Production of docosahexaenoic acid by a novel isolated Aurantiochytrium sp. 6-2 using fermented defatted soybean as a nitrogen source for sustainable fish feed development.

We focused on the production of docosahexaenoic acid (DHA)-containing microbial lipids by Aurantiochytrium sp. using of defatted soybean (DS) as a nitrogen source. Defatted soybean is a plant biomass that could provide a sustainable supply at a low cost. Results showed that Aurantiochytrium sp. could not directly assimilate the DS as a nitrogen source but could grow well in a medium containing DS fermented with rice malt. When cultivated in a fermented DS (FDS) medium, Aurantiochytrium sp. showed vigorous growth with the addition of sufficient sulfate and chloride ions as inorganic nutrients without seawater salt. A novel isolated Aurantiochytrium sp. 6-2 showed 15.8 ± 3.4 g/L DHA productivity (in 54.8 ± 12.1 g/L total fatty acid production) in 1 L of the FDS medium. Therefore, DHA produced by Aurantiochytrium sp. using FDS enables a stable and sustainable DHA supply and could be an alternative source of natural DHA derived from fish oil.

Transcriptome analysis of Aurantiochytrium limacinum under low salt conditions.

Aurantiochytrium limacinum can accumulate high amounts of omega-3 polyunsaturated fatty acids, especially docosahexaenoic acid (DHA). Although salinity affects the DHA content, its impact on the metabolic pathway responsible for DHA production in A. limacinum is not completely understood. To address this issue, we investigated the transcriptional profile of A. limacinum under hypoosmotic stress. We first cultured A. limacinum under typical and low salinity for RNA sequencing, respectively. Transcriptome analyses revealed that 933 genes exhibited significant changes in expression under hypoosmotic conditions, of which 81.4% were downregulated. Strikingly, A. limacinum downregulated genes related to polyketide synthesis and fatty acid synthase pathways, while upregulating ß-oxidation-related genes. In accordance with this, DHA production significantly decreased under hypoosmotic conditions, while antioxidant-related genes were significantly upregulated. Considering that ß-oxidation of fatty acids generates energy and reactive oxygen species (ROS), our results suggest that A. limacinum utilizes fatty acids for energy to survive under hypoosmotic conditions and detoxifies ROS using antioxidant systems.

Protist-Lactic Acid Bacteria Co-Culture as a Strategy to Bioaccumulate Polyunsaturated Fatty Acids in the Protist Aurantiochytrium sp. T66.

Thraustochytrids are aquatic unicellular protists organisms that represent an important reservoir of a wide range of bioactive compounds, such as essential polyunsaturated fatty acids (PUFAs) such as arachidonic acid (ARA), docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), which are involved in the regulation of the immune system. In this study, we explore the use of co-cultures of Aurantiochytrium sp. and bacteria as a biotechnological tool capable of stimulating PUFA bioaccumulation. In particular, the co-culture of lactic acid bacteria and the protist Aurantiochytrium sp. T66 induce PUFA bioaccumulation, and the lipid profile was evaluated in cultures at different inoculation times, with two different strains of lactic acid bacteria capable of producing the tryptophan dependent auxins, and one strain of Azospirillum sp., as a reference for auxin production. Our results showed that the Lentilactobacillus kefiri K6.10 strain inoculated at 72 h gives the best PUFA content (30.89 mg g-1 biomass) measured at 144 h of culture, three times higher than the control (8.87 mg g-1 biomass). Co-culture can lead to the generation of complex biomasses with higher added value for developing aquafeed supplements.

Identification and Functional Analysis of Two Novel Genes-Geranylgeranyl Pyrophosphate Synthase Gene (AlGGPPS) and Isopentenyl Pyrophosphate Isomerase Gene (AlIDI)-from Aurantiochytrium limacinum Significantly Enhance De Novo ß-Carotene Biosynthesis in Escherichia coli.

