The influence of growth rate-controlling feeding strategy on the surfactin production in Bacillus subtilis bioreactor processes.

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 YP/S of 0.21 g/g and YP/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 (YP/S of 0.11 g/g and YP/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 YP/S of 0.15 g/g and YP/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.

Deciphering the influence of NaCl on social behaviour of Bacillus subtilis.

Various environmental signals, such as temperature, pH, nutrient levels, salt content and the presence of other microorganisms, can influence biofilm's development and dynamics. However, the innate mechanisms that govern at the molecular and cellular levels remain elusive. Here, we report the impact of physiologically relevant concentrations of NaCl on biofilm formation and the associated differences in an undomesticated natural isolate of Bacillus subtilis. NaCl exposure and its uptake by bacterial cells induced substantial changes in the architecture of pellicle biofilm and an upsurge in the expansion of biofilm colonies on agar surfaces. We have observed the upregulation of genes involved in motility and the downregulation of genes involved in the biosynthesis of extracellular matrix components through the transcription factor sigD, suggesting the possible underlying mechanisms. To further support these observations, we have used ΔsigD and ΔsrfAC null mutants, which showed compromised NaCl-induced effects. Our results indicate that NaCl induces a lifestyle shift in B. subtilis from a sessile biofilm state to an independent unicellular motile state. Overall, we present evidence that NaCl can reprogramme gene expression and alter cellular morphology and the state of cells to adapt to motility, which facilitates the expansion of bacterial colonies.

Solubilizer of bacterial origin surfactin increases the biological activity of C60 fullerene.

Currently, there exists conflicting data regarding the biological activity of unmodified fullerene C60. Various sources report its toxicity, geroprotective activity, and potential interaction with DNA. Contradictory findings regarding the toxicity of C60 may arise from the use of toxic solvents, as well as the influence of bioavailability and bioactivity on the preparation conditions of C60 suspensions. Furthermore, the microbiota of experimental animals can impact geroprotective activity results by releasing surfactants that facilitate substance penetration through the cell membrane. In this study, we selected conditions for solubilizing fullerene C60 in a solution of surfactin, a surfactant of bacterial origin, as well as in a 2% aqueous solution of TWEEN 80, employing ultrasound. Through bioluminescent analysis using lux biosensors in Escherichia coli MG1655, we observed that C60 in surfactin reduced induced genotoxic and oxidative stress. Given that surfactin enhances membrane permeability to fullerene C60, suspensions of fullerene in designated concentrations of surfactin can be regarded as a DNA protector and antioxidant, warranting further investigation as a promising component of novel drugs.

Development of a microbial dewaxing agent using three spore forming bacteria.

Microbial enhanced oil recovery (MEOR) is a cost effective and efficient method for recovering residual oil. However, the presence of wax (paraffin) in residual oil can substantially reduce the efficiency of MEOR. Therefore, microbial dewaxing is a critical process in MEOR. In this study, a bacterial dewaxing agent of three spore-forming bacteria was developed. Among these bacteria, Bacillus subtilis GZ6 produced the biosurfactant surfactin. Replacing the promoter of the surfactin synthase gene cluster (srfA), increased the titer of surfactin in this strain from 0.33 g/L to 2.32 g/L. The genetically modified strain produced oil spreading rings with diameters increasing from 3.5 ± 0.1 to 4.1 ± 0.2 cm. The LadA F10L/N133R mutant was created by engineering an alkane monooxygenase (LadA) using site-directed mutagenesis in the Escherichia coli host. Compared to the wild-type enzyme, the resulting mutant exhibited an 11.7-fold increase in catalytic efficiency toward the substrate octadecane. When the mutant (pIMPpladA2mu) was expressed in Geobacillus stearothermophilus GZ178 cells, it exhibited a 2.0-fold increase in octadecane-degrading activity. Cultures of the two modified strains (B. subtilis GZ6 (pg3srfA) and G. stearothermophilus GZ178 (pIMPpladA2mu)) were mixed with the culture of Geobacillus thermodenitrificans GZ156 at a ratio of 5:80:15. The resulting composition increased the rate of wax removal by 35% compared to the composition composed of three native strains. This study successfully developed a multi-strain bacterial agent with enhanced oil wax removal capabilities by genetically engineering two bacterial strains.

