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Extraction and Quantification of Alkanes in Cyanobacteria

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Current Microbiology
Jul 2015



Many species of cyanobacteria accumulate alkanes in their cells. It has been reported that these cyanobacteria accumulate mainly 15 long carbon chain alkane, pentadecane (C15H32), or/and 17 long chain alkane, heptadecane (C17H36). Here we describe a protocol of our laboratory for extraction and quantification of cyanobacterial intracellular pentadecane and heptadecane. We have confirmed this protocol was applicable to at least three kinds of cyanobacteria, nitrogen-fixing filamentous cyanobacterium Anabaena sp. PCC7120, non-diazotrophic unicellular cyanobacterium Synechococcus elongatus PCC7942 and halotolerant unicellular cyanobacterium Aphanothece halophytica.

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)


  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


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


  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.
    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).
      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).


  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.


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.


  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.


许多种类的蓝细菌在其细胞中积累烷烃。 据报道,这些蓝细菌主要积累15个长碳链烷烃,十五烷(C 15 H 32),和/或17长链烷烃,十七烷(C 17 36)。 在这里我们描述了我们的实验室提取和定量的蓝藻细胞内十五烷和十七烷的协议。 我们已经证实该方案适用于至少三种蓝细菌,固氮的丝状蓝细菌鱼腥藻 PCC7120,非重氮营养单细胞蓝细菌细长聚球藻PCC7942和耐盐单细胞蓝细菌藻门藻。


  1. 取样管(1.5ml)(Ina-optika corporation,Ina Optica,目录号:CF-0150)
  2. 取样管(50ml)(Corning,Falcon ,目录号:352070)
  3. 玻璃小瓶(Shimadzu Scientific Instruments,目录号:GLC4010-17)
  4. 毛细管柱(15m,内径0.25mm)(Restek Corporation,型号:Rtx-1MS)
  5. 蓝藻( Anabaena sp。PCC7120, Synechococcus elongatus PCC7942和
  6. 硝酸钠(NaNO 3)(Sigma-Aldrich,目录号:28-3440-5)
  7. 磷酸氢二钾(K 2 HPO 4)(Sigma-Aldrich,目录号:24-5240-5)
  8. 硫酸镁七水合物(MgSO 4·7H 2 O·7H 2 O)(KANTO KAGAKU,目录号:25034-00)
  9. 氯化钙脱水物(CaCl 2·2H 2 O 2H 2 O)(Sigma-Aldrich,目录号:05-0590-5)
  10. 柠檬酸(KATAYAMA CHEMICAL,目录号:05-4930)
  11. 柠檬酸铁(Wako Pure Chemical Industries,Siyaku,目录号:097-00835)
  12. 无水乙二胺四乙酸二钠盐(EDTA-Na 2)(Sigma-Aldrich,目录号:09-1420-5)
  13. 碳酸钠(Na 2 CO 3)(Sigma-Aldrich,目录号:28-2180-5)
  14. 硼酸(H 3 BO 3)(KATAYAMA CHEMICAL,目录号:03-2900)
  15. 氯化锰(II)(MnCl 2)(KANTO KAGAKU,目录号:25061-00)
  16. 硫酸锌七水合物(ZnSO 4·7H 2 O 7H 2 O)(KATAYAMA CHEMICAL,目录号:37-0550)
  17. 将脱水钼酸钠(Na 2 MoO 4)2·2H 2 O)(KATAYAMA CHEMICAL,目录号:28- 3400)
  18. 硫酸铜(II)五水合物(CuSO 4+,5H 2 O)(KATAYAMA CHEMICAL,目录号:05-6220)
  19. 六水合硝酸钴(II)[Co(NO 3)2 sub] 6 6H 2 O](KATAYAMA CHEMICAL,目录号:05-5230)
  20. 氯化钠(NaCl)(KANTO KAGAKU,目录号:37144-00)
  21. 氯化钾(KCl)(Sigma-Aldrich,目录号:24-4290-5)
  22. 氯化镁六水合物(MgCl 2·6H 2 O·6H 2 O)(KANTO KAGAKU,目录号:25009-00)
  23. 甲醇(KANTO KAGAKU,目录号:25183-70)
  24. 十五烷(TCI America,目录号:S0287)
  25. 十七烷(TCI America,目录号:S0289)
  26. Anabaena sp。的BG-11培养基。 PCC7120和< em>细长聚球藻PCC7942(Stanier等人,1971)(参见Recipes)
  27. (Hibino et al。,1999)的BG-11培养基+ Turk岛盐溶液(0.5M NaCl)(参见Recipes)