Precursor regulation has been an effective strategy to improve carotenoid production and the availability of novel precursor synthases facilitates engineering improvements. In this work, the putative geranylgeranyl pyrophosphate synthase encoding gene (AlGGPPS) and isopentenyl pyrophosphate isomerase encoding gene (AlIDI) from Aurantiochytrium limacinum MYA-1381 were isolated. We applied the excavated AlGGPPS and AlIDI to the de novo ß-carotene biosynthetic pathway in Escherichia coli for functional identification and engineering application. Results showed that the two novel genes both functioned in the synthesis of ß-carotene. Furthermore, AlGGPPS and AlIDI performed better than the original or endogenous one, with 39.7% and 80.9% increases in ß-carotene production, respectively. Due to the coordinated expression of the 2 functional genes, ß-carotene content of the modified carotenoid-producing E. coli accumulated a 2.99-fold yield of the initial EBIY strain in 12 h, reaching 10.99 mg/L in flask culture. This study helped to broaden current understanding of the carotenoid biosynthetic pathway in Aurantiochytrium and provided novel functional elements for carotenoid engineering improvements.

Effects of glycerol and glucose on docosahexaenoic acid synthesis in Aurantiochyrium limacinum SFD-1502 by transcriptome analysis.

Docosahexaenoic acid (DHA) has numerous functions in adjusting the organic health and pragmatic value in medicine and food field. In this study, we compared glycerol and glucose as the only carbon source for DHA production by Aurantiochytrium. When the glycerol concentration was 120 g/L, the maximum DHA yield was 11.08 g/L, and the DHA yield increased significantly, reaching 47.67% of the total lipid content. When the cells grew in glucose, the DHA proportion was 37.39%. Transcriptome data showed that the glycolysis pathway and tricarboxylic acid cycle in Aurantiochytrium were significantly inhibited during glycerol culture, which promoted the tricarboxylic acid transport system and was conducive to the synthesis of fatty acids by acetyl coenzyme A; glucose as substrate activated fatty acid synthesis (FAS)pathway and produced more saturated fatty acids, while glycerol as substrate activated polyketide synthase (PKS)pathway and produced more long-chain polyunsaturated fatty acids. This laid a foundation for fermentation metabolism regulation and molecular transformation.

Transcriptional responses of Aurantiochytrium limacinum under light conditions.

AIMS: Astaxanthin-producing protist Aurantiochytrium limacinum can accumulate higher amounts of astaxanthin under light conditions; however, little is known about the impact of light exposure on its metabolism. Here, we investigated the transcriptional profile of A. limacinum under light conditions. METHODS AND RESULTS: Transcriptomic analyses revealed that 962 genes of A. limacinum showed a significant change in expression under light conditions, most of which (94.5%) were downregulated. Furthermore, gene ontology enrichment analysis indicated that A. limacinum mainly downregulated genes associated with cell motility, proliferation and gene expression processes, whose activities depend on ATP as an energy source. Additionally, the quantification of carotenoid and its transcripts suggested that ß-carotene and astaxanthin biosynthesis pathways were rate-limiting and tightly regulated steps, respectively. In comparison, these processes were enhanced under light conditions. CONCLUSIONS: Considering that astaxanthin accumulation was highly correlated with reactive oxygen species (ROS) levels in microalgae, our results suggest that A. limacinum reduces ATP consumption to decrease the occurrence of ROS in mitochondria while accumulating astaxanthin to prevent ROS damage. SIGNIFICANCE AND IMPACT OF STUDY: This study provides novel insights into the impact of light exposure on A. limacinum metabolism, thereby facilitating a complete understanding of this protist for efficient astaxanthin production.

Structural and Molecular Characterization of Squalene Synthase Belonging to the Marine Thraustochytrid Species Aurantiochytrium limacinum Using Bioinformatics Approach.

The marine microorganisms thraustochytrids have been explored for their potential in the production of various bioactive compounds, such as DHA, carotenoids, and squalene. Squalene is a secondary metabolite of the triterpenoid class and is known for its importance in various industrial applications. The bioinformatic analysis for squalene synthase (SQS) gene (the first key enzyme in the tri-terpenoid synthesis pathway), that is prevailing among thraustochytrids, is poorly investigated. In-silico studies combining sequence alignments and bioinformatic tools helped in the preliminary characterization of squalene synthases found in Aurantiochytrium limacinum. The sequence contained highly conserved regions for SQS found among different species indicated the enzyme had all the regions for its functionality. The signal peptide sequence and transmembrane regions were absent, indicating an important aspect of the subcellular localization. Secondary and 3-D models generated using appropriate templates demonstrated the similarities with SQS of the other species. The 3-D model also provided important insights into possible active, binding, phosphorylation, and glycosylation sites.