High-throughput evaluation of hemolytic activity through precise measurement of colony and hemolytic zone sizes of engineered Bacillus subtilis on blood agar.

Biosurfactants have remarkable characteristics, such as environmental friendliness, high safety, and excellent biodegradability. Surfactin is one of the best-known biosurfactants produced by Bacillus subtilis. Because the biosynthetic pathways of biosurfactants, such as surfactin, are complex, mutagenesis is a useful alternative to typical metabolic engineering approaches for developing high-yield strains. Therefore, there is a need for high-throughput and accurate screening methods for high-yield strains derived from mutant libraries. The blood agar lysis method, which takes advantage of the hemolytic activity of biosurfactants, is one way of determining their concentration. This method includes inoculating microbial cells onto blood-containing agar plates, and biosurfactant production is assessed based on the size of the hemolytic zone formed around each colony. Challenges with the blood agar lysis method include low experimental reproducibility and a lack of established protocols for high-throughput screening. Therefore, in this study, we investigated the effects of the inoculation procedure and media composition on the formation of hemolytic zones. We also developed a workflow to evaluate the number of colonies using robotics. The results revealed that by arranging colonies at appropriate intervals and measuring the areas of colonies and hemolytic rings using image analysis software, it was possible to accurately compare the hemolytic activity among several colonies. Although the use of the blood agar lysis method for screening is limited to surfactants exhibiting hemolytic activity, it is believed that by considering the insights gained from this study, it can contribute to the accurate screening of strains with high productivity.

Bioprospecting of soil-borne microorganisms and chemical dereplication of their anti-microbial constituents with the aid of UPLC-QTOF-MS and molecular networking approach.

Due to the emergence of drug-resistant microorganisms, the search for broad-spectrum antimicrobial compounds has become extremely crucial. Natural sources like plants and soils have been explored for diverse metabolites with antimicrobial properties. This study aimed to identify microorganisms from agricultural soils exhibiting antimicrobial effects against known human pathogens, and to highlight the chemical space of the responsible compounds through the computational metabolomics-based bioprospecting approach. Herein, bacteria were extracted from soil samples and their antimicrobial potential was measured via the agar well diffusion method. Methanolic extracts from the active bacteria were analyzed using the liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) technique, and the subsequent data was further analyzed through molecular networking approach which aided in identification of potential anti-microbial compounds. Furthermore, 16S rRNA gene sequencing enabled identification of the active bacterial isolates, where isolate 1 and 2 were identified as strains of Bacillus pumilus, whilst isolate 3 was found to be Bacillus subtilis. Interestingly, isolate 3 (Bacillus subtilis) displayed wide-ranging antimicrobial activity against the tested human pathogens. Molecular networking revealed the presence of Diketopiperazine compounds such as cyclo (D-Pro-D-Leu), cyclo (L-Tyr-L-Pro), cyclo (L-Pro-D-Phe), and cyclo (L-Pro-L-Val), alongside Surfactin C, Surfactin B, Pumilacidin E, and Isarrin D in the Bacillus strains as the main anti-microbial compounds. The application of the molecular networking approach represents an innovation in the field of bio-guided bioprospection of microorganisms and has proved to be an effective and feasible towards unearthing potent antimicrobial compounds. Additionally, the (computational metabolomics-based) approach accelerates the discovery of bioactive compounds and isolation of strains which offer a promising avenue for discovering new clinical antimicrobials. Finally, soil microbial flora could serve an alternative source of anti-microbial compounds which can assist in the fight against emergence of multi-drug resistance bacterial pathogens.

