Bacteria Isolation of Kerosene Contaminated Asphalt Waste (Asbuton)

Kerosene is a type of hydrocarbon commonly known as paraffin oil which is usually used as a home heating fuel, lamp and asphalt solvent. The presence of kerosene in the asphalt production can potentially cause environmental pollution, one of which affects soil conditions. Some bacteria that have specific ability to degrade hydrocarbon content in soil are called hydrocarbonoclastic bacteria. This study aims to determine the proportion of the presence of bacteria that have the potential to degrade hydrocarbons, in this case kerosene, in polluted soil at the location of one of the largest asphalt manufacturing companies in Indonesia and to determine the morphological characters of bacterial isolates. Based on the results of the study, Hydrocarbonoclastic bacteria isolated from asbuton had 5 dominant isolates and were identified by PCR analysis. The results of this study were that the five bacterial isolates were Bacillus sp. with 3 of them being Bacillus subtilis and 2 others being Bacillus cereus. This result has an Entiren stiffness of 99.62% - 100%. The type of bacteria Bacillus sp. are capable of reducing the Total Petroleum Hydrocarbon (TPH) and still able to survive and potentially degrade the TPH content in it.


Introduction
Kerosene is a colorless and flammable hydrocarbon liquid that can be produced from the fractional distillation of petroleum at temperatures between 145 -300 o C. Kerosene is also known as paraffin or paraffin oil which is mostly used as a heating fuel for homes, lamps, stoves and jets.In addition, kerosene is also used in asphalt mixtures [1].Some researchers such as [2], [3], [4] revealed that the presence of kerosene in the asphalt mixture will have an impact on the environment, one of which will affect soil conditions.Kerosene has low mobility and some immobility when released to the ground.However, after entering the soil, some of the components of the mixture are firmly attached to the soil [5].This makes kerosene very difficult to biodegrade.Petroleum that pollutes the soil can reach groundwater locations, lakes or water sources that provide water for domestic and industrial needs, so that it becomes a serious problem for areas that rely on groundwater as the main source of clean water life [6].Oil content that is difficult to decompose is hydrocarbon compounds.When these compounds contaminate the soil surface, these substances can evaporate, be washed away by rainwater and enter the soil and then settle as toxic substances and result in disrupting the soil ecosystem and the water cycle [7].
Pollution due to the burning of petroleum has caused serious environmental problems and requires proper management to prevent sustainable environmental damage.The most widely used methods for managing oil-polluted environments are chemical and physical.This method is very effective for shortterm goals, but requires a lot of money and has a negative impact on the environment and ecosystem.Therefore, an environmentally friendly environmental management method is needed, namely biologically, one of which is by utilizing microbes such as bacteria.The characteristic of this bacteria is that it has the ability to utilize carbon compounds as a source carbon and energy needed for its growth, so that it can survive in an environment polluted by petroleum.The degradation process using microbial assistance is relatively easier, low cost and environmentally friendly.Bacterial isolates that have the ability to degrade carbon have previously been found as many as 9 isolates originating from oil and fuel oil dumps in Cilegon, Banten.Bacillus sp.ICBB 7859 and ICBB 9461 are able to reduce Total Petroleum Hydrocarbon (TPH) used oil to below 1% for six weeks, and Bacillus sp.ICBB 5071 is able to reduce the TPH of used oil to below 1% for five weeks [8].Several other researchers have reported on the isolation of bacteria used in degrading carbon in petroleum polluted environments such as waters and soil around oil well areas.However, this bacterium can also be isolated in contaminated soil at automotive repair sites.
Based on the description above, this study aims to determine the proportion of the presence of bacteria that have the potential to degrade hydrocarbons, in this case kerosene, in polluted soil at the location of one of the largest asphalt manufacturing companies in Indonesia and to determine the morphological characters of bacterial isolates.So that later these bacteria will be used as bioremediation of kerosene contaminated soil.

Sample and Media Preparation
The sample used is asbuton waste which has gone through an attrition process with an efficiency percentage of 75% and a TPH concentration in the sample of 58,476 ppm.The sample will be mixed into the growing media.Preparation of growing media, namely Nutrient Agar (NA) (Merck, Germany) and 85% NaCL (Merck, Germany), was then followed by a sterilization process in an autoclave (Hirayama,Japan) at 121 o C for 20 minutes.

Bacterial Isolation
The bacterial isolation process begins with weighing 5 grams of asbuton samples into 85% NaCl and then homogenization using an incubator shaker (Biosan, Latvia) at 150 rpm at 37 o C for 1 hour.Samples were worked on three times to get accurate results.After the homogenization process, the bacterial isolation process was carried out in laminar air flow (Airtech, Japan), starting with dilution from 10 -1 to 10 -4 .The results of the diluted sample were poured into the NA (Merck, Germany) media in the petri dish and the Spread Plate Method was carried out to spread the bacteria across the surface of the media.The NA (Merck, Germany) that had been added to the bacteria was then incubated for 24 hours at 37 o C.

TPC Calculation Process
Calculation of the number of microorganisms is an indicator of the process of biodegradation of hydrocarbons.The number of microorganisms will increase if they are able to live by utilizing the existing substrates in these hydrocarbon compounds.The basis for the calculation is by diluting the suspension with a certain dilution range in stages according to the Colony Forming Unit (CFU) method.The CFU method uses Spread Plate Method [8].

