Journal of Bioscience and Bioengineering VOL. xx No. xx, 1e8, 2014 www.elsevier.com/locate/jbiosc

Extraction of squalene as value-added product from the residual biomass of Schizochytrium mangrovei PQ6 during biodiesel producing process Minh Hien Hoang, Nguyen Cam Ha, Le Thi Thom, Luu Thi Tam, Hoang Thi Lan Anh, Ngo Thi Hoai Thu, and Dang Diem Hong* Department of Algal Biotechnology, Institute of Biotechnology, Vietnam Academy of Science and Technology (VAST), 18 Hoang Quoc Viet Road, Cau Giay, Hanoi, Viet Nam Received 27 January 2014; accepted 20 May 2014 Available online xxx

Today microalgae represent a viable alternative source of squalene for commercial application. The species Schizochytrium mangrovei, a heterotrophic microalga, has been widely studied and provides a high amount of squalene, polyunsaturated fatty acids and has good profiles for biodiesel production. Our work was aimed at examining the squalene contents in Vietnam’s heterotrophic marine microalga S. mangrovei PQ6 biomass and residues of the biodiesel process from this strain. Thin-layer chromatography and high-performance liquid chromatography (HPLC) methods were successfully applied to the determination of squalene in S. mangrovei PQ6. The squalene content and production of S. mangrovei PQ6 reached 33.00 ± 0.02 and 33.04 ± 0.03 mg gL1 of dry cell weight; and 0.992 g LL1 and 1.019 g LL1 in 30 and 150 L bioreactors, respectively after 96 h of fermentation. In addition, squalene was also detected in spent biomass (approximately 80.10 ± 0.03 mg gL1 of spent biomass) from the S. mangrovei PQ6 biodiesel production process. The structure of squalene in residues of the biodiesel process was confirmed from its nuclear magnetic resonance spectra. The results obtained from our work suggest that there is tremendous potential in the exploitation of squalene as a value-added by-product besides biodiesel from S. mangrovei PQ6 to reduce biodiesel price. Ó 2014, The Society for Biotechnology, Japan. All rights reserved. [Key words: Biodiesel; Hydrocarbon; Schizochytrium mangrovei PQ6; Squalene; Value-added co-product]

In the midst of the world energy crisis, third-generation biofuels (derived from algae) have been considered to be viable fuel alternatives (1). Among many types of algae, microalgae seem to be promising because of that they have high growth rate, e.g., doubling in 24 h. Their lipid content could be adjusted through changing growth medium composition (2,3). They could be harvested more than one in a year. Salty or wastewater could be used for their cultivation. They do not compete with traditional agriculture because they are not traditional foods and they can be cultivated in large open ponds or in closed photobioreactors located on nonarable land. They can grow in a wide variety of climate and water conditions (4). Moreover, microalgae can utilize and sequester CO2 from many sources (5). Biodiesel from algal lipid is non-toxic and highly biodegradable and microalgae produce 15e300 times more oil for biodiesel production than traditional crops on an area basis (6,7). However, so far, biofuels derived from microalgae are not economically competitive. Therefore, it is crucial to explore approaches to reduce the costs of algal biodiesel production processes, by using low-cost raw materials and/or coproducing high value-added products. A few experimental works have been

* Corresponding author at: Institute of Biotechnology, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet Road, Cau Giay, Hanoi, Viet Nam. Tel.: þ84 437 911 059; fax: þ84 48 363 144. E-mail addresses: [email protected] (M.H. Hoang), ddhong60vn@ yahoo.com (D.D. Hong).

published using microalgae to obtain biofuel and high value-added products, within this concept (1,8). The valuable co-products present in the microalgal biomass mainly referred are polyunsaturated fatty acids (PUFAs), omega-3 fatty acids, fertilizers, plastics (e.g., polyhydroxyalkanoates, PHAs), recombinant proteins, pigments and hydrocarbon, such as squalene. Squalene (2,6,10,15,19,23-hexamethyltetracosa-2,6,10,14,18,22hexaene) is one of the components of olive oil and can impact human health. It is an unsaponifiable lipid containing six isoprene units that provide the backbone for the biosynthesis of cholesterol, bile acids and steroid. Moreover, squalene is also a basic intermediary metabolite for the biosynthesis of sterols and triterpenes in plants and animals (9). Recent epidemiological studies have indicated that squalene can effectively inhibit chemically-induced lung, colon and skin tumorigenesis in animals under experimental conditions (10). It can protect cells against free radicals, strengthen the body’s immune system, and decrease the risk for various cancers and lower cholesterol levels. Clinical studies have demonstrated that 60e85% of the total amount of squalene from dietary sources is effectively absorbed and distributed to various tissues. Higher squalene intake (around 500 mg per day) appears to be vital for maintaining nutritional health of human beings (11). Currently commercial sources of squalene are often limited to liver oils of deep-sea sharks and plant seed oils (12). However, continuous supply and future availability of these sources are

1389-1723/$ e see front matter Ó 2014, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2014.05.015

