Two-tier vessel for photoautotrophic high-density cultures

Abstract

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.

Appendices

Appendices

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 ).

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

(1)

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

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

(2)

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:

$$ C = 0.2 + \varDelta C $$

(3)

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

(4)

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.

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

(6)

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:

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

(7)

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

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

(8)

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.