The Optimal Supplementation of Fermented Product Produced by Bacillus subtilis Strain LYS1 with High Surfactin Yield for Improving Growth Performance, Intestinal Villi Morphology, and Tibial Bone Strength in Broilers.

This study aimed to investigate the physiochemical characterizations of the fermented product (FP) produced by the high-yield surfactin strain Bacillus subtilis LYS1 (LYS1), as well as its effects on growth performance, carcass traits, intestinal morphology, tibial bone characteristics, and clinical blood biochemistry in broilers. Accordingly, the optimal supplementation of FP for improving growth performance, intestinal villi development, and tibial bone strength in broilers was elucidated using a broken-line quadratic (BLQ) ascending model. Three hundred and sixty 0-day-old Ross 308 broiler chicks, with equal numbers of both sexes, were randomly assigned to dietary supplementation of 2.5% fish meal or 0, 1, 1.5, 2, or 2.5% FP. Each treatment had six replicates, and the experimental period was 5 wk. The LYS1 count, surfactin content, and surfactin composition of the FP were 9.1 log CFU/g, 11.23 mg/g, and C12 to C18, respectively. The FP-supplemented groups improved feed intake, weight gain (WG), and production efficiency factor at 0 to 5 weeks old (p < 0.05) compared with the 0% group. The villus height/crypt depth (V/C) in the jejunum and ileum of the FP-supplemented groups was higher than in the 0% group (p < 0.05). The tibiotarsal index, Ca, and P in the tibia showed a linear effect with increased FP supplementation (p < 0.05). Moreover, the tibiotarsus weight/length index (TWLI) showed a quadratic effect with increased FP supplementation (p < 0.05). The optimal supplementation of FP for WG, V/C in the jejunum and ileum, and TWLI was 1.8, 1.9, and 1.6%, respectively. In conclusion, dietary supplementation with 1 to 2.5% LYS1 FP in broilers can improve their growth performance and the development of intestinal villi. Moreover, 1.9% is the optimal supplementation of LYS1 FP in the diet, based on the fitting results obtained with the BLQ model.

Control of Staphylococcus epidermidis biofilm by surfactins of an endophytic bacterium Bacillus sp. 15 F.

The present paper deals with the preparation and annotation of a surfactin(s) derived from a culture of the endophytic bacterium Bacillus 15 F. The LC-MS analysis of the acetonitrile fraction confirmed the presence of surfactins Leu/Ile7 C15, Leu/Ile7 C14 and Leu/Ile7 C13 with [M+H]+ at m/z 1036.6895, 1022.6741 and 1008.6581, respectively. Various concentrations of the surfactin(s) (hereafter referred to as surfactin-15 F) were used to reduce the adhesion of Staphylococcus epidermidis S61, which served as a model for studying antibiofilm activity on polystyrene surfaces. Incubation of Staphylococcus epidermidis S61 with 62.5 µg/ml of surfactin-15 F resulted in almost complete inhibition of biofilm formation (90.3 ± 3.33 %), and a significant reduction of cell viability (resazurin-based fluorescence was more than 200 times lower). The antiadhesive effect of surfactin-15 F was confirmed by scanning electron microscopy. Surfactin-15 F demonstrated an eradication effect against preformed biofilm, causing severe disruption of Staphylococcus epidermidis S61 biofilm structure and reducing viability. The results suggest that surfactins produced by endophytic bacteria could be an alternative to synthetic products. Surfactin-15 F, used in wound dressings, demonstrated an efficient treatment of the preformed Staphylococcus epidermidis S61 biofilm, and thus having a great potential in medical applications.

Effects of branched-chain amino acids on surfactin structure and antibacterial activity in Bacillus velezensis YA215.

