Schizochytrium Limacinum SR21 Research Articles

Knockout of a PLD gene in Schizochytrium limacinum SR21 enhances docosahexaenoic acid accumulation by modulation of the phospholipid profile

BackgroundThe hydrolysis and transphosphatidylation of phospholipase D (PLD) play important roles in the interconversion of phospholipids (PLs), which has been shown to profoundly impact lipid metabolism in plants. In this study, the effect of the PLD1 gene of Schizochytrium limacinum SR21 (S. limacinum SR21) on lipid metabolism was investigated.ResultsPLD1 knockout had little impact on cell growth and lipid production, but it significantly improved the percentage of polyunsaturated fatty acids in lipids, of which docosahexaenoic acid (DHA) content increased by 13.3% compared to the wild-type strain. Phospholipomics and real-time quantitative PCR analysis revealed the knockout of PLD1 reduced the interexchange and increased de novo synthesis of PLs, which altered the composition of PLs, accompanied by a final decrease in phosphatidylcholine (PC) and an increase in phosphatidylinositol, lysophosphatidylcholine, and phosphatidic acid levels. PLD1 knockout also increased DHA content in triglycerides (TAGs) and decreased it in PLs.ConclusionsThese results indicate that PLD1 mainly performs the transphosphatidylation activity in S. limacinum SR21, and its knockout promotes the migration of DHA from PLs to TAGs, which is conducive to DHA accumulation and storage in TAGs via an acyl CoA-independent pathway. This study provides a novel approach for identifying the mechanism of DHA accumulation and metabolic regulation strategies for DHA production in S. limacinum SR21.

Integrated biorefinery approach for bioconversion of fish manure to volatile fatty acids and its valorization by Schizochytrium sp for docosahexaenoic acid, squalene, and carotenoids production

To meet environmental sustainable goals, an integrated approach was used to generate volatile fatty acids (VFAs) from fish manure through acidogenic fermentation and their valorization by Schizochytrium limacinum SR21 for value-added biomass production. Acidogenic fermentations were conducted at different pH values (5.0–9.0) and total solid (TS) contents (5 %, 8 %, and 12 %). The highest VFA yield from thermally pretreated fish manure was obtained at pH 9.0 and TS 12 %. In batch culture, 84.0 % of the acidogenic fermentation VFAs were utilized by Schizochytrium limacinum SR21. The highest significant biomass, total fatty acids (TFA), and docosahexaenoic acid (DHA) production were 19, 10.1, and 3.0 g/L, respectively, at C/N ratio of 90 in batch culture with yeast extract as a nitrogen source. Analysis of the effects of three nitrogen feeding strategies with simulated VFA in pH-auxostat fed-batch culture shows that DHA concentration of 15.3 g/L and TFA yield of 53.56 g/L were achieved with low nitrogen (10 g/L) feeding. Squalene and carotenoid contents were 85 mg/g and 0.43 mg/g of dry biomass in fed-batch culture. These findings shows that the production of bio-based volatile fatty acids (VFA) is a cost-effective eco-friendly waste management method and provides high-value-added biomass for biofuel and aquaculture feed production.

Using Glycerol to Produce European Sea Bass Feed With Oleaginous Microbial Biomass: Effects on Growth Performance, Filet Fatty Acid Profile, and FADS2 Gene Expression

Using a circular economy concept, the present study investigated the use of crude glycerol, a primary by-product of biodiesel production, as a low-priced nutrient source for heterotrophic cultivation of the fungus-like protist Schizochytrium limacinum SR21 strain. The whole biomass of this oleaginous microorganism, rich in docosahexaenoic acid (DHA) and high-quality proteins, was then paired with a vegetable oil (VO) source and used to replace fish oil (FO) in European sea bass (Dicentrarchus labrax) feeds. Four nutritionally balanced diets were formulated: diet FO (a FO-based diet), diet VO + 0 (a VO-based diet without S. limacinum), and diets VO + 5 and VO + 10 that were VO-based feeds supplemented with 5 and 10% of S. limacinum, respectively. After a 3-month feeding trial, fish of all dietary groups tripled their initial weight, but growth and feeding efficiencies of D. labrax were not significantly different among treatments. Although the formulated diets were balanced for polyunsaturated fatty acids (PUFAs), fish fed with feeds containing either VO or VO plus 5 and 10% of S. limacinum biomass had significantly higher levels of PUFAs in the flesh than fish fed the FO-based diet. Values of health-related lipid indexes, such as atherogenicity index, thrombogenicity index, and flesh lipid quality as well as n-6/n-3 and PUFAs/SFAs ratios confirmed the high nutritional value of sea bass filet, thus representing a healthy product for human consumption. Although the PUFAs/SFAs ratio showed a significantly higher value in fish fed with VO-based diets supplemented with S. limacinum than in those fed with FO diet, suggesting a better filet quality, the n-6/n-3 ratio clearly indicated that filet quality of dietary group FO was best (value of 0.55) and that of group VO + 10 second best (value of 0.98). We also evaluated the nutritional regulation of Δ6-desaturase (or fads2) gene expression in European sea bass liver. European sea bass fed the VO + 0 diet had the highest number of mRNA copies for Δ6-desaturase (or fads2), fish fed with diet VO + 10 the lowest. Our study adds to the growing body of literature concerning the use of thraustochytrid biomass as a valid alternative to marine-derived raw materials for European sea bass feeds.

