- Email: [email protected]
Ettlia sp. YC001 showing high growth rate and lipid content under high CO2
Bioresource Technology 127 (2013) 482–488
Contents lists available at SciVerse ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Ettlia sp. YC001 showing high growth rate and lipid content under high CO2 Chan Yoo a,b, Gang-Guk Choi b, Sun-Chang Kim a, Hee-Mock Oh b,⇑ a b
Department of Biological Science, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 305-701, Republic of Korea Environmental Biotechnology Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 305-806, Republic of Korea
h i g h l i g h t s " Ettlia sp. YC001 showing high biomass productivity even under a high CO2 of 5–10%. " A high lipid content of 42% (dry cell weight) and accumulation of certain carotenoids. " Ettlia sp. YC001 can be a candidate for producing biodiesel and high-value products.
a r t i c l e
i n f o
Article history: Received 10 June 2012 Received in revised form 12 September 2012 Accepted 15 September 2012 Available online 25 September 2012 Keywords: Biodiesel Carotenoid Ettlia Lipid productivity Microalgae
a b s t r a c t Over 100 green-colored colonies were isolated from environmental samples when cultivating on a BG11 agar medium, and 4 strains showing different morphologies were selected based on light microscopic observation. Among these strains, the microalgal species with the highest growth rate under 10% CO2 was identified as Ettlia sp. YC001 using an 18S rDNA-based phylogenetic analysis and morphological comparison. The highest cell density of 3.10 g/L (based on dry cell weight) and biomass productivity of 0.19 g/L/d were obtained under 5% CO2 after 16 days. The lipid content and productivity were also up to 42% of the dry cell weight and 80.0 mg/L/d, respectively. The color of the Ettlia sp. YC001 culture changed from green to red after a month due to the accumulation of certain carotenoids. Therefore, it would seem that Ettlia sp. YC001 is appropriate for mitigating CO2 due to its high biomass productivity, and a suitable candidate for producing biodiesel and high-value products. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction The world is currently confronted by global warming and energy depletion that has led to unexpected climate changes and a serious energy crisis worldwide. This situation is due to the emission of huge amounts of CO2 into the atmosphere from the usage of fossil fuels, such as petroleum, coal, and natural gas (Brennan and Owende, 2010; Chi et al., 2011). The representative emitter of anthropogenic CO2 is coal-fired thermoelectric plants that account for over 7% of global CO2 emissions (De Morais and Costa, 2007). This emission of CO2 can be reduced using various strategies, such as physicochemical absorbents, injection into deep oceans and geological formations, and enhanced biological fixation (Kumar et al., 2010). Microalgae have already been receiving much attention as a promising feedstock to mitigate CO2 and to produce biodiesel. The advantage of microalgae is their potential to fix CO2 from the atmosphere or combustion flue gas ranging from 5% to 30% CO2 (Zeng et al., 2011). Microalgae not only have a high CO2 ⇑ Corresponding author. E-mail address: [email protected] (H.-M. Oh). 0960-8524/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2012.09.046
fixation ability, but also can produce 15–300 times more biodiesel than conventional feedstocks, such as palm oil, corn, and other food crops (Chisti, 2007). Plus, the biodiesel derived from a microalgal biomass is a high-density fuel that is an ideal substitute for petroleum without competing with food crops (Rittmann, 2008). Some microalgae have high content of lipid which can be used as substrate for biodiesel production and have been studied since the 1970s, however it has not been commercialized due to the relatively high production cost (Lv et al., 2010). The production of biodiesel from microalgae consists of selecting the algal strain, mass cultivation, harvesting, lipid extraction, and conversion to biodiesel (Khoo et al., 2011; Scott et al., 2010). However, the choice of a suitable algal strain is generally considered the key factor for the successful biological sequestration of CO2 and production of biodiesel (Pulz and Gross, 2004; Scott et al., 2010). While the estimated number of microalgal species is more than 50,000, only around 3000 have been studied and analyzed (Mata et al., 2010). The desirable microalgal characteristics for mass cultivation are as follows: rapid growth rate, high product content, growth in extreme environments, large cell size, wide tolerance of environmental conditions, CO2 tolerance and uptake, and tolerance of
C. Yoo et al. / Bioresource Technology 127 (2013) 482–488
contaminants (Griffiths and Harrison, 2009). The lipid productivity depends on both the biomass productivity and the lipid content. However, while the lipid content of microalgae increases under stress conditions, such as nitrogen, phosphorus, and silicon deficient conditions, the biomass productivity decreases under these conditions (Griffiths and Harrison, 2009; Rodolfi et al., 2009). For example, in the case of Botryococcus braunii, its lipid content represents 75% of its dry cell weight (DCW), yet its lipid productivity is very low due to its low growth rate (Amaro et al., 2011). Accordingly, the purpose of this study was to isolate microalgae showing high lipid productivity under high CO2 concentration. This included the following: (1) screening diverse microalgae with a high growth rate under high CO2 concentration, (2) determining the lipid productivity of selected species under different CO2 concentrations and (3) analyzing the biochemical compositions, including fatty acids, carotenoid, etc. In addition, the isolated Ettlia sp. was evaluated for lipid production and CO2 sequestration. 2. Methods 2.1. Sampling and screening for high-growth microalgae The environmental water samples used to isolate microalgae were collected from freshwater in Daejeon, South Korea. The samples were suspended in a BG11 medium (Allen and Stanier, 1968), and then spread on a BG11 agar medium for picking up single colonies. After 2 weeks of cultivation, any green-colored single colonies on the BG11 agar medium were transferred to 96-/24-well microtiter plates containing the BG11 medium, incubated at 25 ± 1 °C with continuous illumination of 120 lmol photons/m2/s, and cultivated for 2 weeks. The morphology of the green-color isolates was screened using light-microscope observation, and then a single cell of each isolate was obtained using a Fluorescent-Activated Cell Sorter (FACSA II, Becton–Dickinson, CA, USA) with BD FACSDiva software (ver. 5.0.2). To select the microalgae with a high growth rate under a high concentration of CO2, 4 isolates showing different morphologies were cultivated under 10% CO2 at 0.3 v/v/m with continuous illumination of 120 lmol photons/m2/s for18 days in an 8 L vinyl-bag containing 5 L of the BG11 medium. The growth rate of the 4 isolates was determined based on the change of the DCW, while the CO2-fixation rate was calculated using the following equation which was derived from the report that 100 tons of algal biomass fixes roughly 183 tons of CO2 (Chisti, 2007):
CO2 fixation rate ðg CO2 =L=dÞ ¼ 1:83 biomass productivity ðg=L=dÞ
2.2. Identification of isolated microalgae Three ml of the culture of isolate YC001 was collected by centrifugation at 4000 rpm for 10 min. The genomic DNA was then extracted using a plant Genomic DNA extraction kit (Solgent, Daejeon, Korea) according to manufacturer’s instructions. A 1429 bp segment of the 18S ribosomal RNA gene was then amplified using 165F (forward primer, 50 -CGACTTCTGGAAGGGACGTA30 ) and 1700R (reverse primer, 50 -CTAGGTGGGAGGGTTTAATG-30 ). The reaction mixture for the PCR contained 1 ll of the genomic DNA (10 ng/ll), 1 ll of each primer, and 1 ll of the Maxime PCR Premix Kit (iNtRON Biotechnology, Korea) that contained 2.5 U i-Tag™ DNA polymerase (5 U/ll); 2.5 mM of each dNTP; 10 a reaction buffer; 1 a gel loading buffer. The final volume of the reaction mixture was adjusted to 20 ll by adding distilled water. The PCR was performed using a GeneAmp System 2700 thermal cycler (Applied Biosystems, Foster City, CA, USA). The PCR conditions were 94 °C for 5 min, followed by 30 cycles at 94 °C for
483
1 min, 57 °C for 1 min, and 72 °C for 1 min with a final extension at 72 °C for 5 min. The sequence of the 18S rDNA from the isolated microalgae was compared with sequences in the Genebank database using Basic Local Alignment Search Tool (BLAST) to obtain 18S rDNA sequences of other microalgae showing a high similarity. A phylogenetic tree was constructed using the neighbor-joining method with the genetic distances based on Kimura’s two-parameter model using the MEGA 5 program. The nodal support was estimated by bootstrap values based on 1000 replications. The 18S rRNA gene sequence obtained in this study has since been deposited in the National Center for Biotechnology Information (NCBI) GenBank database under accession number JQ684664. 2.3. Growth measurement of isolate YC001 under various CO2 concentrations To determine the effects of the CO2 concentration on the growth rate, lipid content, and fatty acid composition, isolate YC001 was cultivated under 0.03% (ambient air), 5% and 10% CO2 concentrations at a flow rate of 0.1 v/v/m controlled by a gas flowmeter (Rate-master flowmeter, Dwyer Industrial Inc., USA). CO2 used in this study was 99.9% of purity. Isolate YC001 used was pre-cultivated under ambient air in 250 ml of Erlenmeyer flask containing 100 ml of BG11 medium in the orbital shaking incubator. The cultures were carried out in 2-l bottles containing 1.5 l of a BG11 medium with continuous illumination of 120 lmol photons/m2/s provided by cool white fluorescent lamps at 25 ± 1 °C for 16 days. Agitation was performed with a magnetic stirrer (PC-610, Corning, USA) adjusted to 100 rpm. Cell growth was determined based on the DCW and chlorophyll-a. The DCW was measured by filtering an aliquot of the culture suspension through pre-weighed GF/C filters (Whatman, England). After rinsing with distilled water, the filters were dried at 105 °C for 24 h and reweighed. Meanwhile, the chlorophyll-a was extracted using a chloroform and methanol mixture (2:1, v/v), and determined using a flourometer (Turner 450, Barnstead/Thermolyne, Dubuque, IA). 2.4. Analyses of lipid content and fatty acid composition of isolate YC001 The total lipids were extracted using a modified version of the method developed by Bligh and Dyer (1959). Eight-day and 16day cultures were centrifuged at 4000 rpm for 10 min, and the pellets re-suspended in 4 ml of distilled water. The mixtures were then frozen at 20 °C for 24 h. After adding 5 ml of chloroform, the mixture was crushed by using ultra-sonicator (Vibra-cell VC 100, Sonics & Materials Inc., USA) for 5 min. The lipids were then extracted using chloroform–methanol (2:1, v/v), and separated into chloroform and aqueous methanol layers by the addition of methanol and water to give a final solvent ratio of chloroform:methanol:water of 1:1:0.9. The chloroform layer was washed using 20 ml of a 5% NaCl solution and evaporated to dryness. Thereafter, the total lipids were measured gravimetrically. The fatty acid composition was determined using the protocol supplied by MIDI Inc. (Newark, Del.) and gas chromatography (GC2010, Shimadzu, Koyto, Japan). The saponification involved adding 1 ml of reagent 1 (45 g of sodium hydroxide, 150 ml of methanol, and 150 ml of distilled water) to the each tube containing the samples, followed by heating at 100 °C for 30 min. After cooling the tubes, 2 ml of reagent 2 (325 ml of 6.0 N of HCl and 275 ml of methanol) was added for methylation. To extract the fatty acid methyl esters into the organic phase, 1.25 ml of reagent 3 (200 ml of hexane and 200 ml of methyl tert-butyl ether) was added, followed by gentle shaking. The organic phase containing the fatty acid methyl ester was then extracted after washing with 3 ml of reagent 4 (10.8 g of sodium hydroxide and 900 ml of distilled water). Each fatty acid
484
C. Yoo et al. / Bioresource Technology 127 (2013) 482–488
was identified and quantified based of comparing the retention times and peak areas with FAME Mix, C8-C24 (18918-1AMP, Supelco, Sigma–Aldrich Co. LLC., St. Louis, MO, USA). 2.5. Analysis of carotenoid composition of Ettlia sp.