  1. 玻璃锥形瓶(500ml尺寸)
  3. 恒温室[温度通过调节器(Panasonic,目录号:MCU-201 CPH2)设置为30℃(±0.1℃)]
  4. 荧光灯(光质量:自然白,总光通量:2,950lm)(Yodobashi Camera Co.,TOSHIBA,型号:FL40SN)
  5. 光谱仪(Shimadzu Scientific Instruments,型号:UV-160A)
  6. 离心机(50ml管)(KUBOTA公司,目录号:5911)
  7. 离心机(用于1.5ml管)(KUBOTA Corporation,目录号:3700)
  8. 真空蒸发器(TAITEC CORPORATION,型号:Vc-15s)
  9. 超声波仪(TAITEC CORPORATION,型号:VP-5s)
  10. 冰箱
  11. GC-MS(Shimadzu Scientific Instruments,目录号:GCMS-QP2010)
  12. 氦气


  1. GCMSsolution软件版本2.40(Shimadzu Scientific Instruments,目录号:225-13067-91)


  1. 蓝细菌的生长
    PCC7120和细长聚球藻PCC7942在玻璃锥形瓶中的液体BG-11培养基中在30℃下在恒定的白色荧光灯下以约40μE/m 2/s/s生长。恒温室。将培养物以105rpm摇动。在藻蚜藻的情况下,使用BG-11培养基加特克岛盐溶液(0.5M NaCl)代替BG-11培养基。通过用分光光度计测量730nm处的吸光度来监测蓝细菌细胞的生长 注意:
    1. 其他条件例如非生物胁迫条件(氮饥饿, 高盐度等)(Kageyama et al。,2015)。
    2. 在500ml大小的烧瓶中,培养物体积应小于200ml。

  2. 收获细胞
    1. 指数地生长的蓝藻细胞沉淀 在4℃下从50ml培养物中以4,300×g离心15分钟 (OD 730 =?0.6)。
    2. 在通过倾析弃去培养基后,将细胞悬浮在剩余的培养基(约1ml)中。
    3. 将细胞悬浮液转移到预称重的1.5ml取样管中
    4. 通过在4℃以22,000×g离心10分钟沉淀细胞,然后完全除去培养基。
    5. 在使用前将细胞储存在-80℃。

  3. 萃取烷烃
    1. 冷冻的细胞用真空蒸发器干燥
    2. 测量干细胞重量。
    3. 将甲醇加入干燥的细胞中 注意:我们添加了1毫升甲醇/20毫克干细胞。
    4. 通过超声处理破碎细胞(输出:7,总开启时间:6分钟) 注意:
      1. 在超声处理期间,样品在冰上,12次30秒,随后30秒的间隔冷却。
      2. 注意,在超声处理过程中,甲醇提取物不会扩散。
    5. 将样品在4℃下孵育过夜
    6. 通过在22,000×g离心10分钟沉淀细胞碎片 min,然后将上清液转移至新的1.5ml 管。该甲醇提取物含有烷烃 注意:准备提取物后,我们立即对提取物进行GC-MS分析。

  4. 用GC-MS检测烷烃
    1. 500μl甲醇提取物含有烷烃转移到为GC-MS设计的玻璃小瓶中
    2. 将样品进行GC-MS。以下是一种气体 烷烃(十五烷和十七烷)的色谱法使用a 毛细管柱RESTEK Rtx-1MS(15μm,内径0.25mm):
      1. 使用氦气作为载气,流速为1.3ml min -1 -1。
      2. 在入口温度为300℃下使用1:100分流*比的氦载气与柱注入8μl样品。
      3. 初始烘箱温度为100℃,3分钟。
      4. 以20℃/min的速率升温至320℃ *注意:应使用设计用于分析分析的玻璃插件。