Scale-Up to Pilot of a Non-Axenic Culture of Thraustochytrids Using Digestate from Methanization as Nitrogen Source.

The production of non-fish based docosahexaenoic acid (DHA) for feed and food has become a critical need in our global context of over-fishing. The industrial-scale production of DHA-rich Thraustochytrids could be an alternative, if costs turned out to be competitive. In order to reduce production costs, this study addresses the feasibility of the non-axenic (non-sterile) cultivation of Aurantiochytrium mangrovei on industrial substrates (as nitrogen and mineral sources and glucose syrup as carbon and energy sources), and its scale-up from laboratory (250 mL) to 500 L cultures. Pilot-scale reactors were airlift cylinders. Batch and fed-batch cultures were tested. Cultures over 38 to 62 h achieved a dry cell weight productivity of 3.3 to 5.5 g.L-1.day-1, and a substrate to biomass yield of up to 0.3. DHA productivity ranged from 10 to 0.18 mg.L-1.day-1. Biomass productivity appears linearly related to oxygen transfer rate. Bacterial contamination of cultures was low enough to avoid impacts on fatty acid composition of the biomass. A specific work on microbial risks assessment (in supplementary files) showed that the biomass can be securely used as feed. However, to date, there is a law void in EU legislation regarding the recycling of nitrogen from digestate from animal waste for microalgae biomass and its usage in animal feed. Overall, the proposed process appears similar to the industrial yeast production process (non-axenic heterotrophic process, dissolved oxygen supply limiting growth, similar cell size). Such similarity could help in further industrial developments.

C4-monomethylsterol ß-glucoside and its synthase in Aurantiochytrium limacinum mh0186.

Thraustochytrids, unicellular marine protists, synthesize polyunsaturated fatty acids (PUFAs) and PUFA-containing phospholipids; however, little is known about their glycolipids and their associated metabolism. Here, we report two glycolipids (GL-A, B) and their synthases in Aurantiochytrium limacinum mh0186. Two glycolipids were purified from A. limacinum mh0186, and they were determined by gas chromatography, mass spectrometry and 2D nuclear magnetic resonance to be 3-O-ß-D-glucopyranosyl-stigmasta-5,7,22-triene (GL-A) and 3-O-ß-D-glucopyranosyl-4α-methyl-stigmasta-7,22-diene (GL-B), both of which are sterol ß-glucosides (ß-SGs); the structure of GL-B has not been reported thus far. Seven candidate genes responsible for the synthesis of these ß-SGs were extracted from the draft genome database of A. limacinum using the yeast sterol ß-glucosyltransferase (SGT; EC 2.4.1.173) sequence as a query. Expression analysis using Saccharomyces cerevisiae revealed that two gene products (AlSGT-1 and 2) catalyze the transfer of glucose from uridine diphosphate (UDP)-glucose to sterols, generating sterylglucosides (SGs). Compared to AlSGT-1, AlSGT-2 exhibited wide specificity for sterols and used C4-monomethylsterol to synthesize GL-B. The disruption of alsgt-2 but not alsgt-1 in strain mh0186 resulted in a decrease in the total SG and an almost complete loss of GL-B, indicating that AlSGT-2 is responsible for the synthesis of ß-SGs in A. limacinum mh0186, especially GL-B, which possesses a unique sterol structure.

High-quality genome-scale metabolic model of Aurantiochytrium sp. T66.

