Docosahexaenoic Acid Production Research Articles

Dietary eicosapentaenoic acid and docosahexaenoic acid are linearly retained by common insect crop pests (cabbage looper and bertha armyworm) and alter insect biomass

AbstractThe long‐chain omega‐3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are prevalent in aquatic ecosystems and are not part of the natural diet of herbivorous, terrestrial insects, which generally consume alpha‐linolenic acid (ALA) and linoleic acid (LNA). However, recent advances in genetic engineering have lead to the development of terrestrial crops that express the novel traits of EPA and DHA production. In the present study, we examine the effects of dietary EPA and DHA on the growth, development and fatty acid content of two crop pest insects: bertha armyworm and cabbage looper. Five experimental diets were formulated to include increasing amounts of pure EPA and DHA (in relation to the total diet lipid level), according to the ratios (EPA + DHA relative to a vegetable oil containing ALA and LNA): 0 (control), 0.25 : 0.75 (lowest), 0.5 : 0.5 (low), 0.75 : 0.25 (medium) and 1 : 0 (high). Dietary EPA and DHA had significant effects on development time, mass and fatty acid content in both species. Dietary treatment (interactive with time) had a significant effect on individual mass of both insects, indicating that, over time, EPA and DHA impacted growth. However, insect mass, development and morphology results are not linearly related with increasing dietary EPA and DHA. Both species retained EPA and DHA in adult form, and the body content of EPA and DHA was significantly, positively correlated with EPA and DHA diet treatments in both the bertha armyworm (r2 = 91.3%) and cabbage looper (r2 = 75.8%). Dietary EPA and DHA could have fitness consequences for these organisms and could be nutritionally transferred to higher consumers.

Fed-batch fermentation of mixed carbon source significantly enhances the production of docosahexaenoic acid in Thraustochytriidae sp. PKU#Mn16 by differentially regulating fatty acids biosynthetic pathways.

This study reports comparative evaluation of the growth and DHA productivity of the thraustochytrid strain Thraustochytriidae PKU#Mn16 fermented with seven different substrate feeding strategies. Of these strategies, fed-batch fermentation of the mixed substrate (glucose & glycerol) yielded the maximum growth (52.2 ± 1.5 g/L), DHA yield (Yp/s: 8.65) and productivity (100.7 ± 2.9 mg/L-h), comparable with those of previously reported Aurantiochytrium strains. Transcriptomics analyses revealed that glucose upregulated some genes of the fatty acid synthase pathway whereas glycerol upregulated a few genes of the polyketide synthase pathway. Co-fermentation of the mixed substrate differentially regulated genes of these two pathways and significantly enhanced the DHA productivity. Furthermore, some genes involved in DNA replication, phagosome, carbon metabolism, and β-oxidation were also found to alter significantly during the mixed-substrate fermentation. Overall, this study provides a unique strategy for enhancing growth and DHA productivity of the strain PKU#Mn16 and the first insight into the mechanisms underlying mixed-substrate fermentation.

De Novo Transcriptome Analysis of Polyunsaturated Fatty Acid Metabolism in Marine Protist Thraustochytriidae sp. PKU#Mn16

AbstractThraustochytriidae sp., have drawn the attention as a promising source for producing significant quantities of omega‐3 fatty acids. In this study, Thraustochytriidae sp. PKU#Mn16, with impressive docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) production, was de novo assembled as a complete transcriptome. A total of 59.65 Mb clean reads were assembled into 35,745 transcripts, with a mean size of 991 bp. The results allowed us to annotate 11,924 (45.97%) of the transcripts with putative functions. The potential target genes enriched for specific gene ontology terms, including metabolic process (469 genes) and macromolecular complex (221 genes), were identified. There were 6858 (26.44%) putative proteins classified into 25 categories, of which 459 (6.69%) predicted proteins were assigned to lipid transport and metabolism, closely related to DHA production. The most strongly represented pathways annotated from the kyoto encyclopedia of genes and genomes (KEGG) database were metabolism pathways including carbohydrate (388, 6.99%), amino acid (375, 6.76%), and lipid (330, 5.95%). Of these unigenes with coding sequences, the majority (2950) were 200–300 bp. A total of 7362 simple sequence repeats were identified with the frequency occurrence of 1/4.15 kb. This study reveals a wealth of the most comprehensive transcriptomic resource currently available for Thraustochytriidae sp., which could facilitate further research into the molecular mechanisms of fatty acid production.