Antibiotics are essential for combating pathogens; however, their misuse has led to increased resistance, necessitating the search for effective, low-toxicity alternatives. Surfactin, a cyclic lipopeptide with a C12-C17 ß-hydroxy fatty acid chain, exhibits significant antibacterial activity and resists resistance, making it a research focus. Nonetheless, the effects of branched-chain amino acids (BCAAs) on surfactin's structure and activity are not well understood. This study examines the influence of BCAAs (L-valine, L-leucine, and L-isoleucine) on the lipopeptide (surfactin) produced by Bacillus velezensis YA215. Process optimization shows that adding 1 g/L of L-Leu and L-Ile, and 0.5 g/L of L-Val, maximized surfactin production to 18.59%, 19.23%, and 20.64%, respectively. Surfactin content peaked at 36 h with L-Val and L-Ile, yielding 19.72% and 11.37%. In contrast, L-Leu addition peaked at 24 h, yielding 11.33%. Notably, L-Val supplementation resulted in the highest relative surfactin content. Antimicrobial testing demonstrated that BCAAs significantly enhance the antibacterial effects of lipopeptides against Escherichia coli and Staphylococcus aureus, with Val showing the most pronounced effect. The addition of BCAAs notably altered the composition of surfactin fatty acid chains. Specifically, Val increased the proportions of iso C14 and iso C16 ß-hydroxy fatty acids from 13.3% and 4.216-23.803% and 8.31%, respectively. Additionally, the amino acid composition at the 7th position of the peptide chain changed significantly, especially with Val addition, which increased the proportion of C14 [Val 7] surfactin by 3.29 times. These structural changes are likely associated with the enhanced antibacterial activity of surfactin. These findings provide valuable insights into the roles of BCAAs in microbial fermentation, underscoring their importance in metabolic engineering to enhance the production of bioactive compounds.

Isolation of a surfactin-producing strain of Bacillus subtilis and evaluation of the probiotic potential and antioxidant activity of surfactin from fermented soybean meal.

BACKGROUND: Surfactin, usually produced by microbial metabolism, has many advantages including low toxicity, high biodegradability, and stability at extreme pH levels and temperatures, making it suitable for industry. However, its commercial production has not yet been achieved. RESULTS: A strain with a strong surfactin-producing ability was isolated and identified as Bacillus subtilis SOPC5, based on the appearance of colonies, microscopic observation, and 16S rDNA sequencing. The isolate exhibited significant tolerance to acid, bile, gastric, and intestinal juices, and was sufficiently susceptible to antibiotics. Bacillus subtilis SOPC5 showed high levels of auto-aggregation and surface hydrophobicity, and a strong capacity to secrete protease, amylase, and cellulase. The strain also exhibited antibacterial activity against Staphylococcus aureus 10 306 with a antibacterial circle diameter of 18.0 ± 1.1 mm. The maximal yield of surfactin (1.32 mg mL-1) was obtained by fermenting soybean meal (SBM) using the isolate under the following conditions: SBM 86 g L-1, inoculation 1.5 × 107 CFU mL-1, FeSO4 1.2 mg L-1, MnSO4 2.6 mg L-1, MgSO4 0.5 mg mL-1, L-Glu 4 mg L-1, temperature 33 °C, duration 120 h, and shaking at 210 rpm. The purity of surfactin was 97.42% as measured by high-performance liquid chromatography (HPLC). The half inhibitory concentration (IC50) values for surfactin to scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) radical cation (ABTS·+) were 1.275 ± 0.11 and 0.73 ± 0.08 mg mL-1, respectively. CONCLUSION: This study provides a scientific basis for the application of B. subtilis SOPC5 (as a potential probiotic) and the preparation of its metabolic product (surfactin). © 2024 Society of Chemical Industry.

Production and optimization of surfactin produced from locally isolated Bacillus halotolerans grown on agro-industrial wastes and its antimicrobial efficiency.

