Docosahexaenoic Acid Production Research Articles

Comprehensive analysis of metabolic alterations in Schizochytrium sp. strains with different DHA content

Along with the daily growth of the market requirements for docosahexaenoic acid (DHA) algae oil, a large DHA ingredients are needed to ensure worldwide supply. Undoubtedly a high-productive strain would be the prerequisite for high quality and yield. A comprehensive understanding of the processes of DHA synthesis from glycolysis to the lipid accumulation would be benefit to achieve the final optimization of DHA production. In this study, we comprehensively characterized the metabolic profiles of a Schizochytrium sp. strain, which has higher DHA content and different biomass amino acid composition compared with the wild type to explore the affected pathways and underlying mechanism. Combined with the multivariate statistical analysis, twenty-two differential metabolites were screened as relevant to the discrepancy between two strains. The results showed relatively downregulated glycolysis and saturated fatty acids (SFA) synthesis, and upregulated TCA cycle, amino acids and polyunsaturated fatty acids (PUFA) synthesis in DHA high yield strain. The current study provide a terminal picture of gene regulation from downstream metabolism and demonstrate the advantage of metabolomics in characterizing metabolic status which in turn could provide effective information for the metabolic engineering.

Mining of squalene as a value-added byproduct from DHA producing marine thraustochytrid cultivated on food waste hydrolysate

The commercial production of docosahexaenoic acid (DHA) from oleaginous microorganisms is getting more attention due to several advantages over fish oils. The processing cost became a major bottleneck for commercialization of DHA from microorganisms. The most of cost shares in the feedstock to cultivate the microorganisms and downstream processing. The cost of feedstock can be compensated with the utilization of substrate from waste stream whereas production of value-added chemicals boosts the economic viability of nutraceutical production. In the present study, the docosahexaenoic acid (DHA)-producing marine protist Aurantiochytrium sp. T66 was cultivated on post-consumption food waste hydrolysate for the mining of squalene. After 120 h of cultivation, cell dry weight was 14.7 g/L, of which 6.34 g/L (43.13%; w/w) were lipids. DHA accounted for 2.15 g/L (34.05%) of total extracted lipids or 0.15 g/gCDW. Maximum squalene concentration and yield were 1.05 g/L and 69.31 mg/gCDW, respectively. Hence, utilization of food waste represents an excellent low-cost strategy for cultivating marine oleaginous thraustochytrids and produce squalene as a byproduct of DHA.

Overexpression of an endogenous type 2 diacylglycerol acyltransferase in the marine diatom Phaeodactylum tricornutum enhances lipid production and omega-3 long-chain polyunsaturated fatty acid content

BackgroundOleaginous microalgae represent a valuable resource for the production of high-value molecules. Considering the importance of omega-3 long-chain polyunsaturated fatty acids (LC-PUFAs) for human health and nutrition the yields of high-value eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) require significant improvement to meet demand; however, the current cost of production remains high. A promising approach is to metabolically engineer strains with enhanced levels of triacylglycerols (TAGs) enriched in EPA and DHA.ResultsRecently, we have engineered the marine diatom Phaeodactylum tricornutum to accumulate enhanced levels of DHA in TAG. To further improve the incorporation of omega-3 LC-PUFAs in TAG, we focused our effort on the identification of a type 2 acyl-CoA:diacylglycerol acyltransferase (DGAT) capable of improving lipid production and the incorporation of DHA in TAG. DGAT is a key enzyme in lipid synthesis. Following a diatom based in vivo screen of candidate DGATs, a native P. tricornutum DGAT2B was taken forward for detailed characterisation. Overexpression of the endogenous P. tricornutum DGAT2B was confirmed by qRT-PCR and the transgenic strain grew successfully in comparison to wildtype. PtDGAT2B has broad substrate specificity with preferences for C16 and LC-PUFAs acyl groups. Moreover, the overexpression of an endogenous DGAT2B resulted in higher lipid yields and enhanced levels of DHA in TAG. Furthermore, a combined overexpression of the endogenous DGAT2B and ectopic expression of a Δ5-elongase showed how iterative metabolic engineering can be used to increase DHA and TAG content, irrespective of nitrogen treatment.ConclusionThis study provides further insight into lipid metabolism in P. tricornutum and suggests a metabolic engineering approach for the efficient production of EPA and DHA in microalgae.

