Microalgae maintain sophisticated self-regulatory mechanisms to survive in different environments. Before heterotrophic fermentation period, the usage of MP strategy in this study facilitated physiological acclimation of C. zofingiensis by altering the uptake pattern of carbon source. MP improved the physiological state of C. zofingiensis, stimulated its rapid growth from the first preculture generation, then achieved faster growth rate and higher biomass with altered intracellular biochemical composition in heterotrophic batch culture (Fig. 1). The sharp alternation in biomass demonstrated the superiority of glucose as an organic carbon source for promoting cell growth, and MP further strengthened the absorption and metabolism of glucose (Table 1).
The dramatical change of MP-induced cellular compositions not only covered the adjustment of the ratio of proteins, lipids and carbohydrates (Fig. 1D), but also included the modification in the composition of key target products (Fig. 2). MP obtained great capacity of stimulating protein and carbohydrate degradation towards lipids and promoting the biosynthesis of affiliated high-value components. From the angel of carbon skeleton source proportion for lipid synthesis, the direct MP-induced carbon flux from proteins towards lipids partly replaced the original path flowing along proteins, carbohydrates, and lipids (Table 1). This strategy ensured sufficient utilization of carbon source, avoided unnecessary intermediate metabolic processes and energy loss, which endows it with the capacity to realize efficient industrial production of lipids or other valuable products, thus becoming a competitive strategy in commercial process.
As the culture biomass increased under MP, the Chl a content sharply decreased (Fig. 2A) and the photosynthetic system displayed damage. Meanwhile, the accumulated Chl b and carotenoids could block the oxidative damage of C. zofingiensis cells caused by ROS and provide abundant precursors for astaxanthin biosynthesis, but the decreased astaxanthin accumulation under MP indicated the restriction of this conversion due to reduced photosynthesis (Fig. 1C).
Under MP, the respiration was enhanced, and FAs with same chain length showed higher saturation in general, this trend presented it higher cetane number and stability when applied as materials or propellants of biofuel (Fig. 2B). When MUFAs contribute to improving cold flow properties, the moderate PUFAs help in low-temperature properties and oxidative stability [14]. Moreover, MP effectively promoted the accumulation of C18:1 and C18:2, the crucial intermediate molecules of long-chain FAs synthesis that widely used in dietary supplement and infant food. All these changes of FA composition after MP endowed C. zofingiensis excellent characteristics and great application value as biofuels or healthcare products, thus broadening its commercial develop domain.
Many unsaturated FAs are involved in the esterification of astaxanthin, and most of the intracellular astaxanthin is stored in lipid droplets in the form of astaxanthin ester. Therefore, the synthesis of TAG is closely related to astaxanthin synthesis and accumulation. The transcriptional levels of the genes appertained to TAG and carotenoid synthesis were analyzed, and their metabolic flux was explored by 13C-MFA. As shown in transcriptome results, de novo FA synthesis pathway was up-regulated under MP, thus providing abundant source of acyl substrates. In Kennedy pathway, the expression level of PAP3 and most of DGAT enzymes were significantly upregulated, which could further promote TAG accumulation. However, the expression of GPAT1 and PAP2 were inhibited, which led to the accumulation of AcCoA. Metabolic flux results in FAs synthesis pathway did not completely fit previous assumption (Fig. 4). While AcCoA synthesis was more active under MP, the synthesis of C16 FAs was inhibited for the reason that NADPH from OPP pathway and TCA cycle was used for maintaining cell metabolism and respiration instead of lipid synthesis. Respiration outputs ROS as natural byproducts, but increasing ROS level when exposed to environmental stress, known as oxidative stress, can lead to severe damages, such as the slowdown of ATP synthesis, cell-cycle arrest, and eventually apoptosis. The synthesis pathway of FAs, especially PUFAs, serve as the electron sinks [23], and act as antioxidants to mediate oxidative stress by effectively quenching ROS and adjust the REDOX state of cells. Thus, the enhancing ROS production may be the important factor of the consumption of PUFAs under MP, which can be a reasonable explanation for the overall decrease in FAs saturation [13].
