Extraction and Quantification of Alkanes in Cyanobacteria

Materials and Reagents

1. Sampling tubes (1.5 ml) (Ina-optika corporation, Ina Optica, catalog number: CF-0150 )
   
2. Sampling tubes (50 ml) (Corning, Falcon^®, catalog number: 352070 )
   
3. Glass vial (Shimadzu Scientific Instruments, catalog number: GLC4010-17 )
   
4. Capillary column (15 m, 0.25 mm internal diameter) (Restek Corporation, model: Rtx-1MS )
   
5. Cyanobacteria (Anabaena sp. PCC7120, Synechococcus elongatus PCC7942 and Aphanothece halophytica)
   
6. Sodium nitrate (NaNO₃) (Sigma-Aldrich, catalog number: 28-3440-5 )
   Note: Currently, it is “Sigma-Aldrich, catalog number: 28-3440 ”.
   
7. Potassium phosphate dibasic (K₂HPO₄) (Sigma-Aldrich, catalog number: 24-5240-5 )
   Note: Currently, it is “Sigma-Aldrich, catalog number: 24-5240 ”.
   
8. Magnesium sulfate heptahydrate (MgSO₄^.7H₂O) (KANTO KAGAKU, catalog number: 25034-00 )
   
9. Calcium chloride dihydrate (CaCl₂^.2H₂O) (Sigma-Aldrich, catalog number: 05-0590-5 )
   Note: Currently, it is “Sigma-Aldrich, catalog number: 05-0590 ”.
   
10. Citric acid (KATAYAMA CHEMICAL, catalog number: 05-4930 )
    Note: Currently, it is “Sigma-Aldrich, catalog number: 05-4930 ”.
    
11. Ferric ammonium citrate (Wako Pure Chemical Industries, Siyaku, catalog number: 097-00835 )
    
12. Ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA-Na₂) (Sigma-Aldrich, catalog number: 09-1420-5 )
    
13. Sodium carbonate (Na₂CO₃) (Sigma-Aldrich, catalog number: 28-2180-5 )
    
14. Boric acid (H₃BO₃) (KATAYAMA CHEMICAL, catalog number: 03-2900 )
    Note: Currently, it is “Sigma-Aldrich, catalog number: 03-2900 ”.
    
15. Manganese(II) chloride (MnCl₂) (KANTO KAGAKU, catalog number: 25061-00 )
    
16. Zinc sulfate heptahydrate (ZnSO₄^.7H₂O) (KATAYAMA CHEMICAL, catalog number: 37-0550 )
    Note: Currently, it is “Sigma-Aldrich, catalog number: 37-0550 ”.
    
17. Sodium molybdate dihydrate (Na₂MoO₄^.2H₂O) (KATAYAMA CHEMICAL, catalog number: 28-3400 )
    Note: Currently, it is “Sigma-Aldrich, catalog number: 28-3400 ”.
    
18. Copper(II) sulfate pentahydrate (CuSO₄^.5H₂O) (KATAYAMA CHEMICAL, catalog number: 05-6220 )
    Note: Currently, it is “Sigma-Aldrich, catalog number: 05-6220 ”.
    
19. Cobalt(II) nitrate hexahydrate [Co(NO₃)₂^.6H₂O] (KATAYAMA CHEMICAL, catalog number: 05-5230 )
    Note: Currently, it is “Sigma-Aldrich, catalog number: 05-5230 ”.
    
20. Sodium chloride (NaCl) (KANTO KAGAKU, catalog number: 37144-00 )
    
21. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: 24-4290-5 )
    
22. Magnesium chloride hexahydrate (MgCl₂^.6H₂O) (KANTO KAGAKU, catalog number: 25009-00 )
    
23. Methanol (KANTO KAGAKU, catalog number: 25183-70 )
    
24. Pentadecane (TCI America, catalog number: S0287 )
    
25. Heptadecane (TCI America, catalog number: S0289 )
    
26. BG-11 medium for Anabaena sp. PCC7120 and Synechococcus elongatus PCC7942 (Stanier et al., 1971) (see Recipes)
    
27. BG-11 medium plus Turk Island salt solution (0.5 M NaCl) for Aphanothece halophytica (Hibino et al., 1999) (see Recipes)
    

Equipment

1. Glass conical flask (500 ml size)
   
2. Shaker (TAITEC CORPORATION, model: NR-3 )
   
3. Thermostatic chamber [Temperature was set to 30 °C (±0.1 °C) by the regulator (Panasonic, catalog number: MCU-201 CPH2 )]
   