Please cite this article in press as: Hoang, M. H., et al., Extraction of squalene as value-added product from the residual biomass of Schizochytrium mangrovei PQ6 during biodiesel producing process, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.05.015

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J. BIOSCI. BIOENG., biomass achieved from our obtained results is one of the promising option for lowering the biodiesel cost.

uncertain because of concerns over the preservation of marine wildlife as well as the effects of locality and seasonal variation on crop production (13). In addition, the use of shark liver oil is limited due to the presence of environmental pollutants such as dioxins, polychlorinated biphenyls and heavy metals in shark liver, as well as the unpleasant fishy smell and taste (14,15). As a result, many efforts have been made by scientists and researchers to find alternative squalene sources that have the potential to meet commercial production of high-quality squalene. Production of polyunsaturated fatty acids from oleaginous microorganisms rather than from marine animals such as fish and seals has now been made possible (16). However, currently microbial squalene sources have not been produced enough squalene for commercial applications. In recent studies, microalgae have been explored as an alternative source of squalene (17,18). The green microalga Botryococcus braunii is capable of synthesizing squalene, but the very low growth rate and the obligate autotrophic growth characteristic render it unsuitable for commercial production. Of all the microalgal groups, the heterotrophically-grown thraustochytrids are regarded as a promising cell factory for the production of high-value products such as squalene (19,20). Thraustochytrids can growth rapidly in heterotrophic conditions when supplied with organic carbon. They are considered light-independent because they lack the photosynthetic apparatus for carbon fixation (21). Some thraustochytrids contain high squalene content such as Thraustochytrium ACEM 6063 (0.1 mg g1 of biomass) and Aurantiochytrium mangrovei FB1 (0.162 mg g1 of biomass) (22,23). The study of Fan et al. (13) demonstrated the production potential of squalene by A. mangrovei FB3. The highest cellular squalene content (0.53 mg g1) was achieved in culture supplemented with a glucose concentration of 30 g L1 and 100 mg L1 of terbinafine (terbinafine is an inhibitor of squalene monooxygenase in the sterol biosynthetic pathway). This value was much higher than those previously reported in Saccharomyces cerevisiae (0.041 mg g1 of biomass) and Torulaspora debrueckii (0.24 mg g1 of biomass) (24). Nakazawa et al. (25) reported that when the strain of Aurantiochytrium sp. 18W-13a was grown in the optimum condition (25 C, 25e50% seawater concentration and 2e6% glucose concentration), the squalene content and production of approximately 171 mg g1 of dry cell weight and 0.9 g L1 were much higher than those previously reported in thraustochytrids, plants and yeasts. The production potential of squalene by thraustochytrid Schizochytrium mangrovei PQ6 has been investigated in our laboratory. This microalga was also highlighted the possibility to produce profitable biodiesel as well as the high-value PUFAs (8). Squalene is a non-fuel product extracted from S. mangrovei PQ6. Therefore, studies on how to obtain squalene as another value-added coproduct of biodiesel production can shed lights on potential methods to reduce the cost of biodiesel. In this study, the determination of squalene from Vietnam’s heterotrophic marine microalga S. mangrovei PQ6 biomass and residues of the biodiesel process from this strain were developed. The squalene as a value-added product from biodiesel spent