INTRODUCTION: Optimal exploitation of the huge amounts of agro-industrial residuals that are produced annually, which endangers the ecosystem and ultimately contributes to climate change, is one of the solutions available to produce value-added compounds. AIM AND OBJECTIVES: This study aimed at the economic production and optimization of surfactin. Therefore, the production was carried out by the microbial conversion of Potato Peel Waste (PPW) and Frying Oil Waste (FOW) utilizing locally isolated Bacillus halotolerans. Also, investigating its potential application as an antimicrobial agent towards some pathogenic strains. RESULTS: Screening the bacterial isolates for surfactin production revealed that the strain with the highest yield (49 g/100 g substrate) and efficient oil displacement activity was genetically identified as B. halotolerans. The production process was then optimized utilizing Central Composite Design (CCD) resulting in the amelioration of yield by 11.4% (from 49 to 55.3 g/100 g substrate) and surface tension (ST) by 8.3% (from 36 to 33 mN/m) with a constant level of the critical micelle concentration (CMC) at 125 mg/L. Moreover, the physiochemical characterization studies of the produced surfactin by FTIR, 1H NMR, and LC-MS/MS proved the existence of a cyclic lipopeptide (surfactin). The investigations further showed a strong emulsification affinity for soybean and motor oil (E24 = 50%), as well as the ability to maintain the emulsion stable over a wide pH (4-10) and temperature (10-100 °C) range. Interestingly, surfactin had a broad-spectrum range of inhibition activity against Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa, klebsiella pneumonia, and Candida albicans. CONCLUSION: Subsequently, the screening of the isolates and the utilized food-processing wastes along with the extraction technique resulted in a high yield of surfactin characterized by acceptable ST and CMC levels. However, optimization of the cultural conditions to improve the activity and productivity was achieved using Factor-At-A-Time (OFAT) and Central Composite Design (CCD). In contrast, surface activity recorded a maximum level of (33 mN/n) and productivity of 55.3 g/100 g substrate. The optimized surfactin had also the ability to maintain the stability of emulsions over a wide range of pH and temperature. Otherwise, the obtained results proved the promising efficiency of the surfactin against bacterial and fungal pathogens.

Improving Surfactin Production in Bacillus subtilis 168 by Metabolic Engineering.

Surfactin is widely used in the petroleum extraction, cosmetics, biopharmaceuticals and agriculture industries. It possesses antibacterial and antiviral activities and can reduce interfacial tension. Bacillus are commonly used as production chassis, but wild-type Bacillus subtilis 168 cannot synthesise surfactin. In this study, the phosphopantetheinyl transferase (PPTase) gene sfp* (with a T base removed) was overexpressed and enzyme activity was restored, enabling B. subtilis 168 to synthesise surfactin with a yield of 747.5 ± 6.5 mg/L. Knocking out ppsD and yvkC did not enhance surfactin synthesis. Overexpression of predicted surfactin transporter gene yfiS increased its titre to 1060.7 ± 89.4 mg/L, while overexpression of yerP, ycxA and ycxA-efp had little or negative effects on surfactin synthesis, suggesting YfiS is involved in surfactin efflux. By replacing the native promoter of the srfA operon encoding surfactin synthase with three promoters, surfactin synthesis was significantly reduced. However, knockout of the global transcriptional regulator gene codY enhanced the surfactin titre to 1601.8 ± 91.9 mg/L. The highest surfactin titre reached 3.89 ± 0.07 g/L, with the yield of 0.63 ± 0.02 g/g DCW, after 36 h of fed-batch fermentation in 5 L fermenter. This study provides a reference for further understanding surfactin synthesis and constructing microbial cell factories.

Evaluation of surfactant-aided polycyclic aromatic hydrocarbon biodegradation by molecular docking and molecular dynamic simulation in the marine environment.