The long-chain, ω-3 polyunsaturated fatty acids (PUFAs) (e.g., eicosapentaenoic acid [EPA] and docosahexaenoic acid [DHA]), are essential for humans and animals, including marine fish species. Presently, the primary source of these PUFAs is fish oils. As the global production of fish oils appears to be reaching its limits, alternative sources of high-quality ω-3 PUFAs is paramount to support the growing aquaculture industry. Thraustochytrids are a group of heterotrophic protists with the capability to synthesize and accrue large amounts of DHA. Thus, the thraustochytrids are prime candidates to solve the increasing demand for ω-3 PUFAs using microbial cell factories. However, a systems-level understanding of their metabolic shift from cellular growth into lipid accumulation is, to a large extent, unclear. Here, we reconstructed a high-quality genome-scale metabolic model of the thraustochytrid Aurantiochytrium sp. T66 termed iVS1191. Through iterative rounds of model refinement and extensive manual curation, we significantly enhanced the metabolic scope and coverage of the reconstruction from that of previously published models, making considerable improvements with stoichiometric consistency, metabolic connectivity, and model annotations. We show that iVS1191 is highly consistent with experimental growth data, reproducing in vivo growth phenotypes as well as specific growth rates on minimal carbon media. The availability of iVS1191 provides a solid framework for further developing our understanding of T66's metabolic properties, as well as exploring metabolic engineering and process-optimization strategies in silico for increased ω-3 PUFA production.

A non-canonical Δ9-desaturase synthesizing palmitoleic acid identified in the thraustochytrid Aurantiochytrium sp. T66.

Thraustochytrids are oleaginous marine eukaryotic microbes currently used to produce the essential omega-3 fatty acid docosahexaenoic acid (DHA, C22:6 n-3). To improve the production of this essential fatty acid by strain engineering, it is important to deeply understand how thraustochytrids synthesize fatty acids. While DHA is synthesized by a dedicated enzyme complex, other fatty acids are probably synthesized by the fatty acid synthase, followed by desaturases and elongases. Which unsaturated fatty acids are produced differs between different thraustochytrid genera and species; for example, Aurantiochytrium sp. T66, but not Aurantiochytrium limacinum SR21, synthesizes palmitoleic acid (C16:1 n-7) and vaccenic acid (C18:1 n-7). How strain T66 can produce these fatty acids has not been known, because BLAST analyses suggest that strain T66 does not encode any Δ9-desaturase-like enzyme. However, it does encode one Δ12-desaturase-like enzyme. In this study, the latter enzyme was expressed in A. limacinum SR21, and both C16:1 n-7 and C18:1 n-7 could be detected in the transgenic cells. Our results show that this desaturase, annotated T66Des9, is a Δ9-desaturase accepting C16:0 as a substrate. Phylogenetic studies indicate that the corresponding gene probably has evolved from a Δ12-desaturase-encoding gene. This possibility has not been reported earlier and is important to consider when one tries to deduce the potential a given organism has for producing unsaturated fatty acids based on its genome sequence alone. KEY POINTS: • In thraustochytrids, automatic gene annotation does not always explain the fatty acids produced. • T66Des9 is shown to synthesize palmitoleic acid (C16:1 n-7). • T66des9 has probably evolved from Δ12-desaturase-encoding genes.

Method Development Progress in Genetic Engineering of Thraustochytrids.

Thraustochytrids are unicellular, heterotrophic marine eukaryotes. Some species are known to store surplus carbon as intracellular lipids, and these also contain the long-chain polyunsaturated fatty acid docosahexaenoic acid (DHA). Most vertebrates are unable to synthesize sufficient amounts of DHA, and this fatty acid is essential for, e.g., marine fish, domesticated animals, and humans. Thraustochytrids may also produce other commercially valuable fatty acids and isoprenoids. Due to the great potential of thraustochytrids as producers of DHA and other lipid-related molecules, a need for more knowledge on this group of organisms is needed. This necessitates the ability to do genetic manipulation of the different strains. Thus far, this has been obtained for a few strains, while it has failed for other strains. Here, we systematically review the genetic transformation methods used for different thraustochytrid strains, with the aim of aiding studies on strains not yet successfully transformed. The designs of transformation cassettes are also described and compared. Moreover, the potential problems when trying to establish transformation protocols in new thraustochytrid species/strains are discussed, along with suggestions utilized in other organisms to overcome similar challenges. The approaches discussed in this review could be a starting point when designing protocols for other non-model organisms.