CO2 (Yoo et al., 2010). Therefore, isolate YC001 showing a high growth rate without inhibition even under a high CO2 concentration was finally selected for a further study of growth and lipid production. 3.2. Identification of isolate YC001
To compare the photosynthetic pigments and carotenoids, isolate YC001 was cultivated in an orbital shaking incubator for 30 days until its color changed to red, at which point it was harvested by centrifugation at 4000 rpm for 10 min. A green-colored sample taken under 5% CO2 on day 8 was used as the control. The total carotenoids were extracted and analyzed using the modified method of Kim et al. (2012). An aliquot (10 mg) of lyophilized biomass was homogenized using a pre-chilled mortar and pestle with acetone (5 ml), acid-washed sand (200 mg), anhydrous Na2SO4 (>200 mg), and NaHCO3 (>200 mg). The total carotenoids were repeatedly extracted with acetone until the biomass became colorless and then centrifuged at 5000 rpm at 4 °C for 5 min. The extract was filtered through a 0.45 lm membrane filter (Whatman, PTFE, 13 mm) and subjected to an HPLC analysis using an Agilent 1100 HPLC system (Hewlett–Packard, Palo Alto, CA, USA). All the content levels are expressed as the mean (average content in g DCW) ± SD (standard deviation) of two independent determinations. 3. Results and discussion 3.1. Selection of fast-growing microalgae Over 100 green-colored colonies were picked-up and then incubated in 96-well microtiter plates containing 150 ll of a BG11 medium for 2 weeks. The phenotype of the colonies was mostly round in form, although a few colonies were observed to be filamentous and rod shaped. From over 100 colonies, 4 isolates (YC001–YC004) were selected due to their morphology or growth rate based on their chlorophyll-a concentration (data not shown). In order to confirm a single species, each isolate was re-isolated using fluorescent-activated cell sorting (FACS) and maintained as a single species through subculture with consistent observation of its morphology by using light microscope and molecular technique based on comparison of 18S–28S rDNA ITS sequence. To select the microalgal species showing the highest growth rate under a relatively high concentration of CO2, the 4 isolates were cultivated under 10% CO2 at 0.3 v/v/m for 18 days in a 8-l vinyl-bag containing 5 l of a BG11 medium with continuous illumination of 120 lmol photons/m2/s at 25 ± 1 °C. The cell growth of the 4 isolates continuously increased without inhibition under the high CO2. On day 18, the cell density for each isolate was over 3.40 g/l based on the DCW (Table 1). Additionally, the biomass productivity and CO2 fixation rate in the 4 isolates slightly varied from 0.18 to 0.21 g/l/d and from 0.33 to 0.38 g CO2/l/d, respectively. Among the 4 isolates examined, isolate YC001 showed the fastest growth rate at 0.21 g/l/d and the highest CO2 fixation rate at 0.38 g CO2/l/d. The biomass productivity of isolate YC001 was similar to that Scenedesmus sp. at 0.2 g/l/d when cultivated under 10% Table 1 Characteristics of isolates YC001, YC002, YC003, and YC004 grown under 10% CO2 for 18 days. Characteristics
Isolate YC001
Isolate YC002
Isolate YC003
Isolate YC004
Cell density (g of DCW/L) Biomass productivity (g/L/d) Specific growth rate (/d) Doubling time (d) CO2 fixation rate (g CO2/L/d)
3.99 0.21 0.17 4.12 0.38
3.54 0.19 0.16 4.29 0.35
3.87 0.20 0.16 4.35 0.37
3.40 0.18 0.16 4.46 0.33
For the identification of isolate YC001, its morphological features were observed at each growth stage. At the beginning stage of cultivation, isolate YC001 was spherical in shape, 7–11 lm in diameter, and a green color. Plus, a pyrenoid was invariably observed in each cell. However, during cultivation, its morphology gradually changed to become cylindrical, irregular, and finally cystic red in color. The average size also increased up to around 13 lm in diameter in the red-color culture. During the cell division stage, autospores were observed in the sporangia with 2–8 daughter cells. Thus, based on these features, isolate YC001 was classified as belonging to the Neochloris genus. While Neochloris cells are commonly multinucleate, some can be uninucleate. Thus, the Neochloris genus has been re-divided into Neochloris with multinucleate cells and Ettlia with uninucleate cells (Deason et al., 1991). As a result, 6 species of the Neochloris genus were transferred to the Ettlia genus, while Neochloris terrestris and Neochloris texensis remained in the Neochloris genus. Neochloris species commonly have zoospores with a flagellar apparatus, however, isolate YC001 did not exhibit any zoospores. Neustupa et al. (2011) also reported that zoospores with a flagellar apparatus were not always observed in Parietochloris and Ettlia species. Thus, based on the morphological and reproductive features of isolate YC001, it was finally classified and designated as Ettlia sp. YC001. In the BLAST searches against the GenBank database at NCBI, the sequenced segment (1500 bp) of the 18S rRNA gene of isolate YC001 was found to be most similar to sequences derived from Ettlia texensis SAG 79.80 with a high identity of 98%. A phylogenetic tree was constructed using the 18S rRNA gene sequences of Ettlia sp. YC001 and representatives of class Trebouxiophyceae and Chlorophyceae from the Genebank database (Fig. 1). Class Trebouxiophyceae was composed of Neochloris spp., Parietochloris spp., Ettlia texensis, and Xylochloris irregularis, all of which were originally regarded as the genus Neochloris and subsequently separated into different genera (Deason et al., 1991). Isolate YC001 was found to belong to Trebouxiophyceae and formed a group with Ettlia texensis SAG 79.80. Yet, Ettlia carotinosa, a type species in the Ettlia genus, is not classified as Trebouxiophyceae, but rather as Chlorophyceae. Therefore, it would seem that further study is needed on the taxonomical classification of Ettlia spp. Notwithstanding, on the basis of the morphological and phylogenetic analyses, isolate YC001 was found to be most similar to the genus Ettlia texensis and designated as Ettlia sp. YC001. 3.3. Effect of CO2 concentration on growth of Ettlia sp. YC001 As shown in Fig. 2, the cell densities based on the DCW consistently increased with 2 days of lag phase and reached up to 2.2 g/l for all 3 cultures during the culture period of 16 days. After lag phase, the cell density was dramatically increased under 5% and 10% of CO2 concentration due to acclimation to high CO2 concentrations. Among the 3 cultures, the highest DCW of 3.10 g/l and maximum biomass productivity of 0.29 g/l/d were produced under 5% CO2 after 16 days. Meanwhile, under 10% CO2, the cell density and maximum biomass productivity reached up to 2.93 g/l and 0.31 g/l/d, respectively, after 16 days, plus the CO2 fixation rate was the highest at 0.56 g CO2/l/d. The biomass productivity of Ettlia sp. YC001 with 5% CO2 was 0.19 g/l/d after 16 days, which was similar to the value with 10% CO2 at 0.18 g/l/d, which means that Ettlia sp. YC001 has the capability to grow well without inhibition under
485
C. Yoo et al. / Bioresource Technology 127 (2013) 482–488
100 100
Ettlia_sp._YC001 Ettlia sp. YC001 Ettlia_texensis_SAG_79.80_(GU292343) Ettlia texensis SAG 79.80 (GU292343)
99 99
Neochloris_aquatica_(M62861) Neochloris aquatica (M62861)
67 67
100 100
Neochloris_vigenis_(M74496) Neochloris vigenis (M74496) Parietochloris_cohaerens_UTEX_1707_(EU878372) Parietochloris cohaerens UTEX 1707 (EU878372)
Trebouxiophyceae
Neochloris_aquatica_CCAP_254/5_(FR865697) Neochloris aquatica CCAP 254/5 (FR865697)
99 99 73 73 87 87
Parietochloris_pseudoalveolaris_(M63002) Parietochloris pseudoalveolaris (M63002) Xylochloris_irregularis_CAUP_H_7801_(EU105209) Xylochloris irregularis CAUP H 7801 (EU105209) Heterochlamydomonas_inaequalis_UTEX_1705_(AF367857) Heterochlamydomonas inaequalis UTEX 1705 (AF367857)
Tabris_heimii_IwCl-10_(AB451189) Tabris heimii IwCl-10 (AB451189) Wislouchiella_planctonica_UTEX_LB_1030_(AF252547) Wislouchiella planctonica UTEX LB 1030 (AF252547)
51 51
Ettlia_carotinosa_SAG_213-4_(GU292342) Ettlia carotinosa SAG 213-4 (GU292342)
68 68
Hamakko_caudatus_KzCl-4-1_(AB451188) Hamakko caudatus KzCl-4-1 (AB451188) Pachycladella_umbrina _SAG_10.85_(DQ009775) Pachycladella umbrina SAG 10.85 (DQ009775)
99 99 99 91 91
Chlorophyceae
Protosiphon_botryoides_UTEX_99_(U41177) Protosiphon botryoides UTEX 99 (U41177) Spongiochloris_spongiosa_UTEX _1_(U63107) Spongiochloris spongiosa UTEX 1 (U63107) Dysmorphococcus_globosus_SAG_20-1_(X91629) Dysmorphococcus globosus SAG 20-1 (X91629) Tetracystis_aeria_SAG_89.80_(JN903990) Tetracystis aeria SAG 89.80 (JN903990)
0.01 Fig. 1. Phylogenetic tree based on 18S rRNA gene sequences of Ettlia sp. YC001 and Chlorophyta, with representation of class Trebouxiophyceae and Chlorophyceae from Genbank database. The tree was constructed using the neighbor-joining method and Kimura’s two-parameter model. The bootstrap values are based on 1000 replicates and shown for branches with >50% bootstrap support.
