Genome-scale metabolic modeling of Thraustochytrium sp. RT2316-16: Effects of nutrients on metabolism.

Marine thraustochytrids produce metabolically important lipids such as the long-chain omega-3 polyunsaturated fatty acids, carotenoids, and sterols. The growth and lipid production in thraustochytrids depends on the composition of the culture medium that often contains yeast extract as a source of amino acids. This work discusses the effects of individual amino acids provided in the culture medium as the only source of nitrogen, on the production of biomass and lipids by the thraustochytrid Thraustochytrium sp. RT2316-16. A reconstructed metabolic network based on the annotated genome of RT2316-16 in combination with flux balance analysis was used to explain the observed growth and consumption of the nutrients. The culture kinetic parameters estimated from the experimental data were used to constrain the flux via the nutrient consumption rates and the specific growth rate of the triacylglycerol-free biomass in the genome-scale metabolic model (GEM) to predict the specific rate of ATP production for cell maintenance. A relationship was identified between the specific rate of ATP production for maintenance and the specific rate of glucose consumption. The GEM and the derived relationship for the production of ATP for maintenance were used in linear optimization problems, to successfully predict the specific growth rate of RT2316-16 in different experimental conditions.

Novel nutritionally-enriched gummy jelly infused with nipa palm vinegar powder and nipa palm syrup as functional food ingredients.

The objective of this research was to develop a nutritionally-enriched gummy jelly product incorporating nipa palm vinegar powder (NPVp; a nutrients-rich vinegar) and nipa palm syrup (NPS), a nutrients-rich sweetener with a low glycemic index. A gummy jelly product was developed based on sensory acceptance tests. The water activity and the moisture content of the final product were within the acceptable range for preservation under ambient conditions. The final product had a total phenolic content of 861 µg gallic acid equivalent (GAE) per g and an antioxidant activity (2,2-diphenyl-1-picrylhydrazyl (DPPH) inhibition) of 72.7 %. The final product had the following nutritional attributes (per 100 g dry mass): 319.7 kcal of energy, 8.8 g protein, 0.2 g fats, 70.6 g carbohydrates, 59.9 g total sugars, 0.7 g of total dietary fibers, 34.6 mg calcium, 0.3 mg iron, 168.0 mg sodium, and 774.7 mg vitamin C. The in vitro glycemic index of the product was 27.4. Based on their nutrients-content, NPVp and NPS were suitable for use in other functional food products.

Coenzyme Q in Thraustochytrium sp. RT2316-16: Effect of the Medium Composition.

Coenzyme Q (CoQ; ubiquinone) is an essential component of the respiratory chain. It is also a potent antioxidant that prevents oxidative damage to DNA, biological membranes, and lipoproteins. CoQ comprises a six-carbon ring with polar substituents that interact with electron acceptors and donors, and a hydrophobic polyisoprenoid chain that allows for its localization in cellular membranes. Human CoQ has 10 isoprenoid units (CoQ10) within the polyisoprenoid chain. Few microorganisms produce CoQ10. This work shows that Thraustochytrium sp. RT2316-16 produces CoQ10 and CoQ9. The CoQ10 content in RT2316-16 depended strongly on the composition of the growth medium and the age of the culture, whereas the CoQ9 content was less variable probably because it served a different function in the cell. Adding p-hydroxybenzoic acid to the culture media positively influenced the CoQ10 content of the cell. The absence of some B vitamins and p-aminobenzoic acid in the culture medium negatively affected the growth of RT2316-16, but reduced the decline in CoQ10 that otherwise occurred during growth. The highest content of CoQ9 and CoQ10 in the biomass were 855 µg g-1 and 10 mg g-1, respectively. The results presented here suggest that the thraustochytrid RT2316-16 can be a potential vehicle for producing CoQ10. Metabolic signals that trigger the synthesis of CoQ10 in RT2316-16 need to be determined for optimizing culture conditions.

