Solvent Extraction of Fucoxanthin from Phaeodactylum tricornutum

The extraction yield of fucoxanthin from Phaeodactylum tricornutum, a marine diatom, was investigated for 16 h as the functions of the kind of solvent, temperature, and solvent/diatom weight ratio. Acetone, ethanol, and ethyl acetate were used as solvents for the extractions. The extraction yield increased in a saturation manner with the extraction time when the extraction temperatures were 30°C and 40°C. However, the extraction yield increased with time in the early stage of the extraction; thereafter, it was stagnant or decreased with time in the late stage at 50°C, 75°C, and 85°C. The thermal deterioration would suppress the extraction yield in the late stage at those higher temperatures. The maximum extraction yield was obtained at 40°C and no significant benefit in the extraction yield was obtained when the extraction period was extended over 12 h. The extraction yield markedly increased when the solvent/diatom ratio increased from 3/1 to 6/1, but no marked benefit was obtained when the amount of solvent increased over the ratio of 6/1. Ethanol was the most effective solvent in the extraction of fucoxanthin, and the extraction yield was greater in the order of ethanol > acetone > ethyl acetate.     

Optimization of fatty acid extraction from Phaeodactylum tricornutum UTEX 640 biomass

Fatty acids in the microalga Phaeodactylum tricornutum were isolated using an optimized three-step method: extraction of crude fatty acid potassium salts made by direct saponification of lipids in the microalgal biomass with KOH/ethanol (96%, vol/vol), separation of unsaponifiable lipids by extraction with hexane, and final purification of fatty acids by acidification of the alcoholic solution of potassium soaps followed by extraction of fatty acid into hexane. Direct saponification was carried out in ethanol (96%, vol/vol) using 2.09 mL ethanol (96%) per gram of wet biomass (10 mL/g of dry biomass) mixed with 0.4 g KOH/g of biomass. Under these conditions the fatty acid yield was 87%. The optimal water content of the alcoholic solution for extraction of the unsapononifiables was established as 40%, w/w. Data on equilibrium carotenoid distribution between the alcoholic (40%, w/w water) and hexane phases were determined. These data allow prediction of the carotenoid yields with different volumes of hexane in several extraction steps. The optimal pH of the alcoholic solution before extracting the purified fatty acid was established as pH 6, and the equilibrium fatty acid distribution between the alcoholic and hexane phases was determined. This optimized method permited a 20% reduction in the production costs of highly purified eicosapentaenoic acid (EPA) in the three-step preparative process (extraction of fatty acid, concentration of polyunsaturated fatty acids by the urea method, and EPA fractionation through preparative high-performance liquid chromatography) previously developed by the authors.     

营养元素对三角褐指藻生长和脂类积累的影响

研究了不同营养元素(氮、磷、硅、铁)在不同培养浓度条件下,对三角褐指藻生长以及细胞脂类积累的影响作用。结果显示:藻细胞生长随氮含量的增加而增加,但超过一定浓度后作用不明显,最适合三角褐指藻生长的氮含量为 1.76 mmol/L。缺氮和低氮(0.22~0.44 mmol/L)培养能够明显提高藻细胞脂类含量。缺磷培养能有效促进藻株脂类积累,但是藻细胞生长受到明显抑制。缺硅导致藻细胞的生长减缓,但是对藻细胞的油脂积累影响不显著。在缺铁和高铁(0.048 mmol/L)条件下,藻的总脂含量均高于正常的培养条件。实验证明通过两步培养的方法,先在营养充足的培养基中进行藻细胞生长,然后将藻细胞转移到海水中培养,可以明显提高藻细胞的油脂含量。最终本实验确定海水为诱导三角褐指藻脂类积累的最佳诱导培养基。     

