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Lactic acid bacteria (LABs) are generally recognized as safe (GRAS), and therefore, LAB biosurfactants are beneficial with negligible negative impacts. This study aims to maintain the biosurfactant producing activity of an LAB strain, Weissella cibaria PN3, by immobilizing the bacterial cells on a commercial porous carrier. For biosurfactant production, 2% soybean oil was used as the carbon source. After 72 h, immobilized cells were reused by replacing production medium. The extracellular and cell-bound biosurfactants were extracted from the resulting cell-free broth and cell pellets, respectively. SEM images of used immobilizing carriers showed increased surface roughness and clogged pores over time. Thus, the immobilizing carriers were washed in PBS buffer (pH 8.0) before reuse. To maintain biosurfactant production activity, immobilized cells were reactivated every three production cycles by incubating the washed immobilizing carriers in LB medium for 48 h. The maximum yields of purified extracellular (1.46 g/L) and cell-bound biosurfactants (1.99 g/L) were achieved in the 4th production cycle. The repeated biosurfactant production of nine cycles were completed within 1 month, while only 2 g of immobilized cells/L were applied. The extracellular and cell-bound biosurfactants had comparable surface tensions (31 – 33 mN/m); however, their CMC values were different (1.6 and 3.2 g/L, respectively). Both biosurfactants had moderate oil displacement efficiency with crude oil samples but formed emulsions well with gasoline, diesel, and lavender, lemongrass and coconut oils. The results suggested that the biosurfactants were relatively hydrophilic. In addition, the mixing of both biosurfactants showed a synergistic effect, as seen from the increased emulsifying activity with palm, soybean and crude oils. The biosurfactants at 10 – 16 mg/mL showed antimicrobial activity toward some bacteria and yeast but not filamentous fungi. The molecular structures of these biosurfactants were characterized by FTIR as different glycolipid congeners. The biosurfactant production process by immobilized Weissella cibaria PN3 cells was relatively cheap given that two types of biosurfactants were simultaneously produced and no new inoculum was required. The acquired glycolipid biosurfactants have high potential to be used separately or as mixed biosurfactants in various products, such as cleaning agents, food-grade emulsifiers and cosmetic products.

Drill cuttings from petroleum exploration and production sites can cause diverse environmental problems. Total petroleum hydrocarbons (TPHs) are a major pollutant from the use of polyolefin-based mud. As an alternative to incineration, this study investigated the application of surfactant-enhanced washing technology prior to bioremediation. The washing step was necessary because the initial TPH concentrations were quite high at approximately 15% (w/w). Washing agents were formulated by varying the concentration of lipopeptide biosurfactant (in foamate or cell-free broth), Dehydol LS7TH (fatty alcohol ethoxylate 7EO, oleochemical surfactant) and butanol (as a lipophilic linker) at different salinities. The most efficient formula produced a Winsor Type I microemulsion (oil-in-water microemulsion) with polyolefin and contained only 20% (v/v) foamate and 2% (v/v) Dehydol LS7TH in water. Due to the synergistic behavior between the anionic lipopeptides and non-ionic Dehydol LS7TH, the formula efficiently removed 92% of the TPHs from the drill cuttings when applied in a jar test. To reduce the cost, the concentrations of each surfactant should be reduced; thus, the formula was optimized by the simplex lattice mixture design. In addition, cell-free broth, at a pH of 10, containing 3.0 g/L lipopeptides was applied instead of foamate because it was easy to prepare. The optimized formula removed 81.2% of the TPHs and contained 72.0% cell-free broth and 1.4% Dehydol LS7TH in water. A 20-kg soil washing system was later tested where the petroleum removal efficiency decreased to 70.7% due to polyolefin redeposition during separation of the washing solution. The remaining TPHs (4.5%) in the washed drilled cuttings were further degraded by a mixture of Marinobacter

Biosurfactant-based dispersants were formulated by mixing glycolipids from Weissella cibaria PN3 and lipopeptides from Bacillus subtilis GY19 to enhance the synergistic effect and thereby achieve hydrophilic-lipophilic balance. The proportions of each biosurfactant and dispersant-to-oil ratios (DORs) were varied to obtain the appropriated formulations. The most efficient glycolipid:lipopeptide mixtures (F1 and F2) had oil displacement activities of 81–88% for fuel and crude oils. The baffled flask test of these formulations showed 77–79% dispersion effectiveness at a DOR of 1:25. To reduce the cost of the dispersant, this study optimized the glycolipid production process by using immobilized cells in a stirred tank fermenter. Semicontinuous glycolipid production was carried out conveniently for 3 cycles. Moreover, food wastes, including waste coconut water and waste frying oil, were found to promote glycolipid production. Glycolipids from the optimized process and substrates had similar characteristics but 20–50% lower cost than those produced from basal medium with soybean oil in shaking flasks. The lowest cost dispersant formulation (F2*) contained 10 g/L waste-derived cell-bound glycolipid and 10 g/L lipopeptide and showed high dispersion efficiency with various oils. Therefore, this biosurfactant-based dispersant could be produced on a larger scale for further application.