Utilization POME as growth substrate for local indigenous bacteria Bacillus sp. ALP D1 in producing biosurfactant

Palm Oil Mill Effluent (POME) is a liquid palm oil waste that has beneficial value because of the nutritional content it contains. This research aim to obtain biosurfactant by using POME as a growth substrate. The methods used include biosurfactant production, extraction using acid precipitation, and characterization using TLC, FTIR, and LC-MS. The results showed that the local indigenous bacteria Bacillus sp. ALP D1 can produce biosurfactant under conditions of growth of POME 3%, NaNO3 3%, pH 6, salinity 7% with an IE24 of 69.44%, diameter of oil spreading test at 8.5 cm, and positive at drop collapse test. The extraction stage produces a brownish-yellow crude extract of 0.068 g/L. Biosurfactant characterization using TLC showed pink spots with ninhydrin reagent indicating the presence of nitrogen groups from peptide in the biosurfactant. This is supported by the results of FTIR analysis with the presence of an N-H peak from the peptide group in the absorption area 3384 cm-1. The results of the LC-MS analysis showed that the biosurfactant obtained had Rt 14.64 minutes and 332.22 m/z. Based on the results, it is suspected that the biosurfactant produced by Bacillus sp. ALP D1 is a lipopeptide.


Introduction
Biosurfactants are secondary metabolite products that can be produced by microorganisms [1].Several properties of biosurfactants, such as low toxicity, environmentally friendly, and high biodegradability, make them more attractive than synthetic surfactants [2].Biosurfactants are also widely applied in various industrial fields such as food, beverages, pharmaceuticals, cosmetics, and detergents [3].The other side of interest in the use of biosurfactants is the limited production of biosurfactants.This is because the raw materials for biosurfactant production are still relatively expensive.Therefore, researchers are looking for alternative production raw materials, one of which is by using agroindustrial waste [4].
POME (Palm Oil Mill Effluent) is one of the agro-industrial wastes used in the production of biosurfactants against Bacillus sp.ALP D1.The abundance of POME as palm oil industry waste in Indonesia and the cheaper economic value of POME compared to carbon sources from glucose and fructose make it the reason for it being used as a growth substrate for biosurfactant production in this research [5][6].
2 Previous researcher have reported the biosurfactant activity of the bacteria Bacillus sp.ALP D1 with an emulsification index (IE24) of 60.61%, using a 1% diesel as carbon source, pH 7, salinity of 0.01%, and incubation time of 48 hours [7].However, the report of Bacillus sp.ALP D1 is still produced using diesel carbon sources, which are non-renewable natural resources, without the addition a nitrogen source, which is also a source of nutrients needed to biosurfactants production, and the resulting biosurfactants have not been characterized further.
In this research, biosurfactant production from Bacillus sp.ALP D1 is carried out using POME raw materials as a carbon source, thereby supporting waste utilization.Initial studies have been carried out by adding the optimum parameters of nitrogen source (NaNO3), pH and salinity and obtained optimum conditions for POME production of 3%, NaNO3 3%, pH 6, salinity 7%, and production time of 72 hours.This research provides the opportunity for higher production results compared to previous biosurfactant production.Furthermore, characterization of the biosurfactant produced needs to be carried out to determine the type of biosurfactant, so that the biosurfactant produced can be applied according to its field.A total of 1000 μL of local ALP D1 bacteria was grown in 100 mL MSM medium containing that condition.Next, the bacterial culture was incubated at 30°C, a speed of 110 rpm using an incubator shaker.After that, the culture was centrifuged at 10,000 rpm for 20 minutes and then the supernatant obtained was tested for the emulsification index (IE24), oil spreading, and drop collapse test [8].