富里酸诱导对三角褐指藻EPA合成积累的影响

探究不同富里酸质量浓度对三角褐指藻二十碳五烯酸(EPA)合成积累的影响。结果表明，在质量浓度20 mg/L富里酸诱导下，三角褐指藻EPA含量和产量均达到最大值，为19.81 g/100 g和738.91 mg/L，较无富里酸诱导EPA含量和产量分别提高1.12和1.50倍。外源富里酸作用提高了三角褐指藻胞内过氧化物酶(POD)、超氧化物歧化酶(SOD)、过氧化氢酶(CAT)的酶活，使色素(叶绿素、类胡萝卜素)和总多酚抗氧化组分比例增加，活性氧(ROS)和丙二醛(MDA)水平降低，整体抗氧化能力获得显著提升，有效阻抑EPA的氧化分解；富里酸作用同时还上调了EPA合成通路去饱和酶(Δ⁵-FADS、Δ⁶-FADS、Δ¹²-FADS、Δ¹⁵-FADS、Δ¹⁷-FADS)和延长酶(Δ⁶-ELOVL)等系列关键酶，使其上调表达量较无富里酸作用分别增加0.33、0.94、0.33、0.58、1.15和0.70倍，为EPA高效合成积累提供前驱物和能量。     

富里酸诱导对三角褐指藻EPA合成积累的影响

探究不同富里酸作用浓度对三角褐指藻二十碳五烯酸（EPA）合成积累的影响。结果表明,在20 mg/L富里酸诱导下,三角褐指藻EPA含量和产量均达到最大值,为19.81 g/100 g和738.91 mg/L,较无富里酸诱导EPA含量和产量分别提高2.01和2.54倍。外源富里酸作用提高了三角褐指藻胞内过氧化物酶（POD）、超氧化物歧化酶（SOD）、过氧化氢酶（CAT）酶活,使得色素和多酚抗氧化组分比例增加,活性氧（ROS）和丙二醛（MDA）水平降低,整体抗氧化能力获得显著提升,有效阻抑EPA的氧化分解;富里酸作用同时还上调了EPA合成通路脱饱和酶（Δ5-FADS、Δ6-FADS、Δ12-FADS、Δ15-FADS、Δ17-FADS）和延长酶（Δ6-ELOVL）等系列关键酶,使其上调表达量较无富里酸作用分别增加1.27、1.81、1.24、1.45、1.43和1.46倍,为EPA高效合成积累提供前驱物和能量。富里酸作用效果协同强化了三角褐指藻EPA合成积累效率。     

Eicosapentaenoic acid (20∶5n-3) from the marine microalgaPhaeodactylum tricornutum

Eicosapentaenoic acid (EPA, 20∶5n-3) was obtained from the marine microalgaePhaeodactylum tricornutum by a three-step process: fatty acid extraction by direct saponification of biomass, polyunsaturated fatty acid (PUFA) concentration by formation of urea inclusion compounds, and EPA isolation by semipreparative high-performance liquid chromatography (HPLC). Alternatively, EPA was obtained by a similar two-step process without the PUFA concentration step by the urea method. Direct saponification of biomass was carried out with two solvents that contained KOH for lipid saponification. An increase in yield was obtained because the problems associated with emulsion formation were avoided by separating the biomass from the soap solution before adding hexane for extraction of insaponifiables. The most efficient solvent, ethanol (96%) at 60°C for 1 h, extracted 98.3% of EPA. PUFA were concentrated by the urea method with a urea/fatty acid ratio of 4∶1 at a crystallization temperature of 28°C and by using methanol and ethanol as urea solvents. An EPA concentration ratio of 1.73 (55.2∶31.9) and a recover yield of 78.6% were obtained with methanol as the urea solvent. This PUFA concentrate was used to obtain 93.4% pure EPA by semipreparative HPLC with a reverse-phase, C₁₈, 10 mm i.d.×25-cm column and methanol/water (1% acetic acid), 80∶20 w/w, as the mobile phase. Eighty-five percent of EPA loaded was recovered, and 65.7% of EPA present inP. tricornutum biomass was recovered in highly pure form by this three-step downstream process. Alternatively, 93.6% pure EPA was isolated from the fatty acid extract (without the PUFA concentration step) with 100% EPA recovery yield. This two-step process increases the overall EPA yield to 98.3%, but it is only possible to obtain 20% as much EPA as that obtained by three-step downstream processing.     