  5. 烷烃定量
    对于十五烷和十七烷的定量,我们使用正宗的标准。将这些真实的烷烃标准品用甲醇稀释,然后进行GC-MS以制备标准曲线。通过使用通过GCMSsolution软件计算的峰面积制备标准曲线。使用上述方法的十五烷和十七烷的保留时间分别为?5.6和?7.0min(图1A)。为了鉴定蓝藻提取物中的十五烷和十七烷,将其在色谱图中峰的保留时间和产物峰的断裂模式与真实标准(图1和图2)进行比较。作为代表性数据,使用鱼腥藻显示PCC7120在7分钟时的峰的提取和断裂模式(图2)。为了定量蓝细菌中的十五烷和十七烷,通过GCMS MS软件计算通过GC-MS获得的目标峰的面积,然后使用可信标准的标准曲线测定浓度。如图1C所示,十七烷的标准曲线在0.005-0.5mg/ml的范围内是线性的。对于十五烷也获得类似的线

    图1.真实标准品的GC-MS图谱和标准曲线。显示十五烷和十七烷的色谱图(A),十七烷的断裂模式(B)和标准曲线(C) >

    <图>图2.A鱼腥藻的甲醇提取物的GC-MS图谱。 PCC7120。 A.色谱图显示主峰出现在7.0分钟。在7分钟时主峰的断裂模式类似于十七烷可靠标准的断裂模式(参见图1B)。


  1. Anabaena sp。的BG-11培养基。 PCC7120和< em>细长聚球藻PCC7942(Stanier等人,1971)</em> 制备储备溶液(将这些溶液高压灭菌) 储液#1:6.27g的K 2 HPO 4/200ml H 2 O 2 / 储液#2:15.0g的MgSO 4·7H 2 O 2/200ml H 2 O 2·br/> 储液#3:7.20g的CaCl 2·2H 2 O·2H 2 O/200ml H 2 O·br/> 储液#4:4.00g的Na 2 CO 3·3/200ml H 2 O·
    储液#5:1.20g柠檬酸+ 1.20g柠檬酸铁铵+ 0.20g EDTA 2Na/200ml H 2 O 2 / 储液#6:572mg的H 3 BO 3+ + 362mg的MnCl 2 + 44.4mg的ZnSO 4 > 7H 2 O + 78.0mg的Na 2 MoO 4 2H 2O + 15.8mg的CuSO 4·6H 2 O·5H 2 O + 9.9mg的Co(NO 3) 3 2 6H 2 O/200ml H 2 O
  2. (Hibino et al。,1999)的BG-11培养基+ Turk岛盐溶液(0.5M NaCl)。
    为了制备BG-11培养基加特克岛盐溶液(0.5M NaCl),28.17g NaCl,0.67g KCl,6.92g MgSO 4。将5.50g的MgCl 2·6H 2 O·6H 2 O和1.47g的CaCl 2·6H 2 O混合,在用H 2 O调节至1L之前加入H 2 O 2·2H 2 O 2。值得注意的是,蚜藻(Aphanothece halophytica)可以在从0.25到3.0M NaCl的宽范围的盐度中生长。




  1. Hibino,T.,Kaku,N.,Yoshizawa,T.,Takabe,T.and Takabe,T。(1999)。 DnaK的分子表征来自耐盐藻蓝藻Aphanothece halophytica 用于ATP酶,蛋白质折叠,在各种盐度条件下的铜结合。植物分子生物学 40(3):409-418。
  2. Kageyama,H.,Waditee-Sirisattha,R.,Sirisattha,S.,Tanaka,Y.,Mahakhant,A.和Takabe,T。 通过非生物胁迫改善氮固定和耐盐蓝藻中的烷烃生产烷烃合成基因的遗传操作 Curr Microbiol 71(1):115-120。
  3. Stanier,R.Y.,Kunisawa,R.,Mandel,M。和Cohen-Bazire,G。(1971)。 单细胞蓝绿藻的纯化和性质(Chroococcales订购)。 Bacterial Rev 35(2):171-205。
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引用:Kageyama, H., Waditee-Sirisattha, R., Sirisattha, S., Tanaka, Y., Mahakhant, A. and Takabe, T. (2015). Extraction and Quantification of Alkanes in Cyanobacteria. Bio-protocol 5(24): e1684. DOI: 10.21769/BioProtoc.1684.