Assessment of petroleum biodegradation for Bacillus toyonensis by the using redox indicator 2,6 dichlorophenol indophenol

Petroleum degrading microorganisms have been isolated from different environments with the purpose of being used in bioremediation processes in areas impacted by petroleum spills. The objective of this study was to evaluate the ability of Bacillus toyonensis AM07 strain to metabolize petroleum compounds. The strain was isolated from the effluent dike of the Urucu Petroleum Province, Coari - Amazonas, Brazil. The degrading activity of B. toyonensis was evaluated by the colorimetric method using the redox indicator 2,6-dichlorophenol indophenol (DCPIP). Thus, the microorganism was inoculated into minimal medium with DCPIP, and with petroleum as the sole carbon source. The degradation potential of the microorganism was found by changing the DCPIP staining and absorbance readings 600 nm . The results obtained demonstrated that the bacterial strain was able to degrade petroleum by altering the color of the medium from blue to colorless and by reducing the concentration of the indicator in the absorbance readings. B. toyonensis AM07 strain has shown good performance in the petroleum degradation assays and may be used in the future in remediation technologies for hydrocarbon impacted environments.


Introduction
The microorganisms capable of degrading petroleum have been isolated from different environments with the objective of being used in bioremediation processes of impacted areas (Olajire, & Essien, 2014;Das, & Chandran, 2011). The presence of hydrocarbons on the environment selects microbial populations able to use them as substrate for their maintenance and survival (Darsa, Thatheyus, & Ramya, 2014;Bujang, Ibrahim, & Eh Rak, 2013).
The species of the genus Bacillus have been reported as petroleum compounds degraders, such as pyrene, naphthalene and n-alkenes (Darsa et al., 2014;Sorkhoh, Ibrahim, Ghannoum, & Radwan, 1993). In this genus, a new specie was described by Jiménez et al. (2013a) as part of the phylogenetic group Bacillus cereus, named B. toyonensis. The group consists of Gram-positive, endospore-forming bacteria found in soils, covering the bacterial species B. cereus, Bacillus thuringiensis, Bacillus anthracis, Bacillus mycoides, Bacillus pseudomycoides, Bacillus weihenstephanensis, B. toyonensis and Bacillus cytotoxicus (Jiménez, Blanch, Tamames, & Rosselló-mora, 2013b). The B. toyonensis was isolated for the first time in Japan, in 1966, as B. cereus var. toyoi (BCT-7112 strain), and it has been used as probiotics in the animal nutrition since 1975, when it was officially approved by Japan´s Ministry of Agriculture, Forestry and Fisheries as Toyocerin ® (Jiménez et al., 2013a-b). Okaiyeto, Nwodo, Mabinya, and Okoh (2015) recorded the occurrence of B. toyonensis in samples of sediments from marine environment in South Africa. Adewale, Goh, Lim, and Ting (2015) isolated B. toyonensis, as an endophytic bacteria, from medicinal plants Mentha spicata, with highlighting commercial interest for Asian communities. However, the use of B. toyonensis as a petroleum degrading agent has been little explored.
The method of fast selection of microorganisms that degrade petroleum by using redox indicator DCPIP has been broadly accepted, once it detects an oxidation of NADH to NAD+ (Hanson, Desai, & Desai, 1993;Kubota, Koma, Matsumiya, Chung, & Kubo, 2008). In practice, DCPIP molecule act as electrons acceptor, receiving them from the process of oxidation, mediated by microorganisms which use hydrocarbons as substrate. A test is considered positive when DCPIP changes the original color, from blue (oxidized state) to colorless (reduced state), in the presence of hydrocarbons (Hanson et al., 1993;Bidoia, Montagnolli, & Lopes, 2010). The present study had the aim to assess the petroleum degrading potential by a strain of B. toyonensis, isolated from an effluent dike of Urucu Petrol Basin, Coari -Amazonas, Brazil, by using the technique of rapid selection with redox indicator DCPIP.

Material and methods
The research was performed with the strain of B. toyonensis isolated from samples of water from the dike effluent of Urucu Petrol Basin, located in the Municipality of Coari, Amazonas-Brazil. The bacterial strain AM07 is deposited in the culture collection of the Laboratory of Bacterial Genetics of Universidade Federal do Amazonas -UFAM, Manaus.

