Solvent Extraction of Astaxanthin from Haematococcus Cyst Cells

Table of Contents

1.0 Introduction

Which Citation Styles Can You Handle?

We get a lot of “Can you do MLA or APA?”—and yes, we can! Our writers ace every style—APA, MLA, Turabian, you name it. Tell us your preference, and we’ll format it flawlessly.

2.0 General Background

2.1 Astaxanthin

2.2 Haematococcus pluvialis

3.0 H. pluvialis as a Source of Astaxanthin

Are Writing Services Legal?

Totally! They’re a legit resource for sample papers to guide your work. Use them to learn structure, boost skills, and ace your grades—ethical and within the rules.

3.1 Synthesis Pathway

3.2 Astaxanthin Production

4.0 Solvent Extraction of Astaxanthin from H. pluvialis

What’s the Price for a Paper?

Starts at $10/page for undergrad, up to $21 for pro-level. Deadlines (3 hours to 14 days) and add-ons like VIP support adjust the cost. Discounts kick in at $500+—save more with big orders!

4.1 Cell Disruption

4.2 Solvent Extraction

4.3 Analyses

4.3.1 Spectrophotometric Analysis

Is My Privacy Protected?

100%! We encrypt everything—your details stay secret. Papers are custom, original, and yours alone, so no one will ever know you used us.

4.3.2 HPLC Analysis

5.0 References

1.0 Introduction

Haematococcus pluvialis is one of the potent organisms capable of synthesizing astaxanthin, which is a high value keto-carotenoid. Astaxanthin is believed to accumulate in thick walled cyst cells of Haematococcus, provided that the thick cell wall consists of sporopollenin-like material (algaenan) that can potentially hinders solvent extraction of astaxanthin. Therefore, disruption of cells, either via mechanical or non-mechanical methods, has to be accessed. Subsequently, the extraction of astaxanthin is performed by treating the cells with various solvents, for instance acetone, n-hexane, methanol, dimethyl sulfoxide (DMSO) etc. The effectiveness of these solvent extraction techniques can then be evaluated via two ways: spectrophotometry along with the use of Lichtenthaler equations, and high performance liquid chromatography (HPLC). The overall process flow describing the extraction of astaxanthin from Haematococcus pluvialis is summarized and illustrated as shown in Figure 1 below.

Figure 1: Solvent extraction of astaxanthin.

2.0 General Background

2.1 Astaxanthin

Astaxanthin (3,3’-dihydroxy--carotene-4,4’-dione) is a keto-carotenoid or tetraterpenoid, which belongs to a larger class of chemical compounds known as terpenes. Generally, terpenes are made up of five carbon precursors: isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). As a member of carotenoid compounds, astaxanthin attains oxygen-containing moieties, i.e. hydroxyl (-OH) or ketone (C=O) functional groups as shown in Figure 2 below.

Figure 2: Structure of astaxanthin (Lorenz & Cysewski, 2000).

Why Are You the Best for Research?

Our writers are degree-holding pros who tackle any topic with skill. We ensure quality with top tools and offer revisions—perfect papers, even under pressure.

As can be seen in Figure 2, astaxanthin consists of two asymmetric carbons located at the 3 and 3’ positions of the benzenoid rings on either end of the molecule. Two types of configurations are concerned: R and S configurations which are largely dependent on the exact way of how -OH groups are attached to the carbon atoms at these centers of asymmetry, and hence results in different enantiomers of the astaxanthin molecule. R configuration particularly defines the situation where -OH group is attached and projected above the plane of the molecule, meanwhile the reverse statement implies that astaxanthin attains the S configuration. Thus, three possible enantiomers exist and are designated as 3R,3’R, 3S,3’S and 3R,3’S (Lorenz & Cysewski, 2000). Astaxanthin can be further classified into three: polar free astaxanthin (3 and 3’ -OH groups are not bonded), non-polar astaxanthin monoester (a fatty acid bonded to a 3 position) and diester (a fatty acid bonded to each of the 3 positions) (Todd, 2001).

