On-line estimation of O₂ production, CO₂ uptake, and growth kinetics of microalgal cultures in a gas-tight photobioreactor

Abstract

Growth of the green algae Chlamydomonas reinhardtii and Chlorella sp. in batch cultures was investigated in a novel gas-tight photobioreactor, in which CO₂, H₂, and N₂ were titrated into the gas phase to control medium pH, dissolved oxygen partial pressure, and headspace pressure, respectively. The exit gas from the reactor was circulated through a loop of tubing and re-introduced into the culture. CO₂ uptake was estimated from the addition of CO₂ as acidic titrant and O₂ evolution was estimated from titration by H₂, which was used to reduce O₂ over a Pd catalyst. The photosynthetic quotient, PQ, was estimated as the ratio between O₂ evolution and CO₂ up-take rates. NH₄ ⁺, NO₂ ⁻, or NO₃ ⁻ was the final cell density limiting nutrient. Cultures of both algae were, in general, characterised by a nitrogen sufficient growth phase followed by a nitrogen depleted phase in which starch was the major product. The estimated PQ values were dependent on the level of oxidation of the nitrogen source. The PQ was 1 with NH₄ ⁺ as the nitrogen source and 1.3 when NO₃ ⁻ was the nitrogen source. In cultures grown on all nitrogen sources, the PQ value approached 1 when the nitrogen source was depleted and starch synthesis became dominant, to further increase towards 1.3 over a period of 3–4 days. This latter increase in PQ, which was indicative of production of reduced compounds like lipids, correlated with a simultaneous increase in the degree of reduction of the biomass. When using the titrations of CO₂ and H₂ into the reactor headspace to estimate the up-take of CO₂, the production of O₂, and the PQ, the rate of biomass production could be followed, the stoichiometrical composition of the produced algal biomass could be estimated, and different growth phases could be identified.

Appendix

Distribution of CO₂ in gas tight photobioreactor

To account for the effect of uptake of the nitrogen source on the total content of inorganic carbon in the photobioreactor, the distribution of inorganic carbon between the Pd catalyst, the gas phase, and the liquid medium was calculated. Since batch cultures were grown over relative long periods of time (200–300 h), the calculations were based on pseudo-steady-state conditions

$$ CO_{{2,Pd}} \leftrightarrows CO_{{2,gas}} \leftrightarrows H_{2} CO^{ * }_{{3aq}} \leftrightarrows HCO^{ - }_{{3aq}} + H^{ + }_{{aq}} \leftrightarrows CO^{{2 - }}_{{3aq}} + 2H^{ + }_{{aq}} $$

(A1)

The total amount of inorganic carbon in the photobioreactor, $m_{{C_{i} }}$ was the sum of all pools of inorganic carbon present in the system as described in Eq. A1

$$ m_{{C_{i} }}=m_{{H_{2} CO^{*}_{3} ,aq}} + m_{{HCO^{,}_{3} ,aq}} + m_{{CO^{{2 - }}_{3} ,aq}} + m_{{CO_{2} ,gas}} + m_{{CO_{2} ,Pd}} $$

(A2)

where $m_{{H_{2} CO^{*}_{3} ,aq}} ,\,m_{{HCO^{ - }_{3} ,aq}}$,and $m_{{CO^{{2 - }}_{3} ,aq}}$ are the amounts of dissolved H₂CO₃ (including dissolved CO₂), HCO₃ ⁻, and CO₃ ²⁻, respectively, $m_{{CO_{2} ,gas}}$ is the total amount of CO₂ in the gas phase, and $m_{{CO_{2} ,Pd}}$ is the total amount of CO₂ adsorbed to the Pd catalyst.

At pH 7.5, CO₃ ²⁻ constituted only in the order of 0.1% of the total dissolved inorganic carbon, and the amounts of inorganic carbon in the different pools described in Eqs. A1 and A2 were essentially controlled by the concentration of HCO₃ ⁻ in the growth medium. Since 1 mol of H⁺ was consumed for each mol of NO₃ ⁻ or NO₂ ⁻ taken up by the cells, these protons were regenerated by addition of CO₂, which dissolved as HCO₃ ⁻ and resulted in an equimolar increase of [HCO₃ ⁻]. For each mol of NH₄ ⁺ taken up, 1 mol of H⁺ was produced, and with NH₄ ⁺ as the nitrogen source, less CO₂ than taken up photosynthetically were therefore added to the photobioreactor resulting in an equimolar decrease of [HCO₃ ⁻]. The amount of dissolved HCO₃ ⁻ was therefore estimated by

$$m_{{HCO^{ - }_{3} .aq}}={\left( {{\left[ {HCO^{1}_{3} } \right]}_{{t_{0} }} \pm \Delta N} \right)} \cdot V_{L} $$

(A3)

where ΔN is the total decrease in concentration of the nitrogen source due to consumption by the algae, and V _L is the volume of the liquid medium.