Influence of nitrogen sources on growth of thraustochytrids in waste water from the demineralization of cheese whey

An experimental design was ued to optimize the growth of two thraustochytrids, (Schizochytrium limacinum PA-968 and Japonochytrium marinum AN-4), on different nitrogen sources (yeast extract, corn steep liquor, ammonium sulphate) supplemented into saline waste water from the demineralization of cheese whey. Yeast extract was found to be the most suitable complex nutrient source. Nitrogen limitation was found to increase the lipid content in shake flask cultures of thraustochytrids by 12.7–22.4% w/w. The maximum total lipid content (79.1% w/w) and docosahexaenoic acid productivity (0.465 g/l per day) were achieved by J. marinum AN-4 in shake flask cultures. Fed-batch cultures of J. marinum AN-4, under conditions of nitrogen limitation, yielded biomass with a lower lipid content (72.1% wt.) but higher docosahexaenoic acid productivity (1.43 g/l per day). These results provide proof of concept that fed-batch cultivation of thraustochytrids, combined with nitrogen limitation, can be an appropriate strategy for the productive use of saline waste water from the dairy industry.

Simultaneous production of DHA and squalene from Aurantiochytrium sp. grown on forest biomass hydrolysates

BackgroundRecent evidence points to the nutritional importance of docosahexaenoic acid (DHA) in the human diet. Thraustochytrids are heterotrophic marine oleaginous microorganisms capable of synthesizing high amounts of DHA, as well as other nutraceutical compounds such as squalene, in their cellular compartment. Squalene is a natural triterpene and an important biosynthetic precursor to all human steroids. It has a wide range of applications in the cosmetic and pharmaceutical industries, with benefits that include boosting immunity and antioxidant activity. Apart from its nutritional quality, it can also be utilized for high-grade bio-jet fuel by catalytic conversion.ResultsIn the present study, the potential of thraustochytrid strain Aurantiochytrium sp. T66 to produce DHA and squalene was evaluated. When the strain was cultivated on organosolv-pretreated birch hydrolysate (30 g/L glucose) in flask, it resulted in 10.39 g/L of cell dry weight and 4.98 g/L of total lipids, of which 25.98% was DHA. In contrast, when the strain was grown in a bioreactor, cell dry weight, total lipid, and DHA increased to 11.24 g/L, 5.90 g/L, and 35.76%, respectively. The maximum squalene yield was 69.31 mg/gCDW (0.72 g/L) when the strain was cultivated in flask, but it increased to 88.47 mg/gCDW (1.0 g/L), when cultivation shifted to a bioreactor.ConclusionsThis is the first report demonstrating the utilization of low cost non-edible lignocellulosic feedstock to cultivate the marine oleaginous microorganism Aurantiochytrium sp. for the production of nutraceutical vital compounds. Owing to the simultaneous generation of DHA and squalene, the strain is suitable for industrial-scale production of nutraceuticals.

Molecular and functional characterisation of a putative elovl4 gene and its expression in response to dietary fatty acid profile in Atlantic bluefin tuna (Thunnus thynnus).

Elongation of very long-chain fatty acid 4 (Elovl4) proteins are involved in the biosynthesis of very long-chain (>C24) fatty acids and in many teleost fish species they are key enzymes in the pathway for the production of docosahexaenoic acid (DHA; 22:6n-3) from eicosapentaenoic acid (EPA; 20:5n-3). Therefore, Elovl4 may be particularly important in Atlantic bluefin tuna (ABT; Thunnus thynnus) characterised by having high DHA to EPA ratios. The present study cloned and characterised both the function and expression of an elovl4 cDNA from ABT. The Elovl4 had an open reading frame of 915 base pairs encoding a putative protein of 304 amino acids. Alignment and phylogenetic analyses indicated that the Elovl4 isoform identified in the present study was an Elovl4b. Functional characterisation demonstrated that the Elovl4b enzyme had elongase activity towards all the polyunsaturated fatty acid (PUFA) substrates assayed. The ABT Elovl4b contributed to DHA biosynthesis by elongation of EPA and DPA to 24:5n-3, the latter being desaturated to 24:6n-3 by the action of fads2 (Δ6 desaturase). Additionally, the ABT Elovl4b has a role in the biosynthesis of very long-chain PUFA up to C34, compounds of key structural roles in neural tissues such as eye and brain, which had high levels of elovl4b transcripts. Surprisingly, while the relative expression of fads2, required for the production of DHA from EPA, was increased in liver of ABT fed a diet with reduced levels of EPA and DHA, expression of elovl4b was reduced. Results indicated that ABT has enzymes necessary for endogenous production of DHA from EPA and demonstrate that Elovl4b can effectively compensate for absence of Elovl2.

