STABILIZATION OF BACILLUS SP. MUTANT ISOLATE STRAIN KG7' AND STRAIN KG48 BY ADAPTIVE LABORATORY EVOLUTION (ALE) FOR BIOSURFACTANT PRODUCTION

• Main Author: Rahmatunisa, Nadira
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/70866
• Institution: Institut Teknologi Bandung

Biosurfactants are widely applied in the petroleum industry because they can increase oil recovery. Compared to synthetic surfactant, biosurfactants are more environmentally responsible and have specific activity in extreme environments, but have not been able to compete economically because of the low biosurfactant production capacity. Through the Adaptation Laboratory Evolution (ALE), the metabolism of biosurfactant-producing microorganisms can be engineered, so they can produce more biosurfactants. One of the compounds used as a mutagen in this process is CTAB. In the process, ALE takes time until a stable mutant is obtained to be characterized. This research aims to stabilize the mutant isolates of Bacillus sp. strain KG7’ and KG48 through the ALE strategy and to evaluate the production and emulsification activity of biosurfactants produced by stable mutant. The isolate candidate was stabilized by performing gradual exposure to the LB medium which has given CTAB (a) 3.9 ppm and (b) 4.5 ppm. Mutants are considered to be stable as the frequency of mutants grow on the medium >90%. The characterization of the biosurfactant production of selected mutant isolates was carried out on the SMSSe medium with an incubation time of 96 hours. During the incubation process, the number of microbes, biosurfactant production, and emulsification index (E24) data were collected every 12 hours. The results showed that the KG7'a was stable in the 59th generation (93.11%), KG7'b in the 234th generation (90.33%), while KG48 has not been observed to be stable until the 204th generation (7.08%). Observation of cell morphology shows that wild-type (WT) cells are longer than KG7' (P <0.1) and KG48 (P <0.1). Evaluation of biosurfactant production shows that WT was able to produce biosurfactant as much as 0.123 g/L, while KG7'a only produces 0.107 g/L, and KG7’b produces 0.101 g/L. E24 evaluation showed that in light petroleum, WT biosurfactants able to emulsify 58.9% of oil while the two mutants were only 47.6% (KG7'a) and 51.8% (KG7'b). However, biosurfactants produced by both mutants can emulsify heavy petroleum 74.5% (KG7'a) and 80% (KG7'b), better than WT (71.3%). Biosurfactant E24 activity at different harvest time vary for different oil types. E24 activity of KG7'b in light petroleum was highest at the 12th hour (55%), E24 in the heavy oil was highest at the 24th hour (75.1%), and E24 in cooking oil was highest at the 60th hour (25%). This study shows that the KG7’ mutant isolate was successfully stabilized through the ALE strategy after gradual adaptation to the 59th generation (on CTAB 3.9 ppm) and 234th generation (on CTAB 4.5 ppm) with biosurfactant production not as much as WT but higher emulsification activity in heavy petroleum. Based on these phenomena, it is suspected there are differences in the structure of biosurfactants produced by WT and KG7'b. It can be the basis for further research in molecular structure characterization and biosurfactant production optimization through other approaches besides ALE.