Isolation of fast-growing thraustochytrids and seasonal variation on the fatty acid composition of thraustochytrids from mangrove regions of Navi Mumbai, India.

This study was aimed to isolate fast-growing thraustochytrids and the influence of seasonal variation in fatty acid composition from the mangrove habitat. The thraustochytrids were isolated from fallen yellowish or green mangrove leaves, in four seasons, including winter, summer, rainy, and post rainy season in one year. The thraustochytrids were analyzed for biomass production, total lipid content, and fatty acid profile. The thraustochytrid isolates showed biomass yield and total lipid content in the range of 14.12 ± 0.69 to 22.98 ± 0.53 g/L and 34.98-58.86% per dry cell weight, respectively. The isolates showed two dominant fatty acids, palmitic acid (PA) as saturated fatty acid (SFA) and docosahexaenoic acid (DHA) as long-chain polyunsaturated fatty acids (LC-PUFA) in total fatty acid (TFA) content. The significant differences (P < 0.05) were observed for seasonal variations in SFA and DHA content in summer isolates and winter isolates. The maximum DHA content with 47.12% of TFA, recorded in winter (January) isolates and summer (April) isolates with SFA 68.82% of TFA. The results from this study were verified the hypothesis that the presence of high DHA producing thraustochytrids in lower temperature season in the same habitat. These findings have also emphasized the role of the environmental temperature conditions and the importance of thraustochytrid fatty acid composition as a dietary biomarker. Also, it revealed the ecological significance of thraustochytrid in DHA enrichment in the food web of the marine ecosystem. These findings could be useful while isolating thraustochytrids according to seasons for industrial application for omega 3 fatty acids and biodiesel production.

The strategies to reduce cost and improve productivity in DHA production by Aurantiochytrium sp.: from biochemical to genetic respects.

The marine oleaginous protist Aurantiochytrium sp. (Schizochytrium sp.) is a well-known docosahexaenoic acid (DHA) producer and its different DHA products are the ideal substitute for the traditional fish oil resource. However, the cost of the DHA products derived from Aurantiochytrium sp. (Schizochytrium sp.) is still high, limiting their wide applications. In order to reduce the cost or improve the productivity of DHA from the microbial resource, many researches are focusing on exploring the renewable and low-cost materials as feedbacks, and/or the stimulators for biomass and DHA production. In addition, the genetic engineering is also being used in the Aurantiochytrium sp. (Schizochytrium sp.) system for further improvement. These break the bottleneck of the DHA production by Aurantiochytrium sp. (Schizochytrium sp.) in some degree. In this review, the strategies used currently to reduce cost and improve DHA productivity, mainly from the utilizations of low-cost materials and effective stimulators to the genetic engineering perspectives, are summarized, and the availabilities from the cost perspective are also evaluated. This review provides an overview about the strategies to revolve the production cost and yield of the DHA by Aurantiochytrium sp. (Schizochytrium sp.), a theoretical basis for genetic modification of Aurantiochytrium sp. (Schizochytrium sp.), and a practical basis for the development of DHA industry. KEY POINTS : • Utilizations of various low-cost materials for DHA production • Inducing the growth and DHA biosynthesis by the effective stimulators • Reducing cost and improving DHA productivity by genetic modification • The availability from cost perspective is evaluated.

The Nutritional and Pharmacological Potential of New Australian Thraustochytrids Isolated from Mangrove Sediments.

Mangrove sediments represent unique microbial ecosystems that act as a buffer zone, biogeochemically recycling marine waste into nutrient-rich depositions for marine and terrestrial species. Marine unicellular protists, thraustochytrids, colonizing mangrove sediments have received attention due to their ability to produce large amounts of long-chain ω3-polyunsaturated fatty acids. This paper represents a comprehensive study of two new thraustochytrids for their production of valuable biomolecules in biomass, de-oiled cakes, supernatants, extracellular polysaccharide matrixes, and recovered oil bodies. Extracted lipids (up to 40% of DW) rich in polyunsaturated fatty acids (up to 80% of total fatty acids) were mainly represented by docosahexaenoic acid (75% of polyunsaturated fatty acids). Cells also showed accumulation of squalene (up to 13 mg/g DW) and carotenoids (up to 72 µg/g DW represented by astaxanthin, canthaxanthin, echinenone, and ß-carotene). Both strains showed a high concentration of protein in biomass (29% DW) and supernatants (2.7 g/L) as part of extracellular polysaccharide matrixes. Alkalinization of collected biomass represents a new and easy way to recover lipid-rich oil bodies in the form of an aqueous emulsion. The ability to produce added-value molecules makes thraustochytrids an important alternative to microalgae and plants dominating in the food, pharmacological, nutraceutical, and cosmetics industries.