Fig. 2. Growth curves of Ettlia sp. YC001 cultivated under ambient air (d), 5% (.), and 10% (j) CO2 for 16 days. (a) and (b) in the graph indicates a significant difference at P < 0.05.
a high CO2 concentration. There were significant differences in microalgal productivity between ambient air and 5%, 10% CO2 (P < 0.05), but not significant difference between 5% and 10% CO2 (P > 0.05). It meant that the growth ability of Ettlia sp. YC001 was similar when grown under either 5% or 10% of CO2 concentration suggesting that Ettlia sp. YC001 might grow at an equally fast rate even under higher concentrations of CO2. Hence, it is expected that Ettlia sp. YC001 would not be inhibited by higher CO2 concentration in flue gas as flue gas generally contains CO2 ranged from 10% to 15% (Kumar et al., 2010). Biomass productivity might increase without a lag phase which could be alleviated by pre-adaptation of Ettlia sp. YC001 to high CO2 concentration and larger inoculums (Chiu et al., 2008).
Several reports have already been made on the biomass production by diverse microalgae under high CO2. Under phototrophic culture conditions, the biomass productivities of microalgae have been found to range from 0.02 to 0.63 g/l/d (Chen et al., 2011), while the highest biomass productivity of 0.63 g/l/d was obtained from Neochlorisole oleoabundans under 5% CO2 and a high light intensity (Allen and Stanier, 1968; Li et al., 2008). The biomass productivities of Scenedesmus sp. have been reported as 0.218 g/l/d and 0.203 g/l/d under 10% CO2 and flue gas containing 5.5% CO2, respectively (Yoo et al., 2010). Plus, the biomass productivity of microalgae is generally below 0.1 g/l/d with a CO2 concentration over 10% (Brennan and Owende, 2010). CO2 is essential as a carbon source for photoautotrophic microalgae, yet very high CO2 generally inhibits the growth of microalgae. When compared with ambient air conditions (0.03% CO2), the biomass productivities of Chlorella, Scenedesmus, Botryococcus, and Haematococcus are reduced under a high CO2 concentration ranging from 10% to 18% (Brennan and Owende, 2010). In the case of Chlorella sp., the biomass productivity significantly decreases depending on the CO2 concentration ranging from 2% CO2 to 15% CO2 (Chiu et al., 2008). Therefore, based on these results, Ettlia sp. YC001 would seem to be a potential candidate for mitigating CO2 due to its fast-growing ability and high tolerance of CO2 up to 10%. Microalgae are photosynthetic organisms and their biomass originates from photosynthetic activity. The change in the DCW according to the chlorophyll-a concentration in the cells is plotted in Fig. 3. For all 3 cultures, the DCW according to the chlorophyll-a concentration decreased with the culture time, where the decrease was very steep during the initial 10 days and then became stable. Under all the test conditions, the accumulated biomass productivity consistently increased during the initial 10 days and then reached a plateau. Thus, it would seem that the activity of the chlorophyll-a in producing organic compounds encountered some limitation around this time, such as light intensity, nutrients, etc. Consequently, the optimal culture time of Ettlia sp. YC001 was
486
C. Yoo et al. / Bioresource Technology 127 (2013) 482–488
2.0
0.20
10% CO2 1.5
0.15 1.0
0.10
0.5
0.05
0.0
Accumulated biomass productivity (g/L/d)
Dry cell weight / chlorophyll-a ( 1,000)
0.25 Ambient air 5% CO2
0.00 0
2
4
6
8
10
12
14
16
Culture time (days) Fig. 3. Ratio of DCW to chlorophyll-a (closed symbols) and biomass productivity (open symbols) of Ettlia sp. YC001 cultivated under ambient air (d and s), 5% (. and 5), and 10% (j and h) CO2 for 16 days.
around 10 days for the highest biomass productivity and CO2 fixation. 3.4. Lipid content of Ettlia sp. YC001
3.5. Fatty acid composition of Ettlia sp. YC001
The total lipid content of Ettlia sp. YC001 showed an increasing tendency over a cultivation period of 16 days (Fig. 4). The lipid content of Ettlia sp. YC001 under ambient air increased from 44% on day 8 to 50% on day 16. Under 5% and 10% CO2 conditions, these values slightly decreased from 52% and 47% on day 8 to 42% and 43% on day 16, respectively. Thus, there was no statistically significant difference (p > 0.05) in the lipid content of Ettlia sp. YC001 according to the CO2 concentration on day 16. Conversely, the lipid productivity of Ettlia sp. YC001 under ambient air, 5%, and 10% CO2 was 49.5 mg/l/d, 72.6 mg/l/d, and 62.6 mg/l/d on day 8, respectively (Fig. 4), and then dramatically increased to 66.4 mg/L/d, 80.0 mg/L/d, and 77.0 mg/L/d on day 16, respectively, which was around 1.1–1.3 times higher than that under 5% and 10% CO2 on day 8. The highest lipid productivity obtained was 80.0 mg/L/d under 5% CO2 due to the high biomass productivity and lipid content. The lipid contents of microalgae vary from 1% to 70% of the DCW, and normally range from 20% to 50% (Amaro et al., 2011). Some Ettlia spp. were formerly classified as belonging to the Neochloris
120 Ambient air 5% CO2
100
10% CO2
40
80
30
60
20
40
10
20
0
Lipid productivity (mg/L/d)
Total lipid contents (% of dry cell weight)
60 50
0 Day 0
Day 8
genus, which is well known for the accumulation of large quantities of lipids (Deason et al., 1991; Pruvost et al., 2009). The lipid content of N. oleoabundans ranges from 35% to 54% and is composed of 80% tryglycerides (Tornabene et al., 1983). Large quantities of neutral lipids have also been synthesized and accumulated in the stationary phase and under unfavorable culture conditions, such as nutrient starvation and salinity (Hu et al., 2008). As shown in Fig. 3, the culture conditions in this study reached some limitation on day 10, indicating that a large amount of lipids was accumulated inside the cells after this time. The highest lipid productivity of 204 mg/L/d with an average biomass productivity of 0.30 g/L/d and over 60% lipid content under nitrogen derived media has been obtained from Nanochloropsis sp. F&M-M24, which is a marine microalgal species (Rodolfi et al., 2009). The highest lipid productivity obtained from Neochloris oleoabundans was 133 mg/L/d with 34% 5 mM sodium nitrate and 360 lmol photons/m2/s for 7 days (Li et al., 2008). However, the highest lipid productivity of N. oleoabundans resulted from a high biomass productivity of 0.4 g/L/d under a 3-fold higher light intensity than that used in the current study. Therefore, although the lipid productivity in this study was lower than the highest reported lipid productivity, it is expected that Ettlia sp. YC001 cultivated under real flue gas containing a high CO2 concentration could be applied to biodiesel production due to its high lipid productivity under 10% CO2.
Day 16
Culture time Fig. 4. Total lipid contents based on DCW (outside bar) and lipid productivity (inside bar) of Ettlia sp. YC001 cultivated under ambient air, 5%, and 10% CO2 on day 0, day 8, and day 16. Data are expressed as mean (average total lipid) ± SD (standard deviation).
The fatty acid profile of Ettlia sp. YC001 cultivated under different CO2 concentrations showed similar patterns on day 8 and 16, regardless of the CO2 concentration (Fig. 5). Among the identified fatty acids, the proportions of C16:0, C18:1, C18:2 and C18:3 were similar level, which ranged from 15% to 28% of total identified fatty acids on day 8. The proportions of C16:0 under ambient air, 5%, and 10% of CO2 on day 16 were increased up to 26%, 30%, and 29%, respectively. In particular, C18:1, which is a desirable component of biodiesel, was analyzed as a dominant fatty acid at 37% and 36% of the total identified fatty acids under 5% and 10% CO2 on day 16, respectively. The proportions of C18:1 on day 16 were significantly increased around 2 times than those in all conditions on day 8. No significant difference was found in the fatty acid composition among the different CO2 groups. The proportions of C16:0 and C18:1 increased significantly over the cultivation period from day 8 to day 16. Unidentified fatty acids occupied over 54% of the total fatty acids on day 8 and below 49% on day 16. Although the unidentified fatty acids were occupied around 50% of total fatty acids, several isomers of fatty acids existing between C16:0 and C18:3 were also detected and occupied below 5%, respectively. So, the total of these unidentified fatty acids was estimated over 30% of total fatty acid composition. Therefore, it was expected that total fatty acids to be converted into biodiesel were over 80% in these samples without adverse effects on quality of biodiesel. For the successful production of biodiesel from a microalgal biomass, the fatty acid composition of the total lipids is equally important as the amount of lipids produced (Ho et al., 2010). The biodiesel quality is considerably affected by the composition of fatty acid methyl- or ethyl-ester (FAME/FAEE) in the biodiesel (Knothe, 2008). An important factor that influences the lipid content and fatty acid composition during the batch cultivation of microalgae is the growth phase (Mansour et al., 2003). Monounsaturated fatty acids, such as C16:1 and C18:1, are very important fatty acids for biodiesel, as they apparently improve the quality of biodiesel due to their cetane number, cold-flow characteristics, oxidative stability, and low melting temperature (Knothe, 2008; Stansell et al., 2011). Here, C18:1 was the main component of Ettlia sp. YC001 and its proportion increased with the culture time. Therefore, Ettlia sp. YC001 would seem to be a good candidate for the production of
487
C. Yoo et al. / Bioresource Technology 127 (2013) 482–488
Fig. 5. Fatty acid profiles of Ettlia sp. YC001 cultivated under ambient air, 5%, and 10% CO2 on day 8 and day 16.
Table 2 Comparison of chlorophyll and carotenoid concentrations in green- and red-colored Ettlia sp. YC001. Samples
Green Red a
Chlorophyll (lg/g of DCW)
a
Carotenoids (lg/g DCW)a
Chlorophylla
Chlorophyllb
Total
2645.4±96.2 321.3±47.5
1065.5±57.0 171.2±14.2
4242.0±175.4 563.8±69.1
Lutein 1364.6±211.1 n.d.
Zeaxanthin n.d. n.d.
bCryptoxanthin
a-
n.d. n.d.
n.d. n.d.
b-Carotene
Ketocarotenoid
Total
362.4±36.8 n.d.
n.d. 2864.9±243.2
1727.0±247.9 2864.9±243.2
Carotene
Data are expressed as mean (average value of content for DCW) ± SD (standard deviation value) of two independent experiments.
high quality biodiesel due to its high lipid productivity and high proportion of C18:1.