Regulation of glucose and glycerol for production of docosahexaenoic acid in Schizochytrium limacinum SR21 with metabolomics analysis

Docosahexaenoic acid (DHA), a member of ω−3 polyunsaturated fatty acids (PUFAs), plays an essential effect in the amelioration of human health. Schizochytrium limacinum SR21 is considered as a potential microalgae for DHA production. The carbon source regulates cell metabolism and PUFAs synthesis in S. limacinum. Flask and fed-batch cultivations were conducted to explore the influence of glycerol and glucose on the biomass, growth rate, and DHA content of S. limacinum SR21. In flask cultivation, the highest cell growth rate (12.2 g/L/day) was obtained when glucose was employed as the sole carbon source, whereas the DHA content reached the maximum value (46.88%) in the medium with glycerol as the sole carbon source. Fed-batch fermentation showed that glycerol had comparable biomass 171 g/L, higher DHA yield (40.7 g/L), and higher DHA production rate (223.8 mg/L/h) compared with glucose as the sole carbon source. We combined the advantages of the two carbon sources and developed a new two-stage fed-batch fermentation strategy using mixed carbon sources (glucose and glycerol). This strategy showed an increase in DHA production rate (229.8 mg/L/h) in comparison with the sole carbon sources. Comparative metabolomics analysis indicated that glycerol added in the medium up-regulated the pentose phosphate and glycolysis pathways. Moreover, tricarboxylic acid cycle and mevalonate pathway were dampened. These outcomes provide metabolic basis for enhancing DHA production rate by S. limacinum SR21.

Function of ORFC of the polyketide synthase gene cluster on fatty acid accumulation in Schizochytrium limacinum SR21

BackgroundAs a potential source of polyunsaturated fatty acids (PUFA), Schizochytrium sp. has been widely used in industry for PUFA production. Polyketide synthase (PKS) cluster is supposed to be the primary way of PUFA synthesis in Schizochytrium sp. As one of three open reading frames (ORF) in the PKS cluster, ORFC plays an essential role in fatty acid biosynthesis. However, the function of domains in ORFC in the fatty acid synthesis of Schizochytrium sp. remained unclear.ResultsIn this study, heterologous expression and overexpression were carried out to study the role of ORFC and its domains in fatty acid accumulation. Firstly, ORFC was heterologously expressed in yeast which increased the PUFA content significantly. Then, the dehydratase (DH) and enoyl reductase (ER) domains located on ORFC were overexpressed in Schizochytrium limacinum SR21, respectively. Fatty acids profile analysis showed that the contents of PUFA and saturated fatty acid were increased in the DH and ER overexpression strains, respectively. This indicated that the DH and ER domains played distinct roles in lipid accumulation. Metabolic and transcriptomic analysis revealed that the pentose phosphate pathway and triacylglycerol biosynthesis were enhanced, while the tricarboxylic acid cycle and fatty acids oxidation were weakened in DH-overexpression strain. However, the opposite effect was found in the ER-overexpression strain.ConclusionTherefore, ORFC was required for the biosynthesis of fatty acid. The DH domain played a crucial role in PUFA synthesis, whereas the ER domain might be related to saturated fatty acids (SFA) synthesis in Schizochytrium limacinum SR21. This research explored the role of ORFC in the PKS gene cluster in Schizochytrium limacinum and provided potential genetic modification strategies for improving lipid production and regulating PUFA and SFA content.

