PRODUCTION, CHARACTERIZATION, AND APPLICATION OF BIOSURFACTANTS FROM BIOCONVERSION OF GLYCEROL BY HALOPHILIC BACTERIUM Halomonas elongata BK-AG18 AS AN INHIBITOR OF BACTERIAL

• Main Author: ALVIONITA (NIM : 20515001), MIEKE
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/28914
• Institution: Institut Teknologi Bandung

<p align="justify">The energy demand of biodiesel for fuel has increased since the petroleum stock is limited. The increase in biodiesel production is commonly followed by the increase of glycerol as co-product. One alternative solution to utilise glycerol is converting glycerol into more valuable product. In presence study, glycerol will be bioconverted into biosurfactant by halophilic bacterium Halomonas elongata BKAG18. Four halophilic bacteria, which were Halomonas elongata BK-AG18, Halomonas elongata BK-AB8, Halomonas meridiana BK-AB4, and Chromohalobacter japonicus BK-AB18, indigenous from salty mud crater Bledug Kuwu, Purwodadi, Central Java, were screened their potential to bioconvert glycerol into biosurfactant. Each strain was initially cultivated in LB medium to obtain a preculture. One percent of the pre-culture of each strain was inoculated in the Glis-Ur-MSM containing 3% (w/v) glycerol, 0.3% (w/v) urea, and various salts at 37oC with aeration rate of 150 rpm for 96 hours. Biosurfactant production was monitored by measuring its activity with oil spreading test (OST), which was carried out by dropping the supernatant containing biosurfactant onto two phase system (palm oil-water) placed on a petri dish. The displaced oil by biosurfactant solution was measured its diameter. The longer diameter of oil displaced, the higher the biosurfactant concentration. Concomitanly, the bacterial growth was also inspected by measuring the optical density (OD) with UV-Vis spectrophotometer at 600 nm. The performance of each bacterial strains to convert glycerol into biosurfactant was examined based on their efficiency on producing biosurfactant per bacterial cell. The efficiency was calculated by dividing the diameter of oil spreading from OST with the optical density (OD) the bacterial cell after 96 hours of incubation. The highest efficiency was exhibited by Halomonas elongata BKAG18, which was about 63%. This bacterial strain was then used in the further study for biosurfactant production and characterization. In order to optimize the biosurfactant production by this strain, we varied the concentration of glycerol as the main carbon source, nitrogen sources (NH4Cl, NaNO3, (NH4)2SO4, KNO3, and urea), NaCl concentration, pH and temperature. The optimization result revealed that the best biosurfactant production by H. elongata BK-AG 18 was achieved when this bacterial strain was cultivated in the medium containing 2% (v/v) glycerol, 0.3% (w/v) urea, and 5% (w/v) NaCl at pH 6 and temperature of 35oC. Using this optimized medium, the highest biosurfactant production was observed at 54th hours of cultivation with the diameter of oil spreading from OST was about 3.5 cm. Biosurfactant was obtained by precipitation method using 2.0 M NaOH and extraction with 3V of mix chloroform:methanol (2:1). In order to check the purity of the extracted biosurfactant, thin layer chromatography (TLC) was employed with a solvent system of chloroform:methanol:water (65:25:4). A single spot with Rf value about 0.7 was observed on the TLC plate. Biosurfactants were further characterized to determine its physicochemical properties, including CMC, activity at various pH and salinity, and CTAB-MB test. Biosurfactant has a CMC value of 275 mg/L and showed the best performance as emulsifier at pH 6 with the emulsification index about 48%. Biosurfactants also had a high emulsification activity at a wide range of NaCl concentrations. In order to identify the type of biosurfactant produced by H. elongata BK-AG 18, CTAB-MB test was performed by droping the biosurfactant solution into the CTAB medium. The result was negative confirming that the biosurfactant produced by this bacterial strain was not belong to rhamnolipid group. Futher structural analysis was performed by spectroscopic method with Fourier Transform Infra Red (FTIR) and 1H NMR were carried out. The FTIR spectrum showed the peaks in the wave numbers of 3450, 2962 and 2362 cm-1 which indicates the -OH group, asymmetric vibration of aliphatic stretching of C-H bands, and symmetric vibration of aliphatic stretching of C-H bands, respectively. Carbonyl group (C=O) and C-O-C bonds were also confirmed at 1632 and 1030 cm-1, respectively. Meanwhile, the presence of alkenes was confirmed from stretching mode of CH=CH2 in the wave number of 744 cm-1. 1H NMR spectra of biosurfactant revealed the proton signal of lipid (fatty acid) and sugar (carbohydrate). The multiplet proton at chemical shift of 1.0-2.0 ppm showed the presence of fatty acid moieties. The chemical shift at 3.5-4.2 ppm indicated the proton signal of sugar moieties, whereas the presence of proton anomeric was showed at chemical shift of 5.0 ppm. Based on FTIR and 1H NMR analysis, biosurfactants produced from H. elongata BK-AG18 were predicted to be included in the glycolipid group. The potency of biosurfactant as antibacterial agent was examined toward two bacterial strains, i.e. Escherichia coli and Staphylococcus aureus. Biosurfactants were fail to inhibit E. Coli growth but able to inhibit S. aureus growth with minimal inhibition concentration about 433 mg/L.<p align="justify">