Similar to most of the carotenoids, astaxanthin represents a colorful, lipid-soluble pigment. These characteristics are due to the long chain conjugated double bonds present at the centre of the compound (see Figure 2). In addition to astaxanthin’s role in the coloration of aquatic animals (usually red color), it possesses several essential bioactivities including anti-oxidation, enhancement of immune response and anticancer activities. Numerous studies have confirmed that astaxanthin possesses health-promoting effects in the prevention and treatment of various diseases, for example cancers, chronic inflammatory diseases, metabolic syndrome, diabetes, diabetic nephropathy, cardiovascular diseases, gastrointestinal diseases, liver diseases, neurodegenerative diseases, eye diseases, skin diseases, exercise induced fatigue, male infertility and HgCl₂-induced acute renal failure (Herrero, Orosa García, Abalde, Rioboo Blanco, & Cid, 2012).

Astaxanthin is more commonly detected in some microalgae and phytoplankton, but it can also be found in some fungi, bacteria, yeast, salmon, trout, krill, shrimp, crayfish and crustaceans (Biswal, 2014) as evident in Table 1 below. As in the case of commercial production of astaxanthin, the primary natural sources comprise of:

• Euphausia pacifica (Pacific krill)
• Euphausia superba (Antarctic krill)
• Haematococcus pluvialis (Microalgae)
• Pandalus borealis (Arctic shrimp)
• Xanthophyllomyces dendrorhous, formerly Phaffia rhodozyma (yeast)

The approximate concentrations of astaxanthin in natural sources (Table 1) effectively demonstrate that Haematococcus pluvialis contains the highest level of astaxanthin in nature.

Who Writes My Assignments?

Experts with degrees—many rocking Master’s or higher—who’ve crushed our rigorous tests in their fields and academic writing. They’re student-savvy pros, ready to nail your essay with precision, blending teamwork with you to match your vision perfectly. Whether it’s a tricky topic or a tight deadline, they’ve got the skills to make it shine.

Table 1: Astaxanthin from various natural sources (Biswal, 2014).

2.2 Haematococcus pluvialis

Haematococcus pluvialis (H. pluvialis) is an ubiquitous single cell biflagellate microalgae (Herrero et al., 2012) freshwater species of Chlorophyta from the family of Haematococcaceae. It is often found in temperate regions around the world, particularly in puddles, eaves and birth bathes. An eye-catching red to deep purple color of H. pluvialis as shown in Figure 3 indicates a massive accumulation of the secondary carotenoid astaxanthin, as well as its fatty acid mono or diesters, when the environmental conditions are unfavorable for normal cell growth.

Figure 3: Haematococcus pluvialis cysts (akinete forms in resting stage) full of astaxanthin (Fox, 2011).

Will My Paper Be Unique?

Guaranteed—100%! We write every piece from scratch—no AI, no copying—just fresh, well-researched work with proper citations, crafted by real experts. You can grab a plagiarism report to see it’s 95%+ original, giving you total peace of mind it’s one-of-a-kind and ready to impress.

3.0 H. pluvialis as a Source of Astaxanthin

Present technologies have widely exploit the microalgae Haematococcus pluvialis as the primary industrial source for natural astaxanthin synthesis. As proven in Table 1, H. pluvialis seems to accumulate the highest contents of astaxanthin in nature. According to Mendes-Pinto et al. (2001), Haematococcus can produce up to 8% dry weight of astaxanthin in the form of 3S, 3’S enantiomer, provided that it may consist about 70% monoesters, 10% diesters, 5% free astaxanthin, with the remainder comprising of - carotene, canthaxanthin, and lutein (Todd, 2001).

H. pluvialis has two types of cell morphology depending on the environmental conditions: green motile and non-motile forms. The cells tend to be green under optimal growth conditions, being capable of actively swimming with two flagella and increasing in cell number. However, adverse conditions cause unusual behaviour of green vegetative cells such that they cease to be motile, expand drastically, lose their flagella, form a hard, thick cyst-like wall and eventually enter a resting stage. Within this resting stage, astaxanthin is synthesized in the cyst cells as visualized by a red color due to astaxanthin accumulation (Herrero et al., 2012).

3.1 Synthesis Pathway

By referring to Herrero et al. (2012), higher plants and green algae share the same carotenoid biosynthetic pathway to -carotene. Enzymes of -carotene such as ketolase and hydroxylase catalyze further steps leading to astaxanthin in H. pluvialis.

