TITLE: High Density Cultivation of Synechocystis sp. PCC 6803 using the HDC 6.10B system (CellDeg)
AUTHORS: Oliver Mantovani, Dennis Dienst, Pia Lindberg

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>The </span><a href="http://celldeg.com" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">CellDeg©</span></a><span> high density cultivation system is a revolutionary culturing system for photoautotrophic microorganisms. The culturing vessels come with a semi-permeable membrane on the bottom that allows the diffusion of CO</span><span style = "vertical-align:sub;">2</span><span>, while a membrane in the lid (on top of the vessel) facilitated the passage of O</span><span style = "vertical-align:sub;">2</span><span>. By placing the vessels on top of a highly concentrated carbonate buffer reservoir with high CO</span><span style = "vertical-align:sub;">2</span><span> partial pressure - in combination with constant agitation, nutrient-rich media and high light intesities -  the system allows the obtainment of precedingly unparalleled cell densities [1]. </span></div><div class = "text-block"><span>This protocol has been established in the Lindberg lab at Ångström laboratory (Uppsala University) for highly efficient sesquiterpenoid production with </span><span style = "font-style:italic;">Synechocystis </span><span>sp. PCC 6803 using a dodecane overlay as</span><span style = "font-style:italic;"> in situ </span><span>extractant. </span></div><div class = "text-block">The protocol has been proven successful for small-scale screening procedures over time periods of up to one week.  </div><div class = "text-block">[1] Bähr, L., Wüstenberg, A. & Ehwald, R. J Appl Phycol (2016) 28: 783. https://doi.org/10.1007/s10811-015-0614-5</div></div>

[STEPS]
?. [Preparation of precultures]
Example: 6 well plate preculturesprepare standard polystyrene 6 well plates         → each 3 wells per strain should be sufficient to inoculate 3 replicates in the Celldeg systeminoculate 3 mL standard BG11 medium (dx.doi.org/10.17504/protocols.io.wj5fcq6)       with strains of Synechocystis sp. PCC 6803                                                                                                  if metal induction (e.g. Cu2+ and/or Co2+) is required in the experiment use BG11 w/ modified trace metal composition  → yes, they grow fine without these two trace elements :-)don't forget to add the appropriate antibioticsplace the 6 well plates on a standard orbital shaker, e.g.:     ‘Standard analog shaker, Model 5000 (VWR; orbit: 25 mm; frequency: 120 rpm) incubate at 30 °C under constant light intensities of 50-100 µmol photons ⋅ m−2 ⋅ s−1                     after ~ 4 days go to Step 2        → each 3 wells per strain should be sufficient to inoculate 3 replicates in the Celldeg systeminoculate 3 mL standard BG11 medium (dx.doi.org/10.17504/protocols.io.wj5fcq6)       with strains of Synechocystis sp. PCC 6803                                                                                                if metal induction (e.g. Cu2+ and/or Co2+) is required in the experiment use BG11 w/ modified trace metal composition  → yes, they grow fine without these two trace elements :-)don't forget to add the appropriate antibioticsplace the 6 well plates on a standard orbital shaker, e.g.:     ‘Standard analog shaker, Model 5000 (VWR; orbit: 25 mm; frequency: 120 rpm) incubate at 30 °C under constant light intensities of 50-100 µmol photons ⋅ m−2 ⋅ s−1                     after ~ 4 days go to Step 2don't forget to add the appropriate antibioticsplace the 6 well plates on a standard orbital shaker, e.g.:     ‘Standard analog shaker, Model 5000 (VWR; orbit: 25 mm; frequency: 120 rpm) incubate at 30 °C under constant light intensities of 50-100 µmol photons ⋅ m−2 ⋅ s−1                     after ~ 4 days go to Step 2    ‘Standard analog shaker, Model 5000 (VWR; orbit: 25 mm; frequency: 120 rpm) incubate at 30 °C under constant light intensities of 50-100 µmol photons ⋅ m−2 ⋅ s−1
3 ml
30 °C
?. [Inoculation of Celldeg cultures]
Prepare experimental cultures from preculturesmeasure OD750 in a spectrophotometer (e.g. plate reader)calculate the volume needed for inoculation of 8 mL Celldeg culture:  centrifuge preculture (e.g. 2.0 mL) for 5 min at 2500 g and room temperature                        resuspend pellets in 8 mL CD medium (dx.doi.org/10.17504/protocols.io.2bxgapn)       including appropriate antibiotics                                                                                                           if desired, supply cultures with inducer molecule       → note that higher inducer concentrations might be required under HD conditions:     transfer cell culture to CellDeg vessel centrifuge preculture (e.g. 2.0 mL) for 5 min at 2500 g and room temperature                      resuspend pellets in 8 mL CD medium (dx.doi.org/10.17504/protocols.io.2bxgapn)       including appropriate antibiotics                                                                                                         if desired, supply cultures with inducer molecule       → note that higher inducer concentrations might be required under HD conditions:     transfer cell culture to CellDeg vessel       → note that higher inducer concentrations might be required under HD conditions:    add 2 mL of dodecane to the cultures (only if in situ extraction is desired)
Example:measured OD750 of preculture = 1.2desired  OD750 in Celldeg vessel = 0.3desired volume in Celldeg vessel = 8.0 mLV(preculture)  = (0.3 * 8.0 mL)/1.2 = 2.0 mL
Centrifuge: 2500 34
8 ml
Example:for Cu2+-mediated induction of the PpetE  promoter: add    4 µM CuSO4 every second day for Co2+-mediated induction of the PcoaT  promoter: add 30 µM CoCl2 every day
2 ml
The CellDeg vials, since CO2 is provided from the bottom, do not need a large headspace for gas exchange, and can be filled generously (the ~25 mL vials can easily accommodate 10 mL cultures). If no dodecane overlay is added, 10 mL culture volume should be used to quantitatively minimize evaporation effects.
