Extraction of squalene from yeast by supercritical carbon dioxide

P. Bhattacharjee and R.S. Singhal*Food and Fermentation Technology Department, University Institute of Chemical Technology, Matunga,Mumbai 400 019, India*Author for correspondence: Tel.: þ91-22-24145616, Fax: þ91-22-24145614, E-mail: rekha@foodbio.udct.ernet.in

Received 18 July 2002; accepted 20 March 2003

Keywords: Biomass, carbon dioxide, lipid extract, squalene, supercritical fluid extraction

Summary

Squalene produced under anaerobic conditions, by a strain of Torulaspora delbrueckii was extracted from thebiomass using supercritical carbon dioxide. Minimum use of solvent, lower time of isolation and a higher selectivityof extraction merit use of supercritical fluid extraction (SFE) technique over solvent extraction of squalene, asoptimized and reported previously. A maximum squalene yield of 11.12 lg g)1 (dry weight) of yeast cells wasobtained at a temperature of 60 �C and pressure of 250–255 bar at a constant flow rate of 0.2 l min)1 of carbondioxide. Lyophilization prior to SFE increased the squalene yield to 430.52 lg g)1 dry weight of yeast cells, anamount that is far greater than that obtained by (2:1) chloroform–methanol solvent extraction.

Introduction

Squalene, C30H50, a triterpene hydrocarbon (2,6,10,15,19,23-hexamethyltetracosa-2,6,10,14,18,22-hexaene) is anaturally occurring constituent of plant oils such asolive oil (Roncero & Janer 1962), palm oil and wheatgerm oil, amaranth oil and fish oils as well as humansebum. The richest source of the oil is the liver of theAizame (dogfish) shark (Squalus spp.). It is a clear,brilliant and almost colourless oil with a faint odour andtaste, and is isolated from fish oils by vacuum distilla-tion. It finds extensive use as a detoxification factor, as askin and eye antioxidant, in providing cells with oxygen,as a bactericidal and fungicidal agent, and as anantistatic and emollient in pharmaceuticals and cosme-tics. Other applications are in fine chemicals, magnetictape and low-temperature lubricants. Oryzanol dis-solved in squalene is used as an antioxidant in foods(Ishitani 1980).The limited availability of squalene from convention-

al sources such as shark liver oil compelled manufac-turers to switch over to its substitutes such assqualiforms. This merits its commercial production byfermentation. Various organisms such as Saccharomy-ces (Kamimura et al. 1994; Socaciu et al. 1995;Ciesarova et al. 1996), Pseudomonas (Uragami & Koga1986), Candida (Tsujiwaki et al. 1995a, b), the algaeEuglena (Kawamura & Matsuda 1996) and yeasts ingeneral (Mauricio et al. 1993) have been investigatedfor microbial production of squalene. Previous workfrom our laboratory reported on fermentative produc-tion of squalene by use of commercially available

compressed Baker’s yeast (Saccharomyces cerevisiae)and a strain of Torulaspora delbrueckii isolated frommolasses, under anaerobic conditions (Bhattacharjeeet al. 2001). Isolation of squalene from the lipidextracts, obtained by cell lysis of either strain wascarried out using chloroform–methanol (2:1) mixture.Further downstream processing of squalene i.e., itspurification from the lipid extracts was achieved chro-matographically. The purified squalene was characteri-zed spectroscopically against a standard. Torulasporadelbrueckii gave a higher yield of squalene as comparedto Saccharomyces cerevisiae.Globally, there is an increasing concern to minimize

use of organic solvents, particularly the chlorinatedones. The use of eco-friendly carbon dioxide as analternative to chloroform would be appropriate in theperspective of green technology. Supercritical carbondioxide extraction (SFE) is suitable for extraction ofnon-polar compounds with molecular weights of lessthan 500 (Rizvi et al. 1986). Squalene is a non-polarcompound with a molecular weight slightly above 400.These properties make squalene amenable to supercri-tical carbon dioxide extraction. It is soluble in super-critical CO2 at intermediate pressures of 100–250 bar(Catchpole et al. 1997). This work was aimed atextraction of squalene from Torulaspora delbrueckii bySFE using CO2. Solvent extraction as reported earlier israther time-consuming and requires large volumes oforganic solvents. Squalene was extracted from the driedmicrobial biomass after anaerobic fermentation in shakeflasks under optimum conditions, using supercriticalcarbon dioxide both for cell lysis and isolation of