Iron Deficiency Promotes the Lack of Photosynthetic Cytochrome c550 and Affects the Binding of the Luminal Extrinsic Subunits to Photosystem II in the Diatom Phaeodactylum tricornutum

In the diatom Phaeodactylum tricornutum, iron limitation promotes a decrease in the content of photosystem II, as determined by measurements of oxygen-evolving activity, thermoluminescence, chlorophyll fluorescence analyses and protein quantification methods. Thermoluminescence experiments also indicate that iron limitation induces subtle changes in the energetics of the recombination reaction between reduced Q_B and the S₂/S₃ states of the water-splitting machinery. However, electron transfer from Q_A to Q_B, involving non-heme iron, seems not to be significantly inhibited. Moreover, iron deficiency promotes a severe decrease in the content of the extrinsic PsbV/cytochrome c₅₅₀ subunit of photosystem II, which appears in eukaryotic algae from the red photosynthetic lineage (including diatoms) but is absent in green algae and plants. The decline in the content of cytochrome c₅₅₀ under iron-limiting conditions is accompanied by a decrease in the binding of this protein to photosystem II, and also of the extrinsic PsbO subunit. We propose that the lack of cytochrome c₅₅₀, induced by iron deficiency, specifically affects the binding of other extrinsic subunits of photosystem II, as previously described in cyanobacterial PsbV mutants.     

Supercritical fluid extraction of bioactive lipids from the microalga Nannochloropsis sp.

Marine microalgae are recognised as an important renewable source of bioactive lipids with a high proportion of polyunsaturated fatty acids (PUFA), which have been shown to be effective in preventing or treating several diseases. For the extraction of oil from microalgae, supercritical CO₂ (ScCO₂) is regarded with interest, being safer than hexane and offering a negligible environmental impact, a short extraction time and a high‐quality final product. Whilst some experimental papers are available on the supercritical fluid extraction (SFE) of oil from microalgae, only limited information exists on the kinetics of the process. In such a contest, a mathematical model able to describe the kinetics of the SFE was applied to the recovery with ScCO₂ of lipids from Nannochloropsis sp., a marine microalga commonly used in aquaculture and characterised by a lipid fraction with a high PUFA content. The aim of this paper was to examine the effect of operating conditions on the kinetics of the SFE, on process yields and on the fatty acid composition of lipid extracts.     

Supercritical carbon dioxide extraction of cottonseed with co-solvents

Extraction of cottonseed lipids with supercritical carbon dioxide (SC-CO₂) was conducted with and without a cosolvent, ethanol or 2-propanol (IPA). At 7000 psi and 80°C, the reduced pressure, temperature and density of SC-CO₂ was at 6.5, 1.17 and 1.85, respectively; the specific gravity was 0.87. Under these conditions, CO₂ is denser than most liquid extraction agents such as hexane, ethanol and IPA. The extraction of cottonseed with SC-CO₂ gave a yield of more than 30% (moisture-free basis). This is comparable to yields obtained by the more commonly used solvent, hexane. The crude cottonseed oil extracted by SC-CO₂ was visually lighter than refined cottonseed oil. This was substantiated by colorimetric measurements. No gossypol was detected in the crude oil. However, crude oil extracted by SC-CO₂, to which less than 5% of ethanol or IPA as co-solvent was added, containedca. 200 ppm of gossypol, resulting in the typical dark color of cottonseed crude oil with gossypol. CO₂ extracted a small amount of cottonseed phosphatides, about one-third of that extracted by pure ethanol, IPA or hexane. A second extraction with 100% ethanol or IPA after the initial SC-CO₂ extraction produced a water-soluble lipid fraction that contained a significant amount of gossypol, ranging between 1500 and 5000 ppm. Because pure gossypol is practically insoluble in water, this fraction is believed to be made up of gossypol complexed with polysaccharides and phosphatides. Partially presented at the AOCS 1993 Annual Meeting & Expo in Anaheim, California.     