Production of chemicals and utilities in-house improves the environmental sustainability of phytoplankton-based biorefinery.

Life cycle assessment was used to evaluate the environmental impacts of phytoplanktonic biofuels as possible sustainable alternatives to fossil fuels. Three scenarios were examined for converting planktonic biomass into higher-value commodities and energy streams using the alga Scenedesmus sp. and the cyanobacterium Arthrospira sp. as the species of interest. The first scenario (Sc-1) involved the production of biodiesel and glycerol from the planktonic biomass. In the second scenario (Sc-2), biodiesel and glycerol were generated from the planktonic biomass, and biogas was produced from the residual biomass. The process also involved using a catalyst derived from snail shells for biodiesel production. The third scenario (Sc-3) was similar to Sc-2 but converted CO2 from the biogas upgrading to methanol, which was then used in synthesizing biodiesel. The results indicated that Sc-2 and Sc-3 had a reduced potential (up to 60 % less) for damaging human health compared to Sc-1. Sc-2 and Sc-3 had up to 61 % less environmental impact than Sc-1. Sc-2 and Sc-3 reduced the total cumulative exergy demand by up to 44 % compared to Sc-1. In conclusion, producing chemicals and utilities within the biorefinery could significantly improve environmental sustainability, reduce waste, and diversify revenue streams.

Production of Carotenoids and Phospholipids by Thraustochytrium sp. in Batch and Repeated-Batch Culture.

The carotenogenic thraustochytrid Thraustochytrium sp. RT2316-16 was grown in batch and repeated-batch cultures using different feeds containing glucose, or glycerol, and yeast extract, for the production of lipids, phospholipids and carotenoids. RT2316-16 produced canthaxanthin, astaxanthin and ß-carotene. The effects of biotin, ascorbic acid, light and temperature were evaluated in some of the experiments. In 2-day-old batch cultures, the combined mass percentage of eicosapentaenoic acid and docosahexaenoic acid in total lipids was between 16.5% (glycerol-based medium in the dark; biomass concentration = 4.2 ± 1.1 g L-1) and 42.6% (glucose-based medium under light; biomass concentration = 3.3 ± 0.1 g L-1), decreasing to 3.8% and 6.1%, respectively, after day 4. In repeated-batch cultures, the total lipids in the biomass increased after glucose or glycerol was fed alone, whereas the total carotenoids (168 ± 7 µg g-1 dry weight (DW)) and phospholipids in the biomass increased after feeding with yeast extract. The biomass with the highest content of phospholipids (28.7 ± 4.3 mg g-1 DW) was obtained using a feed medium formulated with glycerol, yeast extract and ascorbic acid. Glycerol was the best carbon source for the production of a biomass enriched with total lipids (467 ± 45 mg g-1 DW). The composition of carotenoids depended strongly on the composition of the feed. Repeated-batch cultures fed with yeast extract contained canthaxanthin as the main carotenoid, whereas in the cultures fed only with glucose, the biomass contained mainly ß-carotene.

Collagen Extraction from Animal Skin.

Collagen is the most abundant structural protein in animals. It is the major component of skin. It finds uses in cosmetics, medicine, yarn production and packaging. This paper reviews the extraction of collagen from hides of most consumed animals for meat with the focus on literature published since 2000. The different pretreatment and extraction techniques that have been investigated for producing collagen from animal skins are reviewed. Pretreatment by enzymatic, acid or alkaline methods have been used. Extraction by chemical hydrolysis, salt solubilization, enzymatic hydrolysis, ultrasound assisted extraction and other methods are described. Post-extraction purification methods are also explained. This compilation will be useful for anyone wishing to use collagen as a resource and wanting to further improve the extraction and purification methods.

Production of polyunsaturated fatty acids by Schizochytrium (Aurantiochytrium) spp.