One-step utilization of inulin for docosahexaenoic acid (DHA) production by recombinant Aurantiochytrium sp. carrying Kluyveromyces marxianus inulinase.

This study aimed to express an inulinase gene from the yeast Kluyveromyces marxianus (KmINU) in Aurantiochytrium sp. and realized one-step utilization of inulin resource for DHA production without any chemical pretreatment. An expression cassette with a length of 6052 bp for expressing the inulinase gene was constructed by a fast two-step PCR method and then was transferred into the Aurantiochytrium sp. cells. The Aurantiochytrium sp. recombinant T39 was selected with an inulinase activity up to 50.1 U/mL in 72 h. In a 5-l fed-batch fermentation, as high as 148.9 g/L of inulin was directly used within 120 h, and only 1.2 g/L of total sugar was left in the medium at the end of fermentation. The biomass of 51.4 g/L with a lipid content of 69.2% DCW and a DHA yield of 14.9 g/L was obtained.

Illustrating and Enhancing the Biosynthesis of Astaxanthin and Docosahexaenoic Acid in Aurantiochytrium sp. SK4.

The marine thraustochytrids are a promising source of docosahexaenoic acid (DHA) and the ketocarotenoid astaxanthin. In this study, the biosynthetic pathways of these two important metabolites in Aurantiochytrium sp. SK4 was illustrated by the analyses of the genome, transcriptome, key enzymes, and pathway products. Two sets of genes were involved in two pathways for the biosynthesis of fatty acids. The absence of Δ-15 desaturase genes and the presence of docosapentaenoic acid (DPA), up to 12% of total fatty acids suggest that Aurantiochytrium sp. SK4 may synthesize DHA mainly via a polyketide synthase (PKS) pathway. Three enzymes, namely geranyl diphosphate synthase (GPPS), farnysyl diphosphate synthase (FPPS), and geranylgeranyle diphosphate synthase (GGPPS) were found to be involved in the formation of GGPP that was subsequently catalyzed to ß-carotene by a trifunctional CrtIBY enzyme. ß-Carotene might be ketolated and then hydroxylated into astaxanthin based on the carotenoid profiles. The formation of GGPP was proposed to be the limiting steps for carotenoid production. Overexpression of the Archaeoglobus GPS together with the Escherichia coli isopentenyl pyrophosphate isomerase, and Vitreoscilla hemoglobin resulted in not only 1.85- and 5.02-fold increases of total carotenoids and astaxanthin, but also 2.40- and 2.74-fold increases of total fatty acids and DHA. This study provides insights into the biosynthesis of carotenoids and fatty acids in Aurantiochytrium.

Increasing dietary levels of docosahexaenoic acid-rich microalgae: Ruminal fermentation, animal performance, and milk fatty acid profile of mid-lactating dairy cows.