Marine oil spills directly cause polycyclic aromatic hydrocarbons (PAHs) pollution and affect marine organisms due to their toxic property. Chemical and bio-based dispersants composed of surfactants and solvents are considered effective oil spill-treating agents. Dispersants enhance oil biodegradation in the marine environment by rapidly increasing their solubility in the water column. However, the effect of dispersants, especially surfactants, on PAHs degradation by enzymes produced by microorganisms has not been studied at the molecular level. The role of the cytochrome P450 (CYP) enzyme in converting contaminants into reactive metabolites during the biodegradation process has been evidenced, but the activity in the presence of surfactants is still ambiguous. Thus, this study focused on the evaluation of the impact of chemical and bio-surfactants (i.e., Tween 80 (TWE) and Surfactin (SUC)) on the biodegradation of naphthalene (NAP), chrysene (CHR), and pyrene (PYR), the representative components of PAHs, with CYP enzyme from microalgae Parachlorella kessleri using molecular docking and molecular dynamics (MD) simulation. The molecular docking analysis revealed that PAHs bound to residues at the CYP active site through hydrophobic interactions for biodegradation. The MD simulation showed that the surfactant addition changed the enzyme conformation in the CYP-PAH complexes to provide more interactions between the enzyme and PAHs. This led to an increase in the enzyme's capability to degrade PAHs. Binding free energy (ΔG||Bind) calculations confirmed that surfactant treatment could enhance PAHs degradation by the enzyme. The SUC gave a better result on NAP and PYR biodegradation based on ΔG||Bind, while TWE facilitated the biodegradation of CHR. The research outputs could greatly facilitate evaluating the behaviors of oil spill-treating agents and oil spill response operations in the marine environment.

Screening Plant Growth-Promoting Bacteria with Antimicrobial Properties for Upland Rice.

This study explores beneficial bacteria isolated from the roots and rhizosphere soil of Khao Rai Leum Pua Phetchabun rice plants. A total of 315 bacterial isolates (KK001 to KK315) were obtained. Plant growth-promoting traits (phosphate solubilization and indole-3-acetic acid (IAA) production), and antimicrobial activity against three rice pathogens (Curvularia lunata NUF001, Bipolaris oryzae 2464, and Xanthomonas oryzae pv. oryzae) were assessed. KK074 was the most prolific in IAA production, generating 362.6 ± 28.0 µg/ml, and KK007 excelled in tricalcium phosphate solubilization, achieving 714.2 ± 12.1 µg/ml. In antimicrobial assays using the dual culture method, KK024 and KK281 exhibited strong inhibitory activity against C. lunata, and KK269 was particularly effective against B. oryzae. In the evaluation of antimicrobial metabolite production, KK281 and KK288 exhibited strong antifungal activities in cell-free supernatants. Given the superior performance of KK281, taxonomically identified as Bacillus sp. KK281, it was investigated further. Lipopeptide extracts from KK281 had significant antimicrobial activity against C. lunata and a minimum inhibitory concentration (MIC) of 3.1 mg/ml against X. oryzae pv. oryzae. LC-ESI-MS/MS analysis revealed the presence of surfactin in the lipopeptide extract. The crude extract was non-cytotoxic to the L-929 cell line at tested concentrations. In conclusion, the in vitro plant growth-promoting and disease-controlling attributes of Bacillus sp. KK281 make it a strong candidate for field evaluation to boost plant growth and manage disease in upland rice.

Computational Methods Reveal a Series of Cyclic and Linear Lichenysins and Surfactins from the Vietnamese Marine Sediment-Derived Streptomyces Strain G222.