Despite the augment of PYR pool with MP, the expression of most genes in MEP pathway showed no obvious promotion. IPP and DMAPP, the precursors for carotenoid synthesis, were significantly their synthesis activities were suppressed due to the lower transcription levels of DXS, CMS, and MCS (Fig. 3D). Similarly, synthesis of astaxanthin and its pigment precursors showed a decline compared to AP, which indicated the restraint of the flux of the terpenoid backbone towards astaxanthin biosynthesis. Transcript abundance of BKT1 and AAT were substantially reduced under MP, which limited β-carotene finally conversed into astaxanthin. Figure 4 showed that carotenoid synthesis activity under MP was significantly declined, which certainly limited the rapid synthesis of astaxanthin. Thus, the lack of precursors and decreased activity of enzymes regulated by genes with lower expression levels jointly resulted in a decline of intracellular astaxanthin content under MP. The decrease of intracellular astaxanthin content can also be explained by ROS accumulation of enhancing respiration. The enhanced activity of TCA cycle (Fig. 4) under MP indicated that C. zofingiensis relies more on the respiration for energy production and carbon availability augmentation. Similar to other antioxidative carotenoids, astaxanthin has been proved to effectively protect algal cells against oxidative damage by quenching the excessive ROS and other free radicals under stress conditions. Therefore, the generation of ROS can lead to consumption of astaxanthin and its precursors. On the other hand, due to the sharp amplification of biomass in MP group, the reduction of effective light intensity caused by the shielding between cells is another reason for the block of astaxanthin accumulation. While C. zofingiensis can produce astaxanthin in darkness, it still shows a typical light-dependent growth and astaxanthin synthesis can still be induced by light intensity, for this process has been identified as a protective response stimulated by photo-oxidative stress [24].
Preculture strategy also plays a critical role in regulating the activity of a multitude of crucial metabolic pathways, which lays a foundation of substance and energy for the production of FA and astaxanthin. Glycolytic pathway outputs PYR and glyceraldehyde 3-phosphate, the former can realize the mutual conversion of basic ingredients central carbon metabolism. Moreover, as an irreplaceable precursor, its accessibility has a decisive influence on the production of carotenoids. AcCoA is the direct downstream of PYR, it serves as the initial block of extending acyl chain [17]. AcCoA participates in TCA circle as the central cellular metabolite of various biochemical reactions. AcCoA is both the precursor for FA and carotenoid synthesis and a product of the degradation pathways of FA and some amino acids, its efficiency and availability are pivotal for FA and carotenoid synthesis. Overall, expression level of genes affecting glycolysis pathway and gluconeogenesis was elevated under MP (Fig. 3E), among which the augmented expression of ENO and PK is responsible for PYR synthesis and its higher availability [19]. PDHC and ACS are main enzymes contribute to AcCoA production, their significant up-regulated expression level under MP strengthened the production and availability of cellular AcCoA (Fig. 3E). Meanwhile, the down-regulated overall expression of genes related to Kennedy pathway inhibited the transformation of glycerol-3-phosphate, which further aggravated the retention of AcCoA. 13C-MFA revealed the strengthened metabolic flux of EMP and anaplerotic reactions, which boosted AcCoA accumulation and NADPH production, provided abundant carbon molecules for carotenoid biosynthesis, and channeled carbon flow rapidly into TCA cycle (Fig. 4).
In addition to an abundance of substrate molecules and enzymes, astaxanthin synthesis also require NADPH and ATP to provide reducing power and energy [19]. With the photosynthesis pathway in C. zofingiensis was hindered, OPP pathway was likely to be the main source of NADPH under MP. OPP pathway generates 2 NADPH molecules for each molecule of glucose-6-phosphate catalyzed. The overexpression of enzymes involved in it, especially 6PGD (Fig. 3E), could catalyze NADPH-producing steps to elevate its content and consequently provide reducing power and energy for biosynthesis process, and the increased reductant pool improved anabolism.
TCA cycle is the crucial pathway that links the metabolism of three major substances and the hub of energy metabolism. As the precursor source for biosynthetic pathways that provide essential substances and sufficient energy for various physiological activities, TCA cycle produce 32 or 30 molecules of ATP in each molecule of glucose with aerobic oxidation. As carbon flow rapidly into TCA cycle and reactions within it were obviously enhanced by MP (Fig. 4), its related genes got generally elevated expression under MP (Fig. 3E), indicated that TCA cycle provided more energy to support the substance synthesis.
Modelling growth is effective for studying and adjusting the performance of microalgae growth, it helps controlling microalgae growth in artificial bioreactors [25]. Exponential fed-batch culture can maximize the growth rate of microalgae through maintaining nutrient content of the system at the most suitable level for cell growth and development. This culture pattern has been proved to be effective in promoting biomass accumulation and increasing cell density. The usage of exponential fed-batch cultures in this study provided C. zofingiensis the highest specific growth rate for maximized biomass production, and resulted in an obvious increase in biomass and astaxanthin productivity (Fig. 5).
As the culture volume expanded to 20 L, the biomass concentration successfully surpassed the highest reported record, MP strategy provided sufficient material for the acquisition of astaxanthin and endowed C. zofingiensis with a greater value for industrialization. While the astaxanthin content of MP cells showed decreasing trend, the final astaxanthin productivity achieved 0.111 g L− 1 day− 1, which was ascribed to the vast biomass accumulation (Fig. 5C). This yield was 7.3-fold higher than the best record of C. zofingiensis [3], and even exceeded that of H. pluvialis [22]. The results strongly demonstrated the superiority of the combination of MP and large-scale fed-batch culture, which has mastered the international pioneering level in industrial production of natural astaxanthin as the optimal strategy for C. zofingiensis.