PRODUCTION, CHARACTERIZATION AND APPLICATION OF RHAMNOLIPID FROM BIO-CONVERTION OF PALM OIL MILL EFFLUENT (POME) WASTE BY HALOFILIC BACTERIA Pseudomonas stutzeri BK-AB12

• Main Author: Rizki Fazli, Rahmad
• Format: Dissertations
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/41775
• Institution: Institut Teknologi Bandung

Rhamnolipid is biosurfactant which is often used as an active ingredient for bioremediation, bioemulsifier, anti microbial, and enhanced oil recovery (EOR) process. However, to obtain rhamnolipid, high costs are still needed so that the utilization of rhamnolipid in some of these fields is still being considered. Research to obtain rhamnolipid with economical production costs continues to grow today. One of them is exploring microbes capable of converting cheap carbon sources, such as waste, into rhamnolipids. In previous studies it was known that the halophilic bacteria Pseudomonas stutzeri BK-AB12 can bioconvert olive oil as a carbon source into rhamnolipid. In this study, the halophil bacteria was further explored in its potential to produce rhamnolipid by replacing olive oil with cheaper carbon sources, namely liquid waste from the palm oil industry known as palm oil liquid waste (POME). In this study it will be shown that P. stutzeri BK-AB12 is a potential halophilic bacterium in converting POME to rhamnolipid to be applied as an antibacterial and surfactant in the enhanced oil recovery (EOR) process. This study was initiated by optimizing bacterial growth in minimum salt medium (MSM) at 37oC, pH 7.5 and 5% NaCl concentration (w/v), by optimizing the three most influential factors in the production process of rhamnolipid using response methodology (RSM). The three factors are carbon source concentration (POME), nitrogen source concentration (urea), and incubation time. RSM results show the best growth in MSM containing 5% (w/v) NaCl and pH 7.5 achieved at POME 20% (v/v), urea 0.2% (w/v), and incubation time of 90 hour. The bacterial growth curve at this optimum condition indicates that the stationary phase occurs at the 48th hour and the death phase at the 72nd hour. Rhamnolipid is produced when bacterial growth enters the stationary final phase towards the death phase, which is from the 72nd hour to the 120th hour, where the highest production is observed at the 96th hour. The resulting rhamnolipid was deposited using 6 M NaOH and then extracted using chloroform: methanol = 2: 1. The obtained rhamnolipid concentration was 3.91 ± 0.24 grams/liter of culture. To identify biosurfactants obtained by P. stutzeri BK-AB12 is rhamnolipid, a qualitative test was carried out using blue agar media containing cetyltrimethylammonium bromide-methylene blue (CTAB-MB). This test gives a positive result which is shown by the formation of a dark blue zone around the part containing rhamnolipid. In addition to qualitative tests, the rhamnolipid structure was also verified by FTIR and 1H-NMR spectroscopic techniques. The FTIR and 1H-NMR spectra show a spectrum pattern that is typical for rhamnolipid. Further analysis was carried out using HRMS to determine the rhamnolipid variant obtained from the bioconversion of POME by P. stutzeri BKAB12. The results show the large variations in rhamnolipid produced for both mono and dirhamnolipid groups. This is because the carbon source used (POME) has quite a variety of fatty acids which have an impact on the metabolic pathway and rhamnolipid biosynthesis. The rhamnolipid sample was further characterized by its physicochemical properties which included the determination of the emulsification index value, the study of rhamnolipid activity at variations in temperature, pH and salinity, and the determination of the value of critical micellar concentration (CMC). The characterization results show that the sample has the highest 24 hours emulsification index value of 71.4% and the highest activity at pH 9, 15% NaCl (w/v) and 55oC temperature. While the CMC value was obtained at the concentration of rhamnolipid 390 mg/L with a decrease in water surface tension of 16 dyne / cm at room temperature. To increase the production of rhamnolipid, this study also tried to provide initial treatment of POME with the ozonation process. This process is carried out by flowing ozone with a fixed flow rate into the POME solution with a certain time variation. From the results of variations in ozonation time, the best POME conditions were observed in the 30 minutes ozonation sample. The results of GCMS analysis showed that the content of POME before and after 30 minutes of ozonation had a difference where pre-ozonation POME contained more components of glycerol than fatty acids, whereas POME after ozonation did not contain glycerol and contained fatty acids as the main component. The profile of the bacterial growth curve in the medium using ozonated POME is different from the bacterial growth curve before ozonation. This growth curve shows the beginning of the stationary phase occurs at 72nd hour (previously 48th hour) and the beginning of the death phase at 120th hour (previously 72nd hour) and rhamnolipid in this situation is produced with the highest concentration at 120th hour (previously 96th hour). Based on this, it can be seen that the ozonation process causes the production of rhamnolipid to be slow but the resulting rhamnolipid product increases. The use of POME from ozonation for 30 minutes can increase rhamnolipid production by 1.17 ± 0.12 grams / liter or about 30% higher than using POME before ozonation. The verification of biosurfactant was done by comparing the FTIR spectrum with the rhamnolipid spectrum before ozonation. The result is that both surfactants provide the same spectrum pattern so that the ozonation POME does not change the biosynthetic pathway and still produces rhamnolipid. Rhamnolipid resulting from bioconversion of POME without and with ozonation is tested for its potential as anti-microbial. Rhamnolipids from non-ozonated POME provide inhibitory growth concentrations (MIC) against Propionibacterium acnes bacteria at 125 ppm and minimum kill concentration (MBC) at 250 ppm. The same results were obtained from rhamnolipids with ozonated POME (there was no difference in the value of MIC and MBC). This concludes that ozonation does not affect the ability of rhamnolipid produced as an anti microbial. Rhamnolipid from bioconversion of POME with ozonation has its potential test as sufactant for the process of increasing oil recovery (EOR). One of the crucial factors in determining the effectiveness of a surfactant for the EOR process is the ability of surfactants to reduce the oil-water layer (IFT) interface tension to 10-3 mN/m at CMC less than 1000 ppm at certain temperatures. The measurement results showed that the obtained rhamnolipid was able to reduce the IFT value to 10-3 mN/m at a temperature of 60oC with CMC around 225 ppm. Therefore, the bioconversed POME rhamnolipids have the potential to be used as EOR surfactants in oil wells with middle temperature categories.