3.6. Carotenoids of Ettlia sp. YC001 During 30 days of cultivation, the culture color of Ettlia sp. YC001 changed from green during the growth stage to red in the senescence stage. This phenomenon was apparently due to the loss of chlorophyll and accumulation of carotenoid inside the cells. As shown in Table 2, the concentrations of chlorophyll-a and -b were 2645.4 ± 96.2 lg/g of the DCW and 1065.5 ± 57.0 lg/g of the DCW, respectively, in the green-colored Ettlia sp. YC001 cultivated under 5% CO2 for 8 days. Conversely, in the red-colored Ettlia sp. YC001, these values were significantly lower at 321.3 ± 47.5 lg/g of the DCW and 171.2 ± 14.2 lg/g of the DCW, respectively. Thus, the total chlorophyll concentration in the red-colored Ettlia sp. YC001 was around 7.5 times lower than that in the green-colored Ettlia sp. YC001. The primary carotenoids lutein and b-carotene were both detected in the green-colored Ettlia sp. YC001 at 1364.6 ± 211.1 lg/g of the DCW and 362.4 ± 36.8 lg/g of the DCW, respectively. Although these carotenoids were not detected in the red-colored Ettlia sp. YC001, the total carotenoid concentration was 2864.9 ± 243.2 lg/g of the DCW. Thus, the total carotenoid concentration in the redcolored Ettlia sp. YC001 was 1.6 times higher than that in the green-colored Ettlia sp. YC001, which was due to the accumulation of red ketocarotenoid confirmed by using HPLC and chromatographic analysis. The total carotenoid concentration in Chlorella vulgaris is known to range from 1.7 mg/g DCW to 4.5 mg/g DCW under salinity stress and nitrogen starvation (Gouveia et al., 1996). Plus, the primary (lutein and b-carotene) and secondary carotenoids in Chlorella zofingiensis have been reported as below 1 mg/g of the DCW and 2.5 mg/g of the DCW, respectively, under oxidative stressed conditions (Ip and Chen, 2005). In another study, the concentrations of chlorophyll and primary carotenoids in C. zofingiensis significantly decreased after exposure to nitrogen and under light stress conditions, whereas the secondary carotenoids, such as astaxanthin, dramatically increased. The red ketocarotenoid
astaxanthin, which is well-known as a strong antioxidant, is largely accumulated under oxidative stress in order to protect the cells against oxidative stress (Ip and Chen, 2005). In this study, the significant accumulation of the red ketocarotenoid was likely to protect the cells from stressful conditions. Only a few microalgal species, such as Chlorella, Dunaliella, Haematococcus, and Spirulina, have been used for the commercial production of carotenoids and pigments. Haematococcus pluvialis and Dunaliella salina are also well-known as a rich natural source of astaxanthin and b-carotene, which account for 1–8% of their DCW and up to 10% of their DCW, respectively (Guerin et al., 2003; Prieto et al., 2011). Although the total carotenoid content in Ettlia sp. YC001 was lower than that in H. pluvialis and D. salina, it is expected that the production of biodiesel using Ettlia sp. YC001 will be accompanied by the production of carotenoids for commercialization. 4. Conclusion In the present study, Ettlia sp. showing a high growth rate and lipid content was successfully isolated from the environment. This strain could grow under a high concentration of CO2 with a high lipid productivity corresponding to the highest lipid productivity reported in previous studies. Among its fatty acids, C18:1 was increased through the culture period and under the various conditions. Plus, certain carotenoids were also produced from this strain. Therefore, the present results suggest that Ettlia sp. YC001 is a suitable strain for mitigating CO2 and producing biodiesel, and a potential candidate for applications producing high-value bioproducts. Acknowledgements This research was supported by a grant from the Advanced Biomass R&D Center, a Global Frontier Program, and the Carbon Dioxide Reduction & Sequestration Research Center, a 21st Century Frontier Program funded by the Korean Ministry of Education, Science & Technology. The authors would like to acknowledge
488
C. Yoo et al. / Bioresource Technology 127 (2013) 482–488
Mi-Jeong Ahn who analyzed the pigments and carotenoids in this study. References Allen, M.M., Stanier, R.Y., 1968. Growth and division of some unicellular blue–green algae. J. Gen. Microbiol. 51, 199–202. Amaro, H.M., Guedes, A.C., Malcata, F.X., 2011. Advances and perspectives in using microalgae to produce biodiesel. Appl. Energy 88, 3402–3410. Bligh, E.G., Dyer, W.J., 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37, 911–917. Brennan, L., Owende, P., 2010. Biofuels from microalgae—a review of technologies for production, processing, and extractions of biofuels and co-products. Renew. Sust. Energy Rev. 14, 557–577. Chen, C.Y., Yeh, K.L., Aisyah, R., Lee, D.J., Chang, J.S., 2011. Cultivation, photobioreactor design and harvesting of microalgae for biodiesel production: a critical review. Bioresour. Technol. 102, 71–81. Chi, Z., O’Fallon, J.V., Chen, S., 2011. Bicarbonate produced from carbon capture for algae culture. Trends Biotechnol. 29, 537–541. Chisti, Y., 2007. Biodiesel from microalgae. Biotechnol. Adv. 25, 294–306. Chiu, S.-Y., Kao, C.-Y., Chen, C.-H., Kuan, T.-C., Ong, S.-C., Lin, C.-S., 2008. Reduction of CO2 by a high-density culture of Chlorella sp. in a semicontinuous photobioreactor. Bioresour. Technol. 99, 3389–3396. De Morais, M.G., Costa, J.A.V., 2007. Isolation and selection of microalgae from coal fired thermoelectric power plant for biofixation of carbon dioxide. Energy Convers. Manage. 48, 2169–2173. Deason, T.R., Silva, P.C., Watanabe, S., Floyd, G.L., 1991. Taxonomic status of the species of the green algal genus Neochloris. Plant Syst. Evol. 177, 213–219. Gouveia, L., Veloso, V., Reis, A., Fernandes, H., Novais, J., Empis, J., 1996. Evolution of pigment composition in Chlorella vulgaris. Bioresour. Technol. 57, 157–163. Griffiths, M., Harrison, S., 2009. Lipid productivity as a key characteristic for choosing algal species for biodiesel production. J. Appl. Phycol. 21, 493–507. Guerin, M., Huntley, M.E., Olaizola, M., 2003. Haematococcus astaxanthin: applications for human health and nutrition. Trends Biotechnol. 21, 210–216. Ho, S.-H., Chen, W.-M., Chang, J.-S., 2010. Scenedesmus obliquus CNW-N as a potential candidate for CO2 mitigation and biodiesel production. Bioresour. Technol. 101, 8725–8730. Hu, Q., Sommerfeld, M., Jarvis, E., Ghirardi, M., Posewitz, M., Seibert, M., Darzins, A., 2008. Microalgal triacylglycerols as feedstocks for biofuel production: perspectives and advances. Plant J. 54, 621–639. Ip, P.F., Chen, F., 2005. Employment of reactive oxygen species to enhance astaxanthin formation in Chlorella zofingiensis in heterotrophic culture. Process Biochem. 40, 3491–3496. Khoo, H.H., Sharratt, P.N., Das, P., Balasubramanian, R.K., Naraharisetti, P.K., Shaik, S., 2011. Life cycle energy and CO2 analysis of microalgae-to-biodiesel: preliminary results and comparisons. Bioresour. Technol. 102, 5800–5807. Kim, S.H., Ahn, Y.O., Ahn, M.J., Lee, H.S., Kwak, S.S., 2012. Down-regulation of betacarotene hydroxylase increases beta-carotene and total carotenoids enhancing
salt stress tolerance in transgenic cultured cells of sweet potato. Phytochemistry 74, 69–78. Knothe, G., 2008. ‘‘Designer’’ biodiesel: optimizing fatty ester composition to improve fuel properties. Energy Fuels 22, 1358–1364. Kumar, A., Ergas, S., Yuan, X., Sahu, A., Zhang, Q., Dewulf, J., Malcata, F.X., van Langenhove, H., 2010. Enhanced CO2 fixation and biofuel production via microalgae: recent developments and future directions. Trends Biotechnol. 28, 371–380. Li, Y., Horsman, M., Wang, B., Wu, N., Lan, C., 2008. Effects of nitrogen sources on cell growth and lipid accumulation of green alga Neochloris oleoabundans. Appl. Microbiol. Biotechnol. 81, 629–636. Lv, J.-M., Cheng, L.-H., Xu, X.-H., Zhang, L., Chen, H.-L., 2010. Enhanced lipid production of Chlorella vulgaris by adjustment of cultivation conditions. Bioresour. Technol. 101, 6797–6804. Mansour, M.P., Volkman, J.K., Blackburn, S.I., 2003. The effect of growth phase on the lipid class, fatty acid and sterol composition in the marine dinoflagellate Gymnodinium sp. in batch culture. Phytochemistry 63, 145–153. Mata, T.M., Martins, A.A., Caetano, N.S., 2010. Microalgae for biodiesel production and other applications: a review. Renew. Sust. Energy Rev. 14, 217–232. Neustupa, J., Eliáš, M., Škaloud, P., Neˇmcová, Y., Šejnohová, L., 2011. Xylochloris irregularis gen. et sp. nov. (Trebouxiophyceae, Chlorophyta), a novel subaerial coccoid green alga. Phycologia 50, 57–66. Prieto, A., Pedro Cañavate, J., García-González, M., 2011. Assessment of carotenoid production by Dunaliella salina in different culture systems and operation regimes. J. Biotechnol. 151, 180–185. Pruvost, J., Van Vooren, G., Cogne, G., Legrand, J., 2009. Investigation of biomass and lipids production with Neochloris oleoabundans in photobioreactor. Bioresour. Technol. 100, 5988–5995. Pulz, O., Gross, W., 2004. Valuable products from biotechnology of microalgae. Appl. Microbiol. Biotechnol. 65, 635–648. Rittmann, B.E., 2008. Opportunities for renewable bioenergy using microorganisms. Biotechnol. Bioeng. 100, 203–212. Rodolfi, L., Chini Zittelli, G., Bassi, N., Padovani, G., Biondi, N., Bonini, G., Tredici, M.R., 2009. Microalgae for oil: strain selection, induction of lipid synthesis and outdoor mass cultivation in a low-cost photobioreactor. Biotechnol. Bioeng. 102, 100–112. Scott, S.A., Davey, M.P., Dennis, J.S., Horst, I., Howe, C.J., Lea-Smith, D.J., Smith, A.G., 2010. Biodiesel from algae: challenges and prospects. Curr. Opin. Biotechnol. 21, 277–286. Stansell, G.R., Gray, V.M., Sym, S.D., 2012. Microalgal fatty acid composition: implications for biodiesel quality. J. Appl. Phycol. 24, 791–801. Tornabene, T.G., Holzer, G., Lien, S., Burris, N., 1983. Lipid composition of the nitrogen starved green alga Neochloris oleoabundans. Enzyme Microb. Technol. 5, 435–440. Yoo, C., Jun, S.-Y., Lee, J.-Y., Ahn, C.-Y., Oh, H.-M., 2010. Selection of microalgae for lipid production under high levels carbon dioxide. Bioresour. Technol. 101, S71– S74. Zeng, X., Danquah, M.K., Chen, X.D., Lu, Y., 2011. Microalgae bioengineering: from CO2 fixation to biofuel production. Renew. Sust. Energy Rev. 15, 3252–3260.