Impact of bubble size on docosahexaenoic acid production by Crypthecodinium cohnii in bubble column bioreactor

Cultivation conditions especially agitation and aeration have critical roles on Crypthecodinium cohnii growth and docosahexaenoic acid (DHA) formation. In this study, for the first time, different air bubble sizes were considered, in a 5-L bubble column bioreactor; and the effect of bubble size on growth and on DHA production by dinoflagellate microalgae C. cohnii was investigated. The cultivation was carried out under 0.5 vvm aeration flow rate, 28 °C, and pH ± 7. The highest yield of DHA (1.4 g DHA L−1) was achieved for 0.36-cm air bubble diameter; the corresponding biomass amount was 17.6 g. The suitable conditions during the logarithmic stage of growth were provided for 0.36-cm air bubble diameter, while oxygen limitation was observed in the culture medium afterwards. The concentration of the microalgal DHA was increased in oxygen limitation conditions. Our results indicate that the air bubble diameter is the most influencing factor on DHA production in a bubble column bioreactor. Current research indicates that optimum bubble size in bubble column bioreactor can lead to optimal conditions for growth and DHA production like two-stage oxygen supply feeding strategy.

Projected declines in global DHA availability for human consumption as a result of global warming

Docosahexaenoic acid (DHA) is an essential, omega-3, long-chain polyunsaturated fatty acid that is a key component of cell membranes and plays a vital role in vertebrate brain function. The capacity to synthesize DHA is limited in mammals, despite its critical role in neurological development and health. For humans, DHA is most commonly obtained by eating fish. Global warming is predicted to reduce the de novo synthesis of DHA by algae, at the base of aquatic food chains, and which is expected to reduce DHA transferred to fish. We estimated the global quantity of DHA (total and per capita) currently available from commercial (wild caught and aquaculture) and recreational fisheries. The potential decrease in the amount of DHA available from fish for human consumption was modeled using the predicted effect of established global warming scenarios on algal DHA production and ensuing transfer to fish. We conclude that an increase in water temperature could result, depending on the climate scenario and location, in a ~ 10 to 58% loss of globally available DHA by 2100, potentially limiting the availability of this critical nutrient to humans. Inland waters show the greatest potential for climate-warming-induced decreases in DHA available for human consumption. The projected decrease in DHA availability as a result of global warming would disproportionately affect vulnerable populations (e.g., fetuses, infants), especially in inland Africa (due to low reported per capita DHA availability). We estimated, in the worst-case scenario, that DHA availability could decline to levels where 96% of the global population may not have access to sufficient DHA.

Inositol as a new enhancer for improving lipid production and accumulation in Schizochytrium sp. SR21.

Lipids produced from agricultural and industrial residues using oleaginous microorganisms for use as biofuels are attracting the attention of researchers due to their environmental benefits. However, low efficiencies and high costs limit their application to a certain extent. The present study is the first to use inositol as an enhancer to improve the production and accumulation of lipids during fermentation by the microalga Schizochytrium sp. SR21. The study aimed to maximize the production of lipids and docosahexaenoic acid (DHA) by optimizing the conditions of inositol addition into the fermentation medium. The corresponding key enzyme and metabolite profiles of SR21 were evaluated. The results indicated that the addition of 120mgL-1 of inositol to the medium at 48h improved lipid and DHA production by 13.90 and 20.82%, resulting in total concentrations of 22.86 and 8.53g/L, respectively. Moreover, the ratio of unsaturated fatty acids (UFAs) to saturated fatty acids (SFAs) increased by 23.38% and is consistent with the results of the metabolomic analysis. The activity of enzymes (i.e., PC, G6PDH, NADPH-ME, and ACL) related to fatty acid synthesis in strain SR21 also increased significantly (43.38%, 28.68%, 37.47%, and 19.87%, respectively). Metabolomic analysis also showed that inositol promoted lipid synthesis in SR21 and significantly increased the relative proportion of UFAs by affecting the citrate cycle and SFA and UFA metabolic pathways. Thus, inositol is an ideal enhancer of lipid production and accumulation by oleaginous microorganisms. Graphical abstract.