Enrichment and isolation
The water samples were enriched in 500 mL Erlenmeyer flasks, containing 90 mL of Bushnell Haas Broth -BH mineral medium (Difco ™) (magnesium sulphate 0.2 g L -1 , calcium chloride 0.02 g L -1 ; potassium nitrate 1.0 g L -1 , ferric chloride 0.05 g L -1 , monopotassium phosphate 1.0 g L -1 , diammonium hydrogen phosphate 1.0 g L -1 ), 10 ml of water, and 1% of petroleum as the only source of carbon. The vials were incubated at 30°C, and at 180 rpm per minute in an orbital shaker (Thermo Scientific MaxQ ™ 4000) for 21 days. The microorganism was isolated on plates containing BH agar medium plus petroleum. The pure culture was transferred to plates containing tryptone medium soybean (TSC) (Difco™) (15 g L -1 casein hydrolyzate; sodium chloride 5 g L -1 ; papaya soybean hydrolyzate 5 g L -1 ; 15 g L -1 agar). The petroleum used in the experiments came from the Coari Petroleum Province, and before being used, it was previously filtered in Millipore™ membranes of 0.22 μm.

Biodegradability test using the DCPIP redox indicator
The qualitative assay was performed in sterile multiwell (24 wells) microplates in triplicate. 1.5 mL of DCPIP [0.01 g L -1 BH] was added to each well, along with 10 μL of petroleum and 25 μL of bacterial inoculum previously cultured in nutrient broth medium (HIMEDIA) at 30°C for 12 hours, at 150 rpm in an orbital shaker (Thermo Scientific MaxQ™ 4000). The cells were centrifuged for 10 min, washed with saline solution (0.9%), and then standardized in a UV-VIS spectrophotometer (Thermo spectronic Biomate 3) at 600 nm O.D = 1 (10 9 cells mL -1 ). The control (1) was prepared with 1.5 mL of DCPIP and 2.5 μL of BH, the control (2) was prepared with 1.5 mL of DCPIP, 2.5 μL of BH, and 10 μL of petroleum. The plates were incubated at 30°C, and the evaluations were performed every 12 hours until the color of the medium changed, from blue (oxidized) to colorless (reduced), indicating a positive result (Hanson et al., 1993;Bidoia et al., 2010;Peixoto et al., 2017).

Quantitative analysis
The quantitative assay was performed in 250 mL Erlenmeyer flasks, in triplicate, incubated at 30°C at 180 rpm in an orbital shaker (Thermo Scientific MaxQ ™ 4000). Aliquots of 1 mL of the culture were collected to read the absorbance at 600 nm in a UV-VIS (Thermo spectronic Biomate 3) spectrophotometer at 24, 48 and 72 hours intervals. For this test, the DCPIP solution was prepared in the concentration of 1 g L -1 in sterile BH medium. The composition of this assay is shown in Table 1.
The quantitative analysis was performed by reading the absorbance, converted to mg of DCPIP, using the standard curve (R2 = 0.9998) (Figure 1)

Biochemical characterization of bacterial isolate
The bacterial isolated was characterized according to its morphology, tinctorial property, and then submitted to biochemical tests having as base Bergey's Manual of Determinative Bacteriology (Holt, Krieg, Sneath, Staley, & Williams, 1994).
Amplification, sequencing and phylogenetic analysis of rRNA 16S gene A bacterial pre-inoculum from pure culture was inoculated in 20 mL of nutrient broth (HIMEDIA™) at 35ºC, at 150 rpm overnight. The isolation and purification of DNA genomic was made based on extraction kit PureLink™ Genomic DNA Mini Kit, from Invitrogen™, using 3 mL of bacterial culture, according to the manufacturer´s instructions. The reaction of amplification was performed by using primers 530F (5ʹ-TGACTGACTGAGTGC CAGCMGCCGCGG-3´) and 1495R (5' TGACTGACTGAGAG CTCTACCTTGTTA-3). The total volume of the reaction was 25 μL having (2.5 μL MgCL 2 [25 mM], 2.5 μL dNTPs [2.5 mM]; 2.0 μL of each primer [5 pMol μL -1 ]; 0.3 μL of Taq DNA polymerase [5 U μL -1 ]; 2.5 μL of buffer 10X). The amplification took place during 35 cycles in Thermal Cycler, Veriti® 96-Well -Applied Biosystems. The thermal profile from the reaction of PCR constituted of denaturation of initial cycle at 95 o C per 1 minute; followed by 35 cycles to 95 o C for 40 seconds, annealing of primers at 58 o C for 40 seconds, and polymerization at 72 o C for 40 seconds (35 cycles); and at final extension step at 72 o C for 7 minutes. For the sequencing of amplicon, it was used the sequencing kit named BigDye® Terminator v3.1 in a capillary sequencer (model 3500 ABI PRISM ® Genetic Analyzer, Applied Biosystems). The sequences generated were treated in programs phred/phrap (Ewing, Hillier, Wendl, & Green, 1998) and CAP 3 available in http://www.biomol.unb.br/phph/, and then, the sequences were analyzed in the program BLASTn National Center for Biotechnology Information -NCBI. The phylogenetic analysis was undertaken based on the evolution distances calculated by algorithms Neighbor-Joining and Jukes-Cantor, by using the program MEGA 7.0. After the building of the phylogenetic tree, it is possible to determine the taxonomical position of the isolate in relation to the species whose corresponding sequences were obtained in NCBI (Kumar, Stecher, & Tamura, 2016;Jukes & Cantor, 1968;Felsenstein, 1985).