World Journal of Microbiology & Biotechnology 19: 605–608, 2003. 605� 2003 Kluwer Academic Publishers. Printed in the Netherlands.

squalene. There are reports on supercritical carbondioxide extraction of microbial lipids from the microal-gae Scenedesmus obliquus (Choi et al. 1987). Freezedrying prior to SFE has been shown to be advantageouswith respect to extraction of lipids from several micro-bial sources. These include eicosapentaenoic acids(EPA) from the filamentous fungus Saprolegnia parasi-tica (Cygnarowicz-Provost et al. 1992); lipids from themicroalga Skeletonema costatum, a marine diatom andOchromonas danica, a freshwater phytoflagellate (Polaket al. 1989) and also that from the fungi of the genusMortierella (Sako et al. 1989; Sakaki et al. 1990). Thiswork investigates the potential of supercritical carbondioxide extraction of squalene from the yeast Torulas-pora delbrueckii, and the effect of lyophilization on thesame.

Materials and methods

Materials

Glucose, yeast extract powder, malt extract powder andpeptone were procured from Himedia Laboratories,Mumbai. Aluminium-coated silica gel 60 (F254) HPTLCplates of E. Merck, Germany were used. All solventsused were of AR grade. A strain of Torulasporadelbrueckii, isolated from molasses, was used for theproduction of squalene. Standard squalene was pro-cured from Sigma-Aldrich Corporation, USA.

Methods

Fermentation and harvesting of microbial biomassAerobic culture, anaerobic production of squalene andrecovery of the same were carried out as reported earlier(Bhattacharjee et al. 2001) on a larger scale in shakeflasks so as to obtain a greater yield of biomass forfurther extraction. One loopful of T. delbrueckii, ob-tained from the Institute of Microbial Technology,Chandigarh was inoculated into 1000 ml of growthmedium having the composition (Long & Ward 1989) asfollows: glucose 20 mg ml)1, yeast extract powder10 mg ml)1 and peptone 20 mg ml)1 (pH 5.5) andincubated at 30 ± 2 �C on a rotary shaker at 44 · g.After 48 h of aerobic growth, 5% of the active culturewas inoculated into a fresh 2500 ml batch of mediumhaving the following composition: glucose 40 mg ml)1,yeast extract powder 10 mg ml)1 and peptone20 mg ml)1 (pH 5.5) (Lodder 1970). The anaerobicconditions were maintained by spreading a layer (about1 cm thick) of sterile light liquid paraffin on the surfaceof the medium and then tightly sealing the flasks. Thebiomass was harvested after 24 h of anaerobic fermen-tation by centrifugation at 20,000 · g for 20 min at28 ± 2 �C. The biomass thus obtained was dried in anoven maintained at 80 �C for 24 h. The dried biomasswas ground in mortar and pestle, and used for SFE.

Supercritical carbon dioxide extractionFor SFE, a model of Applied Separations, Allentown,USA viz. SPEED-SFE was used. The parameters ofSFE as temperature, pressure, time of extraction andflow rate of carbon dioxide during collection phase wereoptimized to selectively extract squalene with minimumsterols and other lipids. Preliminary trials with varyingparameters established pressure and temperature to bethe important variables for extraction. These trials alsoindicated a 30 min static time and 2 h dynamic time tobe best for the extraction and hence were maintained inthis work. Extraction was carried out with 10 g of driedbiomass per batch. Pressure was varied at three levels,150–155, 250–255 and 350–355 bar and temperatures of40 and 60 �C were used. The extracts were collected in50 ml n-hexane in a collection vial kept in an ice bath ata constant flow rate of 0.2 l min)1 of carbon dioxide atthe collection end. Previous work on squalene recoveryfrom olive oil deodorizer distillates had reported nodirect relationship between carbon dioxide used and theextraction yield (Bondioli et al. 1993). Hence the timeand flow rate of CO2 was not increased beyond thelimits specified. An additional factor was that anincrease in operation cost related to the higher amountof CO2 used would not be accompanied by a corre-sponding increase in yield and purity of squalene.