Lipid Distribution Pattern and Transcriptomic Insights Revealed the Potential Mechanism of Docosahexaenoic Acid Traffics in Schizochytrium sp. A-2.

Schizochytrium sp. A-2 is a heterotrophic marine fungus used for the commercial production of docosahexaenoic acid (DHA). However, the pattern of the distribution of DHA and how DHA is channeled into phospholipid (PL) and triacylglycerol (TAG) are unknown. In this study, we systematically analyzed the distribution of DHA in TAG and PL during the growth of the cell. The migration of DHA from PL to TAG was presumed during the fermentation cycle. DHA and docosapentaenoic acid were accumulated in both TAG and phosphatidylcholine (PC), whereas eicosapentaenoic acid was mainly deposited in PC. RNA seq revealed that malic enzyme may provide lipogenic NADPH. In addition, long-chain acyl-CoA synthase and acyl-CoA:lysophosphatidylcholine acyltransferase may participate in the accumulation of DHA in PL. No phosphatidylcholine:diacylglycerol cholinephosphotransferase was identified from the genome sequence. In contrast, phospholipid:diacylglycerol acyltransferase-mediated acyl-CoA-independent TAG synthesis pathway and phospholipase C may contribute to the channeling of DHA from PC to TAG.

Screening for hyper-accumulating lipid mutants in Aurantiochytrium limacinum using high-throughput fluorescence-based method

Docosahexaenoic acid (DHA) has many unique physiological functions such as promoting the development of brain and retina in infants. Therefore, it is widely applied to food, pharmacy, breeding and other industries. To obtain engineered strains of Aurantiochytrium limacinum SR21 suitable for industrial application with increased lipid and DHA production, we designed a simple, fast, accurate and high-throughput screening method based on Nile red staining of oil droplets. First, ultraviolet C (UVC) mutagenesis was used to generate a random mutant library of A. limacinum. Second, screening conditions were optimized including staining conditions of Nile red and the sorting criterion. Thereby, three putative high-lipid mutants (D03432, D05106 and D01521) were selected from the mutant library containing 3 648 mutated clones. The three mutants grew faster and produced higher amounts of lipids and DHA compared to wild type (WT). In 100 mL cultures, the lipid content of D03432 and D05106 mutants reached 64.74% and 63.13% of dry cell weight respectively, whereas the wild strain exhibited only 43.19%. DHA yield in these two mutants were even 2.26-fold and 2.37-fold higher than that of the wild strain. Experiment with 5 L fermentor further confirmed that D03432 and D05106 mutants had better performance than the wild strain on DHA yield (45.51% and 66.46% more than that of the wild strain, respectively), and demonstrated their high potential for industrial application. This work not only generated several high-DHA content mutants with high potential for industrial use, but also provided vital guidance for high-throughput screening of lipid hyper-accumulating mutants in other oil-producing microorganisms.

Screening of Isochrysis strains for simultaneous production of docosahexaenoic acid and fucoxanthin

Microalgae are capable of synthesizing a wide range of value-added compounds such as docosahexaenoic acid (DHA) and fucoxanthin. In the present study, the potential for simultaneous production of DHA and fucoxanthin by the marine alga Isochrysis was investigated via strain screening and culture optimization. A comparative analysis of sixteen Isochrysis strains revealed that CCMP1324 was superior with regard to growth and production of DHA and fucoxanthin. In response to various nutritional and environmental cues, CCMP1324 accumulated DHA and fucoxanthin in a coordinated and growth-dependent manner, which reached ~17.1 and 14.1 mg g−1, respectively. A semi-continuous culture strategy leading to high productivities of DHA (9.05 mg L−1 day−1) and fucoxanthin (7.96 mg L−1 day−1) was developed. Taken together, our study represents a pioneering work studying the accumulation coordination between DHA and fucoxanthin in Isochrysis. These findings provide valuable insights into future exploration for the integrated production of high-value compounds.