Emulsification Index (IE24).
The test was carried out by putting 2 mL of the supernatant that had been obtained into a test tube and adding 2 mL of benzene as a non-polar emulsion-forming substrate.Negative control using distilled water.The mixture was vortexed at high speed for 2 minutes then allowed to stand at room temperature for 24 hours and the emulsion height was measured [9].The emulsification index (IE24) is determined using the formula as in Equation ( 1): (1) 2.2.3.Oil spreading test.The oil spreading test is used to determine the biosurfactant activity of bacteria which was tested by observing the ability of bacteria to remove the coating oil on the surface of the water.The test was carried out using 30 mL of distilled water put in a clean petri dish on a flat place, then add 1 mL of used oil.After that, slowly add 20 μL culture supernatant in the middle of the used oil layer.Negative control use aquades.A positive test can be observed if a clear zone occurs due to elimination used oil coating by biosurfactants [10].Negative control use aquades.The supernatant droplets on the oil will be flat if contains biosurfactants [11].
2.2.5.Extraction of Biosurfactant.Extraction was carried out by acid precipitation method.The first stage is carried out by centrifuging the sample culture at 10,000 rpm for 20 minutes to remove cells.Biosurfactant was obtained by precipitation of cell-free supernatant after adding 6 N HCl to pH 2 and then incubating at 4 °C for 24 hours.Then, the biosurfactant obtained was then freeze dried and then weighed [12].The result of drying in the form of crude biosurfactant extract will be used in characterization.
2.2.6.Thin Layer Chromatography (TLC).The crude biosurfactant was separated on silica gel plates using chloroform, methanol, and water in the ratio (65:25:4, v/v/v) as the expanding solvent system with different color developer reagents.Spots formed were visualized with ninhydrin reagent (250 mg in 50 mL acetone).Furthermore, biosurfactant samples analyzed using a TLC plate were observed in UV light (254 nm, 366 nm), and the addition of ninhydrin reagent to detect the appearance of pink spots indicating a positive test or biosurfactant samples identified as lipopeptides [13].
2.2.7.Fourier Transform InfraRed (FTIR).Crude product of biosurfactant as much as 1 mg mixed with 100 mg KBr.Then the mixture is put into a pellet mold and then pressed using a hydraulic press.
Next, the sample is removed from the mold and analyzed using FTIR with a wave number of 400 to 4000 cm -1 [14].

Liquid Chromatography-Mass Spectrometry (LC-MS).
Preparation for lipopeptide analysis using LC-MS begins with 10 mL of the production supernatant acidified with 6 M HCl to pH 2. Next, stored for 24 hours at 4°C, the precipitate formed is collected via centrifugation (10,000 g, for 20 minutes) and then mixed with 10 ml of distilled water and added 1 M NaOH drop by drop until pH 7.
Then, extract the lipopeptides formed 2 times with a mixture of ethyl acetate and methanol (4: 1, v/v), and add anhydrous sodium sulfate to the phase.collected organics and then filter the organic solvent by evaporate it.
The lipopeptide extract was tested by LC-MS by dissolving it in 1 mL of methanol.A 5 μl sample was injected onto an Allureâ PFP Propyl column (50 x 2.1 mm, particle size 5 μm) maintained at 40°C.For sample separation, a mobile phase consisting of water (A) and methanol (B) supplemented with 2 mmol/L ammonium formate and 0.2% formic acid was used.Run time was 8 minutes with a solvent gradient starting at 60% B. After 1 minute, over the next minute B was increased to 100%, and maintained at 100% for an additional four minutes before returning to the initial solvent composition over 2 minutes.The flow rate is 600 ml min -1 [15].

Biosurfactant Production and Extraction
The results of biosurfactant production were measured for their activity by measuring the emulsification index (IE24), the oil spreading test, and the drop collapse test.The following is the result of the IE24 biosurfactant measurement from Bacillus sp.ALP D1 and biosurfactant activity can be seen by the appearance of the emulsion, which can be seen in Figure 1.In biosurfactant production, a positive test result for emulsification is indicated by the appearance of an emulsion whose emulsification index (IE24) is quantitatively measured at 69.44%, whereas in Figure 1b, a negative test result for biosurfactant the absence of emulsion indicates the absence of biosurfactant activity.Furthermore, the oil spreading test from Bacillus sp.ALP D1 can be seen in Figure 2a.The positive oil spreading test results revealed a clear zone of 8.5 cm as a result removal of used oil films by biosurfactants.Meanwhile, in Figure 2b, the negative control for the oil spreading test used distilled water, the results did not produce a clear zone on the surface of the used oil, which indicates there is no biosurfactant activity.Finally, the biosurfactant activity seen drop collapse test as can seen in Figure 3.The drop collapse test results are positive for biosurfactants if a flat surface is formed when the supernatant is dropped on used oil, as can be seen in Figure 3a.The similar results of other studies on isolates of Bacillus sp.34, 48, and 84 were dripped with used reveal oil that the positive biosurfactant test was characterized by flat and widened droplets [16].Biosurfactant extraction using acid precipitation followed by freeze drying to obtain a crude extract resulted in a brownish-yellow color of 0.068 g/L.

Characterization of Biosurfactant
The biosurfactant characterization stage is one of the significant stages of research carried out to identify whether the biosurfactant compound obtained is the same as the previous type of lipopeptide or not and determine the characteristics of the biosurfactant carried by using three methods, namely Thin Layer Chromatography (TLC), Fourier Transform InfraRed (FTIR), Liquid Chromatography-Mass Spectrometry (LC-MS).

