Nov 18, 2019

Public workspaceHigh Density Cultivation of Synechocystis sp. PCC 6803 using the HDC 6.10B system (CellDeg) V.2

Metabolic Engineering Communications
DOI
dx.doi.org/10.17504/protocols.io.9cgh2tw
  • 1Universität Rostock;
  • 2Department of Chemistry - Microbial Chemistry, Ångström Laboratory, Uppsala, Sweden
Oliver Mantovani
Icon indicating open access to content
QR code linking to this content
DOI: dx.doi.org/10.17504/protocols.io.9cgh2tw
External link: http://celldeg.com
Protocol Citation: Oliver Mantovani, Dennis DD Dienst, Pia Lindberg 2019. High Density Cultivation of Synechocystis sp. PCC 6803 using the HDC 6.10B system (CellDeg). protocols.io https://dx.doi.org/10.17504/protocols.io.9cgh2tw
Manuscript citation:
Dienst D., Wichmann J., Mantovani O., Rodrigues J., Lindberg P. (2019) High density cultivation for efficient sesquiterpenoid biosynthesis in Synechocystis sp. PCC 6803.
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 15, 2019
Last Modified: November 18, 2019
Protocol Integer ID: 29800
Keywords: CellDeg, photoautotrophic organisms, culturing, high-density, cyanobacteria, microalgae
Abstract
The CellDeg© 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 CO2, while a membrane in the lid (on top of the vessel) facilitated the passage of O2. By placing the vessels on top of a highly concentrated carbonate buffer reservoir with high CO2 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].

This protocol has been established in the Lindberg lab at Ångström laboratory (Uppsala University) for highly efficient sesquiterpenoid production with Synechocystis sp. PCC 6803 using a dodecane overlay as in situ extractant.
The protocol has been proven successful for small-scale screening procedures over time periods of up to one week.
[1] Bähr, L., Wüstenberg, A. & Ehwald, R. J Appl Phycol (2016) 28: 783. https://doi.org/10.1007/s10811-015-0614-5
Materials
MATERIALS
ReagentPotassium carbonateP212121
ReagentPotassium bicarbonateMerck MilliporeSigma (Sigma-Aldrich)Catalog #237205
HDC 6.10B Starter Kit (CellDeg), CD media (dx.doi.org/10.17504/protocols.io.2bxgapn)
Preparation of precultures
Preparation of precultures
Example: 6 well plate precultures




  • prepare standard polystyrene 6 well plates
→ each 3 wells per strain should be sufficient to inoculate 3 replicates in the Celldeg system

with strains of Synechocystis sp. PCC 6803 Amount3 mL
  • 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 antibiotics
  • place 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 Temperature30 °C
  • after ~ 4 days go to Step 2



Inoculation of Celldeg cultures
Inoculation of Celldeg cultures
Prepare experimental cultures from precultures

  • measure OD750 in a spectrophotometer (e.g. plate reader)
  • calculate the volume needed for inoculation of 8 mL Celldeg culture:
Note
Example:

  • measured OD750 of preculture = 1.2
  • desired OD750 in Celldeg vessel = 0.3
  • desired volume in Celldeg vessel = 8.0 mL





  • centrifuge preculture (e.g. 2.0 mL) for 5 min at 2500 g and room temperature Centrifigation2500 x g

including appropriate antibiotics Amount8 mL

  • if desired, supply cultures with inducer molecule
→ note that higher inducer concentrations might be required under HD conditions:
Note
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

  • transfer cell culture to CellDeg vessel

  • add 2 mL of dodecane to the cultures (only if in situ extraction is desired) Amount2 mL


Cultivation vessel with dodecane overlay

Note
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
Celldeg System Setup
Bicarbonate-carbonate buffer (reservoir preparation)

Ingredient Concentration (M) Concentration (g/L)
KHCO3 3 270.31
K2CO3 3 41.41
Ingredients of CellDeg carbonate buffer reservoir for a CO2 partial pressure of 90 mbar at 20°C
  • dissolve KHCO3 900 mL H20 Amount900 mL
  • dissolve K2CO3 100 mL H20 Amount100 mL

  • 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

Note
Make sure that the vessel is tightly closed before you stir and heat


  • as the solution doesn’t get in contact with the cultures, it doesn’t need to be sterilized

Vessel kit assembly

  • depending 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 3

  • attach the filled vessels from Step 2 to the tray on top of the reservoir


Celldeg Cultivation
Celldeg Cultivation
Cyanobacteria culturing

  • place 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 Temperature30 °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
Note
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.


  • the bicarbonate-carbonate buffer in the reservoir should be replaced after 4 days of culturing