蓝细菌黑暗、厌氧培养及其分泌物有机酸的HPLC定量检测

Chika  Yasuda; Hiroko  Iijima; Haruna  Sukigara; Takashi   Osanai

doi:10.21769/BioProtoc.2257

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Peer-reviewed

Incubation of Cyanobacteria under Dark, Anaerobic Conditions and Quantification of the Excreted Organic Acids by HPLC

蓝细菌黑暗、厌氧培养及其分泌物有机酸的HPLC定量检测

CY Chika Yasuda

HI Hiroko Iijima

HS Haruna Sukigara

Takashi Osanai email

发布: 2017年05月05日第7卷第9期 DOI: 10.21769/BioProtoc.2257 浏览次数: 8501

评审: Neelanjan BoseAnonymous reviewer(s)

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Abstract

Succinate and lactate are commodity chemicals used for producing bioplastics. Recently, it was found that such organic acids are excreted from cells of the unicellular cyanobacterium Synechocystis sp. PCC 6803 under dark, anaerobic conditions. To conduct the dark, anaerobic incubation, cells were concentrated within a vial that was then sealed with a butyl rubber cap, following which N₂ gas was introduced into the vial. The organic acids produced were quantified by high-performance liquid chromatography via post-labeling with bromothymol blue as a pH indicator. After separation by ion-exclusion chromatography, the organic acids were identified by comparing their retention time with that of standard solutions. These procedures allow researchers to quantify the organic acids produced by microorganisms, contributing to knowledge about the biology and biotechnology of cyanobacteria.

Keywords: Anaerobic condition (厌氧条件)

Synechocystis sp. (集胞藻属)

Background

Cyanobacteria are a group of bacteria that perform oxygenic photosynthesis. Synechocystis sp. PCC 6803 (hereafter Synechocystis 6803) is a non-nitrogen-fixing, unicellular cyanobacterium that is commonly used for basic and applied research studies. Synechocystis 6803 cells are able to excrete organic acids, such as succinate and lactate, under both dark and anaerobic conditions (Osanai et al., 2015). Genetic manipulation and modification of the incubation conditions, such as addition of potassium or NaHCO₃, have succeeded in increasing the levels of organic acids excreted from Synechocystis 6803 cells (Osanai et al., 2015; Hasunuma et al., 2016; Iijima et al., 2016; Ueda et al., 2016). Since succinate and lactate are commodity chemicals used to make various materials, such as bioplastics, their bio-based production is a desirable way to reduce the environmental burden. Herein, we describe the methods of Synechocystis 6803 incubation under dark, anaerobic conditions, and quantification of the organic acids by HPLC via post-labeling with bromothymol blue.