Culturable Diversity and Lipid Production Profile of Labyrinthulomycete Protists Isolated from Coastal Mangrove Habitats of China.

Labyrinthulomycete protists have gained significant attention in the recent past for their biotechnological importance. Yet, their lipid profiles are poorly described because only a few large-scale isolation attempts have been made so far. Here, we isolated more than 200 strains from mangrove habitats of China and characterized the molecular phylogeny and lipid accumulation potential of 71 strains. These strains were the closest relatives of six genera namely Aurantiochytrium, Botryochytrium, Parietichytrium, Schizochytrium, Thraustochytrium, and Labyrinthula. Docosahexaenoic acid (DHA) production of the top 15 strains ranged from 0.23 g/L to 1.14 g/L. Two labyrinthulid strains, GXBH-107 and GXBH-215, exhibited unprecedented high DHA production potential with content >10% of biomass. Among all strains, ZJWZ-7, identified as an Aurantiochytrium strain, exhibited the highest DHA production. Further optimization of culture conditions for strain ZJWZ-7 showed improved lipid production (1.66 g/L DHA and 1.68 g/L saturated fatty acids (SFAs)) with glycerol-malic-acid, peptone-yeast-extract, initial pH 7, 28 °C, and rotation rate 150 rpm. Besides, nitrogen source, initial pH, temperature, and rotation rate had significant effects on the cell biomass, DHA, and SFAs production. This study provides the identification and characterization of nearly six dozen thraustochytrids and labyrinthulids with high potential for lipid accumulation.

Strategies for Enzymatic Synthesis of Omega‐3 Structured Triacylglycerols from Camelina sativa Oil Enriched in EPA and DHA

Enzymatic production of enriched eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) structured triacylglycerols (STAGs) is done in a two‐step reaction from a new source of omega‐3 α‐linolenic acid (ALA), Camelina sativa oil. A fast‐pressurized liquid extraction method with ethyl acetate as green solvent is used to produce camelina oil with a 39.3% yield in less than 20 min. In a first stage, camelina oil with a 29.7% of ALA is used to enzymatically produce 2‐monoacylglycerols (2‐MAGs) with high ALA composition in position sn‐2 (49.8% of ALA) using Lipozyme TL IM at 25 °C. For omega‐3 incorporation into STAGs, several lipases are studied, with 2‐MAG and omega‐3 FAEEs at different ratios (1:5, 1:10, and 1:20 (w/w)), 10% (w/w) of biocatalyst at 35 °C. STAGs yields reached with EPA ethyl esters are notably higher than using DHA‐EE. Lipase from Candida antarctica B (CAL‐B) and Thermomyces lanuginosus (TLL) incorporate EPA into STAGs in the same degree, 95.1 and 95.5%, respectively. However, for enriched DHA STAGs, maximum reaction yield is reached with CAL‐B (81.0%). The transesterification method of 2‐MAGs from vegetable omega‐3 sources like camelina oil, with marine EPA and DHA FAEEs, opens new approaches for developing VLC‐PUFA fortified functional foods.Practical Applications: Results provided in this research not only demonstrate the efficiency of this enzymatic method for enriching camelina oil in EPA and DHA, but offer a very effective strategy for the development of omega‐3‐enriched products from other vegetable oil sources like chia or echium as ingredients for functional foods with recognized health claims for applications in nutraceutical and food industries. Camelina oil proved to be an excellent source to enzymatically produce 2‐MAGs enriched in omega‐3 ALA. Moreover, lipase screening with commercial biocatalysts establish the most appropriate strategy to reach maximum STAGs yields with direct application in industry. Tested lipases exhibit different degree of substrate specificity toward EPA and DHA, omega‐3 acids not present in natural seed oils. Thus, CAL‐B seems to be the most suitable lipase to produce high content EPA and DHA enriched STAGs from camelina oil as new food ingredients for nutraceutical applications.Enzymatic production of structured TAGs enriched in EPA and DHA is done in a two‐step reaction from a natural source of omega‐3 ALA, camelina oil, studying reaction kinetics and characterizing isolated products.