Volatile Fatty Acids (VFAs) Generated by Anaerobic Digestion Serve as Feedstock for Freshwater and Marine Oleaginous Microorganisms to Produce Biodiesel and Added-Value Compounds.

Given an increasing focus on environmental sustainability, microbial oils have been suggested as an alternative to petroleum-based products. However, microbial oil production relies on the use of costly sugar-based feedstocks. Substrate limitation, elevated costs, and risk of contamination have sparked the search for alternatives to sugar-based platforms. Volatile fatty acids are generated during anaerobic digestion of organic waste and are considered a promising substrate for microbial oil production. In the present study, two freshwater and one marine microalga along with two thraustochytrids were evaluated for their potential to produce lipids when cultivated on volatile fatty acids generated from food waste via anaerobic digestion using a membrane bioreactor. Freshwater microalgae Auxenochlorella protothecoides and Chlorella sorokiniana synthesized lipids rich in palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), and linoleic acid (C18:2). This composition corresponds to that of soybean and jatropha oils, which are used as biodiesel feedstock. Production of added-value polyunsaturated fatty acids (PUFA) mainly omega-3 fatty acids was examined in three different marine strains: Aurantiochytrium sp. T66, Schizochytrium limacinum SR21, and Crypthecodinium cohnii. Only Aurantiochytrium sp. T66 seemed promising, generating 43.19% docosahexaenoic acid (DHA) and 13.56% docosapentaenoic acid (DPA) in total lipids. In summary, we show that A. protothecoides, C. sorokiniana, and Aurantiochytrium sp. T66 can be used for microbial oil production from food waste material.

High-Density pH-Auxostat Fed-Batch Culture of Schizochytrium limacinum SR21 with Acetic Acid as a Carbon Source

Schizochytrium limacinum SR21 is an important strain for industrial production of docosahexaenoic acid (DHA), which is an important omega-3 fatty acid used in the nutraceutical and food industry. However, the high cost of carbon sources has limited its further application in the market with much larger volume, such as animal feed for aquaculture, poultry, and livestock. To seek low-cost carbon source, acetic acid is tested in the present study. The effect of different factors, including initial carbon source concentration, pH, aeration rate, and nitrogen sources, on biomass, lipid, and DHA production were tested. With optimized culture conditions, the biomass concentration of 146g/L, total fatty acids (TFAs) of 82.3g/L, and DHA content of 23.0g/L were achieved with a pH-auxostat fed-batch cultivation. These results suggested that acetic acid is a promising feedstock for the low-cost production of DHA. Graphical Abstract.

Use of Biofuel Industry Wastes as Alternative Nutrient Sources for DHA-Yielding Schizochytrium limacinum Production

The simultaneous use of crude glycerol and effluent from anaerobic digestate, both wastes derived from the biofuel industry, were tested in the frame of circular economy concept, as potential low-cost nutrient sources for the cultivation of rich in docosahexaenoic acid (DHA) oil microalgae strain Schizochytrium limacinum SR21. Initially, the optimal carbon and nitrogen concentration levels for high S. limacinum biomass and lipids production were determined, in a culture media containing conventional, high cost, organic nitrogen sources (yeast extract and peptone), micronutrients and crude glycerol at varying concentrations. Then, the effect of a culture media composed of crude glycerol (as carbon source) and effluent digestate at varying proportions on biomass productivity, lipid accumulation, proximate composition, carbon assimilation and fatty acid content were determined. It was shown that the biomass and total lipid content increased considerably with varying effluent concentrations reaching 49.2 g L−1 at 48% (v/v) of effluent concentration, while the lipid yield at the same effluent concentration reached 10.15 g L−1, compared to 17.0 g L−1 dry biomass and 10.2 g L−1 lipid yield when yeast extract and peptone medium with micronutrients was used. Compared to the control treatment, the above production was obtained with 48% less inorganic salts, which are needed for the preparation of the artificial sea water. It was shown that Schizochytrium limacinum SR21 was able to remediate 40% of the total organic carbon content of the biofuel wastes, while DHA productivity remained at low levels with saturated fatty acids comprising the main fraction of total fatty acid content. The results of the present study suggest that the simultaneous use of two waste streams from the biofuel industry can serve as potential nutrient sources for the growth of Schizochytrium limacinum SR21, replacing the high cost organic nutrients and up to one half the required artificial sea water salts, but upregulation of DHA productivity through optimization of the abiotic environment is necessary for industrial application, including aqua feed production.

