Mychonastes Afer HSO-3-1 As a Potential New Source of Biodiesel Cheng Yuan1,2, Junhan Liu1, Yong Fan1, Xiaohui Ren2, Guangrong Hu1 and Fuli Li1*
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Yuan et al. Biotechnology for Biofuels 2011, 4:47 http://www.biotechnologyforbiofuels.com/content/4/1/47 RESEARCH Open Access Mychonastes afer HSO-3-1 as a potential new source of biodiesel Cheng Yuan1,2, Junhan Liu1, Yong Fan1, Xiaohui Ren2, Guangrong Hu1 and Fuli Li1* Abstract Background: Biodiesel is considered to be a promising future substitute for fossil fuels, and microalgae are one source of biodiesel. The ratios of lipid, carbohydrates and proteins are different in different microalgal species, and finding a good strain for oil production remains a difficult prospect. Strains producing valuable co-products would improve the viability of biofuel production. Results: In this study, we performed sequence analysis of the 18S rRNA gene and internal transcribed spacer (ITS) of an algal strain designated HSO-3-1, and found that it was closely related to the Mychonastes afer strain CCAP 260/6. Morphology and cellular structure observation also supported the identification of strain HSO-3-1 as M. afer. We also investigated the effects of nitrogen on the growth and lipid accumulation of the naturally occurring M. afer HSO-3-1, and its potential for biodiesel production. In total, 17 fatty acid methyl esters (FAMEs) were identified in M. afer HSO-3-1, using gas chromatography/mass spectrometry. The total lipid content of M. afer HSO-3-1 was 53.9% of the dry cell weight, and we also detected nervonic acid (C24:1), which has biomedical applications, making up 3.8% of total fatty acids. The highest biomass and lipid yields achieved were 3.29 g/l and 1.62 g/l, respectively, under optimized conditions. Conclusion: The presence of octadecenoic and hexadecanoic acids as major components, with the presence of a high-value component, nervonic acid, renders M. afer HSO-3-1 biomass an economic feedstock for biodiesel production. Background order for microalgae to become an economically viable To meet the rising demand for energy resources and the biofuel feedstock, the cost of producing biodiesel from need to protect the environment, renewable biofuels are microalgae needs to be reduced [6]. Identifying a good needed to replace petroleum-derived transport fuels. strain for oil production, which should feature high lipid Biodiesel, from sources such as microalgae, has received content, high biomass and tolerance to extreme environ- extensive attention in recent years, owing to its biode- ments, remains a difficult prospect [6]. gradability, renewability, and lack of toxicity, among It has been suggested that strains producing valuable other advantages. However, technical and economic bar- co-products, such as feed, fertilizers or pharmaceuticals, riers must be overcome to realize the potential of this would further improve the viability of biofuel production energy source [1-3]. [1,7,8]. To date, omega-3 fatty acid, eicosapentanoic Despite decades of development, the technology acid, decosahexaenoic acid and chlorophyll have been remains in its infancy. For example, problems remain shown to be potentially valuable co-products of microal- with algal culture methods, inefficient harvesting of and gal biodiesel production [9-11]. bioenergy extraction from microalgae cells, and poor The ratios of lipid, carbohydrates and proteins are dif- light penetration in dense microalgal cultures [1,4,5]. In ferent in different microalgal species. In some species, lipids can be up to 60% of the algal dry weight [12,13]. In one exceptional case, a lipid content of 86% dry * Correspondence: [email protected] 1Shandong Provincial Key Laboratory of Energy Genetics, Qingdao Institute weight was reported for the brown resting state colonies of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, of Botryococcus braunii [14]. Factors such as tempera- Qingdao 266101, PR China ture, irradiation, and most markedly, nutrient Full list of author information is available at the end of the article © 2011 Yuan et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Yuan et al. Biotechnology for Biofuels 2011, 4:47 Page 2 of 8 http://www.biotechnologyforbiofuels.com/content/4/1/47 availability, have been shown to be crucial to microalgal strains belonging to the genus Mychonastes clade metabolism, and high lipid productivity can therefore be allowed the recognition of lineages attributable to 10 achieved by optimization of these factors [15,16]. different species (Figure 1). On the 18S rRNA gene tree, The aims of this work were to discover new microal- HSO-3-1 was found to be most similar to the M. afer gae species with high lipid content, yield and suitable strain CCAP 260/6 (GQ477044.1). To confirm the fatty acids for biodiesel production. In our previous results of the 18S rRNA