Different Classes of Phytohormones Act Synergistically to Enhance the Growth, Lipid and DHA Biosynthetic Capacity of Aurantiochytrium sp. SW1.

In the present study, the impact of eight phytohormones from six different classes on the growth, lipid and docosahexaenoic acid (DHA) biosynthetic capacity of Aurantiochytrium sp. SW1 (SW1) was evaluated. Kinetin (KIN), jasmonic acid (JA) and gibberellic acid (GA) significantly enhanced the growth and DHA production of SW1 by 16%–28% and 66%–84% in comparison to the control, respectively. The synergistic effect of these three phytohormones, evaluated by the response surface methodology (RSM), showed that a combination of 3.6 mg/L GA, 2.0 mg/L KIN and 20.0 mg/L JA further increased the growth and DHA production of SW1 by 16% to 28% and 22% to 36%, respectively, in comparison to the individual supplementation. The synergistic effect of these phytohormones was also shown to be time-dependent, where feeding at 24 h of cultivation led to 15%, 26% and 35% further increments in the biomass, lipid and DHA production in comparison to that of 0 h, respectively. The determination of stress markers, antioxidant enzymes and key enzymes involved in fatty acid biosynthesis aided to elucidate the potential mechanism underlying the improvement of growth and DHA production by SW1 at various times of feeding. Supplementation with the phytohormones at 24 h exhibited the maximum impact on reducing the level of reactive oxygen species (ROS) and malondialdehyde (MDA), as well as augmented the antioxidants (superoxide dismutase and catalase) and key metabolic enzymes involved in lipogenesis (malic, glucose-6-phosphate dehydrogenase and ATP-citrate lyase) in comparison to the control and other time points. This study signifies the potential application of phytohormones for improving the growth, lipid and DHA production in Aurantiochytrium spp.

Concentration of Docosahexaenoic and Eicosapentaenoic Acid from Cobia Liver Oil by Acetone Fractionation of Fatty Acid Salts.

The production of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) concentrate from cobia liver oil by acetone fractionation of fatty acid salts was investigated in this study. A three-level-three-factor Box-Behnken design was used to evaluate the effects of reaction time, amount of NaOH added, and acetone ratio on the responses (DHA and EPA content and recovery). The results showed that the amount of NaOH added was the most important factor in the process. The DHA content showed an inverse relation with EPA content and recovery, whereas its content increased proportionally with the amount of NaOH added. With a reaction time of 1.51 h, amount of NaOH added at 0.65 times the molar equivalent of free fatty acid (FFA), and acetone ratio at 13.92, a maximum recovery of DHA + EPA was 98.14%, and the obtained concentrate contained 71.23% DHA + EPA. Finally, the lipase-catalyzed esterification of the DHA + EPA concentrate with glycerol was carried out. The acetone fractionation of fatty acid salts is an efficient technique for producing DHA +EPA concentrate. The DHA +EPA concentrate can be used as starting materials for the production of functional lipids to provide n-3 polyunsaturated fatty acids to the consumers.

Repeated fed-batch strategy and metabolomic analysis to achieve high docosahexaenoic acid productivity in Crypthecodinium cohnii

