Antarctic Thraustochytrids as Sources of Carotenoids and High-Value Fatty Acids.

Eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and carotenoids are needed as human dietary supplements and are essential components in commercial feeds for the production of aquacultured seafood. Microorganisms such as thraustochytrids are potential natural sources of these compounds. This research reports on the lipid and carotenoid production capacity of thraustochytrids that were isolated from coastal waters of Antarctica. Of the 22 isolates, 21 produced lipids containing EPA+DHA, and the amount of these fatty acids exceeded 20% of the total fatty acids in 12 isolates. Ten isolates were shown to produce carotenoids (27.4-63.9 µg/g dry biomass). The isolate RT2316-16, identified as Thraustochytrium sp., was the best producer of biomass (7.2 g/L in five days) rich in carotenoids (63.9 µg/g) and, therefore, became the focus of this investigation. The main carotenoids in RT2316-16 were ß-carotene and canthaxanthin. The content of EPA+DHA in the total lipids (34 ± 3% w/w in dry biomass) depended on the stage of growth of RT2316-16. Lipid and carotenoid content of the biomass and its concentration could be enhanced by modifying the composition of the culture medium. The estimated genome size of RT2316-16 was 44 Mb. Of the 5656 genes predicted from the genome, 4559 were annotated. These included genes of most of the enzymes in the elongation and desaturation pathway of synthesis of ω-3 polyunsaturated fatty acids. Carotenoid precursors in RT2316-16 were synthesized through the mevalonate pathway. A ß-carotene synthase gene, with a different domain organization compared to the gene in other thraustochytrids, explained the carotenoid profile of RT2316-16.

Schizochytrium sp. (T18) Oil as a Fish Oil Replacement in Diets for Juvenile Rainbow Trout (Oncorhynchus mykiss): Effects on Growth Performance, Tissue Fatty Acid Content, and Lipid-Related Transcript Expression.

In this study, we evaluated whether oil extracted from the marine microbe, Schizochytrium sp. (strain T18), with high levels of docosahexaenoic acid (DHA), could replace fish oil (FO) in diets for rainbow trout (Oncorhynchus mykiss). Three experimental diets were tested: (1) a control diet with fish oil (FO diet), (2) a microbial oil (MO) diet with a blend of camelina oil (CO) referred to as MO/CO diet, and (3) a MO diet (at a higher inclusion level). Rainbow trout (18.8 ± 2.9 g fish-1 initial weight ± SD) were fed for 8 weeks and evaluated for growth performance, fatty acid content and transcript expression of lipid-related genes in liver and muscle. There were no differences in growth performance measurements among treatments. In liver and muscle, eicosapentaenoic acid (EPA) was highest in trout fed the FO diet compared to the MO/CO and MO diets. Liver DHA was highest in trout fed the MO/CO diet compared to the FO and MO diets. Muscle DHA was highest in trout fed the MO and MO/CO diets compared to the FO diet. In trout fed the MO/CO diet, compared to the MO diet, fadsd6b was higher in both liver and muscle. In trout fed the FO or MO/CO diets, compared to the MO diet, cox1a was higher in both liver and muscle, cpt1b1a was higher in liver and cpt1a1a, cpt1a1b and cpt1a2a were higher in muscle. Schizochytrium sp. (T18) oil was an effective source of DHA for rainbow trout.

Engineering xylose metabolism in thraustochytrid T18.

BACKGROUND: Thraustochytrids are heterotrophic, oleaginous, marine protists with a significant potential for biofuel production. High-value co-products can off-set production costs; however, the cost of raw materials, and in particular carbon, is a major challenge to developing an economical viable production process. The use of hemicellulosic carbon derived from agricultural waste, which is rich in xylose and glucose, has been proposed as a sustainable and low-cost approach. Thraustochytrid strain T18 is a commercialized environmental isolate that readily consumes glucose, attaining impressive biomass, and oil production levels. However, neither thraustochytrid growth capabilities in the presence of xylose nor a xylose metabolic pathway has been described. The aims of this study were to identify and characterize the xylose metabolism pathway of T18 and, through genetic engineering, develop a strain capable of growth on hemicellulosic sugars. RESULTS: Characterization of T18 performance in glucose/xylose media revealed diauxic growth and copious extracellular xylitol production. Furthermore, T18 did not grow in media containing xylose as the only carbon source. We identified, cloned, and functionally characterized a xylose isomerase. Transcriptomics indicated that this xylose isomerase gene is upregulated when xylose is consumed by the cells. Over-expression of the native xylose isomerase in T18, creating strain XI 16, increased xylose consumption from 5.2 to 7.6 g/L and reduced extracellular xylitol from almost 100% to 68%. Xylose utilization efficiency of this strain was further enhanced by over-expressing a heterologous xylulose kinase to reduce extracellular xylitol to 20%. Moreover, the ability to grow in media containing xylose as a sole sugar was dependent on the copy number of both xylose isomerase and xylulose kinase present. In fed-batch fermentations, the best xylose metabolizing isolate, XI-XK 7, used 137 g of xylose versus 39 g by wild type and produced more biomass and fatty acid. CONCLUSIONS: The presence of a typically prokaryotic xylose isomerase and xylitol production through a typically eukaryotic xylose reductase pathway in T18 is the first report of an organism naturally encoding enzymes from two native xylose metabolic pathways. Our newly engineered strains pave the way for the growth of T18 on waste hemicellulosic feedstocks for biofuel production.