Interaction between Microalgae P. tricornutum and Bacteria Thalassospira sp. for Removal of Bisphenols from Conditioned Media

We studied the efficiency of three culture series of the microalgae Phaeodactylum tricornutum (P. tricornutum) and bacteria Thalassospira sp. (axenic microalgae, bacterial culture and co-culture of the two) in removing bisphenols (BPs) from their growth medium. Bacteria were identified by 16S ribosomal RNA polymerase chain reaction (16S rRNA PCR). The microorganism growth rate was determined by flow cytometry. Cultures and isolates of their small cellular particles (SCPs) were imaged by scanning electron microscopy (SEM) and cryogenic transmission electron microscopy (Cryo-TEM). BPs were analyzed by gas chromatography coupled with tandem mass spectrometry (GC-MS/MS). Our results indicate that some organisms may have the ability to remove a specific pollutant with high efficiency. P. tricornutum in axenic culture and in mixed culture removed almost all (more than 99%) of BPC2. Notable differences in the removal of 8 out of 18 BPs between the axenic, mixed and bacterial cultures were found. The overall removals of BPs in axenic P. tricornutum, mixed and bacterial cultures were 11%, 18% and 10%, respectively. Finding the respective organisms and creating microbe societies seems to be key for the improvement of wastewater treatment. As a possible mediating factor, numerous small cellular particles from all three cultures were detected by electron microscopy. Further research on the mechanisms of interspecies communication is needed to advance the understanding of microbial communities at the nano-level.     

Comparison of isopropanol and hexane for extraction of vitamin E and oryzanols from stabilized rice bran

The effects of solvent-to-bran ratio (2∶1 and 3∶1, w/w), extraction temperature (40 and 60°C), and time (5, 10, 15, 20, and 30 min) were studied for hexane and isopropanol extraction. Increasing the solvent-to-bran ratios and extraction temperature increased the amounts of crude oil, vitamin E and oryzanol recovered for both solvents. An extraction time of 15 min was sufficient for optimum crude oil, vitamin E, and oryzanol extraction. Preheated isopropanol (3∶1 solvent/bran ratio and 60°C) extracted less crude oil (P<.05) but more vitamin E (P<.05) and similar amounts of oryzanol (P>.05) relative to preheated hexane. The data suggest that isopropanol is a promising alternative solvent to hexane for extraction of oil from stabilized rice bran.     

Sugaring-out-assisted aqueous two-phase extraction of fructooligosaccharides from yacon (Smallanthus sonchifolius)

Yacon roots contain about 80 wt% of β-(2-1)-fructooligosaccharides (FOS). This work aims to use water extraction method combined with sugaring-out technology to extract FOS from yacon. Most of FOS was extracted into the acetonitrile (ACN)-rich top phase, when yacon roots were dissolved in an ACN-yacon water extract mixture without extra sugar addition. Experimental results indicated that the partitioning behavior of FOS was closely correlated with the concentration of sugar in water extract of yacon, the system temperature and the volume ratio between ACN solution and yacon water extract.     

