Culturable Diversity of Thraustochytrids from Coastal Waters of Qingdao and Their Fatty Acids

Thraustochytrids have gained significant attention in recent years because of their considerable ecological and biotechnological importance. Yet, the influence of seasons and habitats on their culturable diversity and lipid profile remains poorly described. In this study, a total of 58 thraustochytrid strains were isolated from the coastal waters of Qingdao, China. These strains were phylogenetically close to five thraustochytrid genera, namely Botryochytrium, Oblongichytrium, Schizochytrium, Thraustochytrium, and Sicyoidochytrium. Most of the isolated strains were classified into the genera Thraustochytrium and Oblongichytrium. Further diversity analysis revealed that samples collected from nutrient-rich habitats and during summer/fall yielded significantly higher culturable diversity of thraustochytrids than those from low-nutrient habitats and winter/spring. Moreover, sampling habitats and seasons significantly impacted the fatty acid profiles of the strains. Particularly, the Oblongichytrium sp. OC931 strain produced a significant amount (153.99 mg/L) of eicosapentaenoic acid (EPA), accounting for 9.12% of the total fatty acids, which was significantly higher than that of the previously reported Aurantiochytrium strains. Overall, the results of this study fill the gap in our current understanding of the culturable diversity of thraustochytrids in the coastal waters and the impact of the sampling habitats and seasons on their capacity for lipid accumulation.     

Optimization of Medium Using Response Surface Methodology for Lipid Production by Scenedesmus sp.

Lipid production is an important indicator for assessing microalgal species for biodiesel production. In this work, the effects of medium composition on lipid production by Scenedesmus sp. were investigated using the response surface methodology. The results of a Plackett–Burman design experiment revealed that NaHCO₃, NaH₂PO₄·2H₂O and NaNO₃ were three factors significantly influencing lipid production, which were further optimized by a Box–Behnken design. The optimal medium was found to contain 3.07 g L⁻¹ NaHCO₃, 15.49 mg L⁻¹ NaH₂PO₄·2H₂O and 803.21 mg L⁻¹ NaNO₃. Using the optimal conditions previously determined, the lipid production (304.02 mg·L⁻¹) increased 54.64% more than that using the initial medium, which agreed well with the predicted value 309.50 mg L⁻¹. Additionally, lipid analysis found that palmitic acid (C16:0) and oleic acid (C18:1) dominantly constituted the algal fatty acids (about 60% of the total fatty acids) and a much higher content of neutral lipid accounted for 82.32% of total lipids, which strongly proved that Scenedesmus sp. is a very promising feedstock for biodiesel production.     

Assessment of Chemical and Physico-Chemical Properties of Cyanobacterial Lipids for Biodiesel Production

Five non-toxin producing cyanobacterial isolates from the genera Synechococcus, Trichormus, Microcystis, Leptolyngbya and Chlorogloea were examined in terms of quantity and quality as lipid feedstock for biofuel production. Under the conditions used in this study, the biomass productivity ranged from 3.7 to 52.7 mg·L⁻¹·day⁻¹ in relation to dry biomass, while the lipid productivity varied between 0.8 and 14.2 mg·L⁻¹·day⁻¹. All cyanobacterial strains evaluated yielded lipids with similar fatty acid composition to those present in the seed oils successfully used for biodiesel synthesis. However, by combining biomass and lipid productivity parameters, the greatest potential was found for Synechococcus sp. PCC7942, M. aeruginosa NPCD-1 and Trichormus sp. CENA77. The chosen lipid samples were further characterized using Fourier Transform Infrared spectroscopy (FTIR), viscosity and thermogravimetry and used as lipid feedstock for biodiesel synthesis by heterogeneous catalysis.     

Exogenous Antioxidants Improve the Accumulation of Saturated and Polyunsaturated Fatty Acids in Schizochytrium sp. PKU#Mn4