MATERIALS AND METHODS Algal strain and culture conditions In this work, we used the microalga S. mangrovei PQ6 which was isolated from Phu Quoc Island, Kien Giang province, Vietnam (accession number SPQKG02) and later deposited at the Department of Algal Biotechnology, Institute of Biotechnology belonging to Vietnam Academy of Science and Technology, Vietnam. Fermentation was carried out using a fermentor/ bioreactor (New Brunswick Scientific Co. Inc., Edison, NJ, USA) of two different volumes (30 and 150 L with working volume of 15 and 100 L, respectively), and a M12 medium that contained 90 g L1 glucose, 10 g L1 yeast extract, and 17.5 g L1 artificial seawater (ASW). The inoculum size was 2e3% of the total liquid volume of the bioreactor. Temperature was kept at 28 C. Dissolved oxygen was maintained at above 10% by manually increasing the stirring speed (Rushton blade impellers) from 250 rpm to a maximum of 450 rpm. The aeration rate was kept constant at 0.5 vol air (vol medium)1 min1 after filtering through a 0.2 mm filter. The medium pH was controlled within the range of 6.5e7.5. Addition of antifoam was not necessary during the fermentation. Upon reaching maximum biomass and lipid content, PQ6 biomass was harvested after 96 h of fermentation. In PQ6 strain, there was correlation between biomass and cellular lipid content as described in the report of Hong et al. (26). Algal biomass was harvested by centrifugation at 4000 rpm for 10 min. The algae paste was washed three times with sterile distilled water, dried to a constant weight in an oven at 80 C and then stored in desiccators. To eliminate batch-to-batch variation, all biomass was grown under the same conditions and thoroughly mixed to ensure a homogeneous biomass stock. Cell growth Cell growth was determined by dry weight and cell density as described in the report of Hong et al. (26). Nile Red staining S. mangrovei cells were stained with 15 mM Nile Red [(9(Diethylamino)-5H-benzo [a] phenoxazin 5-one)] obtained from SigmaeAldrich (USA) using protocol of Jara et al. (27). 50 mL of a working solution of Nile Red and acetone (0.1 mg mL1) was added to 1 mL of cell suspension. This mixture was gently vortexed and incubated for 10 min at room temperature in darkness. The stained microalgal cells were observed by using fluorescent microscopy (Nikon eclipse 80i-Japan). The excitation and emission wavelength were 480 and 575 nm, respectively (excitation and emission slits at 5 nm), according to the method recommended by Elsey et al. (28). The epifluorescent images were obtained at 1500 magnification. Preparation of squalene from biomass of S. mangrovei PQ6 The dried biomass of S. mangrovei PQ6 was used for squalene extraction follow two steps process. The first step was lipid extraction. The second step was to remove the saponifiables lipid from total lipid. Analysis of total lipid content was determined as described in the report of Bligh and Dyer (29). Dried biomass (1 g) was placed in a chamber and 5 mL of distilled water was added to the biomass. 6 mL of chloroform and 12 mL of methanol were then added to the chamber. The mixture was homogenized well for 5 min and transferred to a centrifuge tube. Chloroform (6 mL) were used to rinse the chamber and then transferred to the tube, which was thoroughly mixed for 60 s. Distilled water (6 mL) were added to the tube followed by mixing in the same manner. The mixture was centrifuged at 6000 rpm for 10 min. The organic solvent layer containing the algal lipid was then collected. The solvent was evaporated to dryness in a water bath at 60 C. The dried residue was defined as the total extracted lipid. The weight of this residue was recorded for a gravimetric estimate of total yield. Total lipid was placed in a Pyrex flask and mixed with a solution of 5% (w/v) potassium hydroxide in methanolewater (4:1 v/v). The reaction mixture was heated and maintained at 60 C for 3 h and well mixed throughout the experiment. After the reaction, following a cool down time, 4 mL of distilled water were added. The nonsaponifiable lipids were extracted three times with a mixture of hexane-chloroform (4:1 v/v) (10 mL each time). The entire nonsaponifiable lipids in n-hexane phase were combined and the solvent was evaporated to dryness under nitrogen

TABLE 1. Changes of parameters of S. mangrovei PQ6 in 30 L and 150 L bioreactors. Culture time (h)

Cell density (10 cells mL1) 30 L

0 8 24 48 72 96 120

Dry cell weight (g L1)

6

1.56 16.54 50.12 70.40 85.28 124.14 124.12

      

150 L 0.04 0.21 0.12 0.64 0.07 0.21 0.13

1.56 23.49 61.20 78.49 88.22 127.45 126.23

      

0.04 0.17 0.22 0.76 0.06 0.35 0.24

30 L e 4.14  12.13  22.01  26.58  30.05  30.03 

150 L 0.04 0.04 0.12 0.05 0.16 0.11

e 5.88  14.81  24.54  27.50  30.85  30.26 

0.06 0.05 0.27 0.15 0.67 0.35

Total lipid (% dry cell weight) 30 L e 3.18  13.66  42.95  43.74  50.46  50.03 

150 L 0.02 0.03 0.11 0.09 0.08 0.09

e 4.52  16.68  47.89  45.25  51.80  51.06 

0.09 0.08 0.16 0.12 0.08 0.09

Squalene (mg g1 dry cell weight) 30 L e 1.72  6.36  17.12  26.02  33.00  32.43 

150 L 0.03 0.05 0.05 0.03 0.02 0.04

1.87 6.50 17.61 25.14 33.04 32.65

e      

0.05 0.03 0.03 0.04 0.03 0.03

e, not determined. Value are expressed as mean  SD (n ¼ 5).

Please cite this article in press as: Hoang, M. H., et al., Extraction of squalene as value-added product from the residual biomass of Schizochytrium mangrovei PQ6 during biodiesel producing process, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.05.015

VOL. xx, 2014 gas. Then the nonsaponifiable lipids were obtained and dissolved in chloroform to be later analyzed by thin-layer chromatography (TLC). Preparation of squalene from residues of the biodiesel production process Spent biomass was obtained from the biodiesel production process described by Hong et al. (8). Dried biomass (100 g) was mixed with 900 mL of methanol, 100 mL of chlorhydric acid and 500 mL of dichlormethan. The reaction mixture was heated and maintained at 60 C for 3 h and well mixed throughout the experiment. After the reaction, the mixture was allowed to cool to room temperature for 1 h. The reaction mixture was filtered and the residues were collected and used for squalene isolation. The in-situ transesterification producing

SQUALENE AS VALUE-ADDED CO-PRODUCT

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fatty acid methyl esters (FAME) from S. mangrovei PQ6 resulted in a yield of 88% based on algal oil and 44% based on algal biomass. Results obtained by Hong et al. (8) indicated that as 100 g of dried biomass of S. mangrovei PQ6 were used for the biodiesel production, 44 g of FAME and approximately 40 g of spent biomass were obtained. Extraction of squalene from the residues after the biodiesel production was carried out as described for the biomass above in which spent biomass: solvent ratio was 1:2.4 using methanol and chloroform as solvents and methanol: chloroform ratio was 1:2 in the step of lipid extraction. Squalene determination Squalene was separated from unsaponifiable lipids by TLC. Chromatograms were developed on silica gel 60 plates (Merck, Germany)

FIG. 1. Images of S. mangrovei PQ6 cell stained by Nile Red at various fermentation times (0, 8, 12, 24, 48, 72, 96, 120 h) in 30 L and 150 L bioreactor under light (A, C) and fluorescence microscope (B, D). Bars: 10 mm.