Comparison of supercritical CO2 and hexane extraction of lipids from sorghum distillers grains

Total yields and compositions of sorghum dried distillers grains with solubles (DDGS) lipids obtained by supercritical CO₂ (SC‐CO₂) extraction were compared with those obtained by recirculated solvent extraction (RSE) with hexane. The total yield of lipids obtained by SC‐CO₂ extraction at 27.5 MPa and 70 °C was 150 g lipids/kg DDGS, while the yield obtained by RSE with hexane at 69 °C was only 85 g lipids/kg DDGS. The contents of four high‐value compounds, i.e., policosanols, phytosterols, free fatty acids (FFA) and tocols, in the lipids obtained by SC‐CO₂ extraction were 31.2, 15.6, 155.3 and 0.50 mg/g at 27.5 MPa and 70 °C, compared to 26.6, 9.6, 57.3 and 0.03 mg/g for RSE with hexane at 69 °C. The profiles of phytosterols and FFA in the sorghum DDGS lipids were relatively independent of the extraction methods and operating conditions.     

Design and Synthesis of Cross-Linked Micellar Particles to Assist Microalgae Lipid Recovery from Aqueous Extract

A series of cross‐linked micellar particles (CLMP) with internal hydrophobic binding sites was synthesized via a double‐cross‐link concept. A quaternary ammonium surfactant was first synthesized from natural tung oil. With two acrylate groups in the head group and conjugated alkenes in the hydrophobic tail, the surfactant was doubly cross‐linked in the micellar form on both the micellar surface and in the core. Surface cross‐linking at ambient temperature and core cross‐linking at 80 °C yielded 61 % final product (with 40 % space holder). The product and its morphology were characterized by infrared (IR) spectroscopy and scanning electron microscopy (SEM). The chemical nature and functional groups were confirmed by IR, and SEM indicated heterogeneous nanoparticles aggregated into clusters of particles. The binding capacity and selectivity of CLMP for microalgae lipids extracted from Nannochloropsis sp. were investigated and quantified using a turbidity test and mass spectrometry. The best CLMP product was able to extract total algae lipids at 0.8 g lipids/g particles capacity. At a lipid loading above the binding capacity, the CLMP selectively bound non‐charged polar lipids instead of the negatively charged polar lipids. These micellar particles have been demonstrated to be promising materials for extracting lipids from aqueous lipid extract. This paper presents a proof of concept study, and more in‐depth investigation is needed to test lipid extraction from various media and potentials for lipid class separation using these structured particles.     

EPA and DHA in microalgae: Health benefits,biosynthesis, and metabolic engineering advances

Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are ω-3 very long-chain polyunsaturated fatty acids (VLC-PUFAs) that offer a wide range of human health benefits impacting cardiovascular, anti-inflammatory, and neurological health. It is widely known that humans inefficiently synthesize these compounds and as such rely on exogenous dietary sources, such as marine fish oils. Unfortunately, the production of marine fish oils is an unsustainable process and has suffered a dramatic fall in recent years due to overfishing and climate change, as the demand for EPA and DHA continues to rise. Therefore, there is an urgent need to develop alternative, sustainable sources for consumable EPA and DHA. Metabolic engineering of marine microalgae to improve their EPA and DHA productivity is regarded as a promising option that has received increasing commercial attention in recent years. In this mini-review, we describe several notable health benefits of EPA and DHA, summarize the natural sources and biosynthesis of VLC-PUFAS, as well as the recent advances in metabolic engineering of EPA and DHA production in representative microalgal and protist species, including Schizochytrium sp., Phaeodactylum tricornutum, and Nannochloropsis oceanica.     

The Multifaceted Inhibitory Effects of an Alkylquinolone on the Diatom Phaeodactylum tricornutum

The mechanisms underlying interactions between diatoms and bacteria are crucial to understand diatom behaviour and proliferation, and can result in far-reaching ecological consequences. Recently, 2-alkyl-4-quinolones have been isolated from marine bacteria, both of which (the bacterium and isolated chemical) inhibited growth of microalgae, suggesting these compounds could mediate diatom–bacteria interactions. The effects of several quinolones on three diatom species have been investigated. The growth of all three was inhibited, with half-maximal inhibitory concentrations reaching the sub-micromolar range. By using multiple techniques, dual inhibition mechanisms were uncovered for 2-heptyl-4-quinolone (HHQ) in Phaeodactylum tricornutum. Firstly, photosynthetic electron transport was obstructed, primarily through inhibition of the cytochrome b₆f complex. Secondly, respiration was inhibited, leading to repression of ATP supply to plastids from mitochondria through organelle energy coupling. These data clearly show how HHQ could modulate diatom proliferation in marine environments.     

