Double CO₂ fixation in photosynthesis–fermentation model enhances algal lipid synthesis for biodiesel production

Abstract

In this study, a photosynthesis–fermentation model was proposed to merge the positive aspects of autotrophs and heterotrophs. Microalga Chlorella protothecoides was grown autotrophically for CO₂ fixation and then metabolized heterotrophically for oil accumulation. Compared to typical heterotrophic metabolism, 69% higher lipid yield on glucose was achieved at the fermentation stage in the photosynthesis–fermentation model. An elementary flux mode study suggested that the enzyme Rubisco-catalyzed CO₂ re-fixation, enhancing carbon efficiency from sugar to oil. This result may explain the higher lipid yield. In this new model, 61.5% less CO₂ was released compared with typical heterotrophic metabolism. Immunoblotting and activity assay further showed that Rubisco functioned in sugar-bleaching cells at the fermentation stage. Overall, the photosynthesis–fermentation model with double CO₂ fixation in both photosynthesis and fermentation stages, enhances carbon conversion ratio of sugar to oil and thus provides an efficient approach for the production of algal lipid.

Introduction

Lipids including animal fats and plant oils are the main feedstock for biofuel (biodiesel) production. Animals and most microorganisms are heterotrophs. They are able to efficiently synthesize a compact storage of energy-fat whilst releasing a certain amount of CO₂. Plants, including algae, are autotrophs and they function with bulky storage of energy-starch. Oils (lipids) are generally formed in plant seeds and constitute a very small amount of the whole plant. However, absorbance of CO₂ is one of the main advantages of autotrophy, benefiting both the environment and the economy via biomass production.

The study of algae-for-fuel has become a hot topic in recent years with energy prices fluctuating widely and green house gas emissions increasingly becoming a cause for concern (Gouveia and Oliveira, 2009, Jorquera et al., 2009, Pruvost et al., 2009, Yoo et al., 2009). Microalgae are regarded as a good source of biofuel (especially biodiesel) that has the potential to completely displace fossil fuels because of its rapid biomass production, high photosynthetic efficiency and, in some species such as Botryococcus, high lipid content (Haag, 2007). However, cultivation of autotrophic microalgae for biodiesel production still faces some technical challenges. For example, in a photosynthesis growth model (PM), rapidly growing cells contain lower amounts of lipids (<20% of dry weight), whereas algal cells accumulating high lipid contents (40–50% of dry weight) exhibit little growth. Heterotrophic fermentation of Chlorella protothecoides provides an alternative way to solve these problems (Miao and Wu, 2004, Miao and Wu, 2006, Xu et al., 2006). The cell density and lipid content achieved in a 5-L bioreactor was up to 51.2 g/L and 50.3% of dry cell weight (DCW) (Xiong et al., 2008). However, the fermentation growth model (FM) consumes organic carbon (sugar or starch) and is associated with more CO₂ release than the PM.

To develop an integrated strategy for cost-effective and environmentally-friendly production of microalgal biofuels, we adopted a photosynthesis–fermentation model (PFM) for algal cultivation. This model involves the photosynthetic growth of C. protothecoides to increase biomass and subsequent heterotrophic fermentation to maximize cell density and lipid accumulation. In the PFM, not only was CO₂ used for biomass production in the photosynthesis stage, but lipid biosynthesis was also enhanced in the fermentation stage compared with the FM. A theoretical analysis suggested that the CO₂ re-fixation in fermentation stage resulted in enhancing lipid synthesis. This conclusion has been supported by further experimental data, confirming that the PFM is a novel approach for more efficient biodiesel production from microalgae.