蓝藻细菌中烷类的提取和量化

Hakuto Kageyama; Rungaroon Waditee-Sirisattha; Sophon Sirisattha; Yoshito Tanaka; Aparat Mahakhant; Teruhiro Takabe

doi:10.21769/BioProtoc.1684

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Peer-reviewed

Extraction and Quantification of Alkanes in Cyanobacteria

蓝藻细菌中烷类的提取和量化

HK Hakuto Kageyama

RW Rungaroon Waditee-Sirisattha

SS Sophon Sirisattha

YT Yoshito Tanaka

AM Aparat Mahakhant

TT Teruhiro Takabe email

发布: 2015年12月20日第5卷第24期 DOI: 10.21769/BioProtoc.1684 浏览次数: 8690

评审: Maria SinetovaClaudia CatalanottiAnonymous reviewer(s)

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Cover of Current Microbiology, featuring study using the protocol.

Jul 2015

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Abstract

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

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

Equipment

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

Software

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

Procedure

English

中文翻译

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如何引用

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.