Species of Schizochytrium are well known for their remarkable ability to produce lipids intracellularly. However, during their lipid accumulation, reactive oxygen species (ROS) are generated inevitably as byproducts, which if in excess results in lipid peroxidation. To alleviate such ROS-induced damage, seven different natural antioxidants (ascorbic acid, α-tocopherol, tea extract, melatonin, mannitol, sesamol, and butylated hydroxytoluene) were evaluated for their effects on the lipid accumulation in Schizochytrium sp. PKU#Mn4 using a fractional factorial design. Among the tested antioxidants, mannitol showed the best increment (44.98%) in total fatty acids concentration. However, the interaction effects of mannitol (1 g/L) and ascorbic acid (1 g/L) resulted in 2.26 ± 0.27 g/L and 1.45 ± 0.04 g/L of saturated and polyunsaturated fatty acids (SFA and PUFA), respectively, in batch fermentation. These concentrations were further increased to 7.68 ± 0.37 g/L (SFA) and 5.86 ± 0.03 g/L (PUFA) through fed-batch fermentation. Notably, the interaction effects yielded 103.7% and 49.6% increment in SFA and PUFA concentrations in batch fermentation. The possible mechanisms underlining those increments were an increased maximum growth rate of strain PKU#Mn4, alleviated ROS level, and the differential expression of lipid biosynthetic genes andupregulated catalase gene. This study provides an applicable strategy for improving the accumulation of SFA and PUFA in thraustochytrids by exogenous antioxidants and the underlying mechanisms.     

Effects of Structural and Compositional Changes of Nanochloropsis oceania after Enzyme Treatment on EPA-Rich Lipids Extraction

Improved methods for the extraction of eicosapentaenoic acid (EPA), an essential and economically important polyunsaturated fatty acid, are urgently required. However, lipid extraction rates using food-grade solvents such as ethanol are usually low. To improve the ethanol-based extraction rate, and to elucidate the relevant mechanisms, we used cellulase and laccase to treat powdered Nannochloropsis, one of the most promising microalgal sources of EPA. Cellulase and laccase synergistically increased lipid yields by 69.31% and lipid EPA content by 42.63%, by degrading the amorphous hemicellulose and cellulose, improving crystallinity, and promoting the release and extraction of lysodiacylglyceryltrimethylhomoserine. Scanning electron microscopy showed that cell morphology was substantially altered, with cell-wall rupture, loss of cell boundaries, and the release of intracellular substances. In conclusion, Nannochloropsis lipid yields may be directly linked to cell-wall hemicellulose structure, and enzymatic treatment to alter this may improve lipid yields.     

Characterization of biodiesel and bio-oil from Sterculia striata (chicha) oil

This work describes the mechanical and solvent extraction of Sterculia striata seed oil. It was determined that the seeds contain up to 41% in oil, which has an unusual composition. Indeed, up to 50% of the fatty acid contain cyclopropenoid ring. The oil was used as raw material to produce bio-oil and biodiesel and their physical-chemical properties were evaluated. Some of the studied physical-chemical properties of the S. striata biodiesel are in acceptable range for use as biodiesel in diesel engines, showing a promising economic exploitation of this raw material in semi-arid regions. It was also observed that the cyclopropenoid ring remains after transesterification and is decomposed during pyrolysis.     

A Novel Lipid Extraction Method from Wet Microalga Picochlorum sp. at Room Temperature

A novel method using ethanol was proposed for extracting lipids from wet microalga Picochlorum sp. at room temperature and pressure. In this study, Central Composite design (CCD) was applied to investigate the optimum conditions of lipid extraction. The results revealed that the solvent to biomass ratio had the largest effect on lipid extraction efficiency, followed by extraction time and temperature. A high lipid extraction yield (33.04% of the dry weight) was obtained under the following extraction conditions: 5 mL solvents per gram of wet biomass for 37 min with gentle stirring at room temperature. The extraction yield was comparable to that obtained by the widely used Bligh-Dyer method. Furthermore, no significant differences in the distribution of lipid classes and fatty acid composition were observed according to different extraction methods. In conclusion, these results indicated that the proposed procedure using ethanol could extract lipids from wet biomass efficiently and had giant potential for lipid extraction at large scale.     

ARTP Mutagenesis of Schizochytrium sp. PKU#Mn4 and Clethodim-Based Mutant Screening for Enhanced Docosahexaenoic Acid Accumulation

Schizochytrium species are one of the best oleaginous thraustochytrids for high-yield production of docosahexaenoic acid (DHA, 22:6). However, the DHA yields from most wild-type (WT) strains of Schizochytrium are unsatisfactory for large-scale production. In this study, we applied the atmospheric and room-temperature plasma (ARTP) tool to obtain the mutant library of a previously isolated strain of Schizochytrium (i.e., PKU#Mn4). Two rounds of ARTP mutagenesis coupled with the acetyl-CoA carboxylase (ACCase) inhibitor (clethodim)-based screening yielded the mutant A78 that not only displayed better growth, glucose uptake and ACCase activity, but also increased (54.1%) DHA content than that of the WT strain. Subsequent optimization of medium components and supplementation improved the DHA content by 75.5 and 37.2%, respectively, compared with that of mutant A78 cultivated in the unoptimized medium. Interestingly, the ACCase activity of mutant A78 in a medium supplemented with biotin, citric acid or sodium citrate was significantly greater than that in a medium without supplementation. This study provides an effective bioengineering approach for improving the DHA accumulation in oleaginous microbes.     