Please cite this article in press as: Hoang, M. H., et al., Extraction of squalene as value-added product from the residual biomass of Schizochytrium mangrovei PQ6 during biodiesel producing process, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.05.015

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20  20 cm, using n-hexane/diethyl ether/acetic acid (70:30; 4, v/v/v) as mobile phase. The bands on the TLC plates were visualized by spraying 20% sulfuric acid followed by heating in an oven at 100 C for 5 min. The position of squalene bands was determined by comparison with the standard (C30H50, Sigma S3626, Sigma, St. Louis, MO, USA). Squalene bands were scraped off and extracted three times with 30 mL of n-hexane. The solvent was removed and this sample was used for highperformance liquid chromatography assay. For high-performance liquid chromatography analysis, a stock solution of squalene was prepared by dissolving in ethanol to yield standard stock solution, and then storing it in the dark at 20 C. The squalene solutions for making standard calibration curve were prepared using squalene concentration ranging from 100 to 40,000 mg L1 in steps of 300 mg L1 (100e4000 mg L1) and 3000 mg L1 (4000e40,000 mg L1) diluted with acetonitrile. The analyses were performed using the Waters HPLC system and a Thermo Hypersil Gold C18 column (150  2.1 mm, 3 mm) at 30 C with a solution of acetonitrile: distilled water (9:1, v/v) as mobile phase at a flow rate of 250 mL min1 after filtration and degassing by sonication. Squalene was eluted at 19.30 min. Analyses were monitored with a PDA (photodiode array) Accela detector at a wavelength of 198 nm. Identification of squalene was based on UV spectra scanned in the

J. BIOSCI. BIOENG., 190e400 nm range at 1.2 nm sapling interval. The samples were filtered through a 0.45 mm Millipore filter membrane before injection. The retention time was compared with that of standard squalene (C30H50, Sigma S3626). Routine sample calculations were made by comparison of the peak area with that of the standard.

Purification and structural identification of squalene The unsaponifiable lipids were further purified for squalene by column chromatography on a silica gel 60 (24 g, 70e230 mesh ASTM, EMD Millipore, MA, USA) column. A solution of 0.2 g of unsaponifiable lipids in 5 mL of n-hexan was loaded and eluted by washing the column with nhexan, at a flow rate of approximately 1.0 mL/min. Test tubes (10 mL with screw cap) were used for fraction collecting. TLC was used for detection. Squalene appeared completely in the n-hexan elution. The elution was evaporated by vacuum evaporation to give colorless squalene liquid. The residues in the column were washed out by chloroform. The structure of purified squalene was confirmed by the nuclear magnetic resonance (NMR) spectroscopy. NMR experiments were performed using a Bruker Avance e 500 MHz spectrometer (Bruker, Karlsruhe, Germany) at operating frequencies of 500 MHz (1H) and 125 MHz (13C) at the Institute of Chemistry, Vietnam

FIG. 2. Typical chromatogram and PDA UV spectrum of squalene standard solution (A) and squalene isolated by TLC from S. mangrovei PQ6 biomass after 96 h of cultivation in 30 L bioreactor (B).

Please cite this article in press as: Hoang, M. H., et al., Extraction of squalene as value-added product from the residual biomass of Schizochytrium mangrovei PQ6 during biodiesel producing process, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.05.015

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RESULTS

demonstrated a linear correlation between Nile Red fluorescence and the total lipid content of microalgae where an increase of lipid in aging algal cells was primarily due to neutral lipids rather than glycol or phospholipids.