This study aimed to evaluate the effects of increasing dietary levels of microalgae (ALG), rich in docosahexaenoic acid (DHA; All-G-Rich, Alltech, Nicholasville, KY), in isolipidic diets, on animal performance, nutrient digestibility, ruminal fermentation, milk fatty acid profile, energy balance, microbial protein synthesis, and blood serum metabolites in mid-lactating dairy cows. Twenty-four Holstein cows [130.3 ± 15.4 d in milk, and 30.8 ± 0.543 kg/d of milk yield (mean ± standard error)] were used in a 4 × 4 Latin square design experiment to evaluate the following treatments: control diet, without addition of ALG; and increasing levels of ALG [2, 4, and 6 g/kg of dry matter (DM)]. The ALG decreased DM intake and increased total-tract DM apparent digestibility. A tendency was observed for a quadratic effect on total-tract NDF digestibility by ALG inclusion, with peak value of the quadratic response at 4.13 g/kg of DM dose. Moreover, ALG increased ruminal pH and decreased acetate and total volatile fatty acid concentrations. Fat-corrected milk and energy-corrected milk were quadratically affected, and a tendency for a milk yield effect was observed when ALG levels increased, whereas maximal yields were observed with intermediate doses. Milk fat, protein, and lactose concentrations were diminished, whereas productive efficiency was improved by the increase of ALG levels. Saturated fatty acid proportions were decreased, whereas polyunsaturated fatty acid proportions were increased when ALG was fed. There was low DHA transfer into milk; however, ALG inclusion decreased C18:0, C18:1 cis-9, C18:2 cis-9,12, and C18:3 cis-9,12,15 proportions, and increased C18:2 cis-9,trans-11, C18:1 trans-9, and C18:1 trans-11 proportions. Gross energy intake was decreased, whereas no effect was observed on digestible, metabolizable, or net energy intake. The ALG inclusion quadratically affected the microbial protein synthesis, with maximal enhancement at 3.24 g/kg of DM dose, and also increased serum cholesterol concentration. Under the conditions of this experiment, the inclusion of ALG in diets for mid-lactating dairy cows decreased feed intake and increased nutrient digestibility, improving productive efficiency and modifying milk fatty acid profile. Estimated intermediate doses (1.22 to 2.90 g/kg of DM) of DHA-rich ALG may be beneficial to milk, fat-corrected milk, and energy-corrected milk yields, and is recommended for dairy cows.

High-Throughput Biochemical Fingerprinting of Oleaginous Aurantiochytrium sp. Strains by Fourier Transform Infrared Spectroscopy (FT-IR) for Lipid and Carbohydrate Productions.

The traditional biochemical methods for analyzing cellular composition of oleaginous microorganisms are time-consuming, polluting, and expensive. In the present study, an FT-IR method was used to analyze the cellular composition of the marine oleaginous protist Aurantiochytrium sp. during various research processes, such as strains screening, medium optimization, and fermentation, and was evaluated as a green, low-cost, high throughput, and accurate method compared with the traditional methods. A total of 109 Aurantiochytrium sp. strains were screened for lipid and carbohydrate production and the best results were found for the strains No. 6 and No. 32. The yields and productivities could reach up to 47.2 g/L and 0.72 g/L/h for lipid, 21.6 g/L and 0.33 g/L/h for docosahexaenoic acid (DHA) in the strain No. 6, and 15.4 g/L and 0.18 g/L/h for carbohydrate in the strain No. 32, under the optimal conditions, respectively. These results confirmed potentials of the two Aurantiochytrium sp. strains for lipid, DHA, and carbohydrate productions at industrial scales. The FT-IR method in this study will facilitate research on the oleaginous Aurantiochytrium sp., and the obtained two strains for lipid and carbohydrate productions will provide the foundations for their applications in medical, food, and feed industries.

Heterotrophic Aurantiochytrium sp. supplementation to layer diets sustainably increases the omega-3 concentration of eggs.

1. The consumption of adequate amounts of the long-chain polyunsaturated omega-3 fatty acids (n-3 LC-PUFA) has been associated with beneficial effects on human health. Eggs are commonly consumed worldwide, and their omega-3 content can be easily altered by changing the diets of laying hens and so represent an important target for enrichment. 2. In this study, the effect of supplementing laying hens with DHA-rich, Aurantiochytrium limacinum at three different inclusion levels was investigated over a 24-week period. 3. Significant increases in egg DHA concentrations were observed after four weeks and were maintained for the duration of the 24-week study. The supplemented eggs in the current study had a DHA content of 82, 101, and 129 mg/yolk when supplemented with 0.25%, 0.5% and 1% treatments, respectively, which meets the EU criteria to be considered 'high in omega-3'. 4. Using the sustainably grown protist Aurantiochytrium limacinum to supplement layer diets increased the egg DHA concentration and decreased the n-6/n-3 ratio, improving the nutritional value of the eggs for human consumers.