Recycling of lipid-extracted hydrolysate as nitrogen supplementation for production of thraustochytrid biomass.

Efficient resource usage is important for cost-effective microalgae production, where the incorporation of waste streams and recycled water into the process has great potential. This study builds upon emerging research on nutrient recycling in thraustochytrid production, where waste streams are recovered after lipid extraction and recycled into future cultures. This research investigates the nitrogen flux of recycled hydrolysate derived from enzymatic lipid extraction of thraustochytrid biomass. Results indicated the proteinaceous content of the recycled hydrolysate can offset the need to supply fresh nitrogen in a secondary culture, without detrimental impact upon the produced biomass. The treatment employing the recycled hydrolysate with no nitrogen addition accumulated 14.86 g L(-1) of biomass in 141 h with 43.3 % (w/w) lipid content compared to the control which had 9.26 g L(-1) and 46.9 % (w/w), respectively. This improved nutrient efficiency and wastewater recovery represents considerable potential for enhanced resource efficiency of commercial thraustochytrid production.

Sequential recycling of enzymatic lipid-extracted hydrolysate in fermentations with a thraustochytrid.

This study extends the findings of prior studies proposing and validating nutrient recycling for the heterotrophic microalgae, Thraustochytrium sp. (T18), grown in optimized fed-batch conditions. Sequential nutrient recycling of enzymatically-derived hydrolysate in fermentors succeeded at growing the tested thraustochytrid strain, with little evidence of inhibition or detrimental effects upon culture health. The average maximum biomass obtained in the recycled hydrolysate was 63.68±1.46gL(-1) in 90h the first recycle followed by 65.27±1.15gL(-1) in 90h in the subsequent recycle of the same material. These compared to 58.59gL(-1) and 64.92gL(-1) observed in fresh media in the same time. Lipid production was slightly impaired, however, with a maximum total fatty acid content of 62.2±0.30% in the recycled hydrolysate compared to 69.4% in fresh control media.

Nutrient recycling of lipid-extracted waste in the production of an oleaginous thraustochytrid.

Improving the economics of microalgae production for the recovery of microbial oil requires a comprehensive exploration of the measures needed to improve productivity as well as to reduce the overall processing costs. One avenue for cost reduction involves recycling the effluent waste water remaining after lipid extraction. This study investigates the feasibility of recycling those wastes for growing thraustochytrid biomass, a heterotrophic microalgae, where wastes were generated from the enzymatic extraction of the lipids from the cell biomass. It was demonstrated that secondary cultures of the tested thraustochytrid grown in the recycled wastes performed favorably in terms of cell and oil production (20.48 g cells L(-1) and 40.9 % (w/w) lipid) compared to the control (13.63 g cells L(-1) and 56.8 % (w/w) lipid). Further, the significant uptake of solubilized cell material (in the form of amino acids) demonstrated that the recycled waste has the potential for offsetting the need for fresh medium components. These results indicate that the implementation of a nutrient recycling strategy for industrial microalgae production could be possible, with significant added benefits such as conserving water resources, improving production efficiency, and decreasing material inputs.

Nutrient and media recycling in heterotrophic microalgae cultures.

