Potensi Isolate Bakteri Penghasil Biosurfaktan Lokal dalam Penguraian Karbofuran

Abstract

A study about the potency of local bacterial isolates capable of degrading carbofuran has been done. The aims of the study were: to obtain local bacterial isolates from North Sumatra capable of degrading carbofuran, an active compound of pesticide; to study the abilities of the isolates as immobilized cells as well as free cells in carbofuran degradation; to determine what enzyme involve in carbofuran degradation, to find out the best medium for biosurfactant production and to characterize the biosurfactant; and to partially characterize the enzyme. There were 17 isolated have been screened from agricultural land Berastagi and from Belawan Port. Based on the ability to grow on medium containing carbofuran as the sole carbon source and their biorfactant activities, 2 isolates were selected for further studies. Both isolates didn’t grow on Cetrimide Agar and Gram staining confirmed that they were positive Gram staining. Base on molecular analysis, their 16S rRNA, those isolates were Bacillus thuringiensis with percent identity of 99.66% while the other one was B. paranthracis with percent identity of 99.41%. Both strain of B. thuringiensis NF9 and B. paranthracis AB2 were immobilized in alginate and in polyurethane. Alginate beads showed better ability in trapping bacterial cells than that of polyurethane foam. The density of strain NF9 and strain AB2 were 6.60 x 1013 cells/g and 1.94 x 1013 cells/g of alginate respectively. Meanwhile the density of strain NF9 and AB2 were 1.25 x 1012 and 6.60 x 1012 cells/g of polyurethane respectively. As the result, cells which were immobilized within alginate beads had higher ability to degrade carbofuran than that of polyurethane. B. paranthracis AB2 which was immobilized within alginate beads was able to degrade carbofuran completely after 5 days of incubation while others required 15 days of incubation. Both strains NF9 and AB2 degraded carbofuran into carbofuran-7 phenol as the intermediate product. It means that they secreted carbofuran hydrolase in the initial step of carbofuran degradation. The next step was finding out the best medium for biosurfactant production. The combination of glycerol and natrium nitrate with ratio C/N of 20 and olive oil and ammonium nitrate with C/N ratio of 60 were the best medium for B. thuringiensis NF9 and B. paranthracis AB2 respectively to produce biosurfactant. The same medium compositions showed the highest biosurfactant activities for both strains of Bacillus. B. thuringiensis NF9 was able to reduce the surface tension up to 30.0 mN/m, while B. paranthracis AB2 even showed the lower surface tension which was 27.0 mN/m. Characterization of both biosurfactant was done through thin layer chromatography. The result showed that both strains had the same pattern. Both biosurfactants contained lipid and protein. Anthrone test which was used to detect the presence of sugar showed negative result for glucose and rhamnose content. There were other spots instead that weren’t related to those sugars. It was assumed that those spots didn’t belong to sugar groups since the distance were quite far from glucose or rhamnose. Liquid Chromatography Mass Spectra (LCMS) was done for further analysis. The result revealed that biosurfactant from the two strains had significantly different characters. Biosurfctant of strain NF9 consisted of 23 compounds which varied in structure as well as in molecular weight (119,164-851,496 Da). Meanwhile the biosurfactant from strain AB2 consisted of only 9 compounds which varied also in structure and molecular weight (119.164-753.928 Da). Other remarkable difference was the presence of a short chains of peptide. Biosurfactant of strain NF9 contained a heptapeptide with the sequence was Gly-Pro-Phe-Pro-Ile-Ile-Val. That peptide chain was not found in biosurfactant of strain AB2. Those results were astonishing. With limited number of compounds and in the absence of heptapeptide the biosurfactant showed better activity than NF9’s biosurfactant. Besides showing distinct characters, both biosurfactant exhibited some similarities. First, they have the same compound that obviously dominated among others. It was C16H35NO2 which was known as Hexadecanoic acid ammoniate or ammonium palmitate. The compound functioned as emulsifier and also as anticaking, prevent the solid materials such as salt or other powders from being clump when they were exposed to the atmosphere. In addition to ammonium palmitate, they shared four other compounds. One of them was indoline, the simplest compound within biosurfactant with m.w. of 119.64 Da. Indoline played important roles such as: as electron donor, as signal molecule among cells and regulated many features of bacterial cell physiology. The carbofuran hydrolase from previous result was partially characterized. The enzyme from both strains were studied intracellularly as well as extracellularly. The result showed that intracellular carbofuran hydrolase degraded carbofuran better than extracellular one. The enzyme was used to be studied further. Both strains had the same incubation time, 5 hours, which was the best time for the highest activities, which were 23.99 µmol/min and 10.18 µmol/min for strain NF9 and AB2 respectively. The optimum temperature for NF9’s hydrolase was 35°C with the activity of 36,04 µmol/min. increasing temperature up to 40°C decreased the activity by 50%. Meanwhile, strain AB2’s hydrolase resisted more against temperature. It worked best at 50oC with the activity of 46.89 µmol/min. pH optimum for the enzyme of NF9 and AB2 were 7.5-8 and pH 8 with the activities were 35.91 µmol/min and 39.45 µmol/min respectively. Addition of 1 mM metal such as: Zn, Mg, Mn, Ca, Fe, Cu, dan Co promoted enzyme activity. NF9’s hydrolase worked best when Fe was added, while the activity of hydrolase of AB2 showed the best when Mn was added. The presence of triton, tween, CHAPS and others exhibited varied effect. The addition of 0.1% of Triton, Tween dan CHAPS significantly inhibited the activity of NF9’s enzyme, while the addition of DTT promoted the activity up to 163% for strain NF9. Hydrolase’AB2 activity increased when SDS was added. The opposite effect happened when EDTA was added. It could be concluded that carbofuran hydrolase of the two strains had different characters regarding to temperature and pH optimum as well as the presence of some metals and inhibitors.