Hydrophilic: Glucose, sucrose, glycerol, molasses, starch. Generally cheaper and easier to handle. Often leads to lower yields but better cell growth.

Waste substrates (e.g., molasses, whey, industrial effluents, waste cooking oil) are preferred for cost-effectiveness and sustainability.

Nitrogen Source:

Organic (peptone, yeast extract, corn steep liquor) or inorganic (ammonium salts, nitrates).
The C/N ratio is crucial; a high C/N ratio (carbon excess, nitrogen limitation) often triggers secondary metabolite production, including biosurfactants.

Other Nutrients:

Phosphate (e.g., $\text{K}_2\text{HPO}_4$, $\text{KH}_2\text{PO}_4$) for energy metabolism and cell structure.
Trace elements (Mg, Fe, Mn, Zn, Ca) as cofactors for enzymes involved in biosynthesis.

Fermentation Conditions (Bioreactor Operation):

Temperature: Optimal range depends on the microorganism (typically $25-37^\circ\text{C}$ for mesophiles). Affects enzyme activity and cell growth.
pH: Maintained within a specific range (e.g., $6.0-7.5$). Influences enzyme activity, nutrient solubility, and biosurfactant stability. Can be controlled by adding acid/base.
Aeration and Agitation:
- Aeration: Crucial for aerobic microorganisms, providing oxygen for metabolic processes. Airflow rate is a key parameter.
- Agitation: Ensures uniform mixing of nutrients, cells, and oxygen, and reduces mass transfer limitations. Impeller type and speed are important.
Inoculum Size: Sufficient inoculum ensures rapid onset of fermentation.
Fermentation Strategy:
- Batch Fermentation: All nutrients added at the beginning. Simple but can suffer from substrate depletion and product inhibition.
- Fed-Batch Fermentation: Nutrients (especially carbon source) are added incrementally during the process. Allows for higher cell densities and prolonged biosurfactant production, reducing substrate inhibition.
- Continuous Fermentation: Fresh medium is continuously fed, and product is continuously harvested. Achieves high productivity but requires precise control and can be prone to contamination.

Downstream Processing (Separation and Recovery):

Cell Separation: Biosurfactants can be extracellular (secreted into medium) or cell-associated.
- Centrifugation or Microfiltration: Used to separate microbial cells from the cell-free supernatant containing extracellular biosurfactants.
Precipitation:
- Acid Precipitation: Lowering the pH (e.g., to $2.0$) causes many biosurfactants (especially lipopeptides and some glycolipids) to precipitate due to reduced solubility.
- Salt Precipitation: Adding salts (e.g., ammonium sulfate) can cause salting out of biosurfactants.
Solvent Extraction:
- Commonly used to extract biosurfactants from the aqueous phase using organic solvents (e.g., chloroform/methanol mixtures, ethyl acetate, butanol).
- The choice of solvent depends on the polarity of the biosurfactant.
Foam Fractionation:
- Biosurfactants tend