Achieving pH control in microalgal cultures through fed-batch addition of stoichiometrically-balanced growth media

Abstract

Background: Lack of accounting for proton uptake and secretion has confounded interpretation of the stoichiometry of photosynthetic growth of algae. This is also problematic for achieving growth of microalgae to high cell concentrations which is necessary to improve productivity and the economic feasibility of commercial-scale chemical production systems. Since microalgae are capable of consuming both nitrate and ammonium, this represents an opportunity to balance culture pH based on a nitrogen feeding strategy that does not utilize gas-phase CO₂ buffering. Stoichiometry suggests that approximately 36 weight%NH₄⁺ (balance nitrogen as NO₃⁻) would minimize the proton imbalance and permit high-density photoautotrophic growth as it does in higher plant tissue culture. However, algal media almost exclusively utilize nitrate, and ammonium is often viewed as 'toxic' to algae.

Results: The microalgae Chlorella vulgaris and Chlamydomonas reinhardtii exclusively utilize ammonium when both ammonium and nitrate are provided during growth on excess CO₂. The resulting proton imbalance from preferential ammonium utilization causes the pH to drop too low to sustain further growth when ammonium was only 9% of the total nitrogen (0.027 gN-NH₄⁺/L). However, providing smaller amounts of ammonium sequentially in the presence of nitrate maintained the pH of a Chlorella vulgaris culture for improved growth on 0.3 gN/L to 5 gDW/L under 5% CO₂ gas-phase supplementation. Bioreactor pH dynamics are shown to be predictable based on simple nitrogen assimilation as long as there is sufficient CO₂ availability.

Conclusions: This work provides both a media formulation and a feeding strategy with a focus on nitrogen metabolism and regulation to support high-density algal culture without buffering. The instability in culture pH that is observed in microalgal cultures in the absence of buffers can be overcome through alternating utilization of ammonium and nitrate. Despite the highly regulated array of nitrogen transporters, providing a nitrogen source with a balanced degree of reduction minimizes pH fluctuations. Understanding and accommodating the behavior of nitrogen utilization in microalgae is key to avoiding 'culture crash' and reliance on gas phase CO₂ buffering, which becomes both ineffective and cost-prohibitive for commercial-scale algal culture.

Figure 1

pH “toxicity” from nitrogen metabolism in Chlorella vulgaris. Photoautotrophic Chlorella vulgaris cultures were grown in shake flasks with 5% CO₂ (v/v) in air on 0.3 gN/L with 0 to 36% as ammonium (0 to 0.108 gN-NH₄⁺/L) and the remaining as nitrate (0.3 to 0.192 gN-NO₃^-/L). The highest ammonium concentration corresponded to the nitrogen composition of our stoichiometrically-balanced photoautotrophic growth media. The optical density was measured at 550-nm to monitor culture growth as a proxy for dry weight (A). Offline pH samples were taken at 3-hr intervals, degassed, and measured in anticipation of a proton imbalance due to nitrogen metabolism (B).

Figure 2

Inhibition of nitrate assimilation by ammonium in Chlorella vulgaris. Photoautotrophic Chlorella vulgaris cultures were grown in 1.5-L loop air-lift photobioreactors on 0.0135 gN-NH₄⁺/L with chloride and nitrate as the counter-ions. The reactor was supplemented with 5% CO₂ (v/v) in air. The optical density was measured at 550-nm at 3 to 4-hr intervals and was converted to biomass density using a ratio of 0.52 gDW/L/OD₅₅₀ (A). The pH was measured continuously online and averaged at 30-min intervals to track the proton imbalance due to nitrogen utilization (B).

Figure 3

pH instability from nitrogen metabolism in Chlamydomonas reinhardtii. Photoautotrophic Chlamydomonas reinhardtii cultures were grown in 1.5-L loop air-lift photobioreactors on 5% CO₂ (v/v) in air and 0-9% nitrogen from ammonium (0 to 0.027 gN-NH₄⁺/L) with the balance nitrogen as nitrate. The optical density was measured at 550-nm to monitor culture growth and converted to biomass density using the conversion factor of 0.52 gDW/L/OD₅₅₀ determined experimentally (A). The culture pH was monitored online continuously with samples averaged every 5 minutes to monitor nitrogen utilization (B). The nitrate concentration was measured offline using an ion selective electrode (C).

