Efficient Extraction of Starch from Microalgae Using Ultrasonic Homogenizer and Its Conversion into Ethanol by Simultaneous Saccharification and Fermentation ()
1. Introduction
In late years bioethanol has been considered as a candidate of alternative energy of fossil resources [1,2]. Production of bioethanol from corn and sugar cane as raw materials is performed in industrial level, but the remarkable rise of cereals occurs globally because they are also used as food. Recently, many researchers have studies on ethanol production from lignocellulosic material, i.e. wood, bagasse, straw, and etc., which does not compete with food. However, since lignocellulosic materials undergoes lignifications and the pretreatment method, i.e. delignification method, is necessary for the effective saccharification and fermentation of lignocellulosic materials [3-5], it is desired for cost-effective bioethanol production to use other biomass as a source of bioethanol production. On the other hand, microalgae have been attracted as the most promisingly renewable resource for bioethanol production because of their faster growth rate, higher photosynthetic efficiency and polysaccharide production compared with other energy biomass [6-9]. A major obstacle of bioethanol production from microalgae is its hard cell wall that covered starch, a substrate of bioethanol production, contained in microalgae strongly. Therefore, it is necessary to degrade and/or remove the cell wall and enhance accessibility of starch to enzyme and microorganism.
Recently, ultrasonic technologies have been used as pretreatment methods in various industrial fields for decades. For examples, ultrasonic treatments were used to hydrolyze starch contained in corn for increasing enzymatic susceptibility but also to improve melt processing of starch contained in corn [10-12]. Furthermore, Lomboy et al. [13] reported that the total cost of ultrasonic treatment was lower than that of conventional jet cookers for corn dry-grind ethanol plants.
In this work, the efficient extraction of starch from microalgae using ultrasonic homogenizer and its conversion into ethanol by simultaneous saccharification and fermentation (SSF) were attempted. The effect of ultrasonic treatment on extraction of starch was clarified and the optimal condition of SSF was determined for the effective conversion of ultrasonic treated microalgae into ethanol.
2. Materials and Methods
2.1. Microalgae Culture
Chlamydomonas fasciata Ettl NIES-437 provided by MICROBIAL CULTURE COLLECTION at National Institute for Environmental Studies, Japan was used as a microalga in this study. The culture experiment was carried out at 25˚C and pH 7.5 in a 10 L stirred photobioreactor containing 6 L of medium. The carbon source, i.e. CO2, was supplied by bubbling air at an aeration rate of 4 vvm. A light level of 3000 lux was provided by fluorescent lamps continuously. The medium used in this work had the following composition: 150 mg/L Ca(NO3)·4H2O, 100 mg/L KNO3, 50 mg/L β-glycero-phosphoric acid, 40 mg/L MgSO4·7H2O, 0.0001 mg/L vitamine B12, 0.0001 mg/L biotin, 0.01 mg/L thiamin hydrochloride, 0.558 mg/L FeCl3·6H2O, 0.108 mg/L MnCl2·4H2O, 0.066 mg/L ZnSO4·7H2O, 0.012 mg/L CoCl2·6H2O, 0.0075 mg/L Na2MoO4·2H2O, 3 mg/L Na2EDTA·2H2O, and 500 mg/L tris (hydoxymethyl) aminomethane in distilled water. Microalgae and culture were withdrawn from the photobioreactor for measuring the growth amount of microalgae. Microalgae were collected by a centrifugation at 12,000 g during 20 min and then washed with distilled water and methanol for measuring dry cell weight and chlorophyll content in the microalgae, respectively. The chlorophyll content was measured according to the method reported by Grimme and Boardman [14].
2.2. Extraction and Measurement Method of Starch from Microalgae
For efficient extraction of starch from microalgae, dry 2 g of microalgae was added into 20 mL distilled water and then mixed using a vortex followed by ultrasonic treated using a homogenizer (Branson Sonifier 250, Emerson Japan Ltd.) with 30 W and 20 kHz for 0 - 40 min. The sample was taken from the extracted solution obtained from ultrasonic treated microalgae and used for composition analysis. Starch, protein, and ash content were determined using AOAC method [15]. Glucose content was measured by the mutarotase GOD method (Glucose C-Test; Wako Pure Chemical, Osaka, Japan). All analytical determinations were performed in triplicate and average results are shown.
2.3. Alcohol Fermenting Yeast and Inoculum Cultivation
A heat-tolerant alcohol fermenting yeast, Saccharomyces cerevisiae AM12, provided by Bioacademia Co. Ltd., Japan, was used for the SSF experiments. The microorganism was precultured in 100 mL of medium in a 300-mL flask at 40˚C for 24 h using an orbital shaker at 60 rpm. The media for preculture were as follows: 0.1 g/L (NH4)2SO4, 10 g/L glucose, 0.1 g/L KH2PO4, 0.1 g/L MgSO4·7H2O, and 1.0 g/L yeast extract. Thereafter, the cells were collected by centrifugation at 2000 g during 15 min, rinsed thoroughly with sterile distilled water, centrifuged again, and then resuspended in sterile distilled water.
2.4. Simultaneous Saccharification and Fermentation (SSF) Conditions
The medium for SSF contained the extracted solution obtained from ultrasonic treated microalgae (with a substrate concentration of 10% w/v), nutrient medium, 0.05 M acetate buffer (pH 5.0), glutase-AN (0.1% w/v), and 10% v/v preculture solution. Glutase-AN (produced by Aspergillus niger, 13,000 u/g, where u is the amount of enzyme that hydrolyses soluble starch to produce 10 mg of glucose every 30 min at 40˚C and pH 5.0) was provided by HBI Enzymes Inc. The extracted solution, nutrient medium, and buffer were autoclaved at 121˚C for 20 min, but the enzyme solution was added after sterilization using a 0.22-μm-pore size filter. The nutrient medium comprised 1.0 g/L (NH4)2HPO4, 0.05 g/L MgSO4·7H2O, and 2.0 g/L yeast extract [16]. Furthermore, the sterile distilled water containing cells described above was added to the medium and the initial concentration of cells was adjusted to 0.1% w/v. SSF was performed in a 300-mL flask with 100 mL of the medium using the orbital shaker at 100 rpm for 30 h at 40˚C. Aliquots of the samples were then collected and assayed for ethanol and residual glucose concentrations.
2.5. Analytical Methods
Glucose and ethanol concentrations were determined by HPLC using a refractive index (RI) detector and a BioRad HPX-87H column at 65˚C; 5 mM H2SO4 was used as an eluent at a flow rate of 0.6 mL/min, and the injected sample volume was 10 μL. All fermentation experiments were performed in triplicate and average results are shown.
3. Results and Discussion
3.1. Incubation of Microalgae
Figure 1 shows the time courses of dry cell and chlorophyll concentration in the incubation of microalgae using