TITLE: CD Media for High Density Cultivation of Synechocystis sp. PCC 6803
AUTHORS: Oliver Mantovani, Dennis Dienst

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">CellDeg's High Density Cultivation system allows for efficient accumulation of biomass of photosynthetic organisms, such as cyanobacteria and microalgae. The media requires a higher nutrient concentration, to facilitate high growth rates over extended periods. </div><div class = "text-block">This protocol describes the media composition used in the Lindberg lab at Ångström laboratory (Uppsala University). The basic composition was previously described in Lippi et al., [1], and has been slightly modified:</div><div class = "text-block"><ul style = "list-style-type:disc;"><li style = "counter-reset:ol0;list-style-type:disc;">different iron source used</li><li style = "counter-reset:ol0;list-style-type:disc;"><span>NaNO</span><span style = "vertical-align:sub;">3</span><span> as sole (major) nitrogen source (no additional KNO</span><span style = "vertical-align:sub;">3</span><span style = "vertical-align:sub;">)</span></li><li style = "counter-reset:ol0;list-style-type:disc;">no cobalt is added to the standard media </li><li style = "counter-reset:ol0;list-style-type:disc;">no copper is added to the standard media</li></ul></div><div class = "text-block">[1] Lippi L, Bähr L, Wüstenberg A, Wilde A, Steuer R: Exploring the potential of high-density cultivation of cyanobacteria for the production of cyanophycin. Algal Research 2018, 31:363-366</div></div>

[STEPS]
?. Prepare the following 8 stock solutions  ABCD1StockComponentsConcentrationStock concentration (g/L)2Stock AK2HPO44 M696.83Stock BMgSO4 + 7xH2O2 M493.04Stock CCaCl2 + 2xH20500 mM73.55Stock DFe-ammonium-citrate
(Ammonium iron(III) citrate)150 mM39.36Stock ENaNO35 M4257Stock FKHCO32.5 M250.48EDTANa2EDTA150 mM55.89Trace elementsH3BO325 mM1.5510MnCl2 + 4xH2O20 mM4.011ZnSO4 + 2xH2O2 mM0.5812Na2MoO4 + 2xH2O3 mM0.7313CuSO4 + 5xH2O20 µM0.000514Co(NO3)2 + 5xH2O60 µM0.016
ABCD1StockComponentsConcentrationStock concentration (g/L)2Stock AK2HPO44 M696.83Stock BMgSO4 + 7xH2O2 M493.04Stock CCaCl2 + 2xH20500 mM73.55Stock DFe-ammonium-citrate
(Ammonium iron(III) citrate)150 mM39.36Stock ENaNO35 M4257Stock FKHCO32.5 M250.48EDTANa2EDTA150 mM55.89Trace elementsH3BO325 mM1.5510MnCl2 + 4xH2O20 mM4.011ZnSO4 + 2xH2O2 mM0.5812Na2MoO4 + 2xH2O3 mM0.7313CuSO4 + 5xH2O20 µM0.000514Co(NO3)2 + 5xH2O60 µM0.016
If you are working with a promoter that is induced by metals (e.g. Zn2+, Cu2+, Co2+), you can omit that salt from the Trace elements mix and supply it from a sterile filtered stock solution on demand. Synechocystis 6803 can grow to high densities without any Cu2+ and Co2+ in the CD medium!
?. Preparation of 1 L CD mediumfill ~500 mL H2O into an autoclavable 1 L bottle        → do not mix the stock solution without excess H2O to prevent salt precipitationadd the following stock solutions before sterilizationStock A                  -      1 mLStock B                  -      1 mL    Stock C                  -      1 mLStock E                  -    10 mLEDTA                      -      1 mLTrace elements    -      1 mLadd ddH2O  to a final volume of 1 Lautoclave according to your standard procedure       → do not mix the stock solution without excess H2O to prevent salt precipitationadd the following stock solutions before sterilizationStock A                  -      1 mLStock B                  -      1 mL    Stock C                  -      1 mLStock E                  -    10 mLEDTA                      -      1 mLTrace elements    -      1 mLadd ddH2O  to a final volume of 1 Lautoclave according to your standard procedureStock A                  -      1 mLStock B                  -      1 mL    Stock C                  -      1 mLStock E                  -    10 mLEDTA                      -      1 mLTrace elements    -      1 mLadd ddH2O  to a final volume of 1 Lautoclave according to your standard procedure
autoclaving leads to a significant loss of media volume!        ⇒ Fill up the media with autoclaved ddH2O (work under the clean bench)the white precipitate will disappear upon addition of Stock F (Step 3)        ⇒ Fill up the media with autoclaved ddH2O (work under the clean bench)
?. Addition of stocks after autoclavingStock D                -      1 mL Stock F                 -     4 mL  → work under sterile conditions (clean bench)
Stock D contains Fe(III), which tends to precipitate during autoclaving  Stock  F contains HCO3- , which vaporizes under heat treatment