The Streptomyces strain G222, isolated from a Vietnamese marine sediment, was confidently identified by 16S rRNA gene sequencing. Its AcOEt crude extract was successfully analyzed using non-targeted LC-MS/MS analysis, and molecular networking, leading to a putative annotation of its chemical diversity thanks to spectral libraries from GNPS and in silico metabolite structure prediction obtained from SIRIUS combined with the bioinformatics tool conCISE (Consensus Annotation Propagation of in silico Elucidations). This dereplication strategy allowed the identification of an interesting cluster of a series of putative cyclic and linear lipopeptides of the lichenysin and surfactin families. Lichenysins (3-7) were isolated from the sub-fraction, which showed significant anti-biofilm activity against Pseudomonas aeruginosa MUC-N1. Their structures were confirmed by detailed 1D and 2D NMR spectroscopy (COSY, HSQC, HMBC, TOCSY, ROESY) recorded in CD3OH, and their absolute configurations were determined using the modified Marfey's method. The isolated lichenysins showed anti-biofilm activity at a minimum concentration of 100 µM. When evaluated for antibacterial activity against a panel of Gram-positive and Gram-negative strains, two isolated lichenysins exhibited selective activity against the MRSA strain without affecting its growth curve and without membranotropic activity. This study highlights the power of the MS/MS spectral similarity strategy using computational methods to obtain a cross-validation of the annotated molecules from the complex metabolic profile of a marine sediment-derived Streptomyces extract. This work provides the first report from a Streptomyces strain of combined cyclic and linear lichenysins and surfactins, known to be characteristic compounds of the genus Bacillus.

Assessing the feasibility and sustainability of a surfactin production process: a techno-economic and environmental analysis.

Biosurfactants have been profiled as a sustainable replacement for chemical-based surfactants since these bio-based molecules have higher biodegradability. Few research papers have focused on assessing biosurfactant production to elucidate potential bottlenecks. This research aims to assess the techno-economic and environmental performance of surfactin production in a potential scale of 65m3, considering different product yields and involving the European energy crisis of 2021-2022. The conceptual design, simulation, techno-economic, and environmental assessments were done by applying process engineering concepts and software tools such as Aspen Plus v.9.0 and SimaPro v.8.3.3. The results demonstrated the high economic potential of surfactin production since the higher values in the market offset the low fermentation yields, low recovery efficiency, and high capital investment. The sensitivity analysis of the economic assessment elucidated a minimum surfactin selling price between 29 and 31 USD/kg of surfactin, while a minimum processing scale for economic feasibility between 4 and 5 kg/h is needed to reach an equilibrium point. The environmental performance must be improved since the carbon footprint was 43 kg CO2eq/kg of surfactin. The downstream processing and energy demand are the main bottlenecks since these aspects contribute to 63 and 25% of the total emissions. The fermentation process and downstream process are key factors for future optimization and research.

Research advances in the identification of regulatory mechanisms of surfactin production by Bacillus: a review.

Surfactin is a cyclic hexalipopeptide compound, nonribosomal synthesized by representatives of the Bacillus subtilis species complex which includes B. subtilis group and its closely related species, such as B. subtilis subsp subtilis, B. subtilis subsp spizizenii, B. subtilis subsp inaquosorum, B. atrophaeus, B. amyloliquefaciens, B. velezensis (Steinke mSystems 6: e00057, 2021) It functions as a biosurfactant and signaling molecule and has antibacterial, antiviral, antitumor, and plant disease resistance properties. The Bacillus lipopeptides play an important role in agriculture, oil recovery, cosmetics, food processing and pharmaceuticals, but the natural yield of surfactin synthesized by Bacillus is low. This paper reviews the regulatory pathways and mechanisms that affect surfactin synthesis and release, highlighting the regulatory genes involved in the transcription of the srfAA-AD operon. The several ways to enhance surfactin production, such as governing expression of the genes involved in synthesis and regulation of surfactin synthesis and transport, removal of competitive pathways, optimization of media, and fermentation conditions were commented. This review will provide a theoretical platform for the systematic genetic modification of high-yielding strains of surfactin.

The Combined Effects of Azoxystrobin and the Biosurfactant-Producing Bacillus sp. Kol B3 against the Phytopathogenic Fungus Fusarium sambucinum IM 6525.