Functions of Enyolreductase (ER) Domains of PKS Cluster in Lipid Synthesis and Enhancement of PUFAs Accumulation in Schizochytrium limacinum SR21 Using Triclosan as a Regulator of ER.

The polyketide synthase (PKS) cluster genes are supposed to synthesize polyunsaturated fatty acids (PUFAs) in S. limacinum. In this study, two enyolreductase (ER) genes located on PKS cluster were knocked out through homologous recombination to explore their functions. The knock-out of OrfB-ER (located on OrfB subunit) decreased lipid content and had obvious decrease on PUFAs content, indicating OrfB-ER domain played a vital role on PUFAs synthesis; the knock-out of OrfC-ER (located on OrfC subunit) decreased SFAs content and increased total lipid content, indicating OrfC-ER domain was likely to be related with SFAs synthesis, and lipid production could be improved by down-regulating OrfC-ER domain expression. Therefore, the addition of triclosan as a reported regulator of ER domain induced the increase of PUFAs production by 51.74% and lipids yield by 47.63%. Metabolic analysis indicated triclosan played its role through inhibiting the expression of OrfC-ER to reduce the feedback inhibition of SFAs and further to enhance NADPH synthesis for lipid production, and by weakening mevalonate pathway and tricarboxylic acid (TCA) cycle to shift precursors for lipid and PUFAs synthesis. This research illuminates functions of two ER domains in S. limacinum and provides a potential targets for improving lipid production.

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

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

Screening chemical modulators of benzoic acid derivatives to improve lipid accumulation in Schizochytrium limacinum SR21 with metabolomics analysis

BackgroundSchizochytrium sp. is a marine fungus with great potential as an alternative commercial source of lipids rich in polyunsaturated fatty acids (PUFAs). To further increase lipid accumulation in Schizochytrium sp., the effect of exogenous additives has become one of the hotspots of current research. Although benzoic acid derivatives showed positive effects on lipid accumulation in Schizochytrium, the biochemical mechanism needs further investigation.ResultsFour benzoic acid derivatives (sodium benzoate, p-aminobenzoic acid, p-methyl benzoic acid and folic acid) were screened and evaluated for their effect on lipid accumulation in Schizochytrium limacinum SR21. The lipid yield was increased by 56.84% with p-aminobenzoic acid (p-ABA) at a concentration of 200 mg/L among the four tested chemical modulators. The metabolomics analysis showed that 200 mg/L p-ABA was optimal for promoting glucose catabolism in glycolysis with an increase in the mevalonate pathway and a weakening of the tricarboxylic acid (TCA) cycle. Moreover, p-ABA increased NADPH generation by enhancing the pentose phosphate pathway (PPP), ultimately redirecting the metabolic flux to lipid synthesis. Fed-batch fermentation further proved that p-ABA could significantly increase the yield of lipid by 30.01%, reaching 99.67 g/L, and the lipid content was increased by 35.03%, reaching 71.12%. More importantly, the yields of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) were increased by 33.28% and 42.0%, respectively.ConclusionThe addition of p-ABA could promote the synthesis of tetrahydrofolate, enhancing NADPH, which ultimately promoted the flow of carbon flux to lipid synthesis. These findings provide a valuable strategy for improving the lipid accumulation in Schizochytrium by additives.

Formulation and utilisation of spent anaerobic digestate fluids for the growth and product formation of single cell algal cultures in heterotrophic and autotrophic conditions

Spent anaerobically digested effluents of agricultural origin were collected and treated using membrane filtration to achieve three-large particle free-nutrient streams of N:P ratios of 16.53, 3.78 and 14.22. Three algal species were grown on these streams, achieving good levels of bioremediation of digester fluids simultaneously with biomass and associated end product formation. Nannochloropsis oceanica and Scenedesmus quadricuada, where proven highly effective in remediating the streams achieving ammonia and phosphate reduction over 60% while for Schizochytrium limacinum SR21 these serve as an ideal production medium for lipids and biomass reaching 16.70w/w% and 1.42gL−1 correspondingly.These processes thus provide treatment of sludge, avoiding the disposal problems by land spreading. The solid components are nutrient depleted but rich in organic matter as a soil enhancer, while the fluids rich in nutrients can be efficiently utilised for growth to generate high value materials of microalgae facilitating water reclamation.