4. Fluorescent lamp (light quality: natural white, total luminous flux: 2,950 lm) (Yodobashi Camera Co., TOSHIBA, model: FL40SN )
   
5. Spectrometer (Shimadzu Scientific Instruments, model: UV-160A )
   
6. Centrifuge (for 50 ml tubes) (KUBOTA Corporation, catalog number: 5911 )
   
7. Centrifuge (for 1.5 ml tubes) (KUBOTA Corporation, catalog number: 3700 )
   
8. Vacuum evaporator (TAITEC CORPORATION, model: Vc-15s )
   
9. Sonicator (TAITEC CORPORATION, model: VP-5s )
   
10. Fridge
    
11. GC-MS (Shimadzu Scientific Instruments, catalog number: GCMS-QP2010 )
    
12. Helium gas
    

Software

1. GCMSsolution software version 2.40 (Shimadzu Scientific Instruments, catalog number: 225-13067-91)

Procedure

1. Growth of cyanobacteria
   Anabaena sp. PCC7120 and Synechococcus elongatus PCC7942 were grown in liquid BG-11 media in glass conical flasks at 30 °C under constant white fluorescent light at approximately 40 μE/m²/s in thermostatic chamber. The cultures were shaken at 105 rpm. In the case of Aphanothece halophytica, BG-11 medium plus Turk Island salt solution (0.5 M NaCl) was used instead of BG-11 medium. The growth of cyanobacterial cells was monitored by measuring the absorbance at 730 nm with a spectrophotometer.
   Notes:
   
   1. Other conditions such as abiotic stress condition (nitrogen starvation, high salinity, etc.) were applicable (Kageyama et al., 2015).
      
   2. Culture volume should be less than 200 ml in 500 ml size flask.
   
   
2. Harvesting cells
   
   1. Cyanobacterial cells grown exponentially were precipitated by centrifugation at 4,300 x g for 15 min at 4 °C from 50 ml cultures (OD₇₃₀=~0.6).
      
   2. After discarding media by decantation, the cells were suspended in the remaining media (~1 ml).
      
   3. Cell suspensions were transferred to preweighed 1.5 ml sampling tubes.
      
   4. Cells were precipitated by centrifugation at 22,000 x g for 10 min at 4 °C, then media were removed completely.
      
   5. Cells were stored at -80 °C before use.
   
   
3. Extraction of alkanes
   
   1. Frozen cells were dried up using a vacuum evaporator.
      
   2. Dry cell weight was measured.
      Note: Usually ~20 mg of dried cells was obtained.
      
   3. Methanol was added to dried cells.
      Note: We added 1 ml of Methanol/20 mg of dried cells.
      
   4. Cells were disrupted by sonication (output: 7, total on time: 6 min).
      Notes:
      
      1. Samples were on ice during sonication treatment with 12 burst of 30 sec followed by intervals of 30 sec for cooling.
         
      2. Be careful that the methanol extract does not scatter during sonication treatment.
   5. Samples were incubated at 4 °C for overnight.
      
   6. Cell debris was precipitated by centrifugation at 22,000 x g for 10 min at 25 °C, then the supernatants were transferred into new 1.5 ml tubes. This methanol extract contained alkanes.
      Note: After preparing the extracts, we subjected extracts to GC-MS analysis immediately.
      
   
   
4. Detection of alkanes by GC-MS
   
   1. 500 μl of methanol extracts contain alkanes were transferred into glass vials designed for GC-MS.
      
   2. Samples were subjected to GC-MS. The following is a gas chromatography method for alkanes (pentadecane and heptadecane) using a capillary column RESTEK Rtx-1MS (15 m, 0.25 mm internal diameter):
      
      1. Helium was used as the carrier gas at the flow rate of 1.3 ml min^-1.
         
      2. 8 μl of samples were injected using a 1:100 split* ratio of helium carrier gas to column at 300 °C of inlet temperature.
         