Materials and Reagents

50 ml disposable polypropylene tubes
Test tubes for cultivation (Iwaki, catalog number: TEST30NP )
20 ml headspace vials (GL Sciences, catalog number: 1030-46026 )
22 G x 1″ needle (Terumo Medical, catalog number: NN-2225R )
22 G x 1 ½″ needle (Terumo, catalog number: NN-2238S )
Aluminum foil
SupraPure hydrophilic PVDF syringe filters (Recenttec, catalog number: R7-PVDF033S022I )
Target DP vials (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: C4000-1 )
Screw caps (GL Sciences, catalog number: 1030-45261 )
300 µl target polyspring inserts (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: C4010-S630 )
Disposable syringes (Terumo, catalog number: SS-10LZ )
Open-top-style aluminum caps with butyl rubber septa (Systech, catalog number: 98552 )
MF-Millipore membrane filters (Merck Millipore, catalog number: HAWP03700 )
Cyanobacterium Synechocystis sp. PCC 6803
Ammonium chloride (NH₄Cl) (Wako Pure Chemical Industries, catalog number: 017-02995 )
60% perchloric acid (HClO₄) (Wako Pure Chemical Industries, catalog number: 160-05755 )
Trichloroacetic acid
Succinate
Lactate
Citric acid (Nacalai Tesque, catalog number: 09109-85 )
Acetate
Ferric ammonium citrate
Na₂EDTA (Nacalai Tesque, catalog number: 15111-45 )
Sodium nitrate (NaNO₃) (Wako Pure Chemical Industries, catalog number: 195-02545 )
Potassium phosphate dibasic (K₂HPO₄) (Wako Pure Chemical Industries, catalog number: 164-04295 )
Magnesium sulfate heptahydrate (MgSO₄·7H₂O) (Wako Pure Chemical Industries, catalog number: 131-00405 )
Calcium chloride dihydrate (CaCl₂·2H₂O) (Wako Pure Chemical Industries, catalog number: 031-00435 )
Sodium carbonate (Na₂CO₃) (Nacalai Tesque, catalog number: 31311-25 )
Boric acid (H₃BO₃) (Wako Pure Chemical Industries, catalog number: 021-02195 )
Manganese chloride tetrahydrate (MnCl₂·4H₂O) (Wako Pure Chemical Industries, catalog number: 139-00722 )
Zinc sulfate heptahydrate (ZnSO₄·7H₂O) (Wako Pure Chemical Industries, catalog number: 264-00402 )
Pentahydrate copper sulphate (CuSO₄·5H₂O) (Wako Pure Chemical Industries, catalog number: 039-04412 )
Sodium molybdate dehydrate (Na₂MoO₄·2H₂O) (Wako Pure Chemical Industries, catalog number: 196-02472 )
Sulfuric acid (H₂SO₄) (Wako Pure Chemical Industries, catalog number: 199-15995 )
Cobalt dinitrate hexahydrate, Co(NO₃)₂·5H₂O (Wako Pure Chemical Industries, catalog number: 031-03752 )
HEPES (Dojindo Molecular Technologies, catalog number: 342-01375 )
Potassium hydroxide (KOH) (Wako Pure Chemical Industries, catalog number: 168-21815 )
Bromothymol blue (BTB) (Wako Pure Chemical Industries, catalog number: 027-03052 )
Ethanol (Wako Pure Chemical Industries, catalog number: 057-00451 )
Disodium hydrogen phosphate dodecahydrate (Na₂HPO₄·12H₂O) (Wako Pure Chemical Industries, catalog number: 196-02835 )
Succinic acid (Wako Pure Chemical Industries, catalog number: 190-04332 )
DL-Lactic acid lithium salt (MP Biomedicals, catalog number: 100824 )
Sodium acetate (Wako Pure Chemical Industries, catalog number: 190-01071 )
Modified BG-11 liquid medium (see Recipes)
Mobile phase (3 mM HClO₄) (see Recipes)
Reaction solution (0.2 mM BTB in 15 mM Na₂HPO₄) (see Recipes)
500 mM succinate solution (see Recipes)
500 mM lactate solution (see Recipes)
500 mM citrate solution (see Recipes)
500 mM acetate solution (see Recipes)
1 M acetate solution (see Recipes)

Equipment

Cultivation chamber (TOMY Digital Biology, model: CLE-303 )
A gas mixer (KOFLOC, model: RK120XM )
An air pump (Yasunaga, model: LP-30A )
Spectrophotometer (Shimadzu, model: UV-2700 )
High-speed refrigerated microcentrifuge (TOMY Digital Biology, model: MX-305 )
N₂ generator (Sanyo Denshi, model: SN4-4 )
Rotary shaker (Nissin, model: NX-20D )
Freeze dryer (Tokyo Rikaikai, EYELA, model: FDU-2200 )
Ultrasonic cleaning machine (SND, model: US-108 )
Autoclave
The HPLC system (JASCO International, model: LC-2000Plus ) was composed of the following equipment:
1. Photodiode array (PDA) detector (JASCO International, model: MD-2018Plus )
2. Intelligent HPLC pump (JASCO International, model: PU-2080Plus )
3. Line degasser (JASCO International, model: DG-2080-53 )
4. Ternary gradient unit (JASCO International, model: LG-2080-02 )
5. Intelligent column oven (JASCO International, model: CO-2065Plus )
6. Intelligent autosampler (JASCO International, model: AS-2057Plus )
7. Ion-exclusion column (300 x 8.0 mm) (GL Sciences, model: RSpak KC-811, catalog number: 5055-13509 ) and guard column (10 x 4.6 mm) (GL Sciences, model: GPC KF-G, catalog number: 5055-13150 )
8. Reaction coil unit (JASCO International, model: RU-2080-51 )

Software

ChromNAV software (Ver. 1.14)

Procedure

English

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Yasuda, C., Iijima, H., Sukigara, H. and Osanai, T. (2017). Incubation of Cyanobacteria under Dark, Anaerobic Conditions and Quantification of the Excreted Organic Acids by HPLC. Bio-protocol 7(9): e2257. DOI: 10.21769/BioProtoc.2257.