Hexane elimination from soybean oil by continuous packed tower processing with supercritical CO2

Hexane elimination is the most energy-consuming step in the industrial extraction of soybean oil. It utilizes three sets of equipment: two evaporation stages in series followed by a stripper at a pressure of about 0.07 bar. The final hexane residue in the oil is about 1000 ppm. We propose an alternative to the present process for hexane elimination, based on the extraction of the soybean oil/hexane mixture with supercritical CO₂ in a continuous countercurrent packed tower. In this work, we tested a soybean oil/hexane mixture feed containing 10% by weight of hexane. Various pressures and temperatures of the column were tested to reduce hexane residue in the oil. The extraction process was demonstrated to be very effective for hexane separation. Indeed, at the bottom of the column we recovered soybean oil containing quantities of hexane as low as 20 ppm when we operated at 120 bar, 40°C. The effect of process parameters is also discussed.     

In-situ extraction and purification of ethanol using commercial oleic acid

The flash process for the removal of ethanol from commercial oleic acid has been experimentally studied and modelled using a modified Redlich-Kwong-Soave equation of state (RKAES) and the ASPEN process simulator. Using feed ethanol and water concentrations spanning the range of expected liquid-liquid extraction concentrations, it was found that 93.6% by weight of ethanol in the distillate was obtained consuming only 2 MJoules of energy per kilogram of ethanol recovered. The RKAES model was found to reasonably predict the ethanol recovery and purity with errors under 5%. Commercial oleic acid was found to be completely non-inhibitory towards the growth of Saccharomyces cerevisiae cells when used for in-situ extraction at a concentration of 50% of the total fermenter volume.     

The isolation of hydroxy acids from lesquerella oil lipolysate by a saponification/extraction technique

The lipolysate from immobilizedRhizomucor miehei lipase (Lipozyme ^tm )-catalyzed hydrolysis of lesquerella oil contains typically 35% free fatty acid (FFA), 2% monoglyceride, 25% diglyceride (DG), and 38% triglyceride (TG). Of the FFA, 75–80% are hydroxy acids (HFA). Various methods for isolating HFA from the lipolysate were examined, and a novel saponification/extraction method was developed. Lipolysate was mixed with 4 vol equivalents each of KOH/phosphate buffer and polar organic solvent. Hexane was then added to enhance phase separation. Three phases formed: a lower aqueous phase containing nothing of interest, a polar organic solvent middle phase that contained mostly fatty acid soaps, and a hexane-rich upper phase that contained mostly DG and TG, which can be recycled to a relipolysis step. The middle phase, when treated with concentrated hydrochloric acid, NaCl-saturated water, and hexane, released the FFA into the hexane. This fraction, referred to as the “Product” contained >99% of the FFA released in the lipolysis. “Product” consisted of 85–90% FFA, of which 75–80% was HFA. The other 10–15% of the “Product” consisted of partial glycerides and TG. The most critical parameters for the extraction are the pH of the aqueous solution and the polarity of the organic solvent (acetone was found to be the best choice). Additional purification steps for the “Product” are discussed.     

海带中褐藻糖胶的酸提法研究

采用酸提法从海带中提取褐藻糖胶,分别采用MgCl、乙醇分级沉淀法进行纯化.主要探讨了提取时间、温度、pH等因素对提取率的影响.结果表明,在提取温度70 ℃,pH 2.5,70%乙醇浓度,提取3 h,提取率为7.3%.乙醇分级沉淀法提取率高于MgCl法.