Astaxanthin can be synthesized from β-carotene by two different pathways:

Can You Use Any Citation Format?

Yep—APA, Chicago, Harvard, MLA, Turabian, you name it! Our writers customize every detail to fit your assignment’s needs, ensuring it meets academic standards down to the last footnote or bibliography entry. They’re pros at making your paper look sharp and compliant, no matter the style guide.

(a) With the use of diphenylamine (astaxanthin synthesis inhibitor), a synthesis pathway for Haematococcus has been proposed:

β-carotene→echinenone→cantaxanthin→adonirubin→astaxanthin

(b) Alternatively, astaxanthin biosynthesis occurs by:

β-carotene→β-cryptoxanthin→zeaxanthin→adonixanthin→astaxanthin

Can I Change My Order Details?

For sure—you’re not locked in! Chat with your writer anytime through our handy system to update instructions, tweak the focus, or toss in new specifics, and they’ll adjust on the fly, even if they’re mid-draft. It’s all about keeping your paper exactly how you want it, hassle-free.

3.2 Astaxanthin Production

In general, the production of astaxanthin-rich Haematococcus is achieved through a two-step process: vegetative (green) and aplanospore (red) stages (Herrero et al., 2012). Firstly, the mass production of green vegetative cells is achieved under an approximate optimal growth conditions associated with careful control of pH, temperature and nutrient levels. Once a satisfactory viable cell density is accomplished, the broth culture is subjected to stresses to induce the formation of red cyst cells (Lorenz & Cysewski, 2000).

Several induction methods have been developed for the transformation of green cells to red cysts carrying high astaxanthin contents (see Figure 4). They can be divided into two classes according to the role of astaxanthin:

(a) First class utilizes various environmental stresses (including nutrients), except for strong light. This technique is aimed to cause retardation in cell multiplication, preferably via nitrogen starvation or the presence of excessive acetate, salt or other specific inhibitors targeting impaired cell division. Due to the scarcity of some factors necessary for normal cell growth, the cells store astaxanthin possessing an antioxidant function (Herrero et al., 2012).

(b) Second class refers to light induction method which exploits the photo-protective role of astaxanthin under high light intensity. Exposure towards high-intensity light leads to an accelerated astaxanthin accumulation that effectively protects the cells against photo and oxidative damages. Coupled action such as excess acetate addition or a supply of favorable CO₂ concentration can be implemented along with the light induction method to enhance astaxanthin manufacture and accumulation (Herrero et al., 2012).

How Do I Order a Paper?

It’s a breeze—submit your order online with a few clicks, then track progress with drafts as your writer brings it to life. Once it’s ready, download it from your account, review it, and release payment only when you’re totally satisfied—easy, affordable help whenever you need it. Plus, you can reach out to support 24/7 if you’ve got questions along the way!

Figure 4: (a) Green Haematococcus vegetative cells and (b) Red haematocysts as a result of nutrient starvation and sunlight. 400x magnification (Lorenz & Cysewski, 2000).

Typical commercial systems introduce nitrate and phosphate deprivation, high temperature and light intensity, or addition of salt (NaCl) to the culture medium so as to facilitate the manufacture of astaxanthin. Usually within 2-3 days after stressing, red cysts are formed, and they are ready for harvest within 3-5 days following the haematocysts formation (contain about 1.5-3.0% astaxanthin) (Lorenz & Cysewski, 2000).

4.0 Solvent Extraction of Astaxanthin from H. pluvialis

Although Haematococcus pluvialis is one of the most important natural sources of the carotenoid astaxanthin, the thick sporopollenin cell wall in the cyst tends to hinder astaxanthin extraction and its subsequent bio-availability. Therefore, pretreatment of cyst cells (cell disruption) has to be accessed prior to solvent extraction of astaxanthin from H. pluvialis. The efficiency of various solvent extraction methods will then be evaluated through appropriate assays to be mentioned in the following subsections.

How Quick Can You Write?

Need it fast? We can whip up a top-quality paper in 24 hours—fully researched and polished, no corners cut. Just pick your deadline when you order, and we’ll hustle to make it happen, even for those nail-biting, last-minute turnarounds you didn’t see coming.