The cell growth and squalene content of S. mangrovei PQ6 in 30 and 150 L bioreactors Changes in cell density, dry cell weight, total lipid and squalene content during the cell growth of S. mangrovei PQ6 in 30 and 150 L bioreactors are shown in Table 1. At the initial inoculation with 1.56  106 cells mL1, the cell density of PQ6 rapidly increased during the first 24 h and reached 50.12  106 cells mL1, 61.20  0.22  106 cells mL1 respectively in 30 and 150 L bioreactors. The maximum cell density of 124.14  106 cells mL1 and 127.45  0.35  106 cells mL1 was reached after 96 h of fermentation in 30 and 150 L bioreactors, respectively. The analysis of unsaponifiable lipid fraction by TLC revealed the presence of squalene. Squalene bands were scraped off for HPLC analysis. The results of HPLC analysis of squalene contents are shown in Table 1 and Fig. 2. In the 30 L bioreactor, biomass, total lipid and squalene content gradually increased with time after inoculation, reaching 30.05  0.16 g L1, 50.46  0.08% of dry cell weight (DCW) and 33.00  0.02 mg g1 DCW after 96 h respectively while these values in the 150 L bioreactor were 30.85  0.67 g L1, 51.80  0.08% of DCW and 33.04  0.03 mg g1 of DCW, respectively. The squalene yield was estimated to be approximately 0.992 and 1.019 g L1 respectively in 30 and 150 L bioreactors. In both 30 and 150 L bioreactors after 96 h, all analyzed parameters were not significantly different by comparison with those after 120 h of fermentation. Nile Red is a lipophilic fluorescent dye used for intracellular lipid determination in pro- and eukaryotic cells and is capable of detecting neutral lipids. Nile Red permeates all structures within the cell, but the yellow-gold fluorescence only manifests when Nile Red penetrates intracellular neutral lipid globules (30). A Nile Red fluorescence method which was previously applied with success to a number of microalgae (28,30,31) was used for S. mangrovei PQ6. Lipids produced by microalgal cells were observed as bright yellow globules when stained with Nile Red and viewed under epifluorescent light (Fig. 1). No polar lipids including squalene were stained to fluorescent yellow shown in Fig. 1. During a 120 h cultivation of PQ6 strain, fluorescence intensity was correlated with intracellular lipid content (including squalene). Cooksey et al. (30)

Squalene from residues of the biodiesel production process The biggest challenge facing biodiesel production from microalgae is that microalgal biodiesel is not economically competitive compared to fossil fuels at today’s energy prices. Thus, researching efficient methods to reduce the cost of biodiesel is essential to increasing the potential for biodiesel usage in the near future. In our previously reported study (8), biodiesel was obtained from S. mangrovei PQ6 biomass and polyunsaturated fatty acids (C22:5n-3, EPA; C22:6n-3, DHA) as a valuable co-product. The production of fatty acid methyl esters (FAME) from this marine microalga resulted in a yield of 88% based on algal oil and 44% based on algal biomass. The process of separating the obtained FAME into two factions, the first enriched with saturated FAME (SFAME) and the second enriched with unsaturated FAME (UFAME), was then investigated to exploit this valuable coproduct. The obtained results showed that the mass fraction of SFAME and UFAME were 70% and 30%, respectively. The UFAME fraction contains a high content of DHA (accounting for 69.00% of total fatty acids). The test results of the SFAME fraction indicated that specific gravity at 15 C, flash point, water and sediment, kinematic viscosity at 40 C, sulfated ash, sulfur, copper strip corrosion at 50 C, cetane number, carbon residue, iodine number and workmanship all met Vietnam’s Biodiesel B100 Standard. Moreover, the utilization of waste glycerol from the biodiesel process as a carbon source for the cultivation of the microalgae S. mangrovei PQ6 and Spirulina platensis was also investigated (8). In this study, the squalene extracted from the spent biomass of biodiesel production was also another valuable co-product besides biodiesel. The high economic value of squalene would aid in lowering the final fuel production costs. The squalene content in the spent biomass after biodiesel production from S. mangrovei PQ6 biomass was analyzed and determined to be approximately 80.10  0.03 mg g1 of spent biomass by TLC and HPLC. Comparing squalene content in S. mangrovei PQ6 biomass and in spent biomass after biodiesel production indicated that the majority of squalene amount was concentrated in the spent biomass. This result has shown that we can exploit squalene as a potentially valuable by-product besides biodiesel from S. mangrovei PQ6 to reduce the price of biodiesel.

Academy of Science and Technology in Hanoi, Vietnam. In all experiments, CDCl3 was used as the solvent and internal standard.

FIG. 3. Thin-layer chromatography (A) and typical chromatogram of purified squalene from S. mangrovei PQ6 residues of the biodiesel production process (B). Lane 1, squalene standard; lane 2, squalene purified from S. mangrovei PQ6 residues of the biodiesel production process.

Please cite this article in press as: Hoang, M. H., et al., Extraction of squalene as value-added product from the residual biomass of Schizochytrium mangrovei PQ6 during biodiesel producing process, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.05.015

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The structure of squalene from residues of the biodiesel production process To verify structure, the squalene from residues of the biodiesel production process was purified by silica gel column chromatography. Squalene was eluted out completely in the n-hexan fractions. Several fractions rich in squalene were detected by TLC with the Sigma standard, combined, and then the solvent was evaporated by rotary evaporation to yield a colorless

J. BIOSCI. BIOENG., liquid. The squalene in these fractions was then analyzed by HPLC. According to TLC and HPLC data (Fig. 3), we showed that squalene is of good purity (98.6%) and no cross-contaminations were obtained. The structure of the squalene in spent biomass of the biodiesel production process was further confirmed by its 1H and 13C NMR spectroscopic data (Fig. 4). The 1H NMR (500 MHz, CDCl3) (Fig. 4A) showed methyl groups at d 1.60 (s, 18H) and d 1.68 (s, 6H),

FIG. 4. NMR spectra of purified squalene from S. mangrovei PQ6 residues of the biodiesel production process. 1H NMR (500 MHz, CDCl3) spectra (A). 13C NMR (125 MHz, CDCl3) spectra (B).