BackgroundDocosahexaenoic acid (DHA) is essential for human diet. However, high production cost of DHA using C. cohnii makes it currently less competitive commercially, which is mainly caused by low DHA productivity. In recent years, repeated fed-batch strategies have been evaluated for increasing the production of many fermentation products. The reduction in terms of stability of culture system was one of the major challenges for repeated fed-batch fermentation. However, the possible mechanisms responsible for the decreased stability of the culture system in the repeated fed-batch fermentation are so far less investigated, restricting the efforts to further improve the productivity. In this study, a repeated fed-batch strategy for DHA production using C. cohnii M-1-2 was evaluated to improve DHA productivity and reduce production cost, and then the underlying mechanisms related to the gradually decreased stability of the culture system in repeated fed-batch culture were explored through LC– and GC–MS metabolomic analyses.ResultsIt was discovered that glucose concentration at 15–27 g/L and 80% medium replacement ratio were suitable for the growth of C. cohnii M-1-2 during the repeated fed-batch culture. A four-cycle repeated fed-batch culture was successfully developed and assessed at the optimum cultivation parameters, resulting in increasing the total DHA productivity by 26.28% compared with the highest DHA productivity of 57.08 mg/L/h reported using C. cohnii, including the time required for preparing seed culture and fermentor. In addition, LC– and GC–MS metabolomics analyses showed that the gradually decreased nitrogen utilization capacity, and down-regulated glycolysis and TCA cycle were correlated with the decreased stability of the culture system during the long-time repeated fed-batch culture. At last, some biomarkers, such as Pyr, Cit, OXA, FUM, l-tryptophan, l-threonine, l-leucine, serotonin, and 4-guanidinobutyric acid, correlated with the stability of culture system of C. cohnii M-1-2 were identified.ConclusionsThe study proved that repeated fed-batch cultivation was an efficient and energy-saving strategy for industrial production of DHA using C. cohnii, which could also be useful for cultivation of other microbes to improve productivity and reduce production cost. In addition, the mechanisms study at metabolite level can also be useful to further optimize production processes for C. cohnii and other microbes.

Optimization of Rhodomonas sp. under continuous cultivation for industrial applications in aquaculture

The microalgae species Rhodomonas sp. is commonly used in aquaculture for its high nutritional value due to the eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) content. Understanding the effect of cultivation parameters on biomass production rate and composition is presently limited, however essential in further commercialization of this strain. Under nutrient replete conditions, light intensity and temperature are the main factors determining biomass growth and composition. Therefore, the combined effect of light and temperature on the biomass production rate and biomass composition of Rhodomonas sp. was studied using a statistical Design of Experiment approach. Rhodomonas sp. was cultivated under continuous (turbidostat) conditions in lab-scale reactor systems (1.8 l) under different temperature (15–20–25–30 °C) and light conditions (60–195–330–465–600 μmol m−2 s−1). Stable biomass production was observed under all conditions except experiments performed at 30 °C, which led to cell death. Under optimized growth conditions, high growth rates (>1.0d−1) and high biomass production rates, up to 1.5 g l−1 d−1, were obtained in this study. The biomass production rate reported here is >10-fold higher than values reported in literature on Rhodomonas sp. The optimal temperature for maximal growth was found at T = 22–24 °C under all light conditions. The maximum biomass yield on light (Yx,ph – 0.87 g mol−1) was found at light levels between 110 and 220 μmol m−2 s−1. The fatty acid profile was only significantly influenced by temperature, with higher EPA and DHA contents at lower temperatures (15 °C). A total fatty acid (TFA) content of 8–10% of the total dry-weight was found for all tested conditions. The EPA content fluctuated between 9 and 16% of TFA and DHA content between 6 and 9% of TFA, only affected by temperature. A maximum EPA + DHA production rate of 114 mg l‐−1 d−1 was obtained at 20 °C and high light (600 μmol m−2 s−1) conditions.

Comparative Transcriptomic Analysis Uncovers Genes Responsible for the DHA Enhancement in the Mutant Aurantiochytrium sp.

Docosahexaenoic acid (DHA), a n-3 long-chain polyunsaturated fatty acid, is critical for physiological activities of the human body. Marine eukaryote Aurantiochytrium sp. is considered a promising source for DHA production. Mutational studies have shown that ultraviolet (UV) irradiation (50 W, 30 s) could be utilized as a breeding strategy for obtaining high-yield DHA-producing Aurantiochytrium sp. After UV irradiation (50 W, 30 s), the mutant strain X2 which shows enhanced lipid (1.79-fold, 1417.37 mg/L) and DHA (1.90-fold, 624.93 mg/L) production, was selected from the wild Aurantiochytrium sp. Instead of eicosapentaenoic acid (EPA), 9.07% of docosapentaenoic acid (DPA) was observed in the mutant strain X2. The comparative transcriptomic analysis showed that in both wild type and mutant strain, the fatty acid synthesis (FAS) pathway was incomplete with key desaturases, but genes related to the polyketide synthase (PKS) pathway were observed. Results presented that mRNA expression levels of CoAT, AT, ER, DH, and MT down-regulated in wild type but up-regulated in mutant strain X2, corresponding to the increased intercellular DHA accumulation. These findings indicated that CoAT, AT, ER, DH, and MT can be exploited for high DHA yields in Aurantiochytrium.

