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

Abstract

BackgroundLack 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%N-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.ResultsThe 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.ConclusionsThis 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.

Hydrocarbon production in high density Botryococcus braunii race B continuous culture

“…Also, typical algae media formulated for growth of 1–2 gDW/L have very high levels of some inorganics that would not scale to support the target of 20+ gDW/L required of high‐density cultures. The composition included both ammonium and nitrate as used in plant media to provide a pH balance during nitrogen utilization, which also addresses the importance of stoichiometric balance as described recently (Scherholz and Curtis, ). The basic composition for WFAM‐3g is based on a nitrogen level of 0.3 g of nitrogen per liter of media: KNO 3 (0.60 g/L), NH 4 NO 3 (0.61 g/L).…”

“…This procedure was designed as a daily media addition to facilitate multiple effective dilution rates without creating large complexity in feeding with pumps. This daily addition was sufficient to minimize the pH fluctuations that result from preferential uptake of ammonium and associated proton imbalance (Scherholz and Curtis, ). The feed composition was based on OD 550 measurements because they are immediately available at the time of each sampling; however, as noted in the results, there were dramatic changes in dry weight for the same OD 550 , therefore there was a need to correct the gDW/OD 550 ratio continuously during the long‐term culture runs.…”

“…Since it takes a day or more to obtain freeze‐dried weights, this ratio was updated continuously for each culture under study (typically using a prior 4‐sample average). By taking a biomass nitrogen content of 5% (Scherholz and Curtis, ), the amount of nitrogen that needed to be replaced could be immediately calculated. Then, using the 6× WFAM media, an appropriate volume of the concentrate and sterile water was added back to the culture to maintain total culture volume.…”

Sığırlarda Escherchia coli’nin Yüksek Hücre Yoğunluğunda Fermentasyonu İçin En Uygun Kültür Koşullarının Sağlanması

“…pH exerts complex effects because it influences the solubility of nutrients and trace elements, and the cellular metabolism in general [22] . pH homeostasis is important for the function and stability of all cellular enzymes [23] .…”

Ammonium Bicarbonate as Nutrient Substitute for Improving Biomass Productivity of Chlorella variabilis

“…A few microalgae, such as Chlorella vulgaris and Chlamydomonas reinhardtii , exclusively utilize ammonium during their growth 26. This study investigates ammonium bicarbonate as a possible alternative to sodium bicarbonate as the inorganic carbon source for improving biomass productivity and lipid content.…”