Results and discussion
The biodegradability test using DCPIP is a low complexity and low-cost test with proved efficiency by different authors, which highlight the important role of this indicator in the detection of microorganisms, such as fungi, bacteria and yeasts with the potential for degrading petroleum and derivatives (Hanson et al., 1993;Bidoia et al., 2010;Luz, et al., 2011;Mariano, Bonotto, De Angelis, Pirôllo, & Contiero, 2008). When selecting microorganisms with the purpose of using them in bioremediation processes, the DCPIP assay allows the confirmation of the degradation capacity, and in this study, the degradation activity of the bacterial strain AM07 can be observed in the qualitative test, after 16 hours of incubation (Figure 2). The quantitative test of the biodegradability of petroleum, carried out by absorbance readings, showed a reduction in the concentration of DCPIP due to the degrading activity of the bacterial strain AM07 after 24 hours of incubation ( Figure 3, Table 3). The controls were stable due to the lack of carbon source and/or bacterial inoculum. The results obtained represent a remarkable activity when compared to the results described in Varjani and Upasani, (2013). The authors tested the efficiency of the use of petroleum by 69 bacterial strains isolated from enrichment of soil samples. Among the isolates tested, fifteen strains discolored the medium, and six strains recorded the shortest discoloration time of the medium in 120 hours, out of a total of 144 hours. Afuwale and Modi (2012) found hydrocarbon degradation in seven of the 36 bacterial isolates tested, and two of these completely discolored the DCPIP containing medium. The bacterial isolate showed Gram-positive staining, endospore formation, cell diameter > 1 μm, hydrolysis in the starch tests, positive Voges-Proskauer reaction, motility, positive activity for catalase, and its growth patterns in blood agar presented the βhemolytic pattern. In view of the presented results, the microorganism was classified as B. cereus. To confirm the species, the 16S rRNA gene was sequenced, which classified the microorganism as belonging to B. toyonensis species with 99% identity and 100% coverage when compared to the B. toyonensis sequence (BCT-7112) available in the database from GenBank. The nucleotide sequence was deposited on GenBank as B. toyonensis AM07 under accession number KX686603. In order to confirm the taxonomic position of this strain, the phylogenetic analysis of the MEGA 7.0 program (Felsenstein, 1985;Kumar, et al., 2016;Saitou, & Nei 1987), aligning the 16S rRNA gene sequence of the strain isolated in this study, B.toyonensis AM07, along with eight other sequences of the 16S rRNA gene, obtained in Genbank, from different species belonging to the genus Bacillus. The phylogenetic analysis showed that the sequence belonging to the strain isolated in this study, KX686603 B. toyonensis AM07, grouped with robustness to the strain 972704713 in LN995772.1 B. toyonensis, presenting 100% bootstrap, when compared to the sequences of the other species belonging to the Group B. cereus, as shown in Figure 4.  Species belonging to the genus Bacillus isolated from different environments have often been described as degrading hydrocarbons (Darsa et al., 2014;Sorkhoh et al., 1993). Rajasekar et al. (2007) reported the biodegradation of diesel oil and manganese oxidation by B. cereus. Bidoia et al. (2010) verified the potential of isolated strain of B. subtilis in degrading lubricating oil, using the DCPIP colorimetric method. Liu, Liu, Ju, Li, and Yu (2016) isolated strains belonging to the species B. licheniformis that demonstrated their ability to degrade petroleum and produce biosurfactant. Kreischer and Silva (2017) isolated B. toyonensis from soil samples contaminated with agrochemicals and verified the production of biosurfactant. The production of biosurfactant by microorganisms is of paramount importance in the process of biodegradation, as it assists in emulsification, reducing recalcitrance and facilitating the oxidation of hydrocarbons. The present study carried out a preliminary analysis that verified by the colorimetric method with the redox indicator DCPIP the potential of B. toyonensis in degrading petroleum, however no previous records of the association of this related species degradation of this carbon source were found. Acta Scientiarum. Biological Sciences, v. 40, e35640, 2018

Conclusion
The present study demonstrated, through the colorimetric method with DCPIP indicator, the potential of the B. toyonensis AM07 strain isolated from an effluent dike, to use petroleum as a single source of carbon with the possibility of using this strain in remediation technologies of environments impacted with hydrocarbons.