Please cite this article in press as: Hoang, M. H., et al., Extraction of squalene as value-added product from the residual biomass of Schizochytrium mangrovei PQ6 during biodiesel producing process, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.05.015

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methylene groups at d 1.99e2.03 (m, 20H), and internal vinyl signals at d 5.06e5.15 (m, 6H). The 13C NMR (125 MHz, CDCl3) (Fig. 4B) showed methyl carbons at d 16.00, 16.05, 17.67, methylene carbons at d 25.69, 26.69, 26.80, 28.30, 39.75, 39.77, and double bond carbons at d 124.31, 124.34, 124.44, 131.22, 134.89, 135.10. The NMR spectra were in complete agreement with those of the standard squalene material and consistent with the published literature (32).

DISCUSSION The potential of producing biodiesel and valuable co-products such as omega-3 fatty acid (DHA) from the biomass of S. mangrovei PQ6 isolated from Phu Quoc Island, Kien Giang province, Vietnam was explored (8). In this study, we demonstrated that PQ6 strain was a potential producer of squalene. Additionally, we explored the possibility of using spent biomass from the biodiesel production process as a source of squalene. The heterotrophic microalga S. mangrovei PQ6 produced biomass and lipid reaching 30.05  0.16 g L1, 30.85  0.65 g L1 and 50.46  0.08%, 51.80  0.08% of DCW after 96 h of cultivation respectively in 30 and 150 L bioreactors. These results were similar to those previously reported for this alga culture (33,34). In addition, the Nile Red method used was appropriate for the PQ6 strain e it was generally helpful when used in screening programs to discover and select high lipid e and squalene-producing microalgae, especially the heterotrophic marine microalga e S. mangrovei PQ6. Some thraustochytrids contain high squalene content such as Thraustochytrid ACEM 6063 (0.1 mg g1 of biomass) and A. mangrovei FB1 (0.162 mg g1 of biomass) (22,23). The study of Fan et al. (13) explored the production potential of squalene by A. mangrovei FB3. The highest cellular squalene content (0.53 mg g1) was achieved in culture supplemented with a glucose concentration of 30 g L1 and 100 mg L1 of terbinafine. In our study, the values of squalene estimated by TLC and HPLC method were 33.00  0.02 and 33.04  0.03 mg g1 of DCW in 30 and 150 L bioreactors, respectively. These values were several hundred times higher than that of other thraustochytrid strains reported previously (0.002e0.150% of DCW) (12,13,18,22,23). In addition, S. mangrovei PQ6 can grow well in a bioreactor and the obtained results confirm the potential of producing squalene from this strain in the fermentation industry. Recently, Nakazawa et al. (25) reported that when the strain of Aurantiochytrium sp. 18W-13a was grown in the optimum condition (25 C, 25e50% seawater concentration and 2e6% glucose concentration), the squalene content and production of approximately 171 mg g1 dry weight (20% of DCW) and 0.9 g L1 were much higher than those previously reported in thraustochytrids, plants and yeasts. The squalene content in our PQ6 strain was lower compared with that in 18W-13a (33.00  0.02, 33.04  0.03 mg g1 of DCW in 30 and 150 L bioreactors and 171 mg g1 of DCW) but the cell biomass of our PQ6 strain was 6 times higher than that of 18W13a of thraustochytrid (5.0 g dry weight L1 compared with 30.05  0.16e30.85  0.67 g dry weight L1 in 30 and 150 L bioreactors, respectively). Therefore, the squalene production of approximately 0.992 g L1 and 1.019 g L1 in 30 and 150 L bioreactors, respectively, in our PQ6 strain was the same as compared to that of the 18W-13a strain of thraustochytrid (0.9 g L1) (25). The employment of a high-value co-product strategy through the integrated biorefinery approach is expected to significantly enhance the overall cost-effectiveness of microalgal biofuel production. In our work, the high-value co-product exploited was squalene. Although at the moment squalene is mainly used in the cosmetic industries as a moisturizing agent and an emollient, it has garnered more attention as a result of many studies showing its