gene analyses, the ITS1-5.8S- work, we analyzed the biomass, lipid content and fatty ITS2 sequence of 26 strains belonging to the genus acid (FA) composition of 77 microalgae strains isolated Mychonastes clade was studied, and allowed the recogni- from Shandong province and Beijing, China. Strain tion of lineages attributable to eight different species HSO-3-1 produced lipids up to 53.9% of its dry weight. (Figure 2). Strain HSO-3-1 was again found to have a Meanwhile, fatty acid analysis of strain HSO-3-1 by GC- close relationship with M. afer strain CCAP 260/6 MS indicated the presence of C24:1 (nervonic acid, 14.7 (GQ477044.1) using this analysis. mg/g of total lipid, 3.8% of the total fatty acid content), which has been shown to have potential in biomedical Morphology and cellular structure applications. So we carried out further investigations on Cells of M. afer HSO-3-1 are unicellular, solitary and strain HSO-3-1. spheroidal, and the cell size is very homogeneous, vary- ing from 2 to 3 μm in diameter. The cell wall is smooth, Results and Discussion and has a layer of along the central axis when examined Molecular identification under scanning electron microscopy (Figure 3, left). The The 18S rRNA gene and ITS sequences obtained for cell wall consists of a double layer (Figure 3, right). The strain HSO-3-1 were submitted to the National Center outer layer, which is 50 to 200 nm wide, was found to for Biotechnology Information (NCBI) under accession be a trilaminar sheath, consistent with a previous report numbers JF930340 and JF930341, respectively. The [17]. Each cell has a single chloroplast that is crescent- amplified 18S rRNA gene sequence was found to have > shaped and anchorage-dependent (Figure 4A-D). The 99% identity with that of previously sequenced Mycho- chloroplast consists of thylakoid lamellae arranged in six nastes sp. and Pseudodictyosphaerium sp. strains in the almost parallel rows with pyrenoids (Figure 4A-D). NCBI database. The 18S rRNA gene analyses of 26 Reproduction takes place by the formation of two Figure 1 Phylogenetic tree inferred from 18S rRNA gene sequences. The distances within the tree were constructed using the neighbor- joining method based on Kimura’s correction with the CLUSTAL W computer program. The horizontal lengths are proportional to the evolutionary distances. The numbers above or below the internal nodes indicate bootstrap values (1000 replicates). Yuan et al. Biotechnology for Biofuels 2011, 4:47 Page 3 of 8 http://www.biotechnologyforbiofuels.com/content/4/1/47 Figure 2 Phylogenetic tree inferred from internal transcribed spacer (ITS) sequences. The distances within the tree were constructed using the neighbor-joining method based on Kimura’s correction with the CLUSTAL W computer program. The horizontal lengths are proportional to the evolutionary distances. The numbers above or below the internal nodes indicate bootstrap values (1000 replicates)of > 50%. autospores (Figure 4E-F). Lipid bodies are often present [18] and the species Mychonastes homosphaera [19]. in the cytoplast after cultivation for a significant period However, the autospore formation of HSO-3-1 involves (Figure 4G-H). binary fission, and is different to that of M. h20omo- Cells of strain HSO-3-1 are single (seldom found in sphaera. The size of the cells is more homogeneous, colonies) and planktonic. The adult cells are spherical, 2 and the cell wall is thicker than M. homosphaera.In to 5 μm in diameter, with a thin mucilaginous envelope. contrast to the genus Pseudodictyosphaerium,whichis The shape of HSO-3-1 was found to be similar to that characterized as having a parietal, cup-shaped or gir- reported for M. afer [18]. dle-shaped chloroplast without pyrenoids, the chloro- Morphological features of strain HSO-3-1, including plast of HSO-3-1 consists of thylakoid lamellae with the shape of the cells, the composition of the cell wall, pyrenoids. Based on these molecular and morphology and the shape and composition of the chloroplast, are characteristics, strain HSO-3-1 was therefore identified identical to those reported for the genus Mychonastes as M. afer. Figure 3 Microscopy and morphological appearance. (Left) Scanning electron micrograph and (right) cell-wall morphology of Mychonastes afer strain HSO-3-1. Yuan et al. Biotechnology for Biofuels 2011, 4:47 Page 4 of 8 http://www.biotechnologyforbiofuels.com/content/4/1/47 Figure 4 Organelle morphology of Mychonastes afer HSO-3-1. (A-D) Chloroplast (c), starch grains (s), lastoglobule (p) andmitochondrion (m). (E-F) Autospores in parent cells. (G-H) Lipid droplets (l) in M. afer HSO-3-1. Fatty acid identification (honesty), Borago officinalis (borage), hemp, Acer trun- The doubling time of M. afer HSO-3-1 was found to be catum (Purpleblow maple) and Tropaeolum speciosum about 12.9 hours during the exponential growth phase. (Flame flower). Only Lunaria annua L.