13c Metabolic Flux Analysis of the Green Microalga Chlorella vulgaris Under Autotrophic, Mixotrophic, and Heterotrophic Conditions

One of the most pressing grand challenges facing humanity is to sustainably support a human population that is predicted to increase to 9 billion people by mid-century. Meeting this challenge will require the development of new and innovative renewable production systems. One approach to address the problems of dwindling reserves of non-renewable resources and rising levels of carbon dioxide (CO₂) in the atmosphere is to capture concentrated CO₂ emerging from waste treatment facilities, petroleum refineries, coal and other energy sources and convert the CO₂ into useful products, including dietary supplements, medicinals, and biofuels. Microalgae species represent a potential renewable source of such products due to their ability to use light to fix CO₂ into biomolecules.

            Chlorella is a genus of unicellular, non-motile, photosynthetic, eukaryotic microalgae which can survive in fresh and salt water. In regards to microalgal biodiesel production economics, non-polar lipid content is the most important factor and one of the best lipid producing Chlorella species is C. vulgaris. Chlorella vulgaris UTEX 395 is a model green microalga with great biodiesel production potential and a sequenced genome. However, due to inefficiencies in carbon metabolism, critical technical bottlenecks still remain to economic production of desirable products. Therefore, it is important to obtain a quantitative understanding of Chlorella vulgaris UTEX 395 metabolism using ¹³C Metabolic Flux Analysis (¹³C-MFA).

            In this work, we performed labeling switch experiments and took advantage of the fact that C. vulgaris can grow autotrophically, mixotrophically, and heterotrophically and can easily change between these modes of growth. First, cells were grown heterotrophically on fully ¹³C labeled glucose. Next, the fully ¹³C labeled cells were transferred into fresh media. Autotrophic, mixotrophic, and heterotrophic unlabeling conditions were used. No significant turnover of proteinogenic amino acids was observed during CO₂-enriched autotrophic unlabeling or heterotrophic unlabeling. However, during autotrophic unlabeling with air there was significant turnover of proteinogenic amino acids related to photorespiration (alanine, glycine, serine, aspartate, and glutamate) consistent with low concentration of CO₂ near RuBisCO.

           Next, metabolic fluxes were quantified using ¹³C-MFA. In these experiments, we also measured net production and consumption of CO₂ and O₂ in real time using an on-line process mass spectrometer to further improve flux resolution. For heterotrophic and mixotrophic growth, labeled glucose tracers were used. For photosynthetic growth, a combination of labeled and unlabeled carbon dioxide and amino acids were used as substrates. This is in contrast to the current ¹³C-MFA methods for photosynthetic growth which use high concentrations of ¹³C labeled bicarbonate. With our approach, we were able to quantify significant photorespiratory fluxes which are not captured using other methods. The metabolic flux maps enabled us to identify inefficiencies in central carbon metabolism under the different growth conditions and suggest metabolic engineering targets to improve Chorella vulgaris’s metabolic efficiency.