Elvio Henrique Benatto Perino

Endophytic bacteria play a pivotal role in plant health by modulating nutrient availability, growth regulation, and resistance to phytopathogens. However, the functional diversity and ecological significance of the endophytic bacteria in rice (Oryza sativa L.) plants remain incompletely understood. Here, we functionally characterized the diversity of endophytic bacteria isolated from healthy rice plants and evaluated their potential roles in plant growth promotion and fungal pathogen suppression under in vitro conditions. We recovered 23 bacterial isolates from roots, stems, and leaves and identified them, by means of 16S rRNA gene sequencing, as members of the genera Pseudomonas, Enterobacter, and Bacillus. Phylogenetic analysis confirmed the dominance of these genera across plant tissues. Functional characterization revealed pronounced isolate-dependent variability in the production of indole-3-acetic acid (IAA) and ammonia, and in the ability to solubilize essential macro- and micronutrients. Most isolates efficiently solubilized phosphorus (P), while potassium (K) solubilization was particularly prevalent among Bacillus isolates. In contrast, silicon (Si) and zinc (Zn) solubilization were restricted to a smaller subset of isolates, indicating functional specialization within the endophytic community. Antagonistic assays demonstrated that of the 23 isolates tested, 6 isolates inhibited the growth of Bipolaris oryzae, whereas strong suppression of Pyricularia oryzae was observed only for one Pseudomonas and one Enterobacter isolate. Limited inhibitory activity was detected against Rhizoctonia solani, which highlights pathogen-specific interactions. Altogether, our findings indicate that rice harbors functionally diverse endophytic bacteria with complementary in vitro traits related to nutrient mobilization, phytohormone production, and pathogen suppression. These results identify candidate isolates for further validation. In planta, greenhouse, and field studies are now required to establish whether these traits translate into improved plant growth, nutrient uptake, or disease control.

The efficient extraction and purification of recombinant β-casein from Escherichia coli is a crucial step in bioprocess engineering. This study evaluates an adapted high-pressure homogenization (HPH) workflow for recombinant β-casein recovery and compares it with osmotic shock (OS) and standard HPH based on specific protein yield, estimated relative purity, and processing time. In contrast to standard HPH, which requires separate inclusion body recovery, washing, and subsequent denaturant-mediated solubilization, the adapted-HPH workflow integrates mechanical cell disruption with inclusion body recovery during homogenization. Standard HPH was evaluated in two downstream-processing runs, whereas adapted HPH and OS were each evaluated in three downstream-processing runs using separate aliquots from the same bioreactor biomass batch. Adapted HPH demonstrated higher specific yields (4.08 mg_casein/g_CDW) and required 40% less processing time compared to the OS and standard HPH methods. The specific yield obtained with adapted HPH was significantly higher than that obtained with standard HPH (p = 0.026), whereas the difference between adapted HPH and OS was not statistically significant (p = 0.086). Despite the inclusion body washing steps used for OS and standard HPH, adapted HPH achieved a slightly higher average purity than OS (63.46 ± 3.49% versus 59.38 ± 2.79%) and a purity comparable to standard HPH (68.10 ± 6.87%); however, these differences were not statistically significant. Overall, the comparative evaluation of these methods indicates that adapted HPH may provide a simplified alternative for recombinant β-casein recovery, although further validation with a larger number of independent experiments is required.

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.