Contents lists available at SciVerse ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Ettlia sp. YC001 showing high growth rate and lipid content under high CO2 Chan Yoo a,b, Gang-Guk Choi b, Sun-Chang Kim a, Hee-Mock Oh b,⇑ a b
Department of Biological Science, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 305-701, Republic of Korea Environmental Biotechnology Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 305-806, Republic of Korea
h i g h l i g h t s " Ettlia sp. YC001 showing high biomass productivity even under a high CO2 of 5–10%. " A high lipid content of 42% (dry cell weight) and accumulation of certain carotenoids. " Ettlia sp. YC001 can be a candidate for producing biodiesel and high-value products.
a r t i c l e
i n f o
Article history: Received 10 June 2012 Received in revised form 12 September 2012 Accepted 15 September 2012 Available online 25 September 2012 Keywords: Biodiesel Carotenoid Ettlia Lipid productivity Microalgae
a b s t r a c t Over 100 green-colored colonies were isolated from environmental samples when cultivating on a BG11 agar medium, and 4 strains showing different morphologies were selected based on light microscopic observation. Among these strains, the microalgal species with the highest growth rate under 10% CO2 was identified as Ettlia sp. YC001 using an 18S rDNA-based phylogenetic analysis and morphological comparison. The highest cell density of 3.10 g/L (based on dry cell weight) and biomass productivity of 0.19 g/L/d were obtained under 5% CO2 after 16 days. The lipid content and productivity were also up to 42% of the dry cell weight and 80.0 mg/L/d, respectively. The color of the Ettlia sp. YC001 culture changed from green to red after a month due to the accumulation of certain carotenoids. Therefore, it would seem that Ettlia sp. YC001 is appropriate for mitigating CO2 due to its high biomass productivity, and a suitable candidate for producing biodiesel and high-value products. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction The world is currently confronted by global warming and energy depletion that has led to unexpected climate changes and a serious energy crisis worldwide. This situation is due to the emission of huge amounts of CO2 into the atmosphere from the usage of fossil fuels, such as petroleum, coal, and natural gas (Brennan and Owende, 2010; Chi et al., 2011). The representative emitter of anthropogenic CO2 is coal-fired thermoelectric plants that account for over 7% of global CO2 emissions (De Morais and Costa, 2007). This emission of CO2 can be reduced using various strategies, such as physicochemical absorbents, injection into deep oceans and geological formations, and enhanced biological fixation (Kumar et al., 2010). Microalgae have already been receiving much attention as a promising feedstock to mitigate CO2 and to produce biodiesel. The advantage of microalgae is their potential to fix CO2 from the atmosphere or combustion flue gas ranging from 5% to 30% CO2 (Zeng et al., 2011). Microalgae not only have a high CO2 ⇑ Corresponding author. E-mail address: [email protected] (H.-M. Oh). 0960-8524/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2012.09.046
fixation ability, but also can produce 15–300 times more biodiesel than conventional feedstocks, such as palm oil, corn, and other food crops (Chisti, 2007). Plus, the biodiesel derived from a microalgal biomass is a high-density fuel that is an ideal substitute for petroleum without competing with food crops (Rittmann, 2008). Some microalgae have high content of lipid which can be used as substrate for biodiesel production and have been studied since the 1970s, however it has not been commercialized due to the relatively high production cost (Lv et al., 2010). The production of biodiesel from microalgae consists of selecting the algal strain, mass cultivation, harvesting, lipid extraction, and conversion to biodiesel (Khoo et al., 2011; Scott et al., 2010). However, the choice of a suitable algal strain is generally considered the key factor for the successful biological sequestration of CO2 and production of biodiesel (Pulz and Gross, 2004; Scott et al., 2010). While the estimated number of microalgal species is more than 50,000, only around 3000 have been studied and analyzed (Mata et al., 2010). The desirable microalgal characteristics for mass cultivation are as follows: rapid growth rate, high product content, growth in extreme environments, large cell size, wide tolerance of environmental conditions, CO2 tolerance and uptake, and tolerance of
C. Yoo et al. / Bioresource Technology 127 (2013) 482–488
contaminants (Griffiths and Harrison, 2009). The lipid productivity depends on both the biomass productivity and the lipid content. However, while the lipid content of microalgae increases under stress conditions, such as nitrogen, phosphorus, and silicon deficient conditions, the biomass productivity decreases under these conditions (Griffiths and Harrison, 2009; Rodolfi et al., 2009). For example, in the case of Botryococcus braunii, its lipid content represents 75% of its dry cell weight (DCW), yet its lipid productivity is very low due to its low growth rate (Amaro et al., 2011). Accordingly, the purpose of this study was to isolate microalgae showing high lipid productivity under high CO2 concentration. This included the following: (1) screening diverse microalgae with a high growth rate under high CO2 concentration, (2) determining the lipid productivity of selected species under different CO2 concentrations and (3) analyzing the biochemical compositions, including fatty acids, carotenoid, etc. In addition, the isolated Ettlia sp. was evaluated for lipid production and CO2 sequestration. 2. Methods 2.1. Sampling and screening for high-growth microalgae The environmental water samples used to isolate microalgae were collected from freshwater in Daejeon, South Korea. The samples were suspended in a BG11 medium (Allen and Stanier, 1968), and then spread on a BG11 agar medium for picking up single colonies. After 2 weeks of cultivation, any green-colored single colonies on the BG11 agar medium were transferred to 96-/24-well microtiter plates containing the BG11 medium, incubated at 25 ± 1 °C with continuous illumination of 120 lmol photons/m2/s, and cultivated for 2 weeks. The morphology of the green-color isolates was screened using light-microscope observation, and then a single cell of each isolate was obtained using a Fluorescent-Activated Cell Sorter (FACSA II, Becton–Dickinson, CA, USA) with BD FACSDiva software (ver. 5.0.2). To select the microalgae with a high growth rate under a high concentration of CO2, 4 isolates showing different morphologies were cultivated under 10% CO2 at 0.3 v/v/m with continuous illumination of 120 lmol photons/m2/s for18 days in an 8 L vinyl-bag containing 5 L of the BG11 medium. The growth rate of the 4 isolates was determined based on the change of the DCW, while the CO2-fixation rate was calculated using the following equation which was derived from the report that 100 tons of algal biomass fixes roughly 183 tons of CO2 (Chisti, 2007):
CO2 fixation rate ðg CO2 =L=dÞ ¼ 1:83 biomass productivity ðg=L=dÞ
2.2. Identification of isolated microalgae Three ml of the culture of isolate YC001 was collected by centrifugation at 4000 rpm for 10 min. The genomic DNA was then extracted using a plant Genomic DNA extraction kit (Solgent, Daejeon, Korea) according to manufacturer’s instructions. A 1429 bp segment of the 18S ribosomal RNA gene was then amplified using 165F (forward primer, 50 -CGACTTCTGGAAGGGACGTA30 ) and 1700R (reverse primer, 50 -CTAGGTGGGAGGGTTTAATG-30 ). The reaction mixture for the PCR contained 1 ll of the genomic DNA (10 ng/ll), 1 ll of each primer, and 1 ll of the Maxime PCR Premix Kit (iNtRON Biotechnology, Korea) that contained 2.5 U i-Tag™ DNA polymerase (5 U/ll); 2.5 mM of each dNTP; 10 a reaction buffer; 1 a gel loading buffer. The final volume of the reaction mixture was adjusted to 20 ll by adding distilled water. The PCR was performed using a GeneAmp System 2700 thermal cycler (Applied Biosystems, Foster City, CA, USA). The PCR conditions were 94 °C for 5 min, followed by 30 cycles at 94 °C for
483
1 min, 57 °C for 1 min, and 72 °C for 1 min with a final extension at 72 °C for 5 min. The sequence of the 18S rDNA from the isolated microalgae was compared with sequences in the Genebank database using Basic Local Alignment Search Tool (BLAST) to obtain 18S rDNA sequences of other microalgae showing a high similarity. A phylogenetic tree was constructed using the neighbor-joining method with the genetic distances based on Kimura’s two-parameter model using the MEGA 5 program. The nodal support was estimated by bootstrap values based on 1000 replications. The 18S rRNA gene sequence obtained in this study has since been deposited in the National Center for Biotechnology Information (NCBI) GenBank database under accession number JQ684664. 2.3. Growth measurement of isolate YC001 under various CO2 concentrations To determine the effects of the CO2 concentration on the growth rate, lipid content, and fatty acid composition, isolate YC001 was cultivated under 0.03% (ambient air), 5% and 10% CO2 concentrations at a flow rate of 0.1 v/v/m controlled by a gas flowmeter (Rate-master flowmeter, Dwyer Industrial Inc., USA). CO2 used in this study was 99.9% of purity. Isolate YC001 used was pre-cultivated under ambient air in 250 ml of Erlenmeyer flask containing 100 ml of BG11 medium in the orbital shaking incubator. The cultures were carried out in 2-l bottles containing 1.5 l of a BG11 medium with continuous illumination of 120 lmol photons/m2/s provided by cool white fluorescent lamps at 25 ± 1 °C for 16 days. Agitation was performed with a magnetic stirrer (PC-610, Corning, USA) adjusted to 100 rpm. Cell growth was determined based on the DCW and chlorophyll-a. The DCW was measured by filtering an aliquot of the culture suspension through pre-weighed GF/C filters (Whatman, England). After rinsing with distilled water, the filters were dried at 105 °C for 24 h and reweighed. Meanwhile, the chlorophyll-a was extracted using a chloroform and methanol mixture (2:1, v/v), and determined using a flourometer (Turner 450, Barnstead/Thermolyne, Dubuque, IA). 2.4. Analyses of lipid content and fatty acid composition of isolate YC001 The total lipids were extracted using a modified version of the method developed by Bligh and Dyer (1959). Eight-day and 16day cultures were centrifuged at 4000 rpm for 10 min, and the pellets re-suspended in 4 ml of distilled water. The mixtures were then frozen at 20 °C for 24 h. After adding 5 ml of chloroform, the mixture was crushed by using ultra-sonicator (Vibra-cell VC 100, Sonics & Materials Inc., USA) for 5 min. The lipids were then extracted using chloroform–methanol (2:1, v/v), and separated into chloroform and aqueous methanol layers by the addition of methanol and water to give a final solvent ratio of chloroform:methanol:water of 1:1:0.9. The chloroform layer was washed using 20 ml of a 5% NaCl solution and evaporated to dryness. Thereafter, the total lipids were measured gravimetrically. The fatty acid composition was determined using the protocol supplied by MIDI Inc. (Newark, Del.) and gas chromatography (GC2010, Shimadzu, Koyto, Japan). The saponification involved adding 1 ml of reagent 1 (45 g of sodium hydroxide, 150 ml of methanol, and 150 ml of distilled water) to the each tube containing the samples, followed by heating at 100 °C for 30 min. After cooling the tubes, 2 ml of reagent 2 (325 ml of 6.0 N of HCl and 275 ml of methanol) was added for methylation. To extract the fatty acid methyl esters into the organic phase, 1.25 ml of reagent 3 (200 ml of hexane and 200 ml of methyl tert-butyl ether) was added, followed by gentle shaking. The organic phase containing the fatty acid methyl ester was then extracted after washing with 3 ml of reagent 4 (10.8 g of sodium hydroxide and 900 ml of distilled water). Each fatty acid
484
C. Yoo et al. / Bioresource Technology 127 (2013) 482–488
was identified and quantified based of comparing the retention times and peak areas with FAME Mix, C8-C24 (18918-1AMP, Supelco, Sigma–Aldrich Co. LLC., St. Louis, MO, USA). 2.5. Analysis of carotenoid composition of Ettlia sp.