4.1 Cell Disruption

Theoretically, cell disruption involves the application of various effective techniques to open the cell wall in order to obtain the intracellular fluid without disrupting or damaging any of its essential components. Cell type and its cell wall composition remarkably determine the methods that are suitable for the disruption process (Naeem, 2016).

There are effectively two types of cell disruption methods: mechanical methods which utilize forces to separate out intracellular protein, and non-mechanical methods comprising physical, chemical and enzymatic methods (Naeem, 2016) stated in Table 2.

Table 2: Cell disruption methods (Naeem, 2016).

According to the study performed by Mendes-Pinto et al. (2001), a range of physical and chemical processes were tested to promote the disruption of the encysted cells of Haematococcus pluvialis. The astaxanthin-rich biomass were subjected to the following processes:

(i) Autoclave at 121C, 1 atm for 30 minutes

Can You Handle Tough Topics?

Absolutely—bring it on! Our writers, many with advanced degrees like Master’s or PhDs, thrive on challenges and dive deep into any subject, from obscure history to cutting-edge science. They’ll craft a standout paper with thorough research and clear writing, tailored to wow your professor.

(ii) 0.1 M HCl for 15 minutes

(iii) 0.1 M HCl for 30 minutes

(iv) 0.1 M NaOH for 15 minutes

(v) 0.1 M NaOH for 30 minutes

(vi) Enzymatic treatment for 1 hour with a mixture of 0.1% protease K and 0.5% driselase in a phosphate buffer, maintained at pH 5.8 and 30C

How Do You Match Professor Expectations?

We follow your rubric to a T—structure, evidence, tone. Editors refine it, ensuring it’s polished and ready to impress your prof.

(vii) Spray drying with inlet and outlet temperatures of 180C and 115C respectively

(viii) Cell homogenization

Figure 5: SEM of Haematococcus pluvialis (x 2000), bar size 10 μm: (a) control, (b) cell homogenization, (c) spray dry, and (d) autoclave (Mendes-Pinto, Raposo, Bowen, Young, & Morais, 2001).

How Do You Edit My Work?

Send us your draft and goals—our editors enhance clarity, fix errors, and keep your style. You’ll get a pro-level paper fast.

Based on Figure 5, as compared to the intact cells (Figure 5a), obvious cell disruption was observed when H. pluvialis was treated by homogenization (Figure 5b) and autoclave (Figure 5d). An interesting phenomenon was noticed in the spray dried cells (Figure 5c) such that they tended to clump together even though the cells were not damaged by this process. On the other hand, Mendes-Pinto et al. (2001) mentioned that chemical treatment (NaOH or HCl) of H. pluvialis only resulted in slight distortion of some cells unless prolonged exposure was implemented. However, enzymatic treatment showed no visible effect on cell morphology (SEM not shown) (Mendes-Pinto et al., 2001).

Figure 6: Total carotenoid content of Haematococcus cysts: 1. Control; 2. Autoclave; 3. HCl 15 min; 4. HCl 30 min; 5. NaOH 15 min; 6. NaOH 30 min; 7. Enzyme; 8. Cell homogenization; 9. Spray drying (Mendes-Pinto et al., 2001).

Figure 7: Extraction of total carotenoids into acetone from processed Haematococcus biomass. 1. Control; 2. Autoclave; 3. HCl 15 min; 4. HCl 30 min; 5. NaOH 15min; 6. NaOH 30 min; 7. Enzyme; 8. Cell homogenization; 9. Spray drying (Mendes-Pinto et al., 2001).

Can You Brainstorm Topics?

Yep! We’ll suggest ideas tailored to your field—engaging and manageable. Pick one, and we’ll build it into a killer paper.

Distinct cell processing techniques affect the extractable total carotenoids from H. pluvialis as shown in Figures 6 and 7. By referring to Figure 6, the cells that had experienced autoclave, spray dry and homogenization attained the highest recovery of astaxanthin. However, the treatment of Haematococcus cells by acid, alkali or enzyme led to a significant loss of carotenoid in comparison to the unprocessed intact cells (Mendes-Pinto et al., 2001). Upon extraction (Figure 7), high total carotenoid contents were noticed for cells that had been autoclaved or homogenized. This discovery suggests that these two cell disruption methods may be the most suitable and effective ways of rupturing the cell walls and enhancing the extraction of astaxanthin from the lysed cells (Mendes-Pinto et al., 2001).

