Irda Sayuti1, Yusni Ikhwan Siregar1, Bintal Amin1,
Anthoni Agustien
2 and Akmal Djamaan3*

 1Department of Enviromental Science, University of Riau, Riau. Indonesia.
2Department of Biology, Faculty of Mathematic and Natural Science, University of Andalas, Padang-West Sumatra, Indonesia.
3Laboratory of BiotechnologyBiotan Sumatra/Faculty of Pharmacy, University of Andalas, Padang, West Sumatra, Indonesia.

Abstract

This research was conducted to identify the bacterial hydrocarbonclastic in waste tanks, Petapahan, Riau, Indonesia. Bacteria hydrocarbonoclastic are bacteria that have traffic in degrade petroleum. Analysis of 16S rRNA used the primers pair 63 Forward and Reverse 1387 produced 1300 bp. Results of the analysis of the 16Sr RNA sequence in bacterial isolates obtained that IMB-0909 was similar to Pseudomonas tollasii, IMB-10 was similar to Bacillus cereus IMB11 had similarities with Bacillus toyonensis strain BCT-7112, IMB 12 was Lysinobacillus fusiform strainNBRC15717, IMB 15 was Pseudomonas stutzeri strain ATCC 17588 entirely did have the ability to degradepetroleum.

Keywords: 16S RNA, hydrocarbonoclastic, bacteria, waste tank, petroleum

Introduction

As a source of energy, oil and gas have many benefits, it is quite efficient and economical as well as it exists is quite abundant, but when    it is spilled or lost to the environment, the oil  will be impurities that can become harmful pollutants. Petroleum includes hazardous materials and toxic waste. Petroleum pollution can come from drops and spills of petroleum during the activities of drilling, production, refining, and transportation of oil, resulting in the disruption in the balance  of ecosystems, land, water or sea1. One of the contaminants that are difficult to render is the hydrocarbon compounds derived from petroleum or petroleum sludge.These compounds can be toxic if accumulated in the cell.

Biodegradation of hydrocarbons such as petroleum compounds usually require the cooperation of more than one species of bacteria, it is because petroleum is formed from many different hydrocarbon compounds and bacteria can only use hydrocarbon in a specified range. The difference in the ability of the bacteriain the use of hydrocarbon compounds can be used to maximize the biodegradation process, therefore, the ability of a characterization of bacteria can degrade the hydrocarbon compounds became very important to do2,3

The process of degraded petroleum start from inoculated the bacteria sand take interaction with oil. Processing degradation was occure in aerobic conditions. Degradation petroleum in aerobic more quick than in aerobic conditions, because bacteria produce more energy compare anaerobe reactions4.  Interaction between microbe and hydrocarbon by adhetion or emulsification for eliminate anionic heteropolysacarade at wall cell or capsule. All of process ware activated by oxygenase enzyme.

Petroleum degradation be occurred at peripheral to transform hydrocarbon become intermediate compound such as acetyle CoA. CoA was going to come to three carbocsilatecycle and produce Co2 + H2O and energy for growth5,6.  Many factors take impact in petroleum degradation such as petroleum chemistry compounds, microbial community, temperature, oxygen, pH and nutrients.

MATERIAL AND METHODS

Genomic DNA Isolation
All bacteria samples had the ability to degrade petroleum waste, will be tested and analysis of the 16S rRNA(Figure 1). This method had to mixture all samples in the best liquid media. Nutrient Broth (NB) during the 16 hours at 28 ° C with 120 rpm. Genomic extraction using DNA purification KIT Wizard (Invitrogen) follows the KIT instruction: the first stage of Digestion: 1 ml bacterialsuspension1. Around of 5ml microtube put into the centrifuge bacterial suspension with a speed of 5000 rpm for 5 minutes. Add lysozyme digestion buffer as much as 180 µl vortex, incubation temperature 37oC for 30 minutes. Next add 20   µl proteinase K, vortex and then added 200 µl of Lysis buffer and then bindings genomic vortex and incubated for 30 minutes at a temperature of 55oC, absolute ethanol added as many as 200 µl and vortex.

