Materials and Reagents

1. Cyanobacteria
   Note: The cyanobacterium Synechocystis sp. PCC 6803 is grown at 30 °C in BG11 medium (Rippka et al., 1979), buffered with 20 mM TES-KOH (pH 8.0) or 20 mM CHES-KOH (pH 9.0) and bubbled with air for 24 h under continuous illumination using fluorescent lamps.
   
2. Ferric ammonium citrate (Wako Pure Chemical Industries, catalog number: 092-00802 )
   
3. Na₂EDTA·2H₂O (Wako Pure Chemical Industries, catalog number: 345-01865 )
   
4. Sodium nitrate (NaNO₃) (Wako Pure Chemical Industries, catalog number: 192-02555 )
   
5. Dipotassium hydrogenphosphate (K₂HPO₄) (Wako Pure Chemical Industries, catalog number: 164-04295 )
   
6. Magnesium sulfate (MgSO₄) (anhydrous) (Wako Pure Chemical Industries, catalog number: 132-00435 )
   
7. Calcium chloride (CaCl₂) (Wako Pure Chemical Industries, catalog number: 038-24985 )
   
8. Sodium carbonate (Na₂CO₃) (Wako Pure Chemical Industries, catalog number: 199-01585 )
   
9. Boric acid (H₃BO₄) (Wako Pure Chemical Industries, catalog number: 029-02191 )
   
10. Manganese(II) chloride tetrahydrate (MnCl₂·4H₂O) (Wako Pure Chemical Industries, catalog number: 139-00722 )
    
11. Zinc sulfate heptahydrate (ZnSO₄·7H₂O) (Wako Pure Chemical Industries, catalog number: 264-00402 )
    
12. Copper(II) sulfate pentahydrate (CuSO₄·5H₂O) (Wako Pure Chemical Industries, catalog number: 039-04412 )
    
13. Disodium molybdate(VI) dihydrate (Na₂MoO₄·2H₂O) (Wako Pure Chemical Industries, catalog number: 196-02472 )
    
14. Sulfuric acid (Wako Pure Chemical Industries, catalog number: 192-04696 )
    
15. Cobalt(II) nitrate hexahydrate (Co(NO₃)₂·6H₂O) (Wako Pure Chemical Industries, catalog number: 031-03752 )
    
16. TES (Wako Pure Chemical Industries,, catalog number: 340-02655 )
    
17. Potassium hydroxide (KOH) (Wako Pure Chemical Industries, catalog number: 168-21815 )
    
18. 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) (TCI, catalog number: D1328 )
    Note: 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) is an inhibitor of electron transport from QA to QB in photosystem II (PSII), thus oxidizing PQ pool under illumination. DCMU is used to determine maximum chlorophyll fluorescence level (Fm). We dissolve DCMU in ethanol (see below), and the stock solution at the concentration of 10 mM is added to the sample (equivalent to the final concentration of 20 μM) to determine the Fm level. DCMU solution can be stored for months in a freezer at -20 °C.
    
19. Ethanol (Wako Pure Chemical Industries, catalog number: 057-00456 )
    
20. BG11 stock solutions (see Recipes)
    
21. BG11 medium (see Recipes)
    
22. 10 mM DCMU solution in ethanol (see Recipes)
    

Equipment

1. Test tubes (IWAKI, catalog number: TEST30NP )
   
2. Spherical micro quantum sensor (Heinz Walz, model: US-SQS/L )
   Note: The light meter and the spherical micro-quantum sensor are used for measuring photon flux density of growth light and actinic light of WATER-PAM. It is advisable to use spherical micro-sensor for monitoring the photon flux density of actinic light of WATER-PAM, since the light illuminate samples from multiple directions.
   
3. Light meter (LI-COR, model: LI-250A )
   
4. Fluorometer (Heinz Walz, model: WATER-PAM )
   Note: WATER-PAM (Heinz Walz, http://www.walz.com/products/chl_p700/water-pam/introduction.html) is a pulse amplitude modulation (PAM) fluorometer designed for measuring aquatic samples with low chlorophyll content. We use the Red LED type of the fluorometer with the emitter-detector unit of CUVETTE Version. The Red LED type of WATER-PAM is equipped with 3 LEDs peaking at 650 nm for the measuring light, 12 LEDs peaking at 660 nm for the actinic light as well as for the saturating pulse, and 3 LEDs peaking at 460 nm for blue light, which preferentially excite photosystem I (PSI) in cyanobacteria.
   
5. Quartz cuvette for WATER-PAM (Heinz Walz, model: WATER-K )
   
6. Spectrophotometer (JASCO, model: V-650 )
   Note: The optical density of cell cultures at 750 nm is determined by the spectrophotometer. Any other common spectrophotometer can be used for this purpose.
   

Software

1. PC software ‘WinControl’ (WALZ, ver.3.22)
   Note: WATER-PAM is operated from the PC software‘WinControl’.
   

Procedure