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Two-tier vessel for photoautotrophic high-density cultures


Two-tier vessels, developed for culturing of microalgae and cyanobacteria at high cell density on a shaken platform, were assembled from a flat lower chamber to be filled with a CO2 buffer and an upper flat sterile chamber for the culture that was separated from the lower chamber by a porous polypropylene membrane. Diffusive gas exchange with the atmosphere was controlled by the O2 outlet channel. Referred to surface area, rates of CO2 transfer to a shaken weakly alkaline buffer solution across the membrane were higher than those reached on the conventional pathway through the free upper liquid surface. Membrane-mediated CO2 supply enabled rapid growth of Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002 up to ultrahigh cell density. The biomass (dry weight) concentration of Synechococcus cultures reached more than 30 g L−1 on a buffered medium with adequate concentrations of mineral nutrients. An increase of 15 to 20 g L−1 was observed during repeated two-day cycles. Separate pathways for CO2 supply and oxygen outlet prevented significant loss of CO2. Convective gas flow through the oxygen outlet channel enabled the estimation of the O2 generation rate. The permeability of the channel for diffusive O2/N2 exchange limited the O2 concentration to a moderate value. It is concluded that shaken flat cultures using CO2 supply through a porous hydrophobic membrane and diffusive release of O2 through a separate pathway are promising for research on microalgae and cyanobacteria.

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Authors are grateful to Prof. Dr. T. Buckhout and Dr. R. Steuer, Institute of Biology, Humboldt-University, for improving the language and critical comments.

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Correspondence to Rudolf Ehwald.



Appendix 1: Maximum O2 concentration in the culture vessel as dependent on the rate of oxygen production and the design of the oxygen outlet channel

The maximum rate of convective O2 flow through the channel (r o ) obtained with Synechococcus cultures of ultrahigh cell density at a PFD of 900 μmol photons m−2 s−1 was 1.3 mm3 s−1. The chosen cross sectional area (A = 3.14 mm2) and maximum length (l = 10 mm) of the oxygen outlet channel ensured that the gas volume flux (J v  = r o /A = 0.36 mm s−1) through the channel obtained at an even higher rate (r o  = 2 mm3s−1) was smaller than the mean velocity (permeability) of O2 diffusion in the gas flowing through the channel (P = D/l = 2.2 mm s−1). Under this condition the rate of O2 loss from the culture chamber (r l ) can be regarded as the sum of a convective loss rate (r c ) and a diffusive loss rate (r d ).

$$ {r}_l={r}_d+{r}_c $$

When O2 concentrations are expressed as volume fraction of O2 in the gas phase, the rate of diffusional loss of O2 by exchange with N2, (r d ), depends on the concentration difference (∆C) across the oxygen outlet channel by

$$ {r}_d=\varDelta C\kern0.5em A\left(D/l\hbox{--} {J}_v\right)=\varDelta C\kern0.5em A\left(D/l\hbox{--} {r}_o/A\right) $$

The O2 concentration in the culture chamber (C) and the mean concentration in the O2 outlet channel \( \left(\overline{C}\right) \) may be derived from the concentration in the atmosphere (0.2) and the concentration difference across the channel:

$$ C = 0.2 + \varDelta C $$
$$ \overline{C} = 0.2 + \varDelta C/2 $$

The rate of convective O2 loss, r c , is the product of the volume flow rate (r o ) and the mean O2 concentration \( \left(\overline{C}\right) \) within the channel.

$$ {r}_c = {r}_o\left(0.2+\varDelta C/2\right) $$

When the O2 concentration in the gas phase of both chambers is still increasing at a given value of r o ; r l is smaller than r o . A stationary maximum value of the O2 concentration in the culture chamber (C’) is reached when r l approximates to r 0:

$$ {r}_l = \varDelta C'A\left(D/l\hbox{--} {r}_o/A\right) + {r}_0\left(0.2 + \varDelta C'/ 2\right)={r}_o $$

C’ can be obtained by combining Eq. (3) with Eq. (7):

$$ C' = 0.2 + 0.8\kern0.5em {r}_o{\left( AD/l\kern0.15em \hbox{--} \kern0.15em 0.5\ {r}_o\right)}^{\hbox{-} 1} $$

Using Eq. (8), stationary O2 concentrations at rates of O2 generation r o  < 2 AD/l can be derived from the diffusion coefficient of O2 in air D (0.22 cm2s−1) and outlet dimensions A and l.

Appendix 2: Effect of CO2 consumption in the culture chamber on the CO2 buffer in the basal chamber

In the experiments documented in Fig. 7 and Table 2 the DWC of Synechococcus sp. strain PCC 7002 increased by 15 to 20 g L−1 in a volume of 20 mL during a batch cycle. In the culture vessel used (Fig. 1c), two culture chambers were combined with one basal chamber containing a CO2 buffer obtained by mixing 3 M KHCO3 with 3 M K2CO3 in the ratio 9:1 (540 mmol HCO3 and 60 mmol CO3 in 200 mL). If the DWC increased by 20 g L−1 in both culture chambers (culture volume 40 mL), 800 mg of dry biomass were produced. Assuming a carbon content of 50 % in the dry mass (Fontes et al. 1989), this required transfer of c. 400 mg C or 33 mmol CO2 from the basal chamber to the culture chamber. According to the reaction 2 HCO3 ↔CO2 + CO3 2−, absorption of 33 mmol CO2 in the culture chamber induced the formation of 33 mmol CO3 2− in the CO2 buffer, thus reducing the amount of HCO3 by 66 mmol. Hence, the amount of HCO3 in the basal chamber was reduced from initially 540 mmol to c.474 mmol, while the amount of CO3 2−increased from initially 60 mmol to finally 93 mmol. The ratio between KHCO3 and K2CO3 within the basal chamber changed from initially 9:1 to finally c. 5:1 corresponding to a decrease in the CO2 concentration from initially c. 10 % to c. 5 % at 30 °C.

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Bähr, L., Wüstenberg, A. & Ehwald, R. Two-tier vessel for photoautotrophic high-density cultures. J Appl Phycol 28, 783–793 (2016).

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  • Bicarbonate
  • Cyanobacteria
  • Microalgae
  • Oxygen stress
  • Synechocystis sp. PCC 6803
  • Synechococcus sp. PCC 7002