V. DRAGUTINOVI] et al.: New Bacillus circulans Strain from Oil Shale, Food Technol. Biotechnol. 50 (1) 123–127 (2012) 123 ISSN 1330-9862 scientific note (FTB-2480) Characterisation of New Bacillus circulans Strain Isolated from Oil Shale Vesna Dragutinovi}1*, Miroslav M. Vrvi}2,3, Izabela Swiecicka4, Olga Cvetkovi}3, Tanja Beri}5 and Slavi{a Stankovi}5 1Institute of Medical Chemistry, Faculty of Medicine, University of Belgrade, Vi{egradska 26, RS-1000 Belgrade, Serbia 2Faculty of Chemistry, University of Belgrade, Studentski trg 16, RS-11000 Belgrade, Serbia 3Institute of Chemistry, Technology and Metallurgy, Center of Chemistry, University of Belgrade, Njego{eva 12, RS-11000 Belgrade, Serbia 4Department of Microbiology, Institute of Biology, University of Bialystok, PL-15-950 Bialystok, Poland 5Faculty of Biology, University of Belgrade, Studentski trg 16, RS-11000 Belgrade, Serbia Received: March 18, 2010 Accepted: January 18, 2011 Summary A new strain of Bacillus sp. was obtained during experiments of oil shale deminerali- zation, which were carried out in order to get ’pure’ organic matter (kerogen). The demi- neralization efficiency of newly isolated strain was found to be substantially higher in comparison with that of Bacillus circulans Jordan 1890. On the basis of the biochemical characteristics, protein patterns and fatty acid composition, as well as the whole genome profile and 16S rDNA sequencing, the new strain was identified as Bacillus circulans VD01. Key words: Bacillus circulans, oil shale, kerogen, siliceous bacteria B. circulans is a typical chemoorganoheterotrophic bac- Introduction terium using mono-, di- and polysaccharides and poly- hydroxylic alcohols as sources of carbon, energy and elec- Kerogen is a mixture of organic compounds. It re- trons. The efficiency in disintegrating silicates and alumi- presents an insoluble component of oil shale, a compact nosilicates is not equal in all strains of siliceous bacteria, sedimentary rock of homogenous fine-grained compo- and it varies depending on the nature of silicates and the sition. The preparation of pure and unaltered kerogen metabolic products of bacteria, primarily organic acids and concentrate is of geochemical interest (1). Investigations the mucous polysaccharide capsules. The activity of B. cir- of the kerogen chemical composition and structures gener- culans is attributed to its metabolites and their specific re- ally require preliminary isolation of natural organic ma- actions such as acidolysis, alkalysis and complexolysis (4). terial. The isolation of native kerogen is a difficult task due to its complex nature and insolubility, and the fact The aim of this study is to identify newly isolated that in sediments kerogen is mixed with large amounts strain of bacteria from Aleksinac oil shale (Serbia) that of diverse minerals, such as carbonates, silicates and py- exhibited very high demineralization efficiency. rite. Microbial depyritization by the action of chemolitho- autotrophic bacterium Acidithiobacillus ferrooxidans was Materials and Methods found to be quite efficient (2). Siliceous bacterium Bacil- lus circulans was also proposed for bacterial desilicifica- An unidentified strain was obtained from Aleksinac tion of the oil shale (3). oil shale and adapted to grow on modified Ashby’s me- *Corresponding author; Phone: ++381 11 360 7140; Fax: ++381 11 360 7058; E-mail: [email protected] 124 V. DRAGUTINOVI] et al.: New Bacillus circulans Strain from Oil Shale, Food Technol. Biotechnol. 50 (1) 123–127 (2012) dium (3), incubated at 30 °C for 15 days. The strain was cultures according to Gaviria Rivera and Priest (10). The activated by successive seeding and appropriate tests for DNA was digested with 30 U of NotI (MBI Fermentas, identification of Bacillus genus were performed (5). Ac- Vilnius, Lithuania) or 25 U of AscI (New England Bio- tivated culture of B. circulans Jordan 1890, received from Labs GmbH, Frankfurt, Germany) for 4 h. Pulsed field the Center of Microbiology, Sofia, Bulgaria, and adapted gel electrophoresis (PFGE) was performed using the to modified Ashby’s medium, was used for desilicifica- CHEF-DR II System (Bio-Rad Laboratories, Hercules, tion. CA, USA) at 14 oC and 5.3 V/cm, using 1.0 % agarose gel (Sigma-Aldrich) in 0.5×TBE (100 mM Tris, 100 mM Phenotypic analysis borate, 200 mM EDTA). For either NotIorAscI digestion, The morphology of cells was examined by Gram the electrophoresis was carried out for 22 h with pulse from 10 to 130 s. After staining with ethidium bromide staining and the presence of spores was determined 3 using Ziehl-Neelsen stain as previously described (5). In solution (1 mg/cm ), the gels were photographed under brief, culture grown in modified Ashby’s medium was UV light using the Gel Doc 2000 System (Bio-Rad Labo- subjected to high temperature (80 °C) for 10 min and ratories) and analysed with the Quantity One v. 4.1.1 pro- plated on solid modified Ashby’s medium. After over- gram (Bio-Rad). The total molecular mass of the genome night incubation at 37 °C, staining procedure was per- was determined by adding the fragments produced by formed. restriction enzymes, the sizes of which were determined by comparison to yeast marker, as described by Carlson Biochemical characteristics of the new strain et al.