Diverse health benefits are associated with dietary consumption of omega-3 long-chain polyunsaturated fatty acids (ω-3 LC-PUFA), particularly docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). Traditionally, these fatty acids have been obtained from fish oil, but limited supply, variably quality, and an inability to sustainably increase production for a rapidly growing market, are driving the quest for alternative sources. DHA derived from certain marine protists (heterotrophic thraustochytrids) already has an established history of commercial production for high-value dietary use, but is too expensive for use in aquaculture feeds, a much larger potential market for ω-3 LC-PUFA. Sustainable expansion of aquaculture is prevented by its current dependence on wild-caught fish oil as the source of ω-3 LC-PUFA nutrients required in the diet of aquacultured animals. Although several thraustochytrids have been shown to produce DHA and EPA, there is a particular interest in Schizochytrium spp. (now Aurantiochytrium spp.), as some of the better producers. The need for larger scale production has resulted in development of many strategies for improving productivity and production economics of ω-3 PUFA in Schizochytrium spp. Developments in fermentation technology and metabolic engineering for enhancing LC-PUFA production in Schizochytrium spp. are reviewed.

Removal of nitrate and phosphate from simulated agricultural runoff water by Chlorella vulgaris.

Microalgae such Chlorella vulgaris can effectively absorb nitrate and phosphate from contaminated water. This work characterized nitrate and phosphate removal from simulated agricultural runoff using C. vulgaris. Statistically designed experiments were used to model the following responses: (1) algal growth; (2) nitrate removal; (3) phosphate removal; (4) protein in the algal biomass; (5) chlorophyll content of the biomass; (6) the biomass phenolics content; and (7) the free radical scavenging antioxidant activity of the biomass. These response were modelled for the following key experimental factors: initial nitrate concentration in the simulated runoff (1080-3240 mg L-1, as NaNO3), initial phosphate concentration (20-60 mg L-1, as K2HPO4), photoperiod (8-24 h of light/day) and culture duration (5-15 days). The validated models were used to identify the factor levels to maximize the various responses. Nitrate removal was maximized at 85.6% when initial nitrate and phosphate concentrations were 2322 mg L-1 and 38 mg L-1 (N:P atom ratio ≈ 125:1), respectively, with a 17.2 h daily photoperiod in a 13-day culture. Phosphate removal was maximized at 95% when the initial nitrate and phosphate concentrations were 1402 mg L-1 and 56.7 mg L-1 (N:P ≈ 51:1), respectively, with a 15.7 h daily photoperiod in a 14.7-day culture. At least ~14 h of a daily photoperiod and a ~11-day culture period were required to maximize all the studied responses. C. vulgaris is edible and may be used as animal feed. Nutritional aspects of the biomass were characterized. Biomass with more than 24% protein could be produced. Under the best conditions, the chlorophyll (potential food colorants) content of the biomass was 8.5% and the maximum level of total phenolics (antioxidants) in the biomass was nearly 13 mg gallic acid equivalent g-1.

Pathogens and predators impacting commercial production of microalgae and cyanobacteria.

Production of phytoplankton (microalgae and cyanobacteria) in commercial raceway ponds and other systems is adversely impacted by phytoplankton pathogens, including bacteria, fungi and viruses. In addition, cultures are susceptible to productivity loss, or crash, through grazing by contaminating zooplankton such as protozoa, rotifers and copepods. Productivity loss and product contamination are also caused by otherwise innocuous invading phytoplankton that consume resources in competition with the species being cultured. This review is focused on phytoplankton competitors, pathogens and grazers of significance in commercial culture of microalgae and cyanobacteria. Detection and identification of these biological contaminants are discussed. Operational protocols for minimizing contamination, and methods of managing it, are discussed.

Nitrogen Sources Affect the Long-Chain Polyunsaturated Fatty Acids Content in Thraustochytrium sp. RT2316-16.