Influence of Nitrogen Limitation on Lipid Accumulation and EPA and DHA Content in Four Marine Microalgae for Possible Use in Aquafeed

Microalgae are regarded as a promising alternative that can replace fishmeal and fish oil in aquaculture. Under N-limitation, many microalgae species change their carbon storage patterns in favor of neutral lipids (NLs) mainly in the form of triacylglycerol (TAG), but fatty acids in polar lipids (PL) are nutritionally more available for fish than those esterified into NLs. In the present study, the effect of N-limitation on the lipid content and fatty acid profiles in different lipid class of Phaeodactylum tricornutum, Isochrysis aff. galbana clone T-Iso, Rhodomonas baltica and Nannochloropsis oceanica were investigated. The microalgae cells were cultivated by two different methods, batch and semi-continuous culture, to create strong and moderate N-limitation, and this in turn will significantly affect the biomass and lipid productivity. All four species accumulated lipids mainly in the form of TAG in response to strong nitrogen limitation. N. oceanica, however accumulated 51% of the dry weight as lipid in moderate nitrogen limitation and up to 87% of the fatty acid was in TAG. Isochrysis aff. galbana clone T-Iso was the only species where the fraction of polyunsaturated fatty acid (PUFA), especially the fraction of docosahexaenoic acid (DHA) increased with increasing nitrogen limitation. Total lipid productivity showed no increase in batch culture although stronger nitrogen limitation led to lipid accumulation. P. tricornutum had the highest eicosapentaenoic acid (EPA) content, while N. oceanica showed the highest EPA productivity due to the high content of lipid. The highest DHA productivity was found in Isochrysis aff. galbana clone T-Iso from moderate N-limitation mainly due to the high biomass productivity. Based on the results from the current study, N. oceanica and T-Iso are two promising microalgae strains as long-term sustainable source of n-3 long chain -PUFAs under moderate N-limitation. As shown in the present study, increased lipid content in microalgal cells due to strong N-limitation induction may not increase the lipid productivity because biomass production usually is reduced. Therefore, a combination of approaches such as metabolic engineering, conditioning and selection may be needed to further increase the n-3 LC-PUFA productivity without substantial loss of biomass.

Quantitation of seven transmembrane proteins from the DHA biosynthesis pathway in genetically engineered canola by targeted mass spectrometry

Examining tissue-specific expression and the measurement of protein abundance are important steps when assessing the performance of genetically engineered crops. Liquid chromatography-mass spectrometry offers many advantages over traditional methods for protein quantitation, especially when dealing with transmembrane proteins that are often difficult to express or generate antibodies against. In this study, discovery proteomics was used to detect the seven transgenic membrane-bound enzymes from the docosahexaenoic acid (DHA) biosynthetic pathway that had been engineered into canola. Subsequently, a targeted LC-MS/MS method for absolute quantitation was developed and applied to the simultaneous measurement of the seven DHA biosynthetic pathway enzymes in genetically modified canola grown across three sites. The results of this study demonstrated that the enzymatic proteins that drive the production of DHA using seed-specific promoters were detected only in mature and developing seed of DHA canola. None of the DHA biosynthesis pathway proteins were detected in wild-type canola planted in the same site or in the non-seed tissues of the transgenic canola, irrespective of the sampling time or the tissues tested. This study describes a streamlined approach to simultaneously measure multiple membrane-bound proteins in planta.

Production of High-Value Polyunsaturated Fatty Acids Using Microbial Cultures.

Microbes can produce not only commodity fatty acids, such as palmitic acid (16:0) and stearic acid (18:0), but also high-value fatty acids (essential fatty acids). Most high value fatty acids belong to long chain polyunsaturated fatty acids (PUFA), such as omega-3 fatty acids (e.g., eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)) and omega-6 fatty acids (e.g., arachidonic acid (ARA) and γ-linolenic acid (GLA)). EPA (20:5n-3) is a 20-carbon fatty acid with five double bonds, and the first double bond is in the n-3 position. DHA (22:6n-3) is a 22-carbon fatty acid with 6 double bonds and the first double bond is also in the n-3 position. Both EPA and DHA play an essential role in cardiovascular health including prevention of atherosclerotic disease development (Zehr and Walker, Prostaglandins Other Lipid Mediat 134:131-140, 2018). ARA (20:4n-6) is a 20-carbon fatty acid with four double bonds, and the first double bond is in the n-6 position. GLA (18:3n-6) is an 18-carbon fatty acid with three double bonds, and the first double bond is in the n-6 position. ARA and GLA have multiple biological effects, such as lowering blood cholesterol, and lowering cardiovascular mortality (Poli and Visioli, Eur J Lipid Sci Technol 117(11):1847-1852, 2015). This chapter provides details on microbial production of EAP, DHA, ARA, and GLA.