Concentration of the extracts for analysisThe supercritical extracts were concentrated on a rotaryevaporator operating at 500 mm Hg at 45 �C andsubsequently the solvent was totally removed by a gentlenitrogen stream to yield an oily substance. The yield ofthe lipid extract was determined gravimetrically byweighing the oily substance. The squalene content of theoily substance was estimated densitometrically.

Lyophilization as pretreatment for extraction of squalenefrom the dried biomassIn the present work, the fungal biomass obtained afteroven-drying was subjected to pre-freezing at )18 to)20 �C for 30 min and was then lyophilized using aHetoHolten Drywinner III unit at a temperature of )50to )52 �C and a pressure of 8 Pa for 4 h. Squalene wasextracted from the lyophilized biomass at the optimizedcondition of SFE and estimated densitometrically.

Densitometric estimation of squaleneDensitometric assay was carried out after TLC accordingto themethod reported earlier (Bhattacharjee et al. 2001).The concentrated extracts were dissolved in n-hexane(AR grade) and cyclohexane was used as the developingsolvent in which squalene recorded an Rf value of0.60 ± 0.02.

Results and discussion

Triacylglycerols or triglycerides together with phospho-lipids and sterols account for the bulk of the cell lipids

606 P. Bhattacharjee and R.S. Singhal

in Saccharomyces and Torulaspora. Free fatty acids(FFA) rarely account for more than a small fraction ofthe total lipids in these strains. Amongst hydrocarbons,only squalene finds mention. The bulk of the lipids arelocated in membranes and certain triglycerides occur aslipid droplets inside the cells, which are presumed toaccumulate only in the stationary phase of growth(Hunter & Rose 1971). Bondioli et al. (1992) ascertainedthat squalene could not be separated from FFAs andmethyl and ethyl esters by supercritical CO2, althoughthe separation of squalene from a triglyceride matrix isfeasible. But since the percentage of FFAs in cell walllipids of Torulaspora is very small, it was not reasonableto transform the FFAs in the harvested biomass to theircorresponding triglyceride structures as a preliminarystep to SFE.The previously reported solvent extracted lipid extract

obtained from T. delbrueckii was a viscous oily sub-stance that separated into two layers. The upper layerhad a white greasy substance, and the lower layer was apale yellow coloured oily substance. Squalene could bedetected only in the yellow portion of the extract(Bhattacharjee et al. 2001). On the other hand, thesupercritical extracts obtained at 150 and 250 bars werecolourless to pale yellow and less viscous compared tothat obtained by solvent extraction. At 350–355 bar, the

extract was distinctly brownish and more viscoussuggesting the presence of impurities.Table 1 gives the yield of lipid extracts obtained under

different conditions of supercritical carbon dioxideextraction. Table 2 gives the densitometric assay ofsqualene in these lipid extracts. The optimized condi-tions of supercritical carbon dioxide extraction that gavethe best yield of squalene with minimum impurities werea sample size of 10 g dried biomass at a temperature of60 �C and pressure of 250–255 bar for 30 min static and2 h dynamic time at a constant flow rate of 0.2 l min)1

of carbon dioxide. It is evident that the amount of totallipids and squalene extracted increased with increasingpressure from 150 to 350 bar and with increasingtemperature at constant pressure. However, at 350 bar,though apparently there is an increase in total lipidrecovery, there is only a marginal increase in amount ofsqualene extracted compared to that at 250 bar. Underthis condition, the selectivity of supercritical CO2 forsqualene decreases, as there is an increased possibility ofco-extraction of other hydrocarbons, fatty acids, trigly-cerides and sterols at high pressures.On comparison to the previously reported solvent

extraction method, the yield of squalene obtained underthe best conditions of supercritical carbondioxide ex-traction was much lower. This could be attributed to the

Table 1. Yield of lipid extracts obtained from biomass of T. delbrueckii under different supercritical extraction conditions.