Docosahexaenoic acid production from crude glycerol by Schizochytrium limacinum SR21

In our search for a substitute energy source, microalgal biodiesel has presented itself as a potential candidate. However, the development of biodiesel results in the overproduction of crude glycerol, which can cause undesirable environmental issues. The environmental harm can be minimized by converting crude glycerol into value-added products. One solution involves using a microalga Schizochytrium limacinum SR21 to convert crude glycerol into docosahexaenoic acid (DHA). DHA is an essential fatty acid, necessary for developing brain functions in infants and maintaining healthy brain activity in adults. In our study, the highest DHA productivity of 233.73 mg/g biomass was obtained using 3 % crude glycerol in Medium 2 at 20 °C under mixo/heterotrophic cultivation.

Reconstruction and analysis of the genome-scale metabolic model of schizochytrium limacinum SR21 for docosahexaenoic acid production.

BackgroundSchizochytrium limacinum SR21 is a potential industrial strain for docosahexaenoic acid (DHA) production that contains more than 30–40 % DHA among its total fatty acids.MethodsTo resolve the DHA biosynthesis mechanism and improve DHA production at a systematic level, a genomescale metabolic model (GSMM), named iCY1170_DHA, which contains 1769 reactions, 1659 metabolites, and 1170 genes, was reconstructed.ResultsBased on genome annotation results and literature reports, a new DHA synthesis pathway based on a polyketide synthase (PKS) system was detected in S. limacinum. Similarly to conventional fatty acid synthesis, the biosynthesis of DHA via PKS requires abundant acetyl-CoA and NADPH. The in silico addition of malate and citrate led to increases of 24.5 % and 37.1 % in DHA production, respectively. Moreover, based on the results predicted by the model, six amino acids were shown to improve DHA production by experiment. Finally, 30 genes were identified as potential targets for DHA over-production using a Minimization of Metabolic Adjustment algorithm.ConclusionsThe reconstructed GSMM, iCY1170_DHA, could be used to elucidate the mechanism by which DHA is synthesized in S. limacinum and predict the requirements of abundant acetyl-CoA and NADPH for DHA production as well as the enhanced yields achieved via supplementation with six amino acids, malate, and citrate.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2042-y) contains supplementary material, which is available to authorized users.

Docosahexaenoic Acid Production from Crude Glycerol by Schizochytrium Limacinum SR21

The development and promotion of biodiesel, a substitute energy source for fossil fuels, has resulted in the overproduction of crude glycerol, causing undesirable environmental issues. However, the environmental harm can be minimized by converting crude glycerol into useful products. One solution involves using microalga Schizochytrium limacinum SR21 to convert glycerol into docosahexaenoic acid (DHA). DHA is an essential fatty acid, and is necessary to develop and maintain brain functions in infants and adults. The highest DHA productivity of 233.73 mg/g biomass was obtained using 3 % crude glycerol in Medium 2 at 20 °C under mixo/heterotrophic cultivation.

Improved synthesis of docosahexaenoic acid (DHA) using Schizochytrium limacinum SR21 and sustainable media

Docosahexaenoic acid (DHA, C22:6) is an omega-3 fatty acid having beneficial health effects in human beings, though its production is severely hampered due to higher costs and lower yields from the synthesis. The current work concentrates on the intensification in the biomass production and DHA yield using Schizochytrium limacinum SR21. The effect of different media supplements on the DHA yields has been investigated. Citric acid which is a major intermediate in TCA (tricarboxylic acid) cycle proved better among different organic acids used in the work in terms of the yield of both biomass and DHA. The role of arginine and hydrophobic amino acids like tryptophan and tyrosine for the enhanced DHA production has also been illustrated. Monosaccharides (glucose and fructose) and glycerol also resulted in good cell growth and DHA yield. Maximum productivity of 5.7g/l DHA has been obtained when glucose and glycerol were used simultaneously as carbon source. It has also been established that different steep liquors, tomato juice and sweet water can be used as possible cheaper substrates for the production of DHA from S. limacinum SR21.

Growth optimization of thraustochytrid strain 12B for the commercial production of docosahexaenoic acid

High yield of cell, lipid, and docosahexaenoic acid (DHA) from thraustochytrid strain 12B were achieved without the use of a complex medium and at low NaCl concentration which is detrimental to avoid unnecessary corrosion of steel tank equipment during cultivation. Culture medium that contained only 0.1% NaCl and 1% MgSO4 in an organic base solution containing 8% glucose, 1% yeast extract, and 1% peptone, referred here as NM medium, was found to be as good as or superior to the culture medium prepared from 50%(v/v) seawater with percentage lipid/dry cell weight (DCW) of 66.4%(w/w) and DHA yield up to 43.95 mg/g DCW for the thraustochytrid strain 12B. The NM medium was also applicable to the prominently high DHA-accumulating Schizochytrium limacinum SR21, and therefore this medium could probably be used for other thraustochytrid and other types of microbial strains as well.

