cytoplasm. Acetyl-CoA generates cellular fatty acids and subsequently triacylglycerols. SCO produced by Y. lipolytica cultivated under specific growth conditions could be directly converted into biodiesel, as its fatty acid composition is similar to the one from common vegetable oils [13]. Studies related to the production of specialty lipids by the yeast Yarrowia lipolytica during growth on various fatty agroindustrial residues utilized as substrates showed that the potential for growth on stearin (a low-cost industrial derivative of tallow composed of saturated free fatty acids) resulted in significant biomass production. This was accompanied by notable intracellular accumulation of lipid which occurred as a primary anabolic activity regardless of the extracellular nitrogen availability in the medium [14].
In order to reduce the cost of microbial oil production from Y. lipolytica, low-cost raw materials, such as rice hull [15], industrial fats [16], industrial sugars [17], lignocellulosic residues [18], and raw glycerol [19, 20], have been used as substrates. Rice hull, one of the agricultural residues, is used for microbial oil production. According to Economou et al. [15], acid hydrolysis of rice hull using sulfuric acid resulted in hydrolysate that was used as feedstock for microbial lipids production with the oleaginous fungus Mortierella isabellina. Results from kinetic experiments showed the maximum oil accumulation into fungal biomass to be 64.3% and suggested rice hull as cheap source of carbon.
Rice bran is also one of the most abundant agricultural by-products in the world. Typical rice bran is composed of about 15–19.7% lipids, 34.1–52.3% carbohydrates, 7–11.4% fiber, 6.6–9.9% ash, and 10–15%
proteins. After oil is being removed, the residual defatted rice bran (DRB) powder contains significant amount of starchy and cellulosic polysaccharides. Enzymatic and chemical hydrolysis is used to break the polysaccharides into smaller molecules which will be used as carbon sources for microorganisms [21].
The purpose of this study was to investigate the possibility of using cheap and easily available defatted rice bran hydrolysate as a nutrient source for Y. lipolytica Po1g for microbial oil production. The effects of acid concentration, reaction time, and temperature on the hydrolysis of DRB were investigated.
The effects of different types of acid hydrolysates of rice bran on the growth and lipid content were also studied. To the best of our knowledge, this is the first report to use defatted rice bran hydrolysate to culture Y. lipolytica Po1g for microbial oil production.
2. Materials and Methods
2.1. Materials. All solvents and reagents were either high-performance liquid chromatography (HPLC) or analytical reagent grade, obtained from commercial sources. For HPLC analysis, all the standards were purchased from Acros Organics (USA) and Sigma Aldrich (USA). Thin layer chromatography (TLC) aluminium plates (20 × 20 cm) were obtained from Merck KGaA (Darmstadt, Germany). Qualitative filter papers (grade no. 2, 0.26 mm thickness, 80% collection efficiency) were acquired from Advantec, MFS Inc.
(Dublin, CA).
2.2. Defatted Rice Bran Hydrolysate (DRBH) Preparation
2.2.1. Raw Material and Pretreatment. Fresh rice bran was purchased from a local rice mill in Taoyuan County, Taiwan. Bran collected from the mill was stored in a freezer at 4 oC before use. Prior to defatting, it was dried at 50 oC for 24 h until weight was constant. Defatting of rice bran was carried out in a Soxhlet extractor with hexane as the solvent at 60 oC for 6 h. The defatted rice bran was kept at 4 oC for future use.
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2.2.2. Acid Hydrolysis of Defatted Rice Bran. The defatted rice bran was hydrolyzed, following the method of Zhu et al. [4] with minor modification, by using H2SO4 (1%, 2%, 3%, or 4%, v/v) with a bran to acid ratio of 1 : 8 (g/mL). The effect of temperature on hydrolysis was investigated at 60, 70, 80, 90, 100, and 120 oC. The effect of hydrolysis time was also investigated. After hydrolysis, the mixture was subjected to vacuum filtration to obtain the defatted rice bran hydrolysate (DRBH).
2.3. Detoxification of DRBH. To reduce the concentration of inhibitors in DRBH, neutralization with Ca(OH)2 was employed. Ca(OH)2 was slowly added to the DRBH by stirring the mixture at room temperature until the pH was adjusted to 6.5. Then, the hydrolysate was repeatedly vacuum filtrated until the precipitated particles were removed. After its composition was analyzed by HPLC, the detoxified hydrolysate was stored in a refrigerator at 4 oC for further use as nutrient for microbial fermentation.
2.4. Fermentation
2.4.1. Microorganism. Y. lipolytica Po1g cells were obtained from YEASTERN Biotech Co. Ltd. (Taipei, Taiwan). The strain is a derivative of the wild-type strain W29 (ATCC 20460) by a series of genetic modifications. The cells were maintained on a sterilized yeast-, peptone-, and dextrose-(YPD-) agar plate containing 10 g/L yeast extract (Bacto, France), 10 g/L peptone (Bacto, France), 20 g/L glucose (Acros Organics, USA), and 20 g/L agar (Acros Organics, USA) at 4 oC for further microbial fermentation.
2.4.2. Media, Inoculum Preparation, and Large-Scale Fermentation. The preculture of Y. lipolytica Po1g was incubated on a sterilized YPD-agar plate and rejuvenated by incubation in 25 mL YPD medium containing yeast extract (10 g/L), peptone (10 g/L), and D-glucose (20 g/L) for 24 h at 26 oC in an orbital shaker incubator model LM-570 (Chemist Scientific Corp, Taiwan) and then inoculated to cultures in 500 mL Erlenmeyer flasks at an inoculum to medium ratio of 1 : 10 (v/v).