Lab-Scale Optimization of Aurantiochytrium sp. Culture Medium for Improved Growth and DHA Production

Thraustochytrids have gained increasing relevance over the last decades, due to their fast growth and outstanding capacity to accumulate polyunsaturated fatty acids (PUFAs), particularly docosahexaenoic acid (DHA). In this context, the present work aimed to optimize the growth performance and DHA yields by improving the culture medium of Aurantiochytrium sp. AF0043. Accordingly, two distinct culture media were optimized: (i) an inorganic optimized medium (IOM), containing only monosodium glutamate and glucose as nitrogen and carbon sources, respectively; and (ii) an organic and sustainable waste-based optimized medium (WOM), containing corn steep powder and glycerol, added in fed-batch mode, as nitrogen and carbon sources, respectively. Overall, the lab-scale optimization allowed to increase the biomass yield 1.5-fold and enhance DHA content 1.7-fold using IOM. Moreover, WOM enabled a 2-fold increase in biomass yield and a significant improvement in lipid contents, from 22.78% to 31.14%. However, DHA content was enhanced almost 3-fold, from an initial content of 10.12% to 29.66% of total fatty acids contained in the biomass. Therefore, these results strongly suggest, not only that the production pipeline was significantly improved but also confirmed the potential use of Aurantiochytrium sp. AF0043 as a source of DHA.

A new synthetic medium for the optimization of docosahexaenoic acid production in Crypthecodinium cohnii.

The heterotrophic microalgae Crypthecodinium cohnii was usually cultivated in complex medium containing glucose, yeast extract and sea salt. For the preparation of DHA with highest purity, a new defined medium without the yeast extract was developed. Different inoculated densities, C/N ratios, temperatures, culture volumes and glucose additions were investigated to optimize the algal growth rate and DHA production. The growth period in C. cohnii was shortened from 12-14 days to 7-8 days, the OD600 was enhanced from 2.0 to 3.0, the glucose consumption was accelerated and used up on day 3-4, and the DHA content in culture were increased from 10 to 45 nmoles/300 μl batch. It was found that C. cohnii had optimal growth and DHA accumulation in 25 °C, 0.2 inoculated density, 5-10 C/N ratio, 5:1 air/culture volume ratio. This is the first time DHA production using C.cohnii has been optimized in synthetic medium. This allows preparation of uniformly radiolabeled 13C- and 14C-DHA.

Biosynthesis of uniformly carbon isotope-labeled docosahexaenoic acid in Crypthecodinium cohnii

Docosahexaenoic acid (DHA) enriched in brain can yield many important degradation products after the attack of hydroxyl radicals, which is known to serve as a nutraceutical and neuroprotective effects. Oxidative stress is a commonly observed feature of Alzheimer’s disease (AD). Therefore, uniformly radiolabeled DHA plays an important role in studying the oxidative fate of DHA in vivo and vitro. However, carbon isotope labeled DHA isn’t commercially available now. The heterotrophic microalgae Crypthecodinium cohnii (C. cohnii) has been identified as a prolific producer of DHA. In this study, the growth rate and DHA production in C. cohnii were optimized in a new defined media, and the biosynthesis of U-13C-DHA from U-13C-glucose and U-14C-DHA from U-14C-glucose were analyzed by HPLC–MS/MS. Approximately 40 nmoles of U-13C-DHA with higher isotopic purity of 96.8% was produced in a 300 μL batch, and ~ 0.23 μCi of U-14C-DHA with significant specific activity of 5–6 Ci/mol was produced in a 300 μL batch. It was found that C. cohnii had the optimal growth and DHA accumulation at 25 °C in this defined media (C/N = 10). An efficient protocol for the biosynthesis of U-13C-DHA and U-14C-DHA were set up firstly, which provides the basic support for the analysis of oxidative degradation products of DHA in AD.