Castor yield in response to planting date at four locations in the south-central United States

Oilseed crops have potential use in the production of biodiesel. Currently, most oil going into the production of biodiesel is derived from soybean (Gylcine max L.) grown in the central U.S. Inability of the southeastern states to compete with central U.S. soybean production has lead to the search for alternative oilseeds. The high oil content and unique properties of castor (Ricinus communis L.) give it potential for use as biodiesel. However, there is limited information on potential yields, adaptation, and planting dates of adaptation of modern castor cultivars. This study was designed to determine potential yields and ideal planting dates of castor at various locations in Mississippi and Tennessee. A single castor cultivar was sequentially planted at 14 day intervals, at four locations, ranging from Memphis, TN south to Poplarville, MS. Annual planting occurred from 1 April to 1 July at all locations, except the 1 April planting date was excluded from the two northern locations. Mean yield ranged from 89 to 1954 kg ha^?1. Northern most locations and earliest plantings resulted in the greatest yields. Precocious flowering of castor immediately after 1 April planting at Shubuta, MS was associated with a lower yield. This flowering event was also observed at Poplarville, but did not significantly affect yield. The yield reductions due to southern planting coupled with precocious flowering at the southern locations seem to indicate a limited zone of adaptation for castor.     

Biobased Solvents for Pressurized Liquid Extraction of Nannochloropsis gaditana Omega-3 Lipids

To develop greener extraction alternatives for microalgae biomass, ultrasound assisted extraction (UAE) and pressurized liquid extraction (PLE) with different biobased solvents were investigated, demonstrating that both techniques are useful alternatives for algal lipid extraction. Specifically, Nannochloropsis gaditana lipids were extracted by UAE and PLE at different temperatures and extraction times with sustainable solvents like 2-Methyltetrahydrofuran (2-MeTHF) and its mixtures with ethanol and other alcohols. The best oil yields for both PLE and UAE of N. gaditana were achieved with the mixture of 2-MeTHF:ethanol (1:3), reaching yields of up to 16.3%, for UAE at 50 °C and up to 46.1% for PLE at 120 °C. Lipid composition of the extracts was analyzed by HPLC-ELSD and by GC-MS to determine lipid species and fatty acid profile, respectively. Different fractionation of lipid species was achieved with PLE and solvent mixtures of different polarity. Thus, for the extraction of glycolipids, ethanolic extracts contained higher amounts of glycolipids and EPA, probably due to the higher polarity of the solvent. The optimized method was applied to microalgae Isochrysis galbana and Tetraselmis chuii showing the potential of mixtures of biobased solvents like 2-methyl-THF and ethanol in different proportions to efficiently extract and fractionate lipids from microalgal 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 β-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 β-carotene synthase gene, with a different domain organization compared to the gene in other thraustochytrids, explained the carotenoid profile of RT2316-16.     

Variability assessment in Pongamia pinnata (L.) Pierre germplasm for biodiesel traits

Wide variability in oil content was observed in 75 germplasm accessions of Pongamia pinnata (L.) Pierre collected from Telengana region of Andhra Pradesh, India. Out of these, fatty acid profiles of 21 accessions with varying seed oil content were examined. Large variation was observed in stearic, oleic and linoleic fatty acid composition i.e. 1.83–11.50%, 46.66–65.35% and 12.02–32.58% respectively while less variation i.e. 9.25–12.87% was found with palmitic acid content. Saponification number (SN), iodine value (IV) and cetane number (CN) of fatty acid methyl esters of oils varied from 183.3 to 200.91, 74.78 to 100.98 and 50.85 to 59.11 respectively. Fatty acid composition, IV and CN were used to predict the quality of fatty acid methyl esters of oil for use as biodiesel. Fatty acid methyl esters of oils of P. pinnata accessions DORPP 49, 72 and 83 were found most suitable (CN more than 56.6) for use as biodiesel and they meet the major specification of biodiesel standards of USA, Germany and European Standard Organization. The range of variability found for various biodiesel standards in accessions of P. pinnata can be utilized for the establishment of plantations of promising genotypes through clonal means for increased productivity.     