Fermentation was carried out in several 500 mL Erlenmeyer flasks each containing 250 mL detoxified DRBH with initial pH 6.5. To investigate the effect of different nitrogen sources on growth of cells and lipid content, either urea (5 g/L, Acros Organics, USA) or peptone (5 g/L) was added to the DRBH. The effect of the concentration of fermentable sugars in DRBH on microbial growth and lipid content was studied by diluting the sugar concentration obtained at optimum conditions (48.41 g/L) and adjusted to 20 g/L, 30 g/L, or 40 g/L.
The flasks were then incubated in an orbital shaker incubator at 150 rpm and 26 oC. Cell biomass was harvested by centrifugation at 3500 g for 30 min. The cell mass was washed twice with deionized water and dried in an oven at 50 oC until constant weight. All media and flasks were autoclaved at 121 oC for 30 min before the microbial fermentation.
2.5. Analytical Methods
2.5.1. Cell Concentration Determination. For monitoring yeast growth, optical density (OD) of the diluted fermentation broth was measured at 600 nm using a UV/Vis spectrometer V-550 model (Jasco, Japan), and cell concentration was determined from a calibration curve of absorbance versus dry cell weight.
2.5.2. Analysis of Protein, Sugar, and Inhibitor Concentration in DRBH. The solubilized protein content of the hydrolysates was determined by the Bradford method [22]. Absorbance at 595 nm was measured by using a UV-VIS spectrophotometer V-550 model (Jasco, Japan), and then comparison to a
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standard curve provided a relative measurement of protein concentration.
Concentrations of D-glucose, D-xylose, and L-arabinose in the hydrolysates were determined by HPLC (Jasco, Japan) equipped with a PU-1580 pump, a DG-4400 degasser, an Alltech 2000 ELSD detector, and a Zorbax NH 2 column (5 µm particle size, 250 mm × 4.6 mm, Agilent Technologies, USA). The sample was diluted appropriately with deionized water, filtered through a 0.22 µm PVDF syringe filter (Test high) and then injected into the HPLC under the following conditions. The temperature of the ELSD detector was 80 oC, and nitrogen flow rate was 2 mL/min. The column temperature was 25 oC. Acetonitrile:
water (80 : 20, v/v) was the eluent mobile phase with a flow rate of 1 mL/min, and the injection volume was 25 µL.
The concentrations of 5-hydroxymethylfurfural (5-HMF) and furfural in the hydrolysate were determined by HPLC (Jasco, Japan) equipped with a PU-2089 pump combined with degasser, an UV 2077 UV detector, and a Luna C-18 column (5 µm particle size, 250 mm × 4.6 mm, Phenomenex, USA). The sample was diluted appropriately with deionized water, filtered through a 0.22 µm PVDF syringe filter (Test high), and then injected into the column under the following conditions: 25 oC column temperature, acetonitrile : water : acetic acid (11 : 88 : 1, v/v/v) with a flow rate of 0.8 mL/min, injection volume of 25 µL, and the absorption wavelength was 276 nm. The concentrations of these compounds were calculated by using calibration curves obtained from standard D-glucose, D-xylose, L-arabinose, furfural, and 5-HMF solutions.
The amount of total reducing sugars in the hydrolysate was measured by the dinitrosalicylic acid (DNS) method based on a colorimetric reaction between the sugars and dinitrosalicylic acid. DNS reagent (0.5 mL) was added to the appropriately diluted sample (0.5 mL) in a capped brown bottle to prevent DNS from being affected by light. Then, the mixture was heated to 100 oC for 5 min, and after cooling to room temperature in a cold water bath, the absorbance was monitored with a spectrophotometer at 540 nm. The results were calculated based on calibration curve of standard D-glucose.
2.5.3. Lipid Analysis. Extraction of total lipid was performed by using Soxhlet extractor with hexane and methanol (1 : 1, v/v) for 4 h. The extracted lipid was subjected to silica gel thin layer chromatography (TLC) analysis to identify its neutral lipid content. After that, the crude microbial lipid was dewaxed and degummed according to the methods described by Rajam et al. [23], and Vandana et al. [24]. Crude microbial oil was dissolved in hot water at 60 oC, and the water soluble fraction was separated from the insoluble fraction by vacuum filtration. The insoluble fraction was then dissolved in acetone and kept at 60
oC for 1 h to obtain clear solution. After allowing the content to cool to room temperature, the solution was then kept at 5 oC for 24 h to crystallize the remaining waxes. The insoluble fraction was separated by vacuum filtration. The dewaxed and degummed lipid was analyzed by gas chromatography (GC-17A, Shimadzu, Japan) for its neutral lipid composition as well as its fatty acid profile. The GC was equipped with a flame ionization detector and a DB-17HT capillary column (0.25 cm × 30 m, Agilent Technologies Inc., USA). The column temperature was programmed to increase from 80 oC to 365 oC at 10 oC/min and kept at 365 oC for 29 min. Nitrogen was used as the carrier gas at a flow rate of 0.80 mL/min. The split ratio was 1 : 50 (v/v). The temperatures of injector and the detector were both maintained at 370 oC. Twenty milligrams sample was dissolved in 1 mL ethyl acetate, and 0.5 µL sample was taken and injected into the
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GC. Standards of saturated and unsaturated fatty acids (Sigma-Aldrich, USA) were used for the identification of fatty acids in the lipid. All data were averages of triplicate determinations.