The psychrophilic marine microorganism Thraustochytrium sp. RT2316-16 can produce carotenoids as well as lipids containing the omega-3 polyunsaturated fatty acids (PUFA) eicosapentaenoic acid and docosahexaenoic acid. This work reports on the effects of the composition of the culture medium, including certain amino acids, on growth and lipid synthesis by RT2316-16. Compared with the culture on glutamate, the use of lysine, alanine, or serine, increased the content of the omega-3 PUFA in total lipids. In the media that contained yeast extract, glutamate, and glucose, lipid accumulation occurred when organic ammonium was exhausted earlier than glucose. In contrast, lipid mobilization was promoted if glucose was exhausted while organic ammonium (supplied by yeast extract and glutamate) remained in the medium. The total content of carotenoids in the lipid-free biomass decreased during the first 12 to 24 h of culture, simultaneously with a decrease in the total lipid content of the biomass. The experimental data suggested a possible interrelationship between the metabolism of carotenoids and lipids. A high content of omega-3 PUFA in the total lipids could be obtained by growing the thraustochytrid in a medium with a low glucose concentration (6 g L-1) and a high concentration of organic nitrogen (yeast extract 12 g L-1; glutamate 1.06 g L-1), after glucose was exhausted. These observations may guide the development of a strategy to enhance omega-3 PUFA in the biomass.

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.

Immobilization of ß-galactosidase by halloysite-adsorption and entrapment in a cellulose nanocrystals matrix.

BACKGROUND: Immobilization allows easy recovery and reuse of enzymes in industrial processes. In addition, it may enhance enzyme stability, allowing prolonged use. A simple and novel method of immobilizing ß-galactosidase is reported. Effects of immobilization on the enzyme characteristics are explained. ß-Galactosidase is well established in dairy processing and has emerging applications in novel syntheses. METHODS: ß-Galactosidase was immobilized by physical adsorption on halloysite, an aluminosilicate nanomaterial. Optimal conditions for adsorption were identified. The optimally prepared halloysite-adsorbed enzyme was then entrapped in a porous matrix of nanocrystals of sulfated bacterial cellulose, to further enhance stability. RESULTS: Under optimal conditions, 89.5% of the available protein was adsorbed per mg of halloysite. The most active and stable final immobilized biocatalyst had 1 part by mass of the enzyme-supporting halloysite particles mixed with 2 parts of cellulose nanocrystals. Immobilization raised the optimal pH of the catalyst to 7.5 (from 6.0 for the native enzyme) and temperature to 55 °C (40 °C for the native enzyme). During storage at 25 °C, the immobilized enzyme retained 75.8% of initial activity after 60 days compared to 29.2% retained by the free enzyme. CONCLUSION: The immobilization method developed in this work enhanced enzyme stability during catalysis and storage. Up to 12 cycles of repeated use of the catalyst became feasible. GENERAL SIGNIFICANCE: The simple and rapid immobilization strategy of this work is broadly applicable to enzymes used in diverse bioconversions.

Production of lipids by Chaetoceros affinis in media based on palm oil mill effluent.

Biomass and lipid production by the marine diatom Chaetoceros affinis were characterized under continuous light with aeration. Media based on palm oil mill effluent (POME; 10, 20 and 30 % v/v in distilled water) were used together with a standard control medium. The maximum biomass concentration on day 12 of batch cultures in control medium was 821 ± 71 mg L-1. Under identical conditions, in the best POME medium (20 % POME v/v in distilled water with other inorganic components), the biomass concentration was reduced by ∼11 % to 734 ± 66 mg L-1. The lipid content of the biomass grown in the control medium was 50.8 ± 4.5 % by dry weight, but was a little lower (48.9 ± 4.1 % by dry wt) in the above specified best POME medium. In the best POME medium, oleic acid was the major fatty acid (72.3 ± 5.2 % by weight) in the total lipids extracted from the biomass and monounsaturated fatty acids were the main type of fatty acids (74.6 ± 5.2 %). POME levels of >20 % in the medium suppressed both biomass and lipid production relative to the medium with 20 % POME.