CO2 (Yoo et al., 2010). Therefore, isolate YC001 showing a high growth rate without inhibition even under a high CO2 concentration was finally selected for a further study of growth and lipid production. 3.2. Identification of isolate YC001
To compare the photosynthetic pigments and carotenoids, isolate YC001 was cultivated in an orbital shaking incubator for 30 days until its color changed to red, at which point it was harvested by centrifugation at 4000 rpm for 10 min. A green-colored sample taken under 5% CO2 on day 8 was used as the control. The total carotenoids were extracted and analyzed using the modified method of Kim et al. (2012). An aliquot (10 mg) of lyophilized biomass was homogenized using a pre-chilled mortar and pestle with acetone (5 ml), acid-washed sand (200 mg), anhydrous Na2SO4 (>200 mg), and NaHCO3 (>200 mg). The total carotenoids were repeatedly extracted with acetone until the biomass became colorless and then centrifuged at 5000 rpm at 4 °C for 5 min. The extract was filtered through a 0.45 lm membrane filter (Whatman, PTFE, 13 mm) and subjected to an HPLC analysis using an Agilent 1100 HPLC system (Hewlett–Packard, Palo Alto, CA, USA). All the content levels are expressed as the mean (average content in g DCW) ± SD (standard deviation) of two independent determinations. 3. Results and discussion 3.1. Selection of fast-growing microalgae Over 100 green-colored colonies were picked-up and then incubated in 96-well microtiter plates containing 150 ll of a BG11 medium for 2 weeks. The phenotype of the colonies was mostly round in form, although a few colonies were observed to be filamentous and rod shaped. From over 100 colonies, 4 isolates (YC001–YC004) were selected due to their morphology or growth rate based on their chlorophyll-a concentration (data not shown). In order to confirm a single species, each isolate was re-isolated using fluorescent-activated cell sorting (FACS) and maintained as a single species through subculture with consistent observation of its morphology by using light microscope and molecular technique based on comparison of 18S–28S rDNA ITS sequence. To select the microalgal species showing the highest growth rate under a relatively high concentration of CO2, the 4 isolates were cultivated under 10% CO2 at 0.3 v/v/m for 18 days in a 8-l vinyl-bag containing 5 l of a BG11 medium with continuous illumination of 120 lmol photons/m2/s at 25 ± 1 °C. The cell growth of the 4 isolates continuously increased without inhibition under the high CO2. On day 18, the cell density for each isolate was over 3.40 g/l based on the DCW (Table 1). Additionally, the biomass productivity and CO2 fixation rate in the 4 isolates slightly varied from 0.18 to 0.21 g/l/d and from 0.33 to 0.38 g CO2/l/d, respectively. Among the 4 isolates examined, isolate YC001 showed the fastest growth rate at 0.21 g/l/d and the highest CO2 fixation rate at 0.38 g CO2/l/d. The biomass productivity of isolate YC001 was similar to that Scenedesmus sp. at 0.2 g/l/d when cultivated under 10% Table 1 Characteristics of isolates YC001, YC002, YC003, and YC004 grown under 10% CO2 for 18 days. Characteristics
Isolate YC001
Isolate YC002
Isolate YC003
Isolate YC004
Cell density (g of DCW/L) Biomass productivity (g/L/d) Specific growth rate (/d) Doubling time (d) CO2 fixation rate (g CO2/L/d)
3.99 0.21 0.17 4.12 0.38
3.54 0.19 0.16 4.29 0.35
3.87 0.20 0.16 4.35 0.37
3.40 0.18 0.16 4.46 0.33
For the identification of isolate YC001, its morphological features were observed at each growth stage. At the beginning stage of cultivation, isolate YC001 was spherical in shape, 7–11 lm in diameter, and a green color. Plus, a pyrenoid was invariably observed in each cell. However, during cultivation, its morphology gradually changed to become cylindrical, irregular, and finally cystic red in color. The average size also increased up to around 13 lm in diameter in the red-color culture. During the cell division stage, autospores were observed in the sporangia with 2–8 daughter cells. Thus, based on these features, isolate YC001 was classified as belonging to the Neochloris genus. While Neochloris cells are commonly multinucleate, some can be uninucleate. Thus, the Neochloris genus has been re-divided into Neochloris with multinucleate cells and Ettlia with uninucleate cells (Deason et al., 1991). As a result, 6 species of the Neochloris genus were transferred to the Ettlia genus, while Neochloris terrestris and Neochloris texensis remained in the Neochloris genus. Neochloris species commonly have zoospores with a flagellar apparatus, however, isolate YC001 did not exhibit any zoospores. Neustupa et al. (2011) also reported that zoospores with a flagellar apparatus were not always observed in Parietochloris and Ettlia species. Thus, based on the morphological and reproductive features of isolate YC001, it was finally classified and designated as Ettlia sp. YC001. In the BLAST searches against the GenBank database at NCBI, the sequenced segment (1500 bp) of the 18S rRNA gene of isolate YC001 was found to be most similar to sequences derived from Ettlia texensis SAG 79.80 with a high identity of 98%. A phylogenetic tree was constructed using the 18S rRNA gene sequences of Ettlia sp. YC001 and representatives of class Trebouxiophyceae and Chlorophyceae from the Genebank database (Fig. 1). Class Trebouxiophyceae was composed of Neochloris spp., Parietochloris spp., Ettlia texensis, and Xylochloris irregularis, all of which were originally regarded as the genus Neochloris and subsequently separated into different genera (Deason et al., 1991). Isolate YC001 was found to belong to Trebouxiophyceae and formed a group with Ettlia texensis SAG 79.80. Yet, Ettlia carotinosa, a type species in the Ettlia genus, is not classified as Trebouxiophyceae, but rather as Chlorophyceae. Therefore, it would seem that further study is needed on the taxonomical classification of Ettlia spp. Notwithstanding, on the basis of the morphological and phylogenetic analyses, isolate YC001 was found to be most similar to the genus Ettlia texensis and designated as Ettlia sp. YC001. 3.3. Effect of CO2 concentration on growth of Ettlia sp. YC001 As shown in Fig. 2, the cell densities based on the DCW consistently increased with 2 days of lag phase and reached up to 2.2 g/l for all 3 cultures during the culture period of 16 days. After lag phase, the cell density was dramatically increased under 5% and 10% of CO2 concentration due to acclimation to high CO2 concentrations. Among the 3 cultures, the highest DCW of 3.10 g/l and maximum biomass productivity of 0.29 g/l/d were produced under 5% CO2 after 16 days. Meanwhile, under 10% CO2, the cell density and maximum biomass productivity reached up to 2.93 g/l and 0.31 g/l/d, respectively, after 16 days, plus the CO2 fixation rate was the highest at 0.56 g CO2/l/d. The biomass productivity of Ettlia sp. YC001 with 5% CO2 was 0.19 g/l/d after 16 days, which was similar to the value with 10% CO2 at 0.18 g/l/d, which means that Ettlia sp. YC001 has the capability to grow well without inhibition under
485
C. Yoo et al. / Bioresource Technology 127 (2013) 482–488
100 100
Ettlia_sp._YC001 Ettlia sp. YC001 Ettlia_texensis_SAG_79.80_(GU292343) Ettlia texensis SAG 79.80 (GU292343)
99 99
Neochloris_aquatica_(M62861) Neochloris aquatica (M62861)
67 67
100 100
Neochloris_vigenis_(M74496) Neochloris vigenis (M74496) Parietochloris_cohaerens_UTEX_1707_(EU878372) Parietochloris cohaerens UTEX 1707 (EU878372)
Trebouxiophyceae
Neochloris_aquatica_CCAP_254/5_(FR865697) Neochloris aquatica CCAP 254/5 (FR865697)
99 99 73 73 87 87
Parietochloris_pseudoalveolaris_(M63002) Parietochloris pseudoalveolaris (M63002) Xylochloris_irregularis_CAUP_H_7801_(EU105209) Xylochloris irregularis CAUP H 7801 (EU105209) Heterochlamydomonas_inaequalis_UTEX_1705_(AF367857) Heterochlamydomonas inaequalis UTEX 1705 (AF367857)
Tabris_heimii_IwCl-10_(AB451189) Tabris heimii IwCl-10 (AB451189) Wislouchiella_planctonica_UTEX_LB_1030_(AF252547) Wislouchiella planctonica UTEX LB 1030 (AF252547)
51 51
Ettlia_carotinosa_SAG_213-4_(GU292342) Ettlia carotinosa SAG 213-4 (GU292342)
68 68
Hamakko_caudatus_KzCl-4-1_(AB451188) Hamakko caudatus KzCl-4-1 (AB451188) Pachycladella_umbrina _SAG_10.85_(DQ009775) Pachycladella umbrina SAG 10.85 (DQ009775)
99 99 99 91 91
Chlorophyceae
Protosiphon_botryoides_UTEX_99_(U41177) Protosiphon botryoides UTEX 99 (U41177) Spongiochloris_spongiosa_UTEX _1_(U63107) Spongiochloris spongiosa UTEX 1 (U63107) Dysmorphococcus_globosus_SAG_20-1_(X91629) Dysmorphococcus globosus SAG 20-1 (X91629) Tetracystis_aeria_SAG_89.80_(JN903990) Tetracystis aeria SAG 89.80 (JN903990)
0.01 Fig. 1. Phylogenetic tree based on 18S rRNA gene sequences of Ettlia sp. YC001 and Chlorophyta, with representation of class Trebouxiophyceae and Chlorophyceae from Genbank database. The tree was constructed using the neighbor-joining method and Kimura’s two-parameter model. The bootstrap values are based on 1000 replicates and shown for branches with >50% bootstrap support.