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therapeutic effects and possible pharmaceutical applications. Large-scale production of microalgae for biodiesel will increase the availability of this product. After the biodiesel production process from S. mangrovei PQ6 biomass, the squalene content in spent biomass was analyzed and determined to be approximately 80.1  0.03 mg g1 of spent biomass by TLC and HPLC methods. The structure of the squalene in spent biomass of the biodiesel production process was confirmed by its 1H and 13C NMR spectroscopic data. The signals of the 1H and 13 C NMR spectra of the isolated squalene from the residual biomass of S. mangrovei PQ6 during biodiesel producing process were complete agreement with those of the standard squalene material and according to the published literature (32). The majority of squalene in PQ6 strain was concentrated in the spent biomass. This result has shown that apart from omega-3 fatty acid as DHA, glycerol (8), we can exploit squalene as a potential value by-product besides biodiesel from S. mangrovei PQ6 to reduce the price of biodiesel. In summary, this work has shown the great potential in exploiting squalene from Vietnam’s heterotrophic microalga S. mangrovei PQ6 besides biodiesel, and omega-3 fatty acid as DHA and glycerol. While biodiesel is produced from triglyceridessaponifiable lipid fraction, squalene is an unsaponifiable lipid that cannot be used for biodiesel production. Extraction of squalene from the residues of the biodiesel production process proves to be a feasible and effective method to reduce biodiesel price. ACKNOWLEDGMENTS This research is funded by Vietnam National Foundation for Science and Technology Development (NAFOSTED) under grant number 106.03-2012.92 for Assoc./Prof. Dr. Dang Diem Hong; by Ministry of Industry and Trade with program of Biotechnology in processing (01/HD-DT. 01. 13/CNSHCB for Assoc./Prof. Dr. Dang Diem Hong, 2013e2015); and by Vietnam Academy of Science and Technology (VAST. DLT.11/14-15 for Dr. Hoang Thi Minh Hien, 2014e2015). We are grateful for use the facilities of National Key Laboratory, Institute of Biotechnology, VAST. The authors also thank Dr. Hoang Mai Ha, Institute of Chemistry, VAST for his analyses of NMR data and MSc. DTN Mai for her assistant during experiments. References 1. Lopes da Silva, T., Gouveia, L., and Reis, A.: Integrated microbial processes for biofuels and high value-added products: the way to improve the cost effectiveness of biofuel production, Appl. Microbiol. Biotechnol., 98, 1043e1053 (2013). 2. Naik, S. N., Meher, L. C., and Sagar, D. V.: Technical aspects of biodiesel production by transesterification e a review, Renew. Sust. Energy Rev., 10, 248e268 (2006). 3. Rittmann, B. E.: Opportunities for renewable bioenergy using microorganisms, Biotechnol. Bioeng., 100, 203e212 (2008). 4. Li, Q., Horsman, M., Wu, N., Lan, C. Q., and Dubois-Calero, N.: Biofuels from microalgae, Biotechnol. Prog., 24, 815e820 (2008). 5. Schenk, P. M., Thomas-Hall, S. R., Stephens, E., Marx, U. C., Mussgnug, J. H., Posten, C., Kruse, O., and Hankamer, B.: Second generation biofuels: highefficiency microalgae for biodiesel production, Bioenergy Res., 1, 20e43 (2008). 6. Chisti, Y.: Biodiesel from microalgae, Biotechnol. Adv., 25, 294e306 (2007). 7. Gouveia, L. and Oliveira, A. C.: Microalgae as a raw material for biofuels production, J. Ind. Microbiol. Biotechnol., 36, 269e274 (2009). 8. Hong, D. D., Mai, D. T. M., Thom, L. T., Ha, N. C., Lam, B. D., Tam, L. T., Anh, H. T. L., and Thu, N. T. H.: Biodiesel production from Vietnam heterotrophic marine microalga Schizochytrium mangrovei PQ6, J. Biosci. Bioeng., 116, 180e185 (2013). 9. Kohno, Y., Rgawa, Y., Itoh, S., Nagaoka, S., Takahashi, M., and Makai, K.: Kinetic study of quenching reaction of singlet oxygen and scavenging reaction of free radical by squalene in n-butanol, Biochim. Biophys. Acta, 1256, 52e56 (1995). 10. Nergiz, C. and Celikkale, D.: The effect of consecutive steps of refining on squalene content of vegetable oils, J. Food Sci. Technol., 48, 382e385 (2011). 11. Chan, P., Tomlinson, B., Lee, C. B., and Lee, Y. S.: Effectiveness and safety of low-dose pravastatin and squalene, alone and in combination, in elderly patients with hypercholesterolemia, J. Clin. Pharmacol., 36, 422e427 (1996).

Please cite this article in press as: Hoang, M. H., et al., Extraction of squalene as value-added product from the residual biomass of Schizochytrium mangrovei PQ6 during biodiesel producing process, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.05.015