Morphological Analysis Process
Morphological analysis was carried out by observing the characteristics of bacteria macroscopically (TPC, shape, color, bacterial colony margins, colony size) and microscopically (using a microscope and gram staining).

PCR Analysis
The results of pure bacterial isolates that have been obtained are then sent to a third party, namely PT.Genetics Science for PCR analysis to obtain specific types of bacteria.The method used is from the culture results, bacterial colonies are taken and placed in a 1.5 mL tube containing 200 μL Tris EDTA (TE) with pH 8.The tube is then placed in a 100 o C water bath for 10 minutes.Next, the tube was placed in ice and centrifuged (Eba 200 Hettich, Germany) at 8000 rpm for 1 minute.100 μL of the supernatant was taken and stored at -80 o C until ready to use.

1 TPC Analysis Results
The number of bacterial colonies that can be counted using this method ranges from 30 -300 microbes per mL. the results of the TPC calculation from the asbuton samples are shown in Figure 7.

Figure 7. TPC Analysis
The results of the TPC calculation illustrate that the highest number of bacteria in the asbuton sample is at T4 and the lowest number of bacteria is at point T1.If the average of the total bacteria from all points is 5.787 log CFU/g.The increase in the number of bacteria indicates the presence of metabolic processes of microorganisms.The results of screening intermediate compounds showed that there was a process of oxidation of hydrocarbon substrates into simpler compounds and the use of substrates by Bacillus subtilis so that it was suspected that there was a role for catabolic enzymes namely alkane hydroxylase and dioxygenase in the process of degradation of hydrocarbons by Bacillus subtilis.The increase in the number of bacterial colonies that occurs is a logarithmic phase because the bacteria have adapted to oil so they can use the main carbon source, namely kerosene oil, for their growth.Therefore, bacteria can reproduce cells so that the total number of bacterial cells increases [10].The greater the rate of increase in the number of bacterial colonies, the faster the hydrocarbon biodegradation process in the oil.

2 Morphological Analysis of Bacterial Isolates
The halo zone or halo area that is formed can indicate the activity of the lipase enzyme.The decrease in fat content is caused by hydrolysis into free fatty acids and glycerol.The acid formed can break down complex oil components into simpler components.These free fatty acids are easily damaged, thereby reducing fat levels.Bacteria that can form halo areas are bacterial colonies that are thought to be able to degrade hydrocarbons in petroleum-contaminated soils.The larger the halo area produced by a culture, the greater the suspicion that the culture is a hydrocarbon degrading microorganism.Bacterial colonies that form halo areas are bacteria colonies that are tolerant to polluted environments.These bacterial TPC Analysis colonies may be able to degrade hydrocarbons in oil-contaminated soils.Soil is able to support the growth of hydrocarbon degrading bacteria and is known to increase the activity of hydrocarbon degradation due to its ability to bind oxygen.Oxygen (O2) is the most widely used electron receptor in degrading hydrocarbons.Hydrocarbons are broken down more rapidly under aerobic conditions because they help enhance the performance of degrading bacteria.Hydrocarbon degrading bacteria are widely distributed in seas, fresh waters and soil as their place of life.The number of hydrocarbon-degrading bacteria has a positive correlation with the hydrocarbon content of the environment.Oil-degrading bacteria experience very rapid growth and the number of cells is greater in oil-contaminated soil than in non-oil-contaminated soil.Biodegradation by bacteria can occur due to the activity of enzymes that are owned by each bacteria.Through enzymatic processes, bacteria can transform hydrocarbon substances into simpler forms that can be absorbed by bacteria as nutrients for their growth.Oil-degrading bacteria experience very rapid growth and the number of cells is greater in oil-contaminated soil than in non-oilcontaminated soil.The types of bacteria that can survive in high hydrocarbon conditions in this study sample are shown in Table 1 and Figure 8.Based on the results of PCR analysis by PT.Genetics Science, it is known that the five bacterial isolates analyzed were Bacillus sp. with 2 of them being Bacillus cereus and the other 3 being Bacillus subtilis.These PCR results reported that Bacillus sp.ICBB 7859 and ICBB 9461 are capable of reducing the Total Petroleum Hydrocarbon (TPH) of used oil to below 1% for six weeks, and Bacillus sp.ICBB 5071 is able to reduce the TPH of used oil to below 1% for five weeks so that at TPH levels which are still 58,476 ppm, the type of bacteria Bacillus sp.still able to survive and potentially degrade the TPH content in it.

Conclution
Hydrocarbonoclastic bacteria isolated from asbuton had 5 dominant isolates and were identified by PCR analysis.The results of this study were that the five bacterial isolates were Bacillus sp. with 3 of them being Bacillus subtilis and 2 others being Bacillus cereus.These PCR results reported that Bacillus sp.ICBB 7859 and ICBB 9461 are capable of reducing the Total Petroleum Hydrocarbon (TPH) of used oil to below 1% for six weeks, and Bacillus sp.ICBB 5071 is able to reduce the TPH of used oil to below 1% for five weeks so that at TPH levels which are still 58,476 ppm.This result has an Entiren stiffness of 99.62% -100%.The type of bacteria Bacillus sp. are capable of reducing the Total Petroleum Hydrocarbon (TPH) and still able to survive and potentially degrade the TPH content in it.

Figure 1 .
Figure 1.NA media preparation to regrowth bacteria

Figure 2 .
Figure 2. NaCl media preparation for bacterial dilution

Figure 3 .Figure 4 .
Figure 3. Dilution to analyze the number of colony bacteria