Recovery of Red Pigments from Monascus purpureus FTC 5357 by Extraction of Fermented Solids: Operational Conditions and Kinetics

Abstract Solvent extraction of red pigments from fermented solids is reported. The pigments were produced by solid-state fermentation of oil palm frond (OPF) biomass with the food-safe fungus Monascus purpureus FTC 5357. The effects of extraction solvent and other operational conditions (pH, temperature, agitation rate, contact time) on the recovery of pigments are discussed. The recovery was maximized using aqueous ethanol (60% ethanol by vol) as solvent at pH 6, 30 °C, with an extraction time of 16 h and an agitation rate of 180 rpm. A fermented solids dry mass of 1 g was used for each 160 mL of solvent during extraction. The kinetics of extraction were assessed by fitting the experimental data to different models. Peleg's model proved to be the best for describing solid-liquid extraction of the pigments under the above specified conditions. The highest extraction yield of red pigments under the above specified optimal conditions was 207(6.08 AU g(1 dry fermented solids.

Temperature Differentially Affects Gene Expression in Antarctic Thraustochytrid Oblongichytrium sp. RT2316-13.

Oblongichytrium RT2316-13 synthesizes lipids rich in eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). The content of these fatty acids in the total lipids depended on growth temperature. Sequencing technology was used in this work to examine the thraustochytrid's response to a decrease in growth temperature from 15 °C to 5 °C. Around 4% (2944) of the genes were differentially expressed (DE) and only a few of the DE genes (533 upregulated; 206 downregulated) had significant matches to those in the SwissProt database. Most of the annotated DE genes were related to cell membrane composition (fatty acids, sterols, phosphatidylinositol), the membrane enzymes linked to cell energetics, and membrane structure (cytoskeletal proteins and enzymes). In RT2316-13, the synthesis of long-chain polyunsaturated fatty acids occurred through ω3- and ω6-pathways. Enzymes of the alternative pathways (Δ8-desaturase and Δ9-elongase) were also expressed. The upregulation of the genes coding for a Δ5-desaturase and a Δ5-elongase involved in the synthesis of EPA and DHA, explained the enrichment of total lipid with these two long-chain fatty acids at the low temperature. This molecular response has the potential to be used for producing microbial lipids with a fatty acids profile similar to that of fish oils.

Dynamic flux balance analysis of biomass and lipid production by Antarctic thraustochytrid Oblongichytrium sp. RT2316-13.

Production of biomass and lipids in batch cultures of the Antarctic thraustochytrid Oblongichytrium sp. RT2316-13, is reported. The microorganism proved capable of producing nearly 67% docosahexaenoic acid (DHA) and 15% eicosapentaenoic acid (EPA) in its total lipid fraction. Biomass with a maximum total lipid content of 33.5% (wt/wt) could be produced at 15°C in batch culture using a medium containing glucose (20 g/L), yeast extract (10.5 g/L), and other minor components. A lower culture temperature (5°C) reduced biomass and lipid productivities compared to culture at 15°C, but enhanced the DHA and EPA content of the lipids by 6.4- and 3.3-fold, respectively. Both a simple minimally structured mathematical model and a more complex genome-scale metabolic model (GEM) allowed the fermentation profiles in batch cultures to be satisfactorily simulated, but the GEM provided much greater insight in the biochemical and physiological phenomena underlying the observed behavior. Unlike the simpler model, the GEM could be interrogated for the possible effects of various external factors such as oxygen supply, on the expected outcomes. In silico predictions of oxygen effects were consistent with literature observations for DHA producing thraustochytrids.

Simultaneous nitrogen fixation and ethanol production by Zymomonas mobilis.