Surfactin production by Bacillus subtilis is typically performed under aerobic conditions, requiring high aeration and agitation, which leads to mechanical stress for the cells and therefore promotes excessive foaming. As a process-oriented alternative to full aerobic operation, an aerobic to micro-aerobic switching strategy was developed that aims to decouple biomass formation from surfactin synthesis by controlling oxygen availability. Promoter activities relevant to nitrate respiration and surfactin biosynthesis were first characterised in shake flasks using transcriptional reporter strains and online monitoring of dissolved oxygen. The nitrite reductase promoter showed the strongest induction under oxygen-limited conditions and was used to construct an oxygen-responsive production strain in which the native P _srfA promoter was replaced to suppress surfactin formation during aerobic growth and shift production to the micro-aerobic phase. The switching concept was subsequently evaluated in 30-L stirred-tank bioreactor cultivations using stepwise reductions of dissolved oxygen setpoints combined with exponential glucose feeding and nitrate supplementation. The engineered strain B. subtilis MG19 enabled stable transitions into micro-aerobic operation without apparent loss of biomass, while a reference strain B. subtilis MG17 with the native regulation showed surfactin formation already during the aerobic phase and pronounced process disturbance after switching, characterised by glucose accumulation and a strong decrease in surfactin concentration. Overall, the study demonstrates oxygen switching as a scalable process engineering tool for controlling surfactin production phases and highlights key constraints for robust micro-aerobic bioreactor operation. Key points: • Oxygen availability decoupled growth and surfactin formation. • Micro-aerobic process control was evaluated in 30-L bioreactor cultivations. • The engineered strain shifted surfactin production to oxygen-limited conditions.

Two genetically engineered Bacillus subtilis strains, BMV9 and BsB6, were evaluated in terms of culture medium (effect of nutrients on surfactin yield) and potential biotechnological applications of surfactin in agriculture and the petrochemical industry. BMV9 (spo0A3; abrB*; ΔmanPA; sfp+) is, to date, the highest surfactin producer reported scientifically, and BsB6 is a sfp+ laboratory derivative strain that has also demonstrated considerable production potential. To assess their performance, fermentation experiments were conducted in shake flasks using two different culture media, a mineral salt medium and a complex medium, each supplemented with 2% (w/v) glucose. Lipopeptides (surfactin and fengycin) were extracted and quantified at multiple time points (up to 48 h) via high-performance thin-layer chromatography (HPTLC). Optical density, residual glucose, and pH were monitored throughout the cultivation. In parallel, microbial growth in both media were also validated in small-scale cultivation approaches. Antifungal activity of culture supernatants and lipopeptide extracts was tested against two Diaporthe species, key phytopathogens in soybean crops. Given the agricultural relevance of these pathogens, the biocontrol potential of lipopeptides represents a sustainable alternative to conventional chemical fungicides. Additionally, oil displacement tests were performed to evaluate the efficacy of surfactin in enhanced oil recovery (EOR), bioremediation, and related petrochemical processes. High-resolution LC-MS/MS analysis enabled structural characterization and relative quantification of the lipopeptides. Overall, these investigations provide a comprehensive comparison of strain production performance and the associated impact of cultivation media, aiming to define the optimal conditions for economically viable surfactin production and to explore its broader biotechnological applications in agriculture and the petrochemical industry.

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.

Rouxiella badensis DSM 100043^T had been previously proven to produce a novel glucoselipid biosurfactant which has a very low critical micelle concentration (CMC) as well as very good stability against a wide range of pH, temperature, and salinity. In this study, we performed a function-based library screening from a R. badensis DSM 100043^T genome library to identify responsible genes for biosynthesis of this glucoselipid. The identified open reading frames (ORFs) were cloned into several constructs in Escherichia coli for gene permutation analysis and the individual products were analyzed using high-performance thin-layer chromatography (HPTLC). Products of interest from positive expression strains were purified and analyzed by liquid chromatography/electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) and nuclear magnetic resonance (NMR) for further structure elucidation. Function-based screening of 5400 clones led to the identification of an operon containing three ORFs encoding acetyltransferase GlcA (ORF1), acyltransferase GlcB (ORF2), and phosphatase/HAD GlcC (ORF3). E. coli pCAT2, with all three ORFs, resulted in the production of identical R. badensis DSM 100043^T glucosedilipid with Glu-C_10:0-C_12:1 as the main congener. ORF2-deletion strain E. coli pAFP1 primarily produced glucosemonolipids, with Glu-C_10:0,3OH and Glu-C_12:0 as the major congeners, predominantly esterified at the C-2 position of the glucose moiety. Furthermore, fed-batch bioreactor cultivation of E. coli pCAT2 using glucose as the carbon source yielded a maximum glucosedilipid titer of 2.34 g/L after 25 h of fermentation, which is 55-fold higher than that produced by batch cultivation of R. badensis DSM 100043^T in the previous study.

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.

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.

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.