?. [Celldeg System Setup]
Bicarbonate-carbonate buffer (reservoir preparation)  ABC1  Ingredient    Concentration (M)    Concentration (g/L)  2KHCO3  3  270.313K2CO3  3  41.41dissolve KHCO3  900 mL H20                         dissolve K2CO3   100 mL H20                         due to the high final concentrations, gentle heating (≤ 40 °C) of the solutions can accelerate the complete dissolution of the salts (in particular KHCO3) you can easily up- or downscale the buffer amounts                    → the desired mixing ratio for 3M KHCO3: 3M K2CO3 is 9:1        due to the high final concentrations, heating of the solution can accelerate the           complete dissolution of the salts as the solution doesn’t get in contact with the cultures, it doesn’t need to be sterilizedyou can easily up- or downscale the buffer amounts                    → the desired mixing ratio for 3M KHCO3: 3M K2CO3 is 9:1        due to the high final concentrations, heating of the solution can accelerate the           complete dissolution of the salts as the solution doesn’t get in contact with the cultures, it doesn’t need to be sterilized        → the desired mixing ratio for 3M KHCO3: 3M K2CO3 is 9:1due to the high final concentrations, heating of the solution can accelerate the           complete dissolution of the salts as the solution doesn’t get in contact with the cultures, it doesn’t need to be sterilized       complete dissolution of the salts
ABC1  Ingredient    Concentration (M)    Concentration (g/L)  2KHCO3  3  270.313K2CO3  3  41.41
900 ml
100 ml
Make sure that the vessel is tightly closed before you stir and heat
?. [Celldeg System Setup]
Vessel kit assemblydepending on the size of the CellDeg system, the reservoir container has to be filled by 20% of the total volume by the concentrated carbonate solution      → fill a standard reservoir with 200 mL bicarbonate-carbonate buffer from step 3attach the filled vessels from Step 2 to the tray on top of the reservoir      → fill a standard reservoir with 200 mL bicarbonate-carbonate buffer from step 3
?. [Celldeg Cultivation]
Cyanobacteria culturingplace the assembled CellDeg system on an orbital shaker, e.g: IKA KS 130 basic orbital shaker (orbit ø = 4 mm)        → shake at 320 rpm Incubation Chamber: Versatile Environmental Test Chamber (Sanyo) w/o humidifier        → note that the light sources are laterally aligned  temperature: 30 °C                                                                                                                                              sequence of increasing light intensities: 250 µmol photons * m−2 * s−1 (0h-24h),      490 µmol photons * m−2 * s−1 (24h-48h), 750 µmol photons * m−2 * s−1 (tp 48h-xh)    → light intensities were measured with a Licor LI-185B quantum meter           → the values are sums of multi-directional measurements   the bicarbonate-carbonate buffer in the reservoir should be replaced after 4 days of culturing        → shake at 320 rpm Incubation Chamber: Versatile Environmental Test Chamber (Sanyo) w/o humidifier        → note that the light sources are laterally aligned  temperature: 30 °C                                                                                                                                              sequence of increasing light intensities: 250 µmol photons * m−2 * s−1 (0h-24h),      490 µmol photons * m−2 * s−1 (24h-48h), 750 µmol photons * m−2 * s−1 (tp 48h-xh)    → light intensities were measured with a Licor LI-185B quantum meter           → the values are sums of multi-directional measurements   the bicarbonate-carbonate buffer in the reservoir should be replaced after 4 days of culturing        → note that the light sources are laterally aligned  temperature: 30 °C                                                                                                                                            sequence of increasing light intensities: 250 µmol photons * m−2 * s−1 (0h-24h),      490 µmol photons * m−2 * s−1 (24h-48h), 750 µmol photons * m−2 * s−1 (tp 48h-xh)    → light intensities were measured with a Licor LI-185B quantum meter           → the values are sums of multi-directional measurements   the bicarbonate-carbonate buffer in the reservoir should be replaced after 4 days of culturing      490 µmol photons * m−2 * s−1 (24h-48h), 750 µmol photons * m−2 * s−1 (tp 48h-xh)    → light intensities were measured with a Licor LI-185B quantum meter           → the values are sums of multi-directional measurements
30 °C
Note:The incubation chamber used here is not optimized for this cultivation type and has an upper limit in light intensities.  Following the manufacturer's recommendations - particularly regarding the quality of the light source - should distinctly improve the yields.