Profiling of Polar Lipids in Marine Oleaginous Diatom Fistulifera solaris JPCC DA0580: Prediction of the Potential Mechanism for Eicosapentaenoic Acid-Incorporation into Triacylglycerol

The marine oleaginous diatom Fistulifera solaris JPCC DA0580 is a candidate for biodiesel production because of its high lipid productivity. However, the substantial eicosapentaenoic acid (EPA) content in this strain would affect the biodiesel quality. On the other hand, EPA is also known as the essential health supplement for humans. EPAs are mainly incorporated into glycerolipids in the microalgal cell instead of the presence as free fatty acids. Therefore, the understanding of the EPA biosynthesis including the incorporation of the EPA into glycerolipids especially triacylglycerol (TAG) is fundamental for regulating EPA content for different purposes. In this study, in order to identify the biosynthesis pathway for the EPA-containing TAG species, a lipidomic characterization of the EPA-enriched polar lipids was performed by using direct infusion electrospray ionization (ESI)-Q-TRAP-MS and MS/MS analyses. The determination of the fatty acid positional distribution showed that the sn-2 position of all the chloroplast lipids and part of phosphatidylcholine (PC) species was occupied by C16 fatty acids. This result suggested the critical role of the chloroplast on the lipid synthesis in F. solaris. Furthermore, the exclusive presence of C18 fatty acids in PC highly indicated the biosynthesis of EPA on PC. Finally, the PC-based acyl-editing and head group exchange processes were proposed to be essential for the incorporation of EPA into TAG and chloroplast lipids.     

Biological Activities and Chemical Composition of Methanolic Extracts of Selected Autochthonous Microalgae Strains from the Red Sea

Four lipid-rich microalgal species from the Red Sea belonging to three different genera (Nannochloris, Picochlorum and Desmochloris), previously isolated as novel biodiesel feedstocks, were bioprospected for high-value, bioactive molecules. Methanol extracts were thus prepared from freeze-dried biomass and screened for different biological activities. Nannochloris sp. SBL1 and Desmochloris sp. SBL3 had the highest radical scavenging activity against 1,1-diphenyl-2-picrylhydrazyl, and the best copper and iron chelating activities. All species had potent butyrylcholinesterase inhibitory activity (>50%) and mildly inhibited tyrosinase. Picochlorum sp. SBL2 and Nannochloris sp. SBL4 extracts significantly reduced the viability of tumoral (HepG2 and HeLa) cells with lower toxicity against the non-tumoral murine stromal (S17) cells. Nannochloris sp. SBL1 significantly reduced the viability of Leishmania infantum down to 62% (250 µg/mL). Picochlorum sp. SBL2 had the highest total phenolic content, the major phenolic compounds identified being salicylic, coumaric and gallic acids. Neoxanthin, violaxanthin, zeaxanthin, lutein and β-carotene were identified in the extracts of all strains, while canthaxanthin was only identified in Picochlorum sp. SBL2. Taken together, these results strongly suggest that the microalgae included in this work could be used as sources of added-value products that could be used to upgrade the final biomass value.     

Oil productivity and composition of sunflower as a function of hybrid and planting date

Sunflower (Helianthus annuus L.) is a potential cash crop for the southeastern United States for production of cooking oil or biodiesel. Two years (2006 and 2007) of experiments were conducted at each of five locations in Mississippi to evaluate the effect of planting date (April 20, May 20, and June 20), and hybrid (DKF3875, DKF2990, DKF3510, DKF3901, PR63M80, PR62A91, PR63A21, PR63M91, and PR64H41) on seed yield, oil content, and oil composition of sunflower. Seed oil concentration varied from 25 to 47%. The oleic acid concentration in the oil was greater than 85% for DKF3510 and PR64H41, above 65% for PR63M80 and PR63M91, and intermediate for the other hybrids. Total saturated fatty acids (TSFA) concentration in the oil (the sum of palmitic, stearic, arachidic, behenic, and lignoceric acids) ranged from 6.3 to 13.0%, with DKF3510, PR63M91, and PR64H41 having lower concentration of TSFA than the other hybrids. Mean seed yields ranged from 997 to 2096 kg ha⁻¹ depending on location. Mean oil yields at the five locations ranged from 380 to 687 kg ha⁻¹, and calculated biodiesel production ranged from 304 to 550 kg ha⁻¹. Seed and oil yields in this study suggest sunflower in Mississippi should be planted by the last week of May. Later planting (20 June) may significantly decrease both seed and oil yields in the non-irrigated system in Mississippi and in other areas of the southeastern United States with similar environmental conditions.     

