A strategy for the highly efficient production of docosahexaenoic acid by Aurantiochytrium limacinum SR21 using glucose and glycerol as the mixed carbon sources

Highlights

• Glucose facilitated rapid growth and lipid synthesis as sole carbon source.

• Glycerol promoted DHA accumulation but gave low productivity.

• Fed-batch culture was performed using glucose and glycerol as mixed carbon sources.

• The strategy of mixed carbon sources efficiently improved DHA yield and productivity.

Abstract

Glucose and glycerol are useful carbon sources for the cultivation of Aurantiochytrium limacinum SR21. Glucose facilitates rapid growth and lipid synthesis, and glycerol promotes the accumulation of docosahexaenoic acid (DHA) in A. limacinum SR21. To improve the DHA productivity of A. limacinum SR21, shake flask and fed-batch cultures were performed using glucose and glycerol as mixed carbon sources (MCSs). Along with optimization of the MCSs, the best DHA yield and productivity (32.36 g/L and 337.1 mg/L/h) were obtained via fed-batch fermentation with maintenance of a constant air supply. The DHA productivity was 15.24% higher than that obtained using glucose as single carbon source (SCS). This study presents a highly efficient and economic strategy for the production of DHA by A. limacinum SR21.

Introduction

Docosahexaenoic acid (DHA, 22:6) is a particularly important omega-3 fatty acid due to its benefits to human health and commercial use in infant formula (Borowitzka, 2013). Marine fish oil has long been a primary commercial source of DHA, but is associated with many problems, such as unstable quality, a fishy odor, environmental pollution, high processing costs and a tendency to oxidize (Ward and Singh, 2005). Single cell oils (SCOs) avoid these disadvantages of fish oils, and their production may readily be scaled up via biotechnological methods. Algae-derived DHA has thus become the subject of intensive research, and Schizochytrium sp. has been commercially utilized to produce DHA by fermentation (Ratledge, 2004).

Glucose is the most commonly used carbon source for heterotrophic cultures of microalgae, as far higher rates of growth and respiration are obtained than with any other substrate (Perez-Garcia et al., 2011). Bailey et al. (2003) achieved dry cell weight (DCW) of 171.5 g/L and DHA concentration of 35.33 g/L with glucose metabolized by Schizochytrium sp. ATCC20888, proposing the control strategy of stepwise dissolved oxygen (DO). However, turning sugar into algal oil is very expensive because of the high cost of the raw materials and the low conversion rates achieved by the oleaginous microorganisms. Attention has thus focused on ways to decrease production costs. It has been suggested that Aurantiochytrium limacinum SR21, previously called Schizochytrium limacinum SR21 (Yokoyama and Honda, 2007), may be used with cheap carbon sources such as crude glycerol from the biodiesel industry (Ethier et al., 2011). Although previous studies have shown that the yield of DHA from glycerol or crude glycerol is comparable to that from glucose (Chi et al., 2007, Yokochi et al., 1998), the activity of the key enzymes involved in glycerol assimilation, such as glycerol kinase (GK) and glycerol-3-phosphate dehydrogenase (G-3-PDH), may be inhibited by the reduced oxygen availability due to the increasing algal mass and the glycerol concentration expounded in our previous work (Chang et al., 2013b). Strategies to effectively use various substrates and to enhance DHA productivity should therefore be investigated.

Fermentation of the MCSs is an effective strategy for enhancing target metabolic products, and has been widely used in the fermentation industry (Chen et al., 2012). Glucose/glycerol co-fermentation has been used to produce propionic acid by Propionibacterium freudenreichii (Wang and Yang, 2013), 1,3-propanediol by Clostridium butyricum (Abbad-Andaloussi et al., 1998), ε-poly-l-lysine by Streptomyces sp. (Chen et al., 2012), H₂ by Escherichia coli (Trchounian and Trchounian, 2013) and β-carotene by Blakeslea trispora (Mantzouridou et al., 2008). Easterling et al. (2009) indicated that mixing glycerol with either dextrose or xylose served to decrease the saturation of fatty acid methyl esters produced by Rhodotorula glutinis, and dextrose/glycerol co-fermentation achieved the highest accumulation of triacylglycerol. When glucose was mixed with the same amount of glycerol in the medium for producing polyunsaturated fatty acids by Thraustochytrium sp., glycerol was consumed preferentially in the early stages of culture (Scott et al., 2011).

However, various strategies to improve the production of DHA by Schizochytrium sp. using glucose or glycerol as individual carbon sources have been explored, few reports on glucose and glycerol as the MCSs have been published. In the current study, we attempt to develop a glucose and glycerol co-fermentation strategy that is conducive to the enhancement of DHA yield and productivity by A. limacinum SR21.