Fig. 2. Growth curves of Ettlia sp. YC001 cultivated under ambient air (d), 5% (.), and 10% (j) CO2 for 16 days. (a) and (b) in the graph indicates a significant difference at P < 0.05.
a high CO2 concentration. There were significant differences in microalgal productivity between ambient air and 5%, 10% CO2 (P < 0.05), but not significant difference between 5% and 10% CO2 (P > 0.05). It meant that the growth ability of Ettlia sp. YC001 was similar when grown under either 5% or 10% of CO2 concentration suggesting that Ettlia sp. YC001 might grow at an equally fast rate even under higher concentrations of CO2. Hence, it is expected that Ettlia sp. YC001 would not be inhibited by higher CO2 concentration in flue gas as flue gas generally contains CO2 ranged from 10% to 15% (Kumar et al., 2010). Biomass productivity might increase without a lag phase which could be alleviated by pre-adaptation of Ettlia sp. YC001 to high CO2 concentration and larger inoculums (Chiu et al., 2008).
Several reports have already been made on the biomass production by diverse microalgae under high CO2. Under phototrophic culture conditions, the biomass productivities of microalgae have been found to range from 0.02 to 0.63 g/l/d (Chen et al., 2011), while the highest biomass productivity of 0.63 g/l/d was obtained from Neochlorisole oleoabundans under 5% CO2 and a high light intensity (Allen and Stanier, 1968; Li et al., 2008). The biomass productivities of Scenedesmus sp. have been reported as 0.218 g/l/d and 0.203 g/l/d under 10% CO2 and flue gas containing 5.5% CO2, respectively (Yoo et al., 2010). Plus, the biomass productivity of microalgae is generally below 0.1 g/l/d with a CO2 concentration over 10% (Brennan and Owende, 2010). CO2 is essential as a carbon source for photoautotrophic microalgae, yet very high CO2 generally inhibits the growth of microalgae. When compared with ambient air conditions (0.03% CO2), the biomass productivities of Chlorella, Scenedesmus, Botryococcus, and Haematococcus are reduced under a high CO2 concentration ranging from 10% to 18% (Brennan and Owende, 2010). In the case of Chlorella sp., the biomass productivity significantly decreases depending on the CO2 concentration ranging from 2% CO2 to 15% CO2 (Chiu et al., 2008). Therefore, based on these results, Ettlia sp. YC001 would seem to be a potential candidate for mitigating CO2 due to its fast-growing ability and high tolerance of CO2 up to 10%. Microalgae are photosynthetic organisms and their biomass originates from photosynthetic activity. The change in the DCW according to the chlorophyll-a concentration in the cells is plotted in Fig. 3. For all 3 cultures, the DCW according to the chlorophyll-a concentration decreased with the culture time, where the decrease was very steep during the initial 10 days and then became stable. Under all the test conditions, the accumulated biomass productivity consistently increased during the initial 10 days and then reached a plateau. Thus, it would seem that the activity of the chlorophyll-a in producing organic compounds encountered some limitation around this time, such as light intensity, nutrients, etc. Consequently, the optimal culture time of Ettlia sp. YC001 was
486
C. Yoo et al. / Bioresource Technology 127 (2013) 482–488
2.0
0.20
10% CO2 1.5
0.15 1.0
0.10
0.5
0.05
0.0
Accumulated biomass productivity (g/L/d)
Dry cell weight / chlorophyll-a ( 1,000)
0.25 Ambient air 5% CO2
0.00 0
2
4
6
8
10
12
14
16
Culture time (days) Fig. 3. Ratio of DCW to chlorophyll-a (closed symbols) and biomass productivity (open symbols) of Ettlia sp. YC001 cultivated under ambient air (d and s), 5% (. and 5), and 10% (j and h) CO2 for 16 days.
around 10 days for the highest biomass productivity and CO2 fixation. 3.4. Lipid content of Ettlia sp. YC001
3.5. Fatty acid composition of Ettlia sp. YC001
The total lipid content of Ettlia sp. YC001 showed an increasing tendency over a cultivation period of 16 days (Fig. 4). The lipid content of Ettlia sp. YC001 under ambient air increased from 44% on day 8 to 50% on day 16. Under 5% and 10% CO2 conditions, these values slightly decreased from 52% and 47% on day 8 to 42% and 43% on day 16, respectively. Thus, there was no statistically significant difference (p > 0.05) in the lipid content of Ettlia sp. YC001 according to the CO2 concentration on day 16. Conversely, the lipid productivity of Ettlia sp. YC001 under ambient air, 5%, and 10% CO2 was 49.5 mg/l/d, 72.6 mg/l/d, and 62.6 mg/l/d on day 8, respectively (Fig. 4), and then dramatically increased to 66.4 mg/L/d, 80.0 mg/L/d, and 77.0 mg/L/d on day 16, respectively, which was around 1.1–1.3 times higher than that under 5% and 10% CO2 on day 8. The highest lipid productivity obtained was 80.0 mg/L/d under 5% CO2 due to the high biomass productivity and lipid content. The lipid contents of microalgae vary from 1% to 70% of the DCW, and normally range from 20% to 50% (Amaro et al., 2011). Some Ettlia spp. were formerly classified as belonging to the Neochloris
120 Ambient air 5% CO2
100
10% CO2
40
80
30
60
20
40
10
20
0
Lipid productivity (mg/L/d)
Total lipid contents (% of dry cell weight)
60 50
0 Day 0
Day 8
genus, which is well known for the accumulation of large quantities of lipids (Deason et al., 1991; Pruvost et al., 2009). The lipid content of N. oleoabundans ranges from 35% to 54% and is composed of 80% tryglycerides (Tornabene et al., 1983). Large quantities of neutral lipids have also been synthesized and accumulated in the stationary phase and under unfavorable culture conditions, such as nutrient starvation and salinity (Hu et al., 2008). As shown in Fig. 3, the culture conditions in this study reached some limitation on day 10, indicating that a large amount of lipids was accumulated inside the cells after this time. The highest lipid productivity of 204 mg/L/d with an average biomass productivity of 0.30 g/L/d and over 60% lipid content under nitrogen derived media has been obtained from Nanochloropsis sp. F&M-M24, which is a marine microalgal species (Rodolfi et al., 2009). The highest lipid productivity obtained from Neochloris oleoabundans was 133 mg/L/d with 34% 5 mM sodium nitrate and 360 lmol photons/m2/s for 7 days (Li et al., 2008). However, the highest lipid productivity of N. oleoabundans resulted from a high biomass productivity of 0.4 g/L/d under a 3-fold higher light intensity than that used in the current study. Therefore, although the lipid productivity in this study was lower than the highest reported lipid productivity, it is expected that Ettlia sp. YC001 cultivated under real flue gas containing a high CO2 concentration could be applied to biodiesel production due to its high lipid productivity under 10% CO2.
Day 16
Culture time Fig. 4. Total lipid contents based on DCW (outside bar) and lipid productivity (inside bar) of Ettlia sp. YC001 cultivated under ambient air, 5%, and 10% CO2 on day 0, day 8, and day 16. Data are expressed as mean (average total lipid) ± SD (standard deviation).
The fatty acid profile of Ettlia sp. YC001 cultivated under different CO2 concentrations showed similar patterns on day 8 and 16, regardless of the CO2 concentration (Fig. 5). Among the identified fatty acids, the proportions of C16:0, C18:1, C18:2 and C18:3 were similar level, which ranged from 15% to 28% of total identified fatty acids on day 8. The proportions of C16:0 under ambient air, 5%, and 10% of CO2 on day 16 were increased up to 26%, 30%, and 29%, respectively. In particular, C18:1, which is a desirable component of biodiesel, was analyzed as a dominant fatty acid at 37% and 36% of the total identified fatty acids under 5% and 10% CO2 on day 16, respectively. The proportions of C18:1 on day 16 were significantly increased around 2 times than those in all conditions on day 8. No significant difference was found in the fatty acid composition among the different CO2 groups. The proportions of C16:0 and C18:1 increased significantly over the cultivation period from day 8 to day 16. Unidentified fatty acids occupied over 54% of the total fatty acids on day 8 and below 49% on day 16. Although the unidentified fatty acids were occupied around 50% of total fatty acids, several isomers of fatty acids existing between C16:0 and C18:3 were also detected and occupied below 5%, respectively. So, the total of these unidentified fatty acids was estimated over 30% of total fatty acid composition. Therefore, it was expected that total fatty acids to be converted into biodiesel were over 80% in these samples without adverse effects on quality of biodiesel. For the successful production of biodiesel from a microalgal biomass, the fatty acid composition of the total lipids is equally important as the amount of lipids produced (Ho et al., 2010). The biodiesel quality is considerably affected by the composition of fatty acid methyl- or ethyl-ester (FAME/FAEE) in the biodiesel (Knothe, 2008). An important factor that influences the lipid content and fatty acid composition during the batch cultivation of microalgae is the growth phase (Mansour et al., 2003). Monounsaturated fatty acids, such as C16:1 and C18:1, are very important fatty acids for biodiesel, as they apparently improve the quality of biodiesel due to their cetane number, cold-flow characteristics, oxidative stability, and low melting temperature (Knothe, 2008; Stansell et al., 2011). Here, C18:1 was the main component of Ettlia sp. YC001 and its proportion increased with the culture time. Therefore, Ettlia sp. YC001 would seem to be a good candidate for the production of
487
C. Yoo et al. / Bioresource Technology 127 (2013) 482–488
Fig. 5. Fatty acid profiles of Ettlia sp. YC001 cultivated under ambient air, 5%, and 10% CO2 on day 8 and day 16.
Table 2 Comparison of chlorophyll and carotenoid concentrations in green- and red-colored Ettlia sp. YC001. Samples
Green Red a
Chlorophyll (lg/g of DCW)
a
Carotenoids (lg/g DCW)a
Chlorophylla
Chlorophyllb
Total
2645.4±96.2 321.3±47.5
1065.5±57.0 171.2±14.2
4242.0±175.4 563.8±69.1
Lutein 1364.6±211.1 n.d.
Zeaxanthin n.d. n.d.
bCryptoxanthin
a-
n.d. n.d.
n.d. n.d.
b-Carotene
Ketocarotenoid
Total
362.4±36.8 n.d.
n.d. 2864.9±243.2
1727.0±247.9 2864.9±243.2
Carotene
Data are expressed as mean (average value of content for DCW) ± SD (standard deviation value) of two independent experiments.
high quality biodiesel due to its high lipid productivity and high proportion of C18:1.