Efficient docosahexaenoic acid production by Schizochytrium sp. via a two-phase pH control strategy using ammonia and citric acid as pH regulators

Abstract The cell growth and docosahexaenoic acid (DHA) synthesis of Schizochytrium sp. are closely related to the culture pH. A two-phase pH control strategy based on nitrogen consumption was developed in which pH 7.0 was used for biomass accumulation and pH 5.0 for DHA synthesis. Using this strategy, the cell dry weight and DHA content reached 98.07 and 25.85 g/L, respectively. Furthermore, ammonia and citric acid were used as pH regulators. Application of citric acid further resulted in 7.88 and 4.87% improvements of total lipids and the ratio of DHA to total fatty acids, respectively. Ammonia, as a suitable nitrogen source, promoted non-lipid biomass accumulation. Using this method, a maximum DHA yield of 32.75 g/L was obtained with non-lipid biomass (58.01 g/L) and the ratio of DHA to total fatty acids (52.36%). This study provides an easy strategy for large-scale industrial production of DHA via high-cell-density fermentation of Schizochytrium sp.

Production of Lipids and Proteome Variation in a Chilean Thraustochytrium striatum Strain Cultured under Different Growth Conditions.

Total lipids and docosahexaenoic acid (DHA) production by a Chilean isolated thraustochytrid were evaluated under different growth conditions in shake flasks. The analyzed strain was identified as Thraustochytrium striatum according to an 18S rRNA gene sequence analysis. The strain (T. striatum AL16) showed negligible growth in media prepared with artificial seawater at concentrations lower than 50% v/v and pH lower than 5. Maltose and starch were better carbon sources for growth than glucose. DHA content of the biomass grown with maltose (60gL-1) was doubled by increasing the agitation rate from 150 to 250rpm. The DHA (0.8-6%) and eicosapentaenoic acid (0.2-21%) content in the total lipids varied depending on culture conditions and culture age. Lipid and DHA concentration increased (up to 5gL-1 and 66mgL-1, respectively) by regularly feeding the culture with a concentrated starch solution. Carotenoid accumulation was detected in cells grown with maltose or starch. Contrasting conditions of starch and glucose cultures were selected for comparative proteomics. Total protein extracts were separated by two-dimensional gel electrophoresis; 25 spots were identified using ESI-MS/MS. A protein database (143,006 entries) for proteomic interrogation was generated using de novo assembling of Thraustochytrium sp. LLF1b - MMETSP0199_2 transcriptome; 18 proteins differentially expressed were identified. Three ATP synthases were differentially accumulated in cultures with glucose, whereas malate dehydrogenase was more abundant in cells cultured with starch.

The hidden systems in the human brain

Soybean curd wastewater (SCWW) is a by-product of the compression of protein coagulants performed during production of soybean curd (tofu). In this study, SCWW was used as a low-cost nutrient source for the cultivation of an Aurantiochytrium strain known to produce significant amounts of polyunsaturated fatty acids (PUFAs), such as docosahexaenoic acid (DHA). Instead of sterilization of the SCWW medium by heating (which promotes the Maillard reaction and production of hydroxymethylfurfural), the SCWW medium was sterilized using peracetic acid (PAA) followed by neutralization with a ferric ion solution. The growth of cells was similar in PAA-sterilized SCWW medium and sterile-filtered or autoclaved M7D20 medium that contained commercial components (glucose, yeast extract, and peptone). Surprisingly, cells grown in PAA-sterilized SCWW medium had more than 4-fold greater production of DHA than cells grown in autoclaved or filtered M7D20 medium.