This work reports on production of ethanol with simultaneous fixing of nitrogen (N2) using the anaerobic bacterium Zymomonas mobilis DSM 473. A batch fermentation with an initial glucose concentration of 50 g L-1, an initial pH of ∼5.5, an inoculum size of 10% by volume and a N2 feeding rate of 50 mL min-1 without mechanical agitation was found to provide the highest ethanol productivity (0.401 g L-1 h-1). Ethanol yield on glucose exceeded 97% of the theoretical maximum. The nitrogen content of the microbial biomass was 10.4% w/w at 65 h and all of it was derived by fixation of dinitrogen. Repeated-batch fermentations were investigated for ethanol production using simultaneous nitrogen fixation. A 2-cycle repeated-batch fermentation lasting 71 h gave a maximum ethanol yield on glucose of 0.475 g g-1 and an ethanol productivity of 0.675 g L-1 h-1. The yield (0.415 g g-1) and productivity (0.638 g L-1 h-1) were reduced in a 3-cycle repeated batch operation lasting 94 h. The need to fix nitrogen did not reduce the final achievable ethanol concentration, or the ethanol yield on glucose, relative to fermentations provided with fixed nitrogen, but did reduce the ethanol productivity by ∼82% because less cell mass was produced.

Theabrownin from Pu-erh tea together with swinging exercise synergistically ameliorates obesity and insulin resistance in rats.

PURPOSE: Theabrownin (TB)-containing Pu-erh tea has been shown to be hypolipidemic in rats fed a high-fat diet. Physical exercise such as swinging is also known to reduce obesity. We hypothesized that TB in combination with swinging can synergistically ameliorate obesity and insulin resistance in rats with metabolic syndrome. METHODS: TB, rosiglitazone, or lovastatin (controls) was administered by gavage to rats fed a diet high in fat, sugar, and salt. A subgroup of the rats was subjected to a 30-min daily swinging exercise regimen, whereas the other rats did not exercise. RESULTS: Theabrownin in combination with swinging was found to significantly improve serum lipid status and prevent development of obesity and insulin resistance in rats. Liver transcriptomics data suggested that theabrownin activated circadian rhythm, protein kinase A, the adenosine monophosphate-activated protein kinase, and insulin signaling pathways by enhancing cyclic adenosine monophosphate levels and, hence, accelerating nutrient metabolism and the consumption of sugar and fat. The serum dopamine levels in rats increased significantly after exercise. In parallel work, intraperitoneal dopamine injections were shown to significantly reduce weight gain and prevent the elevation in triglyceride levels that would otherwise be induced by the high fat-sugar-salt diet. Theabrownin prevented obesity and insulin resistance mainly by affecting the circadian rhythm, while swinging exercise stimulated the overproduction of dopamine to accelerate metabolism of glucose and lipid. CONCLUSIONS: Theabrownin and exercise synergistically ameliorated metabolic syndrome in rats and effectively prevented obesity.

Biomass and lipid production by Rhodococcus opacus PD630 in molasses-based media with and without osmotic-stress.

Rhodococcus opacus PD630 was used to produce biomass and lipids in molasses-based media with and without osmotic stress. In a 7-day aerobic batch culture at 30 °C, the biomass and lipid concentrations were maximized using an initial molasses concentration of 80 g/L and ammonium acetate (nitrogen source) concentration of 2.14 g/L. At a fixed initial molasses concentration of 80 g/L, the concentration of the nitrogen source was further fine-tuned to 2.25 g/L, to maximize the lipid content of the biomass to around 30% by dry mass. This medium was used to test the effects of stressing salts (sodium acetate, magnesium sulfate, sodium chloride) on production of lipids and biomass. A two-step bolus feeding with magnesium sulfate and sodium acetate, enhanced the final biomass concentration to around 19 g/L (a 50% increase relative to control), but the lipid content in the biomass was reduced to around 16% w/w. A 33% enhancement in lipid concentration relative to control, was achieved by feeding magnesium sulfate and sodium acetate. Sugarcane molasses could be effectively used to produce biomass and lipids instead of using the much more expensive pure carbon sources such as glucose and sucrose.