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12. Chen, G., Fan, K. W., Lu, F. P., Li, Q., Aki, T., Chen, F., and Jiang, Y.: Optimization of nitrogen source for enhanced production of squalene from thraustochytrid Aurantiochytrium sp. New Biotechnol., 27, 382e389 (2010). 13. Fan, K. W., Aki, T., and Chen, F.: Enhanced production of squalene in the thraustochytrid Aurantiochytrium mangrovei by medium optimization and treatment with terbinafine, World J. Microbiol. Biotechnol., 26, 1303e1309 (2010). 14. Turoczy, N. J., Laurenson, L. J., Allinson, G., Nishikawa, M., Lambert, D. F., Smith, C., Cottier, J. P., Irvine, S. B., and Stagnitti, F.: Observation on metal concentration in three species of shark (Deania calcea, Centroscymnus crepidater, and Centroscymnus owstoni) from southeaster Australian water, J. Agric. Food Chem., 48, 4357e4364 (2000). 15. Storelli, M. M., Ceci, E., Storelli, A., and Marcotrigiano, G. O.: Polychlorinated biphenyl, heavy metal and methylmercury residues in hammerhead sharks: contaminant status and assessment, Mar. Pollut. Bull., 46, 1035e1039 (2003). 16. Ward, O. P. and Singh, A.: Omega-3/6 fatty acids: alternative sources of production, Process Biochem., 40, 3627e3652 (2005). 17. Okada, S., Devarenne, T. P., and Chappell, J.: Molecular characterization of squalene synthase from the green microalga Botrycoccus braunii, race B, Arch. Biochem. Biophys., 373, 307e317 (2000). 18. Li, Q., Chen, G. Q., Fan, K. W., Lu, F. P., Aki, T., and Jiang, Y.: Screening and characterization of squalene-producing thraustochytrids from Hong Kong mangroves, J. Agric. Food Chem., 57, 4267e4272 (2009). 19. Gupta, A., Barrow, C. J., and Puri, M.: Omega-3 biotechnology: thraustochytrids as a novel source of omega-3 oils, Biotechnol. Adv., 30, 1733e1745 (2012). 20. Adarme-Vega, T. C., Thomas-Hall, S. R., and Schenk, P. M.: Towards sustainable sources for omega-3 fatty acids production, Curr. Opin. Biotechnol., 26, 14e18 (2014). 21. Fan, K. W. and Chen, F.: Production of high-value products by the marine microalgae thraustochytrids, pp. 293e323, in: Yang, S. T. (Ed.), Bioprocessing for value-added products from renewable resources: new technologies and applications. Elsevier Science, New York (2007). 22. Lewis, T. E., Nichols, P. D., and McMeekin, T. A.: Sterol and squalene content of a docosahexaenoic acid producing thraustochytrid: influence of culture age, temperature and dissolved oxygen, Mar. Biotechnol., 3, 439e447 (2001).

J. BIOSCI. BIOENG., 23. Jiang, Y., Fan, K. W., and Wong, R. T. Y.: Fatty acids composition and squalene content of the marine microalga Schizochytrium mangrovei, J. Agric. Food Chem., 52, 1196e1200 (2004). 24. Bhattacharjee, P., Shukla, V. B., Singhal, R. S., and Kulkarni, P. R.: Studies on fermentative production of squalene, World J. Microbiol. Biotechnol., 17, 811e816 (2001). 25. Nakazawa, A., Matsura, H., Kose, R., Kato, S., Honda, D., Inouye, I., Kaya, K., and Watanabe, M. M.: Optimization of culture of the thraustochytrid Aurantiochytrium sp. strain 18W-13a for squalene production, Bioresour. Technol., 109, 287e291 (2012). 26. Hong, D. D., Anh, H. T. L., and Thu, N. T. H.: Study on biological characteristics of heterotrophic marine microalga-Schizochytrium mangrovei PQ6 isolated from Phu Quoc Island, Kien Giang province, Vietnam, J. Phycol., 47, 944e954 (2011). 27. Jara, A. D. L., Mendoza, H., Martel, A., and Molina, C.: Flow cytometric determination of lipid content in a marine dinoflagellate, Crypthecodinium cohnii, J. Appl. Phycol., 15, 433e438 (2003). 28. Elsey, D., Jameson, D., Raleigh, B., and Cooney, M. J.: Fluorescent measurement of microalgal neutral lipids, J. Microbiol. Methods, 68, 639e642 (2007). 29. Bligh, E. G. and Dyer, W. J.: A rapid method of total lipid extraction and purification, Can. J. Biochem. Physiol., 37, 911e917 (1959). 30. Cooksey, K. E., Guckert, J. B., Williams, S. A., and Callis, P. R.: Fluorometric determination of the neutral lipid-content of microalgal cells using Nile red, J. Microbiol. Methods, 6, 333e345 (1987). 31. Lee, S. J., Yoon, B. D., and Oh, H. M.: Rapid method for the determination of lipid from the green alga Botryococcus braunii, Biotechnol. Tech., 12, 553e556 (1998). 32. Pouchert, C. J. and Behnke, J.: The Aldrich library of 13C and 1H FTNMR spectra, pp. 46. Aldrich Chemical Co., Milwaukee, WI (1993). 33. Yokochi, T., Honda, D., Higashihara, T., and Nakahara, T.: Optimization of docosahexaenoic acid production by Schizochytrium limacinum SR21, Appl. Microbiol. Biotechnol., 49, 72e79 (1998). 34. Johnson, M. B. and Wen, Z. Y.: Production of biodiesel fuel from the microalgae Schizochytrium limacinum by direct transesterification of algal biomass, Energy Fuels, 23, 5179e5183 (2009).

Please cite this article in press as: Hoang, M. H., et al., Extraction of squalene as value-added product from the residual biomass of Schizochytrium mangrovei PQ6 during biodiesel producing process, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.05.015