4.2 Solvent Extraction

Partition of components or compounds in a mixture can be easily accomplished by applying solvent extraction or liquid-liquid extraction. This process works based on the solubility of the desired component(s) in two different immiscible liquids: aqueous and organic phases. Several studies or research papers are focused in relation to the astaxanthin extraction methods from green alga Haematococcus pluvialis:

(a) An Efficient Method for Extraction of Astaxanthin from Green Alga Haematococcus pluvialis (Sarada, Vidhyavathi, Usha, & Ravishankar, 2006).

This study had introduced three methodologies in the extraction of astaxanthin from H. pluvialis:

(i) Mortar, pestle and neutral glass powder were used to homogenize H. pluvialis, subsequently extracted with acetone.

(ii) Separate cell samples were treated or extracted with acetone (1 h and 24 h), methanol (24 h) and dimethylsulfoxide (DMSO) with 5 drops of glacial acetic acid.

(iii) Biomass were separately treated with HCl (1-10 N), and 2 N formic acid, citric acid, acetic acid and tartaric acid, followed by extraction in acetone for 1 h.

Do You Offer Fast Edits?

Yes! Need a quick fix? Our editors can polish your paper in hours—perfect for tight deadlines and top grades.

The results obtained from this study are shown in Table 3 and Figure 8, such that in this case the selected Haematococcus cells contained 2% total carotenoid (w/w) with astaxanthin constituted 88% of total carotenoids and 1.76% w/w of dry biomass (Sarada et al., 2006).

Table 3: Astaxanthin extractability in terms of different solvents with and without acid treatment (Sarada et al., 2006).

Treating H. pluvialis with solvents alone (methodology (ii)) yielded low astaxanthin extractability, except for DMSO in which the enhanced extractability was aided by the presence of glacial acetic acid. This observation is also proved by the fact that Aquasearch in-house research  had confirmed and selected DMSO as the best choice among extractants for green algae or more specifically Haematococcus algae meal and cysts (Aquasearch, 1999a). On the other hand, among the acids tested (methodology (iii)), HCl treatment followed by acetone extraction gave the highest astaxanthin extractability (Table 3).

Figure 8: Astaxanthin extractability from Haematococcus encysted cells after treatment with HCl at 70 °C for 2 min (Sarada et al., 2006).

Can You Start With an Outline?

Sure! We’ll sketch an outline for your approval first, ensuring the paper’s direction is spot-on before we write.

By referring to Figure 8, the astaxanthin extractability was also proved to increase with increasing HCl concentration. Yet, the extractability was observed to decline significantly at higher concentrations (> 4 N HCl) due to cell floating (probably dead). In addition, the astaxanthin extracted via methodology (i) and methodology (iii) were analyzed by both TLC and HPLC (data not shown), eventually demonstrating that both samples were identical, i.e. indicating that efficient extraction of astaxanthin can be achieved without homogenization by mechanical means (Sarada et al., 2006).

(b) Four Different Methods Comparison for Extraction of Astaxanthin from Green Alga Haematococcus pluvialis (Dong, Huang, Zhang, Wang, & Liu, 2014).

In this research article, four different extraction methods were implemented and intensively evaluated for the extraction of astaxanthin from H. pluvialis:

(i) HCl pretreatment followed by acetone extraction (HCl-ACE Extraction)

(ii) Hexane/isopropanol (6:4, v/v) mixture solvent extraction (HEX-IPA Binary Solvents Extraction)

(iii) Methanol extraction followed by acetone extraction (MET-ACE 2-Step Extraction)

(iv) Soy-oil extraction (or Oil-Soy Extraction)

The outcomes of the study are tabulated in Table 4, showing the effect of different extraction methods on oil yield and total astaxanthin content (TAC).

Can You Add Charts or Stats?

Definitely! Our writers can include data analysis or visuals—charts, graphs—making your paper sharp and evidence-rich.