3.6. Carotenoids of Ettlia sp. YC001 During 30 days of cultivation, the culture color of Ettlia sp. YC001 changed from green during the growth stage to red in the senescence stage. This phenomenon was apparently due to the loss of chlorophyll and accumulation of carotenoid inside the cells. As shown in Table 2, the concentrations of chlorophyll-a and -b were 2645.4 ± 96.2 lg/g of the DCW and 1065.5 ± 57.0 lg/g of the DCW, respectively, in the green-colored Ettlia sp. YC001 cultivated under 5% CO2 for 8 days. Conversely, in the red-colored Ettlia sp. YC001, these values were significantly lower at 321.3 ± 47.5 lg/g of the DCW and 171.2 ± 14.2 lg/g of the DCW, respectively. Thus, the total chlorophyll concentration in the red-colored Ettlia sp. YC001 was around 7.5 times lower than that in the green-colored Ettlia sp. YC001. The primary carotenoids lutein and b-carotene were both detected in the green-colored Ettlia sp. YC001 at 1364.6 ± 211.1 lg/g of the DCW and 362.4 ± 36.8 lg/g of the DCW, respectively. Although these carotenoids were not detected in the red-colored Ettlia sp. YC001, the total carotenoid concentration was 2864.9 ± 243.2 lg/g of the DCW. Thus, the total carotenoid concentration in the redcolored Ettlia sp. YC001 was 1.6 times higher than that in the green-colored Ettlia sp. YC001, which was due to the accumulation of red ketocarotenoid confirmed by using HPLC and chromatographic analysis. The total carotenoid concentration in Chlorella vulgaris is known to range from 1.7 mg/g DCW to 4.5 mg/g DCW under salinity stress and nitrogen starvation (Gouveia et al., 1996). Plus, the primary (lutein and b-carotene) and secondary carotenoids in Chlorella zofingiensis have been reported as below 1 mg/g of the DCW and 2.5 mg/g of the DCW, respectively, under oxidative stressed conditions (Ip and Chen, 2005). In another study, the concentrations of chlorophyll and primary carotenoids in C. zofingiensis significantly decreased after exposure to nitrogen and under light stress conditions, whereas the secondary carotenoids, such as astaxanthin, dramatically increased. The red ketocarotenoid
astaxanthin, which is well-known as a strong antioxidant, is largely accumulated under oxidative stress in order to protect the cells against oxidative stress (Ip and Chen, 2005). In this study, the significant accumulation of the red ketocarotenoid was likely to protect the cells from stressful conditions. Only a few microalgal species, such as Chlorella, Dunaliella, Haematococcus, and Spirulina, have been used for the commercial production of carotenoids and pigments. Haematococcus pluvialis and Dunaliella salina are also well-known as a rich natural source of astaxanthin and b-carotene, which account for 1–8% of their DCW and up to 10% of their DCW, respectively (Guerin et al., 2003; Prieto et al., 2011). Although the total carotenoid content in Ettlia sp. YC001 was lower than that in H. pluvialis and D. salina, it is expected that the production of biodiesel using Ettlia sp. YC001 will be accompanied by the production of carotenoids for commercialization. 4. Conclusion In the present study, Ettlia sp. showing a high growth rate and lipid content was successfully isolated from the environment. This strain could grow under a high concentration of CO2 with a high lipid productivity corresponding to the highest lipid productivity reported in previous studies. Among its fatty acids, C18:1 was increased through the culture period and under the various conditions. Plus, certain carotenoids were also produced from this strain. Therefore, the present results suggest that Ettlia sp. YC001 is a suitable strain for mitigating CO2 and producing biodiesel, and a potential candidate for applications producing high-value bioproducts. Acknowledgements This research was supported by a grant from the Advanced Biomass R&D Center, a Global Frontier Program, and the Carbon Dioxide Reduction & Sequestration Research Center, a 21st Century Frontier Program funded by the Korean Ministry of Education, Science & Technology. The authors would like to acknowledge
488
C. Yoo et al. / Bioresource Technology 127 (2013) 482–488
Mi-Jeong Ahn who analyzed the pigments and carotenoids in this study. References Allen, M.M., Stanier, R.Y., 1968. Growth and division of some unicellular blue–green algae. J. Gen. Microbiol. 51, 199–202. Amaro, H.M., Guedes, A.C., Malcata, F.X., 2011. Advances and perspectives in using microalgae to produce biodiesel. Appl. Energy 88, 3402–3410. Bligh, E.G., Dyer, W.J., 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37, 911–917. Brennan, L., Owende, P., 2010. Biofuels from microalgae—a review of technologies for production, processing, and extractions of biofuels and co-products. Renew. Sust. Energy Rev. 14, 557–577. Chen, C.Y., Yeh, K.L., Aisyah, R., Lee, D.J., Chang, J.S., 2011. Cultivation, photobioreactor design and harvesting of microalgae for biodiesel production: a critical review. Bioresour. Technol. 102, 71–81. Chi, Z., O’Fallon, J.V., Chen, S., 2011. Bicarbonate produced from carbon capture for algae culture. Trends Biotechnol. 29, 537–541. Chisti, Y., 2007. Biodiesel from microalgae. Biotechnol. Adv. 25, 294–306. Chiu, S.-Y., Kao, C.-Y., Chen, C.-H., Kuan, T.-C., Ong, S.-C., Lin, C.-S., 2008. Reduction of CO2 by a high-density culture of Chlorella sp. in a semicontinuous photobioreactor. Bioresour. Technol. 99, 3389–3396. De Morais, M.G., Costa, J.A.V., 2007. Isolation and selection of microalgae from coal fired thermoelectric power plant for biofixation of carbon dioxide. Energy Convers. Manage. 48, 2169–2173. Deason, T.R., Silva, P.C., Watanabe, S., Floyd, G.L., 1991. Taxonomic status of the species of the green algal genus Neochloris. Plant Syst. Evol. 177, 213–219. Gouveia, L., Veloso, V., Reis, A., Fernandes, H., Novais, J., Empis, J., 1996. Evolution of pigment composition in Chlorella vulgaris. Bioresour. Technol. 57, 157–163. Griffiths, M., Harrison, S., 2009. Lipid productivity as a key characteristic for choosing algal species for biodiesel production. J. Appl. Phycol. 21, 493–507. Guerin, M., Huntley, M.E., Olaizola, M., 2003. Haematococcus astaxanthin: applications for human health and nutrition. Trends Biotechnol. 21, 210–216. Ho, S.-H., Chen, W.-M., Chang, J.-S., 2010. Scenedesmus obliquus CNW-N as a potential candidate for CO2 mitigation and biodiesel production. Bioresour. Technol. 101, 8725–8730. Hu, Q., Sommerfeld, M., Jarvis, E., Ghirardi, M., Posewitz, M., Seibert, M., Darzins, A., 2008. Microalgal triacylglycerols as feedstocks for biofuel production: perspectives and advances. Plant J. 54, 621–639. Ip, P.F., Chen, F., 2005. Employment of reactive oxygen species to enhance astaxanthin formation in Chlorella zofingiensis in heterotrophic culture. Process Biochem. 40, 3491–3496. Khoo, H.H., Sharratt, P.N., Das, P., Balasubramanian, R.K., Naraharisetti, P.K., Shaik, S., 2011. Life cycle energy and CO2 analysis of microalgae-to-biodiesel: preliminary results and comparisons. Bioresour. Technol. 102, 5800–5807. Kim, S.H., Ahn, Y.O., Ahn, M.J., Lee, H.S., Kwak, S.S., 2012. Down-regulation of betacarotene hydroxylase increases beta-carotene and total carotenoids enhancing
salt stress tolerance in transgenic cultured cells of sweet potato. Phytochemistry 74, 69–78. Knothe, G., 2008. ‘‘Designer’’ biodiesel: optimizing fatty ester composition to improve fuel properties. Energy Fuels 22, 1358–1364. Kumar, A., Ergas, S., Yuan, X., Sahu, A., Zhang, Q., Dewulf, J., Malcata, F.X., van Langenhove, H., 2010. Enhanced CO2 fixation and biofuel production via microalgae: recent developments and future directions. Trends Biotechnol. 28, 371–380. Li, Y., Horsman, M., Wang, B., Wu, N., Lan, C., 2008. Effects of nitrogen sources on cell growth and lipid accumulation of green alga Neochloris oleoabundans. Appl. Microbiol. Biotechnol. 81, 629–636. Lv, J.-M., Cheng, L.-H., Xu, X.-H., Zhang, L., Chen, H.-L., 2010. Enhanced lipid production of Chlorella vulgaris by adjustment of cultivation conditions. Bioresour. Technol. 101, 6797–6804. Mansour, M.P., Volkman, J.K., Blackburn, S.I., 2003. The effect of growth phase on the lipid class, fatty acid and sterol composition in the marine dinoflagellate Gymnodinium sp. in batch culture. Phytochemistry 63, 145–153. Mata, T.M., Martins, A.A., Caetano, N.S., 2010. Microalgae for biodiesel production and other applications: a review. Renew. Sust. Energy Rev. 14, 217–232. Neustupa, J., Eliáš, M., Škaloud, P., Neˇmcová, Y., Šejnohová, L., 2011. Xylochloris irregularis gen. et sp. nov. (Trebouxiophyceae, Chlorophyta), a novel subaerial coccoid green alga. Phycologia 50, 57–66. Prieto, A., Pedro Cañavate, J., García-González, M., 2011. Assessment of carotenoid production by Dunaliella salina in different culture systems and operation regimes. J. Biotechnol. 151, 180–185. Pruvost, J., Van Vooren, G., Cogne, G., Legrand, J., 2009. Investigation of biomass and lipids production with Neochloris oleoabundans in photobioreactor. Bioresour. Technol. 100, 5988–5995. Pulz, O., Gross, W., 2004. Valuable products from biotechnology of microalgae. Appl. Microbiol. Biotechnol. 65, 635–648. Rittmann, B.E., 2008. Opportunities for renewable bioenergy using microorganisms. Biotechnol. Bioeng. 100, 203–212. Rodolfi, L., Chini Zittelli, G., Bassi, N., Padovani, G., Biondi, N., Bonini, G., Tredici, M.R., 2009. Microalgae for oil: strain selection, induction of lipid synthesis and outdoor mass cultivation in a low-cost photobioreactor. Biotechnol. Bioeng. 102, 100–112. Scott, S.A., Davey, M.P., Dennis, J.S., Horst, I., Howe, C.J., Lea-Smith, D.J., Smith, A.G., 2010. Biodiesel from algae: challenges and prospects. Curr. Opin. Biotechnol. 21, 277–286. Stansell, G.R., Gray, V.M., Sym, S.D., 2012. Microalgal fatty acid composition: implications for biodiesel quality. J. Appl. Phycol. 24, 791–801. Tornabene, T.G., Holzer, G., Lien, S., Burris, N., 1983. Lipid composition of the nitrogen starved green alga Neochloris oleoabundans. Enzyme Microb. Technol. 5, 435–440. Yoo, C., Jun, S.-Y., Lee, J.-Y., Ahn, C.-Y., Oh, H.-M., 2010. Selection of microalgae for lipid production under high levels carbon dioxide. Bioresour. Technol. 101, S71– S74. Zeng, X., Danquah, M.K., Chen, X.D., Lu, Y., 2011. Microalgae bioengineering: from CO2 fixation to biofuel production. Renew. Sust. Energy Rev. 15, 3252–3260.