      3. Initial oven temperature of 100 °C for 3 min.
         
      4. Ramp to 320 °C at a rate of 20 °C/min.
         *Note: Glass insert designed for split analysis should be used.
   
   
5. Quantification of alkanes
   For Quantification of pentadecane and heptadecane, we used authentic standards. These authentic standards of alkane were diluted with methanol, and then subjected to GC-MS to make standard curves. Standard curves were made by using the peak area calculated by GCMSsolution software. Retention time of pentadecane and heptadecane using the method described above was ~5.6 and ~7.0 min, respectively (Figure 1A). For identification of pentadecane and heptadecane in cyanobacterial extracts, their retention times of the peaks in chromatogram and fragmentation patterns of product peaks were compared with authentic standards (Figure 1 and Figure 2). As a representative data, chromatogram using Anabaena sp. PCC7120 extract and fragmentation pattern of the peak at 7 min were shown (Figure 2). For quantification of pentadecane and heptadecane in cyanobacteria, area of the target peak obtained by GC-MS was calculated by GCMSsolution software, and then concentration was determined using standard curve of authentic standards. As shown in Figure 1C, standard curve of heptadecane was linear at the range of 0.005 - 0.5 mg/ml. A similar line was also obtained for pentadecane.
   
   
   Figure 1. GC-MS profile and standard curve of authentic standards. Chromatogram of pentadecane and heptadecane (A), and fragmentation pattern (B) and standard curve (C) of heptadecane are shown.
   
   
   Figure 2. GC-MS profile of methanol extract of Anabaena sp. PCC7120. A. Chromatogram indicated the main peak appeared at 7.0 min. B. Fragmentation pattern of the main peak at 7 min was similar to that of heptadecane authentic standard (see Figure 1B).
   

Recipes

1. BG-11 medium for Anabaena sp. PCC7120 and Synechococcus elongatus PCC7942 (Stanier et al., 1971)
   Preparation of stock solutions (These solutions were autoclaved.)
   Stock #1: 6.27 g of K₂HPO₄/200 ml H₂O
   Stock #2: 15.0 g of MgSO₄^.7H₂O/200 ml H₂O
   Stock #3: 7.20 g of CaCl₂^.2H₂O/200 ml H₂O
   Stock #4: 4.00 g of Na₂CO₃ / 200 ml H₂O
   Stock #5: 1.20 g of citric acid + 1.20 g of Ferric ammonium citrate + 0.20 g of EDTA 2Na/200 ml H₂O
   Stock #6: 572 mg of H₃BO₃ + 362 mg of MnCl₂ + 44.4 mg of ZnSO₄^.7H₂O + 78.0 mg of Na₂MoO₄^.2H₂O + 15.8 mg of CuSO₄^.5H₂O + 9.9 mg of Co(NO₃)₂^.6H₂O/200 ml H₂O
   For preparation of BG-11 medium, firstly 1.5 g of NaNO₃ was dissolved in H₂O (~700 ml). Then the stock solutions above were added 1 ml each. Final volume was adjusted to 1 L with H₂O. This medium was distributed into flasks and autoclaved for culturing cyanobacteria, Anabaena sp. PCC7120 and Synechococcus elongatus PCC7942.
   
2. BG-11 medium plus Turk Island salt solution (0.5 M NaCl) for Aphanothece halophytica (Hibino et al., 1999)
   For preparation of BG-11 medium plus Turk Island salt solution (0.5 M NaCl), 28.17 g of NaCl, 0.67 g of KCl, 6.92 g of MgSO₄^.7H₂O, 5.50 g of MgCl₂^.6H₂O and 1.47 g of CaCl₂^.2H₂O were added before adjusted to 1 L with H₂O. It is noted that Aphanothece halophytica can grow in a wide range of salinity from 0.25 to 3.0 M NaCl.
   

Acknowledgments

This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan and the International Research Center for Natural Environmental Science of Meijo University.

References

1. Hibino, T., Kaku, N., Yoshizawa, T., Takabe, T. and Takabe, T. (1999). Molecular characterization of DnaK from the halotolerant cyanobacterium Aphanothece halophytica for ATPase, protein folding, copperbinding under various salinity conditions. Plant Mol Biol 40(3): 409-418.
   
2. Kageyama, H., Waditee-Sirisattha, R., Sirisattha, S., Tanaka, Y., Mahakhant, A. and Takabe, T. (2015). Improved alkane production in nitrogen-fixing and halotolerant Cyanobacteria via abiotic stresses and genetic manipulation of alkane synthetic genes. Curr Microbiol 71(1): 115-120.
   
3. Stanier, R. Y., Kunisawa, R., Mandel, M. and Cohen-Bazire, G. (1971). Purification and properties of unicellular blue-green algae (order Chroococcales). Bacterial Rev 35(2): 171-205.