This study aimed to evaluate how the combined presence of the synthetic fungicide azoxystrobin (AZ) and the biosurfactant-producing Bacillus sp. Kol B3 influences the growth of the phytopathogenic fungus Fusarium sambucinum IM 6525. The results showed a noticeable increase in antifungal effectiveness when biotic and abiotic agents were combined. This effect manifested across diverse parameters, including fungal growth inhibition, changes in hyphae morphology, fungal membrane permeability and levels of intracellular reactive oxygen species (ROS). In response to the presence of Fusarium and AZ in the culture, the bacteria changed the proportions of biosurfactants (surfactin and iturin) produced. The presence of both AZ and/or Fusarium resulted in an increase in iturin biosynthesis. Only in 72 h old bacterial-fungal co-culture a 20% removal of AZ was noted. In the fungal cultures (with and without the addition of the bacteria), the presence of an AZ metabolite named azoxystrobin free acid was detected in the 48th and 72nd hours of the process. The possible involvement of increased iturin and ROS content in antifungal activity of Bacillus sp. and AZ when used together are also discussed. Biosurfactants were analyzed by liquid chromatography with tandem mass spectrometry (LC-MS/MS). Microscopy techniques and biochemical assays were also used.

Regulation mechanism and bioactivity characteristic of surfactin homologues with C14 and C15 fatty acid chains.

BACKGROUND: Surfactin, a green lipopeptide bio-surfactant, exhibits excellent surface, hemolytic, antibacterial, and emulsifying activities. However, a lack of clear understanding of the synthesis regulation mechanism of surfactin homologue components has hindered the customized production of surfactin products with different biological activities. RESULTS: In this study, exogenous valine and 2-methylbutyric acid supplementation significantly facilitated the production of C14-C15 surfactin proportions (up to 75% or more), with a positive correlation between the homologue proportion and fortified concentration. Subsequently, the branched-chain amino acid degradation pathway and the glutamate synthesis pathway are identified as critical pathways in regulating C14-C15 surfactin synthesis by transcriptome analysis. Overexpression of genes bkdAB and glnA resulted in a 1.4-fold and 1.3-fold increase in C14 surfactin, respectively. Finally, the C14-rich surfactin was observed to significantly enhance emulsification activity, achieving an EI24 exceeding 60% against hexadecane, while simultaneously reducing hemolytic activity. Conversely, the C15-rich surfactin demonstrated an increase in both hemolytic and antibacterial activities. CONCLUSION: This study presents the first evidence of a potential connection between surfactin homologue synthesis and the conversion of glutamate and glutamine, providing a theoretical basis for targeting the synthesis regulation and structure-activity relationships of surfactin and other lipopeptide compounds.

Solubilization of Phospholipid by Surfactin Leading to Lipid Nanodisc and Fibrous Architecture Formation.

Nanodiscs belong to a category of water-soluble lipid bilayer nanoparticles. In vivo nanodisc platforms are useful for studying isolated membrane proteins in their native lipid environment. Thus, the development of a practical method for nanodisc reconstruction has garnered consider-able research interest. This paper reports the self-assembly of a mixture of bio-derived cyclic peptide, surfactin (SF), and l-α-dimyristoylphosphatidylcholine (DMPC). We found that SF induced the solubilization of DMPC multilamellar vesicles to form their nanodiscs, which was confirmed by size-exclusion chromatography, dynamic light scattering, and transmission electron microscopy analyses. Owing to its amphiphilic nature, the self-assembled structure prevents the exposure of the hydrophobic lipid core to aqueous media, thus embedding ubiquinol (CoQ10) as a hydrophobic model compound within the inner region of the nanodiscs. These results highlight the feasibility of preparing nanodiscs without the need for laborious procedures, thereby showcasing their potential to serve as promising carriers for membrane proteins and various organic compounds. Additionally, the regulated self-assembly of the DMPC/SF mixture led to the formation of fibrous architectures. These results show the potential of this mixture to function as a nanoscale membrane surface for investigating molecular recognition events.