The second stage of binding: put spin Klum into the tube, pour the liquid from the binding process into spin Klum and centrifuge with a speed of 10,000 rpm for 2 minutes. Then remove the collection tube, replace with a new one. The third stage of washing: Add 500 µl wash buffer I, centrifuge with a speed of 10.000 rpm for 2 minutes, then remove the collection tube, and replaced with a new one. Add 500 µl wash buffer 2, centrifuges with speed 12.000 rpm for 3 minutes. Then taken spin Klum, enter into a sterile 1.5 ml micro tube. The fourth stage Elusi: add 200 µl genomicelusion buffer, leave for 1 minute centrifuge with speed 12.000 rpm for 3 minutes, then remove spin Klum, next electrophoresis agarose gel with 1.5% and see in UV light.

DNA amplification

Amplification using 16 srRNA Primer from
Promega, stages that are conducted inaccordance with the instructions of the manufacturer of the kit Protocol.DNA had extracted (Eppendorf, Westbury, NY) for 15 ¼l of 100 minutes then added DNA rehydration (Promega). Amplification of the 16 S gener RNA had done in 50 µl containing 10 pmol 63f primer (5 ‘-AGAGTTTGATC (A/C) TGGCTCAG-3 ‘) and 1387r (5 ‘-GG (C/T) TACCTTGTTACGACTT-3 ‘).  Around of 25 µl green tag Promega, DNA template 3 µl (10 ng/µl), the addition of a nuclease-free water 20 µl of PCR Amplification cycle was 35. beginning with the denaturation temperature was 940C for 3, then proceed with stage 940C denaturation for 1 minute. 550C temperature Annealing lasted for 30 seconds, the expansions for 30 seconds at a temperature of 720C and end sat the end of the expansions at 720C for5seconds. PCR cycle lastsasmuc has 35cycles, 3 hours. 7 µl of PCR results are examined by gel electrophoresis. Visualization of results of PCR was performed on agarose electrophoresis 75grin 50 ml of 0,5TBE.

RESULTS AND DISCUSSION

Genomic DNA Isolation
The genomic DNA isolation using DNA isolation kit from INVITROGEN beginning from the taking of the pellet suspension of bacteria in culture liquid (Nutrient Broth) using the centrifuge. Isolation of DNA results can be seen from Figure 1 below.

Each sample of DNA isolation result showed the results good enough, this is because the isolation technique that is used is theisolation techniques already adopted from the isolation kit used, namely INVITROGEN. Samples of IMB and IMB-10 shows the results of isolation are not good, look at the picture that the very thin ribbon, possibly this is due to a number of pellets obtained from bacterial suspensions IMB and IMB-10 bit. The results of this DNA isolation continued to amplify with aPCR.

Amplified genomic DNA with primer 16s rRNA
Amplification Genomic DNA with the primer 16s rRNA Amplification using PCR machine Thermo. The PCR cycle run is 35cycles. The results of PCR with primer 63F and1387 R can be seen in Figure 2 below.

The results of PCR using primer16s rRNAcouples (63F and 1387R) indicates the length of the product PCR obtained i.e. 1,300 BP. Ribbon-Ribbon amplicons result from PCR primer pairs match showed 63F and 1387R against bacterial genome samples are used. The intensity of the PCR product obtained variation each sample, samples of IMB-9andIMB-10,this is due to the concentration of the temple used appropriate low, due to the thin insulation results (Figure 10).

Samples  IMB-09,  IMB-10,IMB-11, IMB-12,IM Band IMB-15show there sults of the nice amplicon and assertive. It also had the same correlations with genomic DNA isolation results obtained as a template on the activities of the PCR. This bacteria had capability to degrade compound in human body including aromatic hydrocarbon, inhibition mushroom fungi pathogens7.

