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.

Soil-borne fungal pathogens such as Sclerotium spp., Rhizoctonia spp., and Macrophomina spp. pose significant threats to global agriculture, with soybean crops among the most severely affected due to damping-off disease. These pathogens cause substantial yield losses, making their management a critical concern. In this study, we investigated the potential of Bacillus siamensis BCL, a novel Neotropical strain, as an eco-friendly solution for managing Sclerotium, Rhizoctonia, and Macrophomina species. The strain exhibited strong antifungal activity, significantly inhibiting fungal growth in vitro, with the greatest suppression observed against Macrophomina spp., reaching up to 81%. In vivo assays further confirmed the biocontrol potential of B. siamensis. When applied at 10⁶ colony-forming units (CFU)/mL, the strain reduced disease symptoms and improved plant growth parameters—including root length, shoot biomass, and leaf number—compared to untreated, infected controls. The protective effect varied by pathogen, with the most significant recovery in root length observed against Macrophomina spp. (85%) and Sclerotium spp. (78%). In preventive treatments, fermentation extracts of the B. siamensis strain suppressed disease progression, although they did not promote seedling growth. A genomic analysis of B. siamensis BCL revealed genes encoding antimicrobial secondary metabolites, including terpenes, fengycins, and surfactins. These findings highlight B. siamensis BCL as a promising candidate for sustainable crop protection and a valuable resource for developing novel antimicrobial strategies in agriculture.

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: Cow’s milk represents an important protein source. Here, especially casein proteins are important components, which might be a promising source of alternative protein production by microbial expression systems. Nevertheless, caseins are difficult-to-produce proteins, making heterologous production challenging. However, the potential of genome-reduced Bacillus subtilis was applied for the recombinant production of bovine α_S1-casein protein. Methods: A plasmid-based gene expression system was established in B. subtilis allowing the production of his-tagged codon-optimized bovine α_S1-casein. Upscaling in a fed-batch bioreactor system for high cell-density fermentation processes allowed for efficient recombinant α_S1-casein production. After increasing the molecular abundance of the recombinant α_S1-casein protein using immobilized metal affinity chromatography, zeta potential and particle size distribution were determined in comparison to native bovine α_S1-casein. Results: Non-sporulating B. subtilis strain BMV9 and genome-reduced B. subtilis strain IIG-Bs-20-5-1 were applied for recombinant α_S1-casein production. Casein was detectable only in the insoluble protein fraction of the genome-reduced B. subtilis strain. Subsequent high cell-density fed-batch bioreactor cultivations using strain IIG-Bs-20-5-1 resulted in a volumetric casein titer of 56.9 mg/L and a yield of 1.6 mg_casein/g_CDW after reducing the B. subtilis protein content. Comparative analyses of zeta potential and particle size between pre-cleaned recombinant and native α_S1-casein showed pH-mediated differences in aggregation behavior. Conclusions: The study demonstrates the potential of B. subtilis for the recombinant production of bovine α_S1-casein and underlines the potential of genome reduction for the bioproduction of difficult-to-produce proteins.

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.

Treatment of potato plants with the pathogen-associated molecular pattern Pep-13 leads to the activation of more than 1200 genes. One of these, StPIP1_1, encodes a protein of 76 amino acids with sequence homology to PAMP-induced secreted peptides (PIPs) from Arabidopsis thaliana. Expression of StPIP1_1 is also induced in response to infection with Phytophthora infestans, the causal agent of late blight disease. Apoplastic localization of StPIP1_1-mCherry fusion proteins is dependent on the presence of the predicted signal peptide. A synthetic peptide corresponding to the last 13 amino acids of StPIP1_1 elicits the expression of the StPIP1_1 gene itself, as well as that of pathogenesis related genes. The oxidative burst induced by exogenously applied StPIP1_1 peptide in potato leaf disks is dependent on functional StSERK3A/B, suggesting that StPIP1_1 perception occurs via a receptor complex involving the co-receptor StSERK3A/B. Moreover, StPIP1_1 induces expression of FRK1 in Arabidopsis in an RLK7-dependent manner. Expression of an RLK from potato with high sequence homology to AtRLK7 is induced by StPIP1_1, by Pep-13 and in response to infection with P. infestans. These observations are consistent with the hypothesis that, upon secretion, StPIP1_1 acts as an endogenous peptide required for amplification of the defense response.