Figure 4

Schematic of cellular regulation of nitrogen assimilation in the algae Chlamydomonas reinhardtii. The number of identified transporters is in indicated on the respective membrane (i.e. 3x = three ammonium transporters on the chloroplast membrane). Dashed lines in the figure legend represent the regulatory elements involved in nitrogen metabolism. Additional key notations: Amt = ammonium transporter, Nit = nitrate transporter, NR = nitrate reductase, NiR = nitrite reductase, CA = carbonic anhydrase, CCM = carbon concentration mechanisms.

Figure 5

Behavioral model of nitrogen utilization which facilities photobioreactor pH control. This schematic represents the control elements that give rise to the observed pH response that can be controlled with incremental addition of ammonium. The presence of ammonium inhibits transport of nitrate into the algal cell and results in a net efflux of protons to the culture media. Following depletion of ammonium from the media, nitrate is transported into the cell and reduced to ammonium prior to assimilation, resulting in a net influx of protons from the culture media. Notations: Amt = ammonium transporter, Nit = nitrate transporter, NR = nitrate reductase.

Figure 6

Fed-batch addition of nitrogen for pH control in Chlorella vulgaris. A photoautotrophic Chlorella vulgaris culture was grown in a trickle film photobioreactor on balanced growth media (0.3 gN/L at 36%N-NH₄⁺ and balance as nitrate) under 5% CO₂ (v/v) in air and subjected to a diurnal light cycle with a 16-hr photoperiod. The culture was initially grown on potassium nitrate with five ammonium nitrate additions made over the course of the growth period. Online pH was monitored continuously and averaged every minute to demonstrate pH fluctuations due to the ammonium nitrate additions indicated by the arrows and the concentration of ammonium in the reactor following each feeding (A). The red arrow signifies the unexpected pH response following the fifth ammonium nitrate addition. The optical density was measured at 550-nm to monitor biomass growth and converted to culture density using the biological conversion factor of 0.53 gDW/L/OD₅₅₀ determined experimentally (B). The inset of (B) shows the relative change in proton concentration during ammonium metabolism following the first four NH₄NO₃ additions.

Figure 7

pH control maintained during nitrogen limitation in Chlamydomonas reinhardtii. A photoautotrophic Chlamydomonas reinhardtii culture was grown on balanced growth media under 5% CO₂ (v/v) in air in a trickle film bioreactor and subjected to a diurnal light cycle with a 16-hr photoperiod. The culture was initially started on potassium nitrate until ammonium nitrate additions began after 10 hours of growth with a total of 0.6 gN/L added over the 75-hr growth period. The pH was measured online to monitor the proton imbalance from nitrogen metabolism before and after nitrogen depletion (indicated by red line at ~25 hrs) with black arrows indicating the time of each NH₄NO₃ addition, and the red arrow indicating the addition of NH₄NO₃ supplemented with a balance of non-nitrogen nutrient salts (A). The optical density was measured at 550-nm and converted to biomass density using the conversion factor of 0.51 gDW/L/OD₅₅₀ determined experimentally (B). Also shown in (B inset) is the change in proton concentration per utilized nitrate.

Figure 8

Air-grown cultures demonstrate pH control through stoichiometry-balanced nitrogen feed prior to carbon limitation. A photoautotrophic Chlamydomonas reinhardtii culture was grown on balanced growth media under 5% CO₂ (v/v) in baffled shake flasks and subjected to a diurnal light cycle with a 16-hr photoperiod. The culture was started on potassium nitrate followed by ammonium nitrate additions (indicated by arrows and given in mgN-NH₄⁺/L) began after 5-hrs of growth with a total of 0.3 gN/L added over the 48-hr growth period. The culture was allowed to grow past the onset of carbon limitation as indicated by the dotted red line (~21 hrs). The optical density was measured at 550-nm to monitor culture growth (A). The pH was measured online to monitor the proton imbalance from nitrogen metabolism (B). The inset of (B) shows the change in proton concentration per ammonium assimilated for the second to fifth NH₄NO₃ additions prior to carbon limitation.