In order for microalgae-based processes to reach commercial production for biofuels and high-value products such as omega-3 fatty acids, it is necessary that economic feasibility be demonstrated at the industrial scale. Therefore, process optimization is critical to ensure that the maximum yield can be achieved from the most efficient use of resources. This is particularly true for processes involving heterotrophic microalgae, which have not been studied as extensively as phototrophic microalgae. An area that has received significant conceptual praise, but little experimental validation, is that of nutrient recycling, where the waste materials from prior cultures and post-lipid extraction are reused for secondary fermentations. While the concept is very simple and could result in significant economic and environmental benefits, there are some underlying challenges that must be overcome before adoption of nutrient recycling is viable at commercial scale. Even more, adapting nutrient recycling for optimized heterotrophic cultures presents some added challenges that must be identified and addressed that have been largely unexplored to date. These challenges center on carbon and nitrogen recycling and the implications of using waste materials in conjunction with virgin nutrients for secondary cultures. The aim of this review is to provide a foundation for further understanding of nutrient recycling for microalgae cultivation. As such, we outline the current state of technology and practical challenges associated with nutrient recycling for heterotrophic microalgae on an industrial scale and give recommendations for future work.

Comparative analysis of ß-carotene hydroxylase genes for astaxanthin biosynthesis.

Astaxanthin (3,3'-dihydroxy-4,4'-diketo-ß-carotene) (1) is a carotenoid of significant commercial value due to its superior antioxidant potential, application as a component of animal feeds, and ongoing research that links its application to the treatment and prevention of human pathologies. The high commercial cost of 1 is also based upon its complex synthesis. Chemical synthesis has been demonstrated, but produces a mixture of stereoisomers with limited applications. Production from biological sources is limited to natural producers with complex culture requirements. The biosynthetic pathway for 1 is well studied; however, questions remain that prevent optimized production in heterologous systems. Presented is a direct comparison of 12 ß-carotene (2) hydroxylases derived from archaea, bacteria, cyanobacteria, and plants. Expression in Escherichia coli enables a comparison of catalytic activity with respect to zeaxanthin (3) and 1 biosynthesis. The most suitable ß-carotene hydroxylases were subsequently expressed from an efficient dual expression vector, enabling 1 biosynthesis at levels up to 84% of total carotenoids. This supports efficient 1 biosynthesis by balanced expression of ß-carotene ketolase and ß-carotene hydroxylase genes. Moreover, our work suggests that the most efficient route for astaxanthin biosynthesis proceeds by hydroxylation of ß-carotene to zeaxanthin, followed by ketolation.

A high-throughput screen for the identification of improved catalytic activity: ß-carotene hydroxylase.

Astaxanthin is a natural product of immense value. Its biosynthesis has been investigated extensively and typically requires the independent activity of two proteins, a ß-carotene ketolase and ß-carotene hydroxylase. Rational engineering of this pathway has produced limited success with respect to the biological production of astaxanthin. Random mutagenesis of the ß-carotene ketolase has also been pursued. However, to date, no suitable method has been developed for the investigation of the ß-carotene hydroxylase because ß-carotene and zeaxanthin cannot be differentiated visually, unlike ß-carotene and canthaxanthin. Thus, random mutagenesis and efficient selection of improved ß-carotene hydroxylase clones is not feasible. Presented here are the steps required for the efficient generation of a ß-carotene hydroxylase random mutagenesis library in Escherichia coli. Subsequently presented is a novel high-throughput screening method for the rapid identification of clones with enhanced ß-carotene hydroxylase activity. The validity of the presented method is confirmed by functional expression of the mutated proteins, combined with accurate quantification of produced carotenoids. The developed method has potential applications in the development of biological systems for improved carotenoid biosynthesis, as well as robust astaxanthin production.

Efficient extraction of canthaxanthin from Escherichia coli by a 2-step process with organic solvents.

Canthaxanthin has a substantial commercial market in aquaculture, poultry production, and cosmetic and nutraceutical industries. Commercial production is dominated by chemical synthesis; however, changing consumer demands fuel research into the development of biotechnology processes. Highly productive microbial systems to produce carotenoids can be limited by the efficiency of extraction methods. Extraction with hexane, acetone, methanol, 2-propanol, ethanol, 1-butanol, tetrahydrofuran and ethyl acetate was carried out with each solvent separately, and subsequently the most efficient solvents were tested in combination, both as mixtures and sequentially. Sequential application of methanol followed by acetone proved most efficient. Extraction efficiency remained stable over a solvent to biomass range of 100:1 to 55:1, but declined significantly at a ratio of 25:1. Application of this method to a canthaxanthin-producing Escherichia coli production system enabled efficient canthaxanthin extraction of up to 8.5 mg g(-1) dry biomass.

Use of raw glycerol to produce oil rich in polyunsaturated fatty acids by a thraustochytrid.