Proteins Expressed Differently Between Glucose and Glycerol for Schizochytrium limacinum SR21

Due to rapid expansion of biodiesel production, the quantity of crude glycerol on the global market has been surging. One marine microalga, Schizochytrium limacinum SR21 has great capability in transforming crude glycerol to intracellular lipids, especially docosahexaenoic acid (DHA). To elucidate the mechanisms involved in glycerol uptake and conversion, the proteomic profiles of cells grown on either glycerol or glucose were obtained using 2-Dimensional fluorescence difference gel electrophoresis (2-D DIGE) and compared. Out of 24 proteins that were made differentially between glycerol and glucose-grown cells at statistically significant levels, 11 proteins strongly related to the metabolism of glycerol were identified. A glycerol conversion pathway was proposed based on key enzymes determined. Keywords: 2-Dimensional fluorescence difference gel electrophoresis (2-D DIGE), actin, biodiesel, differentially expressed proteins, glucose, glycerol, glycerol metabolism, glycerol conversion pathway, glycerol 3-phosphate dehydrogenase, glycerol kinase, microalgal oil, peroxidase, protein identification, proteomic, Schizochytrium limacinum SR21

Integrated production of long chain polyunsaturated fatty acids (PUFA)-rich Schizochytrium biomass using a nutrient supplemented marine aquaculture wastewater

Schizochytrium limacinum SR21 was used to remove waste nutrients from a marine recirculating aquaculture wastewater while simultaneously producing a valuable biomass containing PUFA enriched with long chain fatty acids which may have potential as an aquafeed additive.Clean seawater and marine aquaculture wastewater supplemented with yeast extract (1.0–10gL−1) and glycerol (10–90gL−1) were used for a comparative study of growth and PUFA production using S. limacinum in batch, continuous and in a packed bed reactor (pall rings, surface area 340m2/m3) that allow surface growth and cell recycling for a high cell density culture. Good growth rates (μ=0.013h−1) were obtained in batch and continuous culture modes. Biomass of S. limacinum was increased from 4.0gL−1 as DCW in batch culture to 57.9gL−1 in retention culture (D=0.062h−1). Retention culture of S. limacinum in a packed bed reactor also produced total lipids of 36.5gL−1 (53–65%, w/w as DCW) and long chain fatty acids (>C22) of 12.6gL−1 (21.8%, w/w as DCW) in 350h. Long chain fatty acids of C22, C18 and C16, as the main fatty acids, were produced and production was enhanced when supplemented wastewater was used in growth medium. Changes to the relative concentrations of glycerol and yeast extract (C:N ratio) alter the amounts of oil produced and types of fatty acid produced.S. limacinum in continuous culture mode (D=0.013h−1) enabled significant reductions (<90%) in phosphorous, nitrogen and ammonia concentrations of marine aquaculture wastewater and efficiency of wastewater treatment was further improved in retention culture operating at D=0.062h−1. This system would be well suited to treating nutrient concentrated effluent from sludge sedimentation/dewatering tanks in recirculating aquaculture systems (RAS). The treatment capacity of the reactor was considerable and in this current study, 420g N, 190g P and 10g total NH3day−1m−3 reactor could be removed using the retention culture system.

Fermentable sugar release from Jatropha seed cakes following lime pretreatment and enzymatic hydrolysis

Composition change of Jatropha seed cake samples was evaluated upon lime pretreatment at 100 °C with different parameters. With a lime dose of 0.2 g and a water content of 10 ml per gram of cake and a treatment period of 1 h, 38.2 ± 0.6% of lignin was removed. However, 65 ± 16% of hemicellulose was also lost under this condition. For all the treatments tested, cellulose content was not affected by lime supplementation. Through further examining total reducing sugar (TRS) release by enzymatic hydrolysis after lime pretreatment, we have found that 0.1 g of lime and 9 ml of water per gram of cake and 3 h pretreatment produced the maximal 68.9% conversion of cellulose. Without lime pretreatment, the highest cellulose conversion was 33.3%. One microalgal species, Schizochytrium limacinum SR21 was able to grow on the hydrolyzates and generate a biomass density of 3.2 g/l in 4 days.