Thin Layer Chromatography (TLC).
In this study, the principle of sample separation in TLC carried using methanol, chloroform and distilled water with a volume ratio of (65:25:4, v/v).Visualization results of biosurfactant testing from Bacillus sp.ALP D1 uses ninhydrin reagent, which can be seen in Figure 4. Based on the results of layer chromatography in Figure 4, the crude extract of biosurfactant from Bacillus sp.ALP D1 shows spots on the silica gel surface at wavelengths 254 and 366 nm.The biosurfactant compound forms a red color when sprayed with ninhydrin and placed in the oven at a temperature of 80 o C, which indicates that it contains free amino acids.This indicates the presence of biosurfactant compounds which belong to the lipopeptide group.The presence of spots that are not completely round in shape indicates that the biosurfactant compound is not yet pure and is still in the form of a crude extract so further purification is needed [17].
Based on the results of TLC analysis, it was obtained that biosurfactant was produced from the bacteria Bacillus sp.ALP D1 is lipopeptide type, to ensure the presence of amino functional groups and other functional groups in the biosurfactant, FTIR characterization was carried out.

Fourier Transform InfraRed (FTIR)
. FTIR is a characterization method used to identify all functional groups contained in biosurfactant samples from bacteria Bacillus sp.ALP D1.The following are the FTIR results of biosurfactants from bacteria Bacillus sp.ALP D1, can be seen in Figure 5. Based on Figure 5, several main absorption peaks were obtained, namely at wavelengths 3384 and 3280 cm -1 indicating the presence of an amino group obtained from the stretch between the N-H groups, 2922 cm -1 indicating the stretch of the -OH group from the -COOH (carboxyl) group, 1632 cm -1 resulting from the stretching of the CO-N group, and 1543 cm -1 resulting from the aliphatic carbon chain -C-H.The results of the FTIR spectrum obtained from the bacteria Bacillus sp.ALP D1 indicates that the biosurfactant obtained is a lipopeptide type.
The results of FTIR spectrum obtained from the bacteria Bacillus sp.ALP D1 was compared with several references to identify the type of biosurfactant obtained.Following are several comparisons of the results of the FTIR spectrum of biosurfactants, which can seen in Table 1.The molecular weight was processed using the Masslynx application and identified with the structural formula C15H29N3O5 which is thought to be a low molecular weight of lipopeptide biosurfactant.Based on molecular weight, biosurfactant divided into two groups, namely low molecular weight and high molecular weight [21].Some researchers reported that high molecular weight lipopeptide biosurfactant from the bacterium Bacillus licheniformis with the surfactin type appeared at a retention time of 21.22 minutes and a high molecular weight of 1058 m/z [20].Meanwhile, low molecular weight lipopeptide biosurfactant from Bacillus thrungiensis bacteria is thulomycin with a low molecular weight of 349.2545 m/z [22].The value obtained is similar to that obtained from Bacillus sp.ALP D1.It is suspected that the biosurfactant obtained is a type of low molecular weight lipopeptide.

Conclusion
Biosurfactant of bacteria Bacillus sp.ALP D1 obtained under conditions of 3% POME concentration, 72 hours production time, 3% NaNO3 concentration, pH 6, and 7% salinity produced biosurfactant activity in the emulsification test of 69.44%, the diameter of the oil spreading is 8.5 cm, flat and widened surfaces in the drop collapse test.The crude extract produced is brownish-yellow color with a weight of 0.068 g/L.The characterization results in TLC expressed as pink spots using ninhydrin reagent.FTIR characterization of biosurfactants showed absorption peaks of 3384 and 3280 cm -1 indicating the presence of amino groups obtained from the stretch between the N-H groups, and 1632 cm -1 resulting from the stretching of the CO-N group indicating the presence amide in lipopeptide structure.LC-MS characterization using ESI(+) appeared at a retention time of 14.64 minutes and a molecular weight of 332.22 m/z.Based on the results of the characterization of biosurfactants from Bacillus sp.ALP D1 can indicated as a biosurfactant a type of low molecular weight lipopeptide.

Figure 2a .
Figure 2a.Positive test result for oil spreading bacteria Bacillus sp.ALP D1 Figure 2b.Negative control of oil spreading bacteria Bacillus sp.ALP D1

Figure 3a .
Figure 3a.Positive test result for drop collapse bacteria Bacillus sp.ALP D1 Figure 3b.Negative control of drop collapse bacteria Bacillus sp.ALP D1

7 3. 2 . 3 .
Liquid Chromatography-Mass Spectrometry (LC-MS).Characterization of biosurfactants using LC-MS was carried out to support data from the results of the TLC and FTIR tests carried out on lipopeptide biosurfactant compounds.Following are the absorption peaks produced by LC-MS for the retention time shown in Figure6and the molecular weight in Figure7of the bacteria Bacillus sp.ALP D1.

Table 1 .
Comparison of FTIR Biosurfactant Spectrum Result