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Biomass and Oil Content of Microalgae Under Mixotrophic Conditions

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Biomass and Oil Content of Microalgae Under Mixotrophic Conditions Environ. Eng. Res. 2015 Research Paper http://dx.doi.org/10.4491/eer.2014.043 pISSN 1226-1025 eISSN 2005-968X In Press, Uncorrected Proof Biomass and Oil Content of Microalgae under Mixotrophic Conditions Hee-Jeong Choi†, Seung-Mok Lee Department of Environmental Engineering, Catholic Kwandong University, Gangneung 210-701, Korea Abstract The growth of the algae strains Chlorella vulgaris, Botryococcus Braunii and Scenedesmus sp. under mixotrophic conditions in the presence of different concentrations of crude glycerol was evaluated with the objective of increasing the biomass growth and algal oil content. A high biomass concentration was characteristic of these strains when grown on crude glycerol compared to autotrophic growth, and 5 g/L glycerol yielded the highest biomass concentration for these strains. Mixotrophic conditions improved both the growth of the microalgae and the accumulation of triacylglycerols (TAGs). The maximum amount of TAGs in the algal strains was obtained in the 5 g/L glycerol growth medium. The fatty acid profiles of the oil for the cultures met the necessary requirements and are promising resources for biofuel production. Keywords: Biomass, Glycerol, Microalgae, Mixotrophic, Oil content This is an Open Access article distributed under the terms Received July 15, 2014 Accepted December 27, 2014 of the Creative Commons Attribution Non-Commercial Li- † cense (http://creativecommons.org/licenses/by-nc/3.0/) Corresponding Author which permits unrestricted non-commercial use, distribution, and repro- E-mail: [email protected] duction in any medium, provided the original work is properly cited. Tel: +82-33-649-7297 Fax: +82-33-647-7535 Copyright © 2014 Korean Society of Environmental Engineers http://eeer.org 1 1. Introduction 2 Algae cultures have primarily been developed as an important source of aquaculture feeds, 3 human food supplements and pharmaceutical [1], and algae have been proposed as a good 4 candidate for fuel production [2]. Algae strains that are robust and highly productive are 5 selected for the conversion of biomass into energy [3], and strains with relatively high lipid 6 contents are highly attractive for biodiesel fuel production [4]. Microalgae have high growth 7 rates and produce lipids for biofuel production, which is essential for increasing biomass 8 production and amount of lipids that decrease the cost of biodiesel production [5]. Microalgae 9 as a source of renewable energy have received considerable interest; however, further 10 optimization of mass culture conditions is necessary for microalgal biofuel production to be 11 economically viable and sustainable [6]. 12 The ability of microalgae to transition from photoautotrophic to mixotrophic growth is a 13 phenomenon that exists in a number of genera and species throughout the major taxonomic 14 divisions [7, 8]. Many algal organisms can use either autotrophic or heterotrophic metabolic 15 processes for growth; therefore, they can photosynthesize and utilize organic materials [9]. In 16 heterotrophy, algae grow in darkness where cells get energy completely from organic carbon 17 in the media, while in mixotrophy, algae can obtain energy from both organic carbon and 18 light. Such a condition is suitable for algal species that cannot grow in complete darkness but 19 require low light or agitation [10, 11]. Growth rate and biomass production for some algae in 20 mixo- or heterotrophic conditions can be several times higher than those in a photoautotrophic 21 condition alone [11]. Moreover, the synthesis of metabolic products such as lipids and 22 pigments is influenced by the quality and quantity of organic carbon. The use of organic 23 carbon in mixotrophic culture would also reduce the need for carbon dioxide in the culture 24 and facilitate the growth of algal species sensitive to agitation [12]. Bouarab et al. [13] 25 reported that Micractinium pusillum grew in the presence of organic substrates, such as 1 26 glucose and acetate under both mixotrophic and heterotrophic conditions. It can be concluded 27 from the above that mixotrophism is an ideal nutritional mode for high density cultivation of 28 microalgae for the production of biofuels and functional components. However, even though 29 the biomass and lipid productivities are significantly higher compared with those from 30 authotrophic growth, the cost of the organic carbon sources (usually in the form of glucose or 31 acetate) is high when compared against all other added nutrients. To overcome this high 32 carbon cost, a cheap resource must be found. Crude glycerol, which is derived from biodiesel 33 production process, is capable of providing such a supply. As biodiesel production continues 34 to increase, the market is being flooded with crude glycerol [14]. Crude glycerol prices have 35 dropped from $0.25/1b in 2004 to $0.025-0.05/1b in 2006 [15,16]. The increase supply and 36 low demand for crude glycerol have pushed biodiesel producers into eagerly seeking ways to 37 dispose this by-product. Therefore, development of sustainable processes for utilizing this 38 organic raw material is imperative. 