Synthesis of Jatropha curcas oil-based biodiesel in a pulsed loop reactor

Jatropha curcas oil (JCO) has a high content of free fatty acids and has been used extensively as a feedstock in biodiesel production. In the present study, the transesterification reaction of JCO to Jatropha curcas methyl ester (biodiesel) was performed in a continuous pulsed loop reactor under atmospheric conditions. The JCO was pre-treated prior to the reaction to reduce the free fatty acid content to below 1% (w/w). The operating parameters of the loop reactor were optimised based on the conversion of the JCO to Jatropha curcas biodiesel and included reaction temperature, molar ratio of oil to MeOH, reaction time and oscillation frequency. The findings show that the highest reaction conversion of 99.7% (w/w) was achieved using KOH catalyst and 98.8% conversion was obtained using NaOCH₃ catalyst. The optimal operating conditions were a molar ratio of 6:1, an oscillation frequency of 6 Hz, temperature of 60 °C, feedstock FFA content of 0.5% (w/w) and only 10 min of reaction time. As a commercial commodity, the physical properties of biodiesel were analysed, and they compared well with the characteristics of fossil-based diesel fuel.     

Combined Effects of Nitrogen Concentration and Seasonal Changes on the Production of Lipids in Nannochloropsis oculata

Instead of sole nutrient starvation to boost algal lipid production, we addressed nutrient limitation at two different seasons (autumn and spring) during outdoor cultivation in flat panel photobioreactors. Lipid accumulation, biomass and lipid productivity and changes in fatty acid composition of Nannochloropsis oculata were investigated under nitrogen (N) limitation (nitrate:phosphate N:P 5, N:P 2.5 molar ratio). N. oculata was able to maintain a high biomass productivity under N-limitation compared to N-sufficiency (N:P 20) at both seasons, which in spring resulted in nearly double lipid productivity under N-limited conditions (0.21 g L⁻¹ day⁻¹) compared to N-sufficiency (0.11 g L⁻¹ day⁻¹). Saturated and monounsaturated fatty acids increased from 76% to nearly 90% of total fatty acids in N-limited cultures. Higher biomass and lipid productivity in spring could, partly, be explained by higher irradiance, partly by greater harvesting rate (~30%). Our results indicate the potential for the production of algal high value products (i.e., polyunsaturated fatty acids) during both N-sufficiency and N-limitation. To meet the sustainability challenges of algal biomass production, we propose a dual-system process: Closed photobioreactors producing biomass for high value products and inoculum for larger raceway ponds recycling waste/exhaust streams to produce bulk chemicals for fuel, feed and industrial material.     

The Marine-Derived Fungus Clonostachys rosea,Source of a Rare Conjugated 4-Me-6E,8E-hexadecadienoic Acid Reducing Viability of MCF-7 Breast Cancer Cells and Gene Expression of Lipogenic Enzymes

A marine-derived strain of Clonostachys rosea isolated from sediments of the river Loire estuary (France) was investigated for its high lipid production. The fungal strain was grown on six different culture media to explore lipid production changes. An original branched conjugated fatty acid, mainly present in triglycerides and mostly produced when grown on DCA (23% of total fatty acid composition). It was identified as 4-Me-6E,8E-hexadecadienoic on the basis of spectroscopic analyses. This fatty acid reduced viability of MCF-7 breast cancer cells in a dose dependent manner (up to 63%) at physiological free fatty acid human plasma concentration (100 μM). Reduction of gene expression of two lipogenic enzymes, the acetyl CoA carboxylase (ACC) and the fatty acid synthase (FAS) was evaluated to explore the mechanisms of action of 4-Me-6E,8E-16:2 acid. At 50 μM, 50% and 35% of mRNA gene expression inhibition were observed for ACC and FAS, respectively.