Table 4: Oil yield and TAC of  H. Pluvialis cell extracts (Dong et al., 2014).

It was observed that HCl-ACE extraction method presented the highest extraction oil yield and TAC. Although HEX-IPA and MET-ACE methods showed the similar extraction oil yield, the latter possessed a higher TAC extraction. In view of oil-soy method, the resultant TAC was significantly lower than those of other three extraction methods even though the extraction oil yield was relatively satisfactory (Dong et al., 2014).

The total oil yield and TCA extraction from H. pluvialis by four different techniques presented a strongly significantdifference, which are technically viable depending on theastaxanthin content of extracts. The best extraction method,in terms of oil yield, TAC and antioxidant of extract, wasHCl-ACE procedure. SEM (Scanning Electron Microscopy), NMR (Nuclear Magnetic Resonance), and DPPH (1,1-diphenyl-2-picrylhydrazyl) Radical Scavenging Activity assays andreducing power experiments could be carried out to further confirm that HCl-ACE procedure was suitable for extractionof astaxanthin from H. pluvialis biomass (Dong et al., 2014).

(c) Extraction of Astaxanthin via Supercritical CO₂ (Majewski, Perrut, & Valderrama, 2000)

Instead of extraction using organic solvents, supercritical CO₂ can be an alternative for alga treatment due to its some unique characteristics and being an environmental friendly solvent. Majewski et al. (2000) had performed three runs involving the processing of samples by:

(1) Milling followed by supercritical CO₂ extraction.

(2) Milling, then grinding with dry ice (solid CO₂), followed by supercritical CO₂ extraction.

(3) Similar to (2) but extracted using supercritical CO₂ and 9.4% ethanol (co-solvent).

Figure 9: Extract yield of astaxanthin by supercritical CO₂ (60C/300 bar). Run 1 (); Run 2 (); Run 3 () (Majewski et al., 2000).

Sample preparation and extraction conditions determine the astaxanthin contents in the extract. Figure 9 shows that the yield increased significantly when the samples were twice grinded (run (1) vs. run (2)). This fact reveals the importance of cellular wall breaking (subsection 4.1) on extraction efficiency. More efficient milling procedure yielded more extract, which in turns resulted in higher concentration of astaxanthin in samples. The addition of co-solvent (9.4% ethanol) into supercritical CO₂ only increases the extraction yield to a small extent, but does improved the extraction and recovery of astaxanthin (Majewski et al., 2000).

4.3 Analyses

According to Cysewski (2006), analysis of astaxanthin content of a product can be conducted in two ways: spectrophotometric analysis and high performance liquid chromatography (HPLC) analysis.

4.3.1 Spectrophotometric Analysis

Via the usage of spectrophotometer (e.g. UV-VIS spectrophotometer), the maximum absorbance of astaxanthin (indicated by the wavelength in between 470-480 nm) is accessed accordingly. However, spectrophotometric assay method contributes to a problem such that astaxanthin cannot be distinguished from other carotenoids which also absorb light at the mentioned wavelength, hence leading to a false or rather an inaccurate results (Cysewski, 2006).

Instead, the efficiency of solvent extraction is approximately quantified by determining the carotenoid and chlorophyll contents through the usage of Lichtenthaler equations as mentioned in the following research papers.

(a) The study conducted by Şükran et al. (1998) involved the determination of pigment levels of four algae species by using various extraction solvents (methanol, acetone and diethyl ether). Absorbances were read and the amount of chlorophyll a, chlorophyll b and carotenoids were calculated according to the formulas of Lichtenthaler and Wellburn (1985) as shown in Table 5 (Şükran, GÜNEŞ, & Sivaci, 1998).

Table 5: Lichtenthaler equations for different solvents (Şükran et al., 1998).

(b) Based on the paper written by Sumanta et al. (2014), the solvents defined in Table 6 were used and compared to investigate their extraction capabilities of pigments from fern leaves. Noticed that there is a trade-off between choosing the best solvent for efficient quantitative extraction of chlorophylls and use of a solvent best suited for spectrophotometric assay (Sumanta, Haque, Nishika, & Suprakash, 2014).

Table 6: Solvent properties (Sumanta et al., 2014).