains the srfA operon but lacks the sfp gene. Our analysis included theoretical calculations of ATP and NADPH + H ⁺ requirements for de novo surfactin synthesis, and we measured growth behavior and carbon and nitrogen source consumption. Results indicated that surfactin production significantly reduces biomass yield ( Y _X/S ) and specific growth rates due to the metabolic costs of expressing the non-ribosomal peptide synthetase (NRPS) and the diversion of key precursors. Notably, BMV12 exhibited higher growth rates compared to the surfactin-producing strain. Proteome analyses further revealed differential protein abundance in non-surfactin-producing strains, indicating altered metabolic pathways that may relieve the metabolic burden associated with surfactin synthesis. These findings highlight the complex trade-offs between secondary metabolite production and cellular growth, providing a foundation for metabolic engineering strategies aimed at optimizing surfactin yields while minimizing metabolic costs.