Phylogenetic analysis shows that the IMB-10 sample was very similar to the 16s sequencerRNA with Bacillus cereus with valueNR 074540.1. Analysiswas done using Phylogenetic analysis shows rRNA with Bacillus cereus with valueNR 074540.1. Analysis using boosstrap 1,000. This figure states that the sequence stability rate will be estimated use this analysis. Bacillus cereus does haveability to degrade petroleum. The influence of oil contamination the earth in the water will change the response of the membrane bacterial cells thus affecting absorption and the need for bacterial oxygen, so influential to the degradation process8. Bacillus cereus a lot found in wastewater tanks at refinerie soil9.

Bacciluscereushadreported could degradation the phenolic compound in petroleum until 95% and more8.Bacillus cereus strainJMG-01inenhancedanthracenadegradation along the utilization of other hydrocarbons10.Another reported, bacillus cereus can remove contaminated petroleum in soil or about 30 days11. IMB 11 had similarities with Bacillus toyonensis strain BCT-7112. Bacillus toyonensishadbeenreportedthatitisabletosurviveinareas that are contaminated by petroleum.

Fig. 1. Genomic DNA isolation

Fig. 2. Results of PCR using primer pairs 63F and 1387R (M = Marker 1000bp; IMB (Isolates Petroleum)

Petroleum use as carbon sources12.Analysis of the sequence of the bases of a sample of IMB-11 with all isolates of bacteria from the Blast using a program bio edit and phylogenetic tree analysis using the program MEGA 6.The discovery two isolates that can be degrade petroleum and the two isolates utilized the source petroleum as the only source carbon, as for the two bacterial isolates13. Bacteria degrade petroleum and obtained type of bacteria 94% similarity for Bacillus thuringiensis and Bacillus bombysepticus, and  95%  similarity  for  Bacillus toyonensis BCT-711213,14.

This bacteria had been had researched to produce antimicrobial or antibiotics15. Research had been reported Bacillus toyonensis had capability animals probiotic16,17.

16srRNAsequenceofsamplesofIMB-12 had similarities with Lysinibacillus fusiform strain NBRC15717 obtained from the results of the blast. This bacteria was reported to be used to protect the hulls of the activities of biofouling17. From the results of research that had been done that the data obtained, a sample of IMB-12 has the ability to degrade petroleum of 62.61%. Data that had been retrieved it states that Lysinibacillus fusiform also had the ability to degrade the oil, which can be applied in the future to handlecases of environmental pollution due to oilspills.

Fig. 3. Phylogenetic tree sample IMB-09 using Mega 6

Fig. 4. Phylogenetic tree sample IMB-10 using Mega 6

The results of sequencing samples of IMB-15’s16srRNAsequence an alyzed using blast, obtained that samples of IMB-15 had similarities with Pseudomonas stutzeri strain ATCC 17588. Samples of the IMB-15 has the ability to degrade petroleum valued at 76.63%. Pseudomonas stutzeri had been reported to have the ability to degrade petroleum18. These bacteria were also reported to have the same capabilities19. Reported that this bacteria opportunity pathogen of human20.This bacteria have been reported in patients undergoing continuous ambulatory peritoneal dialysis (CAPD)21,22.

Fig. 5. Phylogenetic tree sample IMB-11 using Mega 6

Fig. 6. Phylogenetic tree Sample IMB-12 using Mega 6

Fig. 7. Phylogenetic tree sample IMB-15 using Mega 6

Over all according to the isolation and identification, bacteria degradation petroleum in waste tank show that bacteria isolate can obtain nutrient from hydrocarbon in waste tank via degradation process. This process come from hydrocarbon was degradated by peripheral pathway by oxygenase enzyme eto become a simple compounds like acetyleCoA. Five bacteria found in waste tank can surfive because their can degradate hydrocarbon becoming nutrition for the life. Microbial community had big impact for degrading petroleum.

CONCLUSIONS

Fragment of PCR results obtained 16SrRNAs were 1300bp. Blast shown samples  oftheIMB- 09 was similar to Pseudomonas tollasii, IMB- 10 was similar to Bacillus cereus, IMB-11 was similar to Bacillus toyonensis, IMB- 12 was similar to the sample and the fusiform Lysinibacillus fusiformis and IMB-15 Pseudomonas stutzeri. The all bacteria have the ability to degrade petroleum.

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