Effect of sequential recycling of spent media wastewater on docosahexaenoic acid production by newly isolated strain Aurantiochytrium sp. ICTFD5

The main aim of this work was the optimization of recycled spent media wastewater (SMW) concentration for high biomass production of Aurantiochytrium sp. ICTFD5. Further, optimization for growth patterns and lipid accumulation capacity with three subsequent recycling runs was also performed. The biomass production after 96 h fermentation for recycling with 50% SMW was; 21.3 ± 1.5, 19.1 ± 1.3, 19 ± 1.2, and 23 ± 1.2 g/L for the first, second, third recycle runs, and control respectively. All the recycle runs were carried out with the same media and cultivation conditions. Subsequent recycling affected lipid accumulation, and it was decreased by ~4 to 9% compared to the control. The compositional shift of fatty acids was observed with sequential recycle runs, changing more towards saturated fatty acids content, suggesting it to be a new potential source for biodiesel feedstock.

Co-production of DHA and squalene by thraustochytrid from forest biomass

Omega-3 fatty acids, and specifically docosahexaenoic acid (DHA), are important and essential nutrients for human health. Thraustochytrids are recognised as commercial strains for nutraceuticals production, they are group of marine oleaginous microorganisms capable of co-synthesis of DHA and other valuable carotenoids in their cellular compartment. The present study sought to optimize DHA and squalene production by the thraustochytrid Schizochytrium limacinum SR21. The highest biomass yield (0.46 g/gsubstrate) and lipid productivity (0.239 g/gsubstrate) were observed with 60 g/L of glucose, following cultivation in a bioreactor, with the DHA content to be 67.76% w/wtotal lipids. To reduce costs, cheaper feedstocks and simultaneous production of various value-added products for pharmaceutical or energy use should be attempted. To this end, we replaced pure glucose with organosolv-pretreated spruce hydrolysate and assessed the simultaneous production of DHA and squalene from S. limacinum SR21. After the 72 h of cultivation period in bioreactor, the maximum DHA content was observed to 66.72% w/wtotal lipids that was corresponded to 10.15 g/L of DHA concentration. While the highest DHA productivity was 3.38 ± 0.27 g/L/d and squalene reached a total of 933.72 ± 6.53 mg/L (16.34 ± 1.81 mg/gCDW). In summary, we show that the co-production of DHA and squalene makes S. limacinum SR21 appropriate strain for commercial-scale production of nutraceuticals.

Biofilm cultivation of marine dinoflagellates under different temperatures and nitrogen regimes enhances DHA productivity

Dinoflagellates contain large amounts of omega-3 fatty acids, including the nutritionally important docosahexaenoic acid (DHA). However, their cultivation in suspensions is characterized by low growth rates. Twin-layer porous substrate photobioreactors (TL-PSBRs) have been shown to support growth of different microalgal species, including the robust dinoflagellate Symbiodinium voratum. In the present study, the potential of cultivating marine autotrophic dinoflagellate species in a TL-PSBR for DHA production was explored. Based on initial screening experiments, two Symbiodinium species with high biomass and DHA productivities were selected: the symbiotic Symbiodinium microadriaticum CCAC 2475 B and the free-living Symbiodinium voratum CCAC 3869 B. The effects of three different temperatures (17, 22 and 27 °C) and nitrogen regimes (nitrate, ammonium and nitrogen-free) on biomass growth, total lipid accumulation and fatty acid methyl esters (FAMEs) content, with emphasis on DHA, were evaluated. The two lower temperatures (17 and 22 °C) enhanced growth and total lipid accumulation of S. microadriaticum CCAC 2475 B and S. voratum CCAC 3869 B. Cultivation at 22 °C and nitrogen limitation led to a significant positive effect on DHA productivity. Symbiodinium. microadriaticum CCAC 2475 B reached a DHA productivity of 145.4 mg m−2 day−1 and DHA content in the dry biomass of 2% (w/w) after 4 days of nitrogen depletion. The results of the present study demonstrated that autotrophic dinoflagellates, when cultivated on a TL-PSBR, produce comparable amounts of lipids and fatty acids to other commercially used microalgal species including the valuable DHA.