Run no. Weight of biomass charged

for extraction (g)

Extraction conditions Yield of lipid extract

obtained (g)Pressure (bar) Temperature (�C)

1 10.3812 150–155 40 0.0121

2 9.9393 150–155 60 0.0211

3 10.5757 250–255 40 0.0384

4 10.6856 250–255 60 0.0475

5 9.9090 350–355 40 0.1432

6 9.9458 350–355 60 0.1983

7a 3.6688 250–255 60 0.0847

a This run was with lyophilized biomass.

Table 2. Densitometric assay of squalene extracted from T. delbrueckii supercritical carbon dioxide.

SCFE

run no.

lg of lipid

extract

applied on

the plate

Y1a Y a2 X b

1 ¼ðY1=mÞ�10�3

X b2 ¼

ðY2=mÞ�10�3

Mean

· 10)3SD ðn� 1;

n ¼ 2Þ�10�6

% RSD

= (SD

/mean)

· 100

lg of

squalene in

the total

lipid extract

Yield of squalene

in lg obtained/g

partially dried

biomass charged

for extractionc

1 12.10 18,627 18,955 57.521 58.534 58.028 716 1.23 58.028 5.589

2 21.10 22,343 22,859 68.996 70.589 69.793 1126 1.61 69.793 7.022

3 38.40 34,366 34,819 106.123 107.522 106.823 989 0.93 106.823 10.101

4 47.50 38,243 38,682 118.096 119.452 118.774 959 0.81 118.774 11.115

5 143.20 36,257 36,588 111.964 112.985 112.475 722 0.64 112.475 11.351

6 198.30 37,452 37,817 115.653 116.780 116.217 797 0.69 116.217 11.685

7d 1694 511,051 512,003 1578.146 1581.086 1579.62 1470 0.093 1579.5 430.522

a Y1 and Y2 are areas under the curves for squalene when the lipid extract is spotted in replica in one TLC plate.b X1 and X2 are lg of squalene in the lipid extract, evaluated from standard curve of pure squalene with slope, m = 323830, R2 = 0.9927.c In comparison to solvent extraction method where the yield of squalene from the microbial biomass was 27.492 lg g)1 of dried biomass of

T. delbrueckii.d This run was with lyophilized biomass.

Supercritical carbon dioxide extraction of squalene 607

fungal cell walls, which impeded efficient extraction.Pretreatment of the cell wall could improve recoveryefficiency of squalene averting cumbersome downstreamprocessing. There are reports on pretreatment of Mor-tierella ramanniana var. angulispora fungus for enhancedrecovery of c-linolenic acid containing lipids by grindingthe cells in presence of hot ethanol prior to SFE (Sakoet al. 1989; Sakaki et al. 1990). For extraction of EPAfrom the fungus S. parasitica (Cygnarowicz-Provostet al. 1992), lipids from microalga Skeletonema costatumand Ochromonas danica, (Polak et al. 1989) and thatfrom Chlorella vulgaris (Mendes et al. 1995), lyophiliza-tion prior to SFE has been reported. The yield ofsqualene from the lyophilized biomass of yeast at theoptimized conditions of SFE was found to be muchhigher at 430.52 lg g)1 dry weight of yeast cells ascompared to that obtained with oven-dried biomass(Table 2). Moreover, the yield was also significantlyhigher than that obtained by chloroform–methanolextraction as previously reported. Thus SFE is an eco-friendly technique that gives higher extraction efficiencyof squalene in lesser time from lyophilized biomass ofTorulaspora delbrueckii.

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