Glucose is the typical carbon source for producing microbial polyunsaturated fatty acids (PUFA) with single cell microorganisms such as thraustochytrids. We assessed the use of a fish oil derived glycerol by-product (raw glycerol), produced by a fish oil processing plant, as a carbon source to produce single cell oil rich in polyunsaturated fatty acids (PUFA), notably docosahexaenoic acid (DHA). These results were compared to those obtained when using analytical grade glycerol, and glucose. The thraustochytrid strain tested produced similar amounts of oil and PUFA when grown with both types of glycerol, and results were also similar to those obtained using glucose. After 6 days of fermentation, approximately 320 mg/g of oil, and 145 mg/g of PUFA were produced with all carbon sources tested. All oils produced by our strain were 99.95% in the triacylglycerol form. To date, this is the first report of using raw glycerol derived from fish oil for producing microbial triglyceride oil rich in PUFA.

Stability studies on astaxanthin extracted from fermented shrimp byproducts.

To the best of our knowledge, stability studies on astaxanthin contained in carotenoproteins extracted from lactic acid fermented shrimp byproduct have never been reported. Carotenoprotein powder, containing 1% free astaxanthin, was subjected to oxidation factors of illumination, oxygen availability, and temperature, using synthetic astaxanthin as a control. The individual effects as well as first and second degree interactions were studied on natural and synthetic free astaxanthin stability. Air and full light were the two individual factors with the highest effects on astaxanthin oxidation. Sixty-two and 46% natural and synthetic astaxanthin, respectively, oxidized when exposed to air for 8 weeks of storage, whereas 35 and 28% of natural and synthetic astaxanthin, respectively, oxidized under full light. Ninety-seven and 88% of natural and synthetic astaxanthin, respectively, oxidized under a combination of full light, air, and 45 degrees C at 8 weeks of storage. Storage in the dark, nonoxygen, and 25 degrees C were the treatments that efficiently minimized astaxanthin oxidation. Natural astaxanthin from fermented shrimp byproduct presented moderate stability levels. Although natural astaxanthin oxidized faster than the synthetic pigment, its stability may improve by antioxidant and polymer addition.

Evaluation of fatty acid extraction methods for Thraustochytrium sp. ONC-T18.

Various extraction methods were assessed in their capacity to extract fatty acids from a dried biomass of Thraustochytrium sp. ONC-T18. Direct saponification using KOH in ethanol or in hexane:ethanol was one of the most efficient techniques to extract lipids (697 mg g(-1)). The highest amount of fatty acids (714 mg g(-1)) was extracted using a miniaturized Bligh and Dyer extraction technique. The use of ultrasonics to break down cell walls while extracting with solvents (methanol:chloroform) also offered high extraction yields of fatty acids (609 mg g(-1)). Moreover, when the transesterification mixture used for a direct transesterification method was doubled, the extraction of fatty acids increased approximately 77% (from 392 to 696 mg g(-1)). This work showed that Thraustochytrium sp. ONC-T18 has the ability to produce over 700 mg g(-1) of lipids, including more than 165 mg g(-1) of docosahexaenoic acid, which makes this microorganism a potential candidate for the commercial production of polyunsaturated fatty acids. Finally, other lipids, such as myristic, palmitic, palmitoleic, and oleic acids, were also produced and recovered in significant amounts (54, 196, 123, and 81 mg g(-1)), respectively.

Critical assessment of various techniques for the extraction of carotenoids and co-enzyme Q10 from the Thraustochytrid strain ONC-T18.

A variety of techniques for extracting carotenoids from the marine Thraustochytrium sp. ONC-T18 was compared. Specifically, the organic solvents acetone, ethyl acetate, and petroleum ether were tested, along with direct and indirect ultrasonic assisted extraction (probe vs bath) methods. Techniques that used petroleum ether/acetone/water (15:75:10, v/v/v) with 3 h of agitation, or 5 min in an ultrasonic bath, produced the highest extraction yields of total carotenoids (29-30.5 microg g-1). Concentrations up to 11.5 microg g-1 of canthaxanthin and 17.5 microg g-1 of beta;-carotene were detected in extracts stored for 6 weeks. Astaxanthin and echinenone were also detected as minor compounds. Extracts with and without antioxidants showed similar carotenoid concentration profiles. However, total carotenoid concentrations were approximately 8% higher when antioxidants were used. Finally, an easy-to-perform and inexpensive method to detect co-enzymes in ONC-T18 was also developed using silica gel TLC plates. Five percent methanol in toluene as a mobile phase consistently eluted co-enzyme Q10 standards and could separate the co-enzyme fractions present in ONC-T18.