Optimization of the production of docosahexaenoic fatty acid by the heterotrophic microalga Crypthecodinium cohnii utilizing a dark fermentation effluent

Dark fermentation is an anaerobic digestion process of biowaste, used to produce hydrogen as a fuel, which however releases high amounts of polluting volatile fatty acids in the environment. In order for the process to become more competitive, the acids stream can be utilized through conversion to high added-value docosahexaenoic acid by the microalga Crypthecodinium cohnii. Docosahexaenoic acid is one of the two main omega-3 fatty acids, necessary for human nutrition. The purpose of this work was to optimize the production of omega-3 fatty acids by the cells, utilizing the organic content of a dark fermentation effluent. For that purpose, the effect of different fermentation conditions was examined, such as incubation temperature, nitrogen source and concentration, the addition of chemical modulators, as well as the feeding composition. The volatile fatty acid content of the effluent was totally depleted in a fed-batch culture of the microalga, while the cells accumulated DHA in a percentage of 35.6% of total lipids, when fed with yeast extract or 34.2% when fed with ammonium sulfate. Taking into consideration the economic feasibility of the culture conditions proposed it was concluded that the use of yeast extract could be substituted by the much economic ammonium sulfate.

Nutrient recovery from tofu whey wastewater for the economical production of docosahexaenoic acid by Schizochytrium sp. S31

Docosahexaenoic acid plays a vital role in human health as it is essential for the proper function of the nervous system and for visual functions. To decrease the cost of docosahexaenoic acid production by Schizochytrium, the cost of the medium should be further decreased. In this study, the use of tofu whey wastewater to culture Schizochytrium sp. for docosahexaenoic acid production was tested, with the goal of reducing the medium cost. The results indicated that tofu whey wastewater presented a better culture performance with respect to biomass, lipid, and docosahexaenoic acid production compared with three traditional media. Through simple pH adjustment, the biomass and docosahexaenoic acid productivity reached 1.89 and 0.24 g/L/day, respectively, which were much higher than those obtained using traditional medium. The removal efficiency of chemical oxygen demand, total nitrogen, and total phosphorus reached 64.7, 66.0, and 59.3%, respectively. Due to the rich nutrients in tofu whey wastewater, the use of extra nitrogen source was avoided and the total medium cost for docosahexaenoic acid production in cultures using tofu whey wastewater was <1/3 of that of traditional media. This result indicated that tofu whey wastewater is an effective and economic basal medium for docosahexaenoic acid production by Schizochytrium sp.

Identity and Safety of a Novel &amp;lt;i&amp;gt;Aurantiochytrium sp.&amp;lt;/i&amp;gt; for Terrestrial Heterotrophic Docosahexaenoic Acid Production

The objective of the studies in this paper was to expand on the published toxicological assessment of Aurantiochytrium limacinum (AURA) with further strain characterization and to investigate the potential for the biomass or extracted oil to have antimicrobial properties or undesirable substances. AURA is being investigated as a novel source of the omega-3 long-chain polyunsaturated fatty acid docosahexaenoic acid (DHA) for enriching foods of animal origin by means of feed supplementation. In the first studies, we provided the 18S rRNA identification of the novel marine isolated thraustochytrid, established the nutritional composition of AURA biomass for application as a food or feed ingredient including proximate analysis and fatty acid profiling, and confirmed the DHA production potential of the strain. We determined through minimum inhibitory concentration (MIC) analysis that the unextracted AURA biomass was safe, showing no antimicrobial influence and no evidence of any deleterious effects of this product or its extracts at concentrations up to 1% w/w on the reference human intestinal bacteria tested. This would indicate that AURA should not stimulate selective pressure on the commensal microbiota and is therefore unlikely to aid development of antimicrobial resistance and the concomitant harm to humans and animals. Further analysis revealed that the AURA biomass produced through industrial heterotrophic fermentation was free from undesirables; toxic marine microalgal metabolites, heavy metals, pesticides, microbial contaminants, and mycotoxins. Including heterotrophically-grown AURA in food or feed, up to 1% w/w, is a safe and environmentally beneficial strategy for DHA supplementation.