39 Recently, a process using crude glycerol as a substrate for the fermentation of the 40 microalga Schizochytrium limacinum was developed. The oleaginous Schizochytrium 41 limacinum can produce significant amounts of total lipids and docosahexaenoic acid, 42 particularly when grown on a variety of carbon sources, such as glucose, glycerol or fructose 43 [17,18]. The above findings suggest that biodiesel-derived crude glycerol is a potential 44 substrate for the mixotrophic cultivation of oleaginous microalgae to utilize crude glycerol 45 and reduce the production cost of microalgal biodiesel. However, there are few reports 46 examining the effects of crude glycerol, particularly on biomass production and algal cell 47 components under mixotrophic conditions. In this study, the effects of various concentrations 48 of crude glycerol on the biomass growth and oil content of Chlorella vulgaris, Botryococcus 49 Braunii and Scenedesmus sp. under mixotrophic conditions were evaluated. 50 2 51 2. Materials and Methods 52 53 2.1. Microalgae Cultures and Medium 54 The investigated microalgae isolated from KMCC (Korea Marine Microalgae Culture Center). 55 The seed cultures of Chlorella vulgaris, Botryococcus Braunii and Scenedesmus sp. were 56 cultivated in Jaworski’s medium (JM) under LED lamps at ambient temperature. JM 57 comprises 4.0 g Ca(NO3)2∙H2O, 2.48 g KH2PO4, 10.0 g MgSO4∙7H2O, 3.18 g NaHCO3, 0.45 58 g EDTAFeNa, 0.45 g EDTANa2, 0.496 g H3BO3, 0.278 g MnCl2∙4H2O, 0.20 g 59 (NH4)6Mo7O24∙4H2O, 0.008 g cyanocobalamin, 0.008 g thiamine HCl, 0.008 g biotin, 16.0 g 60 NaNO3 and 7.2 g Na2HPO4∙12H2O in 200 ml deionized water. The microalgae were cultured 61 in 200 mL conical flask containing 100 mL of JM pH (7.2 ± 0.3), and then 10 mL Chlorella 62 vulgaris, Botryococcus Braunii and Scenedesmus sp. were added. The cultures were 63 maintained in a dark and light cycle of 8 and 16 h, respectively. 64 65 2.2. Experimental Design 66 2.2.1. Optical panel photobioreactor (OPPBR) construction and operation 67 A schematic diagram of the OPPBR is shown in Figure 1a. The OPPBR was operated at a 68 15-L working volume and was equipped with an OP in each reactor. The initial 69 concentrations of the inoculated microalgae were Chlorella vulgaris, 0.357± 0.7 g/L, 70 Botryococcus Braunii, 0.342 ± 0.7 g/L and Scenedesmus sp., 0.367 ± 0.6 g/L. The 71 experiments were conducted at neutral pH (7.2 ± 0.3) under dark and light cycles of 8 and 16 72 h, respectively. The temperature was maintained at 23 ± 1C using LEDs for 20 days. The 73 OPPBRs were aerated continuously at an aeration rate of 0.5 L/min. CO2 at the equivalent 3 74 aeration rate of 0.02 vvm (ca. 2%) was used for cultivation. The OPPBR was designed such 75 that the light source (22 light-emitting diodes (LEDs)), an LED panel (bar type), was placed 76 in the OPPBR. A v-grooved OP was inserted underneath this in the PBR. The thickness of the 77 OP was 6 mm (cf. Figure 1b). The light incident was uniformly distributed across both sides 78 of the OP in the reactor and provides greater functionality. The LED light source was used 79 because it was efficient and provided the required wavelength light from 430 nm to 670 nm, 80 which is selective for microalgal growth. Moreover, the light intensity, which represents the 81 amount of light used for photosynthesis, was ~250 µmol photons/m2/s. The mixotrophic 82 conditions for algae cultivation were achieved with crude glycerol purchased as a byproduct 83 of biodiesel production. The corresponding amount of crude glycerol was added to the JM 84 growth medium to achieve to the desired mixotrophic medium. 85 86 (a) 87 88 89 (b) 4 90 91 Fig. 1. (a) A schematic of OPPBR, (b) Optical panel with LEDs. 92 93 2.2.2. Optical Panel in Photobioreactor 94 The characteristics of the OP are listed in Table 1. The OP exhibited 93% transmittance and 95 1.19 g/cm2 specific gravity. The OP dimensions were 210 mm (L) 290 mm (H) 6 mm (W) 96 as shown in Figure 1b and were constructed from a transparent panel of pure PMMA 97 (poly-methylmethacrylate). This material has good transparency and its light absorption in the 98 visible region is negligible [19]. 99 100 Table 1. Characteristics of optical panel (OP) Parameters Method Unit v-cut OP Specific gravity ISO 1183 _ 1.19 Transmittance ISO 13468 % 93 Heat distortion temperature ISO 75 ℃ 94 Melt flow rate ISO 1133 g/10 min 1.5 Tensile strength ISO 527 MPa 75 Mold shrinkage MRC method % 0.2-0.6 101 102 In this study, a v-cut OP (Fig.
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