high-resolution LC-MS/MS analysis

Stages

1. 1.
   Shake-flask fermentation under two media conditions(functional_characterization)

   This stage establishes how the two engineered strains perform under different nutrient conditions before downstream application testing.

   Selection: compare strain performance and nutrient effects on surfactin yield in mineral salt versus complex medium, each with 2% glucose

2. 2.
   Time-course lipopeptide quantification and cultivation monitoring(secondary_characterization)

   This stage provides analytical and process readouts needed to compare strains and media over time.

   Selection: quantify surfactin and fengycin and monitor optical density, residual glucose, and pH throughout cultivation

3. 3.
   Small-scale growth validation(confirmatory_validation)

   This stage confirms growth behavior observed in the main cultivation comparison.

   Selection: validate microbial growth in both media using small-scale cultivation approaches

4. 4.
   Agricultural antifungal testing(confirmatory_validation)

   This stage evaluates whether the produced lipopeptides have practical biocontrol activity against soybean phytopathogens.

   Selection: test culture supernatants and lipopeptide extracts against two Diaporthe species

5. 5.
   Petrochemical oil displacement testing(confirmatory_validation)

   This stage tests whether the produced surfactin shows function relevant to enhanced oil recovery and related uses.

   Selection: evaluate surfactin efficacy in oil displacement tests relevant to enhanced oil recovery, bioremediation, and related petrochemical processes

6. 6.
   LC-MS/MS lipopeptide characterization(secondary_characterization)

   This stage adds structural and compositional detail to the production and application comparisons.

   Selection: structurally characterize and relatively quantify lipopeptides by high-resolution LC-MS/MS

Steps

1. 1.
   Cultivate BMV9 and BsB6 in shake flasks with mineral salt or complex medium supplemented with 2% glucoseengineered producer strains under comparison

   Generate biomass and lipopeptides under defined media conditions for comparative analysis.

   Cultivation is required before any production, growth, or application measurements can be made.

2. 2.
   Extract lipopeptides and quantify surfactin and fengycin at multiple time points by HPTLC while monitoring optical density, residual glucose, and pHquantification assay

   Measure production dynamics and cultivation state across strains and media.

   Analytical monitoring follows cultivation so the authors can compare output and process behavior over time.

3. 3.
   Validate microbial growth in both media using small-scale cultivation approaches

   Confirm growth behavior observed in the main cultivation experiments.

   This confirmatory step follows the main fermentation measurements to validate growth observations across media.

4. 4.
   Test culture supernatants and lipopeptide extracts against two Diaporthe species

   Assess agricultural biocontrol potential of the produced lipopeptides.

   Application testing is performed after production samples are available from fermentation and extraction.

5. 5.
   Perform oil displacement tests to evaluate surfactin efficacy for enhanced oil recovery, bioremediation, and related petrochemical processes

   Assess petrochemical application potential of surfactin-containing preparations.

   This downstream application assay uses produced surfactin after fermentation and extraction workflows have generated material to test.

6. 6.
   Use high-resolution LC-MS/MS to structurally characterize and relatively quantify the lipopeptidesstructural characterization assay

   Define lipopeptide structural profiles and relative abundance patterns associated with the compared strains and media.

   This analytical step follows production and application testing to provide deeper compositional interpretation of the observed outputs.