# Two Engineered Bacillus subtilis Surfactin High-Producers: Effects of Culture Medium, and Potential Agricultural and Petrochemical Applications

## TL;DR

This study shows that two engineered bacteria can produce surfactin, a natural compound useful for controlling crop fungi and cleaning oil spills, under specific growth conditions.

## Contribution

The study introduces two high-yield surfactin-producing Bacillus subtilis strains and evaluates their performance in different media for agricultural and petrochemical applications.

## Key findings

- Surfactin effectively controlled soybean crop fungi and separated oil and water.
- BMV9 is the highest surfactin producer reported to date.
- Optimal surfactin production was achieved in specific culture media conditions.

## Abstract

This study explores ecological solutions for agriculture and the petroleum industry. The goal was to evaluate the potential of two strains of bacteria, named Bacillus subtilis, which were genetically modified to produce a natural soap-like molecules known as surfactin. We evaluated the surfactin production in different types of nutrients, named culture media, and then explored two practical applications. First, we checked if surfactin could work as a biological control, meaning a natural pesticide, against two types of fungi that cause diseases in soybean crops. Second, we investigated the effectiveness of surfactin in helping to clean up oil spills or improve oil extraction in industrial processes. As a result, the produced surfactin proved to be effective both against the soybean fungi and in separating oil and water. In conclusion, this research defines the ideal cultivation conditions to produce large quantities of surfactin in an economically viable way. This work is valuable to society, since it offers a sustainable alternative to traditional agricultural chemicals and contributes to the development of cleaner and more efficient methods for oil recovery and environmental cleanup.

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.