(11). Bacilli, fulfilling the morphology and general bio- SDS-PAGE of whole cell proteins chemical characteristics of genus Bacillus, were assayed using commercial kits API 20E and 50CHB (API System, Sodium dodecyl sulphate polyacrylamide gel elec- bioMérieux, Marcy l’Etoile, France) as described by Berke- trophoresis (SDS-PAGE) was performed as described by ley et al. (6). The strain was identified by analysing the Costas (12). Electrophoresis was performed at the con- test results (obtained according to the manufacturer’s stant current of 20 mA, using a 10 % separating gel (pH= protocol) with API Lab Plus software and named B. 8.8) anda5%stacking gel (pH=6.8). The mini gel electro- circulans VD01. phoresis equipment SE260 was purchased from Hoefer Scientific Instruments (San Francisco, CA, USA). Mole- DNA analysis cular mass standard (Mr=15–250 kDa) was purchased from GE Healthcare (Little Chalfont, UK). For DNA extraction, cells were grown for 24 h in nutrient medium (7) on a rotary shaker at 200 rpm and 30 °C. DNA was extracted and purified by the conven- Determination of fatty acid composition tional method (8). The purity and quality of each DNA The lyophilized cell fatty acids (composition of 100 preparation were determined by monitoring the ratio of mg in 5 mL of NaOH-CH3OH) were heated for 15 min absorbance at 260 nm to that at 280 nm (1.8 to 2.1) and at 100 oC. The saponified material was cooled and acid- the ratio of absorbance at 260 nm to that at 230 nm (1.8 ified to pH=2. Boron trifluoride-methanol (BF3–CH3OH) to 2.1). The DNA samples were thermally denaturated reagent was added to 4 mL of saponified material, and by setting the temperature from 60 to 100 °C; at a rate of the mixture was reheated at 100 °C for 5 min. The con- 1 °C per minute. The standard sample contained calf thy- tent of the tube was added to 10 cm3 of saturated sodi- mus DNA (SERVA GmbH, Heidelberg, Germany). The um chloride solution and the methyl esters were extract- changes in absorbance were measured at 260 nm. All de- ed twice with an equal volume of 1:4 trichlormethane/ terminations were done by absorbance spectrophoto- hexane (13). ® meter Gilford G-250 (Gilford Instrument Laboratories Determination of fatty acid composition of bacterial Inc., Oberlin, OH, USA). The guanine-plus-cytosine (G+C) biomass was performed on Agilent 6890 chromatograph composition of the samples was calculated as previously with Agilent 5973 mass detector (Agilent Technologies, described (9). Santa Clara, CA, USA). Amplification of 16S rDNA by PCR and sequence Bacterial desilicification determination The newly isolated strain of B. circulans was activat- In order to obtain 16S rDNA sequences, polymerase ed by growing on a modified Ashby’s medium. The ex- 3 chain reaction (PCR) was carried out using primers P116S periments were carried out in the 500-cm Erlenmeyer (5’-GAATCTTCCACAATGGACG-3’) and P216S (5’-TGA- flask with a solid/liquid medium ratio of 1:50 (by mass CGGGCGGTGTGTACAAG-3’) according to the follow- per volume), on a rotary shaker at 200 rpm on 30 °C for ing protocol: 94 °C for 2 min, 30 cycles at 94 °C for 30 s, 30 days. Every third day fresh medium was added to 50°Cfor30s,68°Cfor30sand72°Cfor10min.The the substrate. Several parameters, such as pH, the num- purified PCR fragments were sequenced with both pri- ber of microorganisams as well as the amount of resi- mers and compared to 16S rRNA gene sequences in the dual silicon dioxide, aluminium oxide and ferric oxide public database using BLAST (NCBI, Bethesda, MD, USA). in the substrate ash were monitored throughout the ex- periment. The number of microorganisms was deter- PFGE of genomic DNA mined by the method of serial dilutions on a potato The low melting point agarose gel (Sigma-Aldrich, dextrose agar plate (pH=7). St. Louis, MO, USA) embedded chromosomal DNA of At the end of the leaching period the samples were the newly isolated strain was prepared from 6-hour-old treated with distilled water and hydrochloric acid (1:1, V. DRAGUTINOVI] et al.: New Bacillus circulans Strain from Oil Shale, Food Technol. Biotechnol. 50 (1) 123–127 (2012) 125 by mass per volume). The solid residue was used to pre- B. circulans VD01 strain exhibits a unique chromo- pare the ash (800 °C) by the analysis of Si, Al and Fe by somal profile as it was detected with PFGE analysis (Fig. spectrophotometry on a Varian 475 atomic apsorption 1).