Exergy analysis for docosahexaenoic acid production by fermentation and strain improvement by adaptive laboratory evolution for Schizochytrium sp.

Exergy analysis is powerful tool for process optimization and mechanism analysis. In this study, exergy analysis was performed for docosahexaenoic acid (DHA) fermentation process. More than 86% of input exergy was contributed by glucose. The exergy of biomass was about 64.66% of the total output exergy when the phosphate concentration was 4 g L−1. The exergy efficiencies of DHA (ηDHA) for the starting strains and the evolved strains under high oxygen concentration, low temperature, and two-factor conditions were also investigated. The ηDHA in the collected experimental data was not more than 20.9%. It was proved that there was a positive correlation between ηDHA and the biomass yield. It was indicated that adaptive laboratory evolution (ALE) improved biomass yield which had the most important effect on enhancing ηDHA and DHA yield (or DHA productivity). It is necessary to improve ηDHA through process optimization and ALE in future.

Lipid and DHA-production in Aurantiochytrium sp. \u2013 Responses to nitrogen starvation and oxygen limitation revealed by analyses of production kinetics and global transcriptomes

Thraustochytrids of the genera Schizochytrium and Aurantiochytrium accumulate oils rich in the essential, marine n3 fatty acid docosahexaenoic acid (DHA). DHA production in Aurantiochytrium sp T66 was studied with the aim to provide more knowledge about factors that affect the DHA-productivities and the contributions of the two enzyme systems used for fatty acid synthesis in thraustochytrids, fatty acid synthetase (FAS) and PUFA-synthase. Fermentations with nitrogen starvation, which is well-known to initiate lipid accumulation in oleaginous organisms, were compared to fermentations with nitrogen in excess, obtained by oxygen limitation. The specific productivities of fatty acids originating from FAS were considerably higher under nitrogen starvation than with nitrogen in excess, while the specific productivities of DHA were the same at both conditions. Global transcriptome analysis showed significant up-regulation of FAS under N-deficient conditions, while the PUFA-synthase genes were only marginally upregulated. Neither of them was upregulated under O2-limitation where nitrogen was in excess, suggesting that N-starvation mainly affects the FAS and may be less important for the PUFA-synthase. The transcriptome analysis also revealed responses likely to be related to the generation of reducing power (NADPH) for fatty acid synthesis.

Biological Carbon Recovery from Sugar Refinery Washing Water into Microalgal DHA: Medium Optimization and Stress Induction

Sugar refinery washing water (SRWW) contains abundant levels of carbon sources and lower levels of contaminants than other types of wastewater, which makes it ideal for heterotrophic cultivation of microalgae. Here, carbon sources in SRWW were utilized for conversion into the form of value-added docosahexaenoic acid (DHA) using Aurantiochytrium sp. KRS101. Since SRWW is not a defined medium, serial optimizations were performed to maximize the biomass, lipid, and DHA yields by adjusting the nutrient (carbon, nitrogen, and phosphorus) concentrations as well as the application of salt stress. Optimum growth performance was achieved with 30% dilution of SRWW containing a total organic carbon of 95,488 mg L−1. Increasing the nutrient level in the medium by supplementation of 9 g L−1 KH2PO4 and 20 g L−1 yeast extract further improved the biomass yield by an additional 14%, albeit at the expense of a decrease in the lipid content. Maximum biomass, lipid, and DHA yields (22.9, 6.33, and 2.03 g L−1, respectively) were achieved when 35 g L−1 sea salt was applied on a stationary phase for osmotic stress. These results demonstrate the potential of carbon-rich sugar refinery washing water for DHA production using Aurantiochytrium sp. KRS101 and proper cultivation strategy.