The amount of dissolved H₂CO₃ ^* in the growth medium was described by

$$ m_{{H_{2} CO^{*}_{{_{3} }} ,aq}}={\left( {\frac{{{\left[ {HCO^{ - }_{3} } \right]} \cdot {\left[ {H^{ + } } \right]}}} {{K_{1} }}} \right)} \cdot V_{L} $$

(A4)

where K ₁ is the equilibrium constant between H₂CO₃ ^* and HCO₃ ⁻ + H⁺ (10^−6.3 M, Stumm and Morgan 1995). The amount of dissolved CO₃ ²⁻ is described by

$$ m_{{CO^{{2 - }}_{3} ,aq}}={\left( {\frac{{K_{2} \cdot {\left[ {HCO^{ - }_{3} } \right]}}} {{{\left[ {H^{ + } } \right]}_{{t_{0} }} }}} \right)} \cdot V_{L} $$

(A5)

where K ₂ is the equilibrium constant between HCO₃ ⁻ and CO₃ ²⁻ + H⁺ (10^−10.3 M, Stumm and Morgan 1995).

The relationship between the partial pressure of CO₂ in the headspace, $p_{{CO_{2} }}$ and the concentration of H₂CO₃ ^* in the medium was described by Henry’s law

$$p_{{CO_{2} }}=K_{H} \cdot {\left[ {H_{2} CO^{ * }_{3} } \right]} $$

(A6)

where K _H is Henry’s constant (3.0 • 10³ kPa M⁻¹, Atkins 1980). The total amount of CO₂ in the headspace was calculated from $p_{{CO_{2} }}$ using the gas law

$$ m_{{CO_{2} ,gas}}=\frac{{p_{{CO_{2} }} \cdot V_{G} }} {{R \cdot T}} $$

(A7)

where V _G is the volume of the gas in the headspace and the closed gas loop, R is the gas constant, and T is the absolute temperature.

The amount of CO₂ reversibly adsorbed onto the Pd catalyst was described by a Langmuir binding isotherm

$$ m_{{CO_{2} ,Pd}}=\frac{{c_{{CO_{2} ,Pd,\max }} \cdot W_{{Pd}} \cdot p_{{CO_{2} ,gas}} }} {{a + p_{{CO_{2} ,gas}} }} $$

(A8)

where $c_{{CO_{2} ,Pd,\max }}$ is the maximal surface-cover of CO₂ on the Pd catalyst (60 μmol g⁻¹), W _Pd is the mass of Pd catalyst in the catalytic column (25 g), and a is the half saturation constant (2.1 kPa). The parameters, $c_{{CO_{2} ,Pd,\max }}$ and a were estimated by measuring the increase of partial pressure after adding known amounts of CO₂ to a closed chamber containing the Pd catalyst.

If the nitrogen uptake is measured or modelled, it is now possible to calculate the relationship between the overall change in total inorganic carbon content and nitrogen content in the photobioreactor, $\frac{{\Delta m_{{C_{i} }} }} {{\Delta N \cdot V_{L} }}$

$$\frac{{\Delta m_{{C_{i} }} }}{{\Delta N \cdot V_{L} }}=\frac{{m_{{C_{i} }} - m_{{C_{i} t_{0} }} }}{{{\left( {N - N_{{t_{0} }} } \right)} \cdot V_{L} }} $$

(A9)

With NO₃ ⁻ and NO₂ ⁻ as nitrogen sources, $\frac{{\Delta m_{{C_{i} }} }} {{\Delta N \cdot V_{L} }}$ is negative. With NH₄ ⁺ as nitrogen source, $\frac{{\Delta m_{{C_{i} }} }} {{\Delta N \cdot V_{L} }}$ is positive. In the experiments described in this paper, ΔN was estimated from Eq. 13.