Advanced
Screening and Characterization of Oleaginous Microalgal Species from Northern Xinjiang
Screening and Characterization of Oleaginous Microalgal Species from Northern Xinjiang
Journal of Microbiology and Biotechnology. 2015. Jun, 25(6): 910-917
Copyright © 2015, The Korean Society For Microbiology And Biotechnology
  • Received : November 28, 2014
  • Accepted : January 15, 2015
  • Published : June 28, 2015
Download
PDF
e-PUB
PubReader
PPT
Export by style
Article
Author
Metrics
Cited by
TagCloud
About the Authors
Lei Wu
University of Chinese Academy of Sciences, Beijing 100039, P.R. China
Liangliang Xu
University of Chinese Academy of Sciences, Beijing 100039, P.R. China
Chunxiang Hu
Key Laboratory of Algal Biology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, P.R. China
cxhu@ihb.ac.cn

Abstract
A total of 646 strains, including green algae and diatoms, were isolated from 220 samples to screen microalgae with high lipid productivity (LP). The samples were obtained from nine habitats in Northern Xinjiang, China in June 2013. This study initially identified eight lipidrich strains, namely, Desmodesmus intermedius XJ-498, D. intermedius XJ-145, D. intermedius XJ-99, Monoraphidium pusillum XJ-489, M. dybowskii XJ-435, M. dybowskii XJ-151, Mychonastes homosphaera XJ-488, and Podohedriella falcata XJ-176, based on 18S rDNA sequencing. The strains were cultured in a photobioreactor for the same period. Results showed that the specific growth rate (day -1 ) of M. pusillum XJ-489 was the highest (1.14 ± 0.06), and the biomass concentration (g/l) of D. intermedius XJ-99 was the highest (2.84 ± 0.3). Futhermore, the lipid content (%) of M. dybowskii XJ-151 was the highest (33.5 ± 4.38), and the lipid productivity (mg l -1 day -1 ) of My. homosphaera XJ-488 was the highest (86.41 ± 9.04). C16 to C18 accounted for 86% to 98% of the total lipid, and the biodiesel qualities of the selected algae corresponded to international standards. This study suggests that My. homosphaera XJ-488, D. intermedius XJ-99, and M. dybowskii XJ-151 are the most potential strains for biodiesel production among all the isolated strains.
Keywords
Introduction
With the increasing energy crisis and deteriorating environment induced by using fossil energy, exploring renewable, environmentally friendly, and economically viable alternative fuels has become an important strategic direction of world energy [20] . Third-generation biodiesel feedstocks derived from microalgae have emerged as one of the most promising alternative sources [2] . With the deep understanding of microalgae, the problem of microalgae germplasm resources is becoming increasingly prominent; algal species almost become the main factors that restrict the development of the bioenergy industry [7 , 23] . Therefore, screening for indigenous oleaginous microalgae with a rapid growth rate and high lipid productivity (LP) is the key to support this technique [24] .
Good strains are not only known to meet the requirement of high lipid content (LC), high biomass concentration, and ease in harvesting, but also known to have strong environmental tolerance and good biodiesel quality [10 , 11 , 22] .
Xinjiang Uygur Autonomous Region is the largest province of China. This region lies in the northwest part of China, and is the center of the Eurasian continent. Given its location in the inland and mountain barrier surrounding the area, the region has to a typical temperate continental arid climate. Considering the vast territory, complicated terrain, and different climate along with different heights and longitudes, Northern Xinjiang was chosen as the study region. Different types of water (lakes, rivers, ditches, ponds, and reservoirs) and soil (deserts, meadows, farmlands, and shallows) samples were collected to obtain sufficient representative samples and more excellent strains. After separation and purification of microalgae, the biomass concerntration and lipid accumulation of the purified strains were immediately examined. Strains with LP higher than 40 mg l -1 day -1 were identified by 18S rDNA sequence analysis. This study also analyzed the fatty acid (FA) profiles and biodiesel quality of the selected algae.
Materials and Methods
- Survey Region and Sampling
Northern Xinjiang, including the Junggar Basin, lies in the north of the Tianshan Mountain and south of the Altai Mountain, with a total area of more than 60 km 2 . Snowmelt from the mountain forms several continent rivers, flowing into depressions of the basin, except the Eltrix River, which flows into the Arctic Ocean. The altitude of the territory is variable, with an average altitude of less than 500 m, and Aydingkol Lake has the lowest altitude (161 m below sea level). The central part of the basin is the Gurbantunggut Desert, which is the second largest desert in China. The majority of this area is fixed or semi-fixed sand dunes. The western basin is the Ili area, with more mountains than plains and cold temperate semi-arid continental climate.
In this study, a total of 220 algae samples (185 water samples and 35 soil samples) were collected from nine habitats. The samples in the same habitat were collected at a distance of at least 15 km. The location, pH, temperature, soil property, and vegetation condition of the sampling site were also recorded.
- Isolation and Purification of Microalgae
BG11 medium was used to incubate freshwater microalgae and most soil microalgae. F/2 medium was used to incubate saline microalgae. Agar plates and micropipettes were employed to isolate algae strains. Each purified strain was initiallycultured in a test tube (30 ml) at 25 ± 1℃ under continuous illumination using white fluorescent light at an intensity of 70 µmol s -1 m -2 for 1 to 3 weeks. The strains were then incubated into a 100 ml flask with sterile air at 25 ± 1℃ under an intensity of 80 µmol s -1 m -2 for 10 days.
- Identification by 18S rDNA Sequence
Microalgae strains were initially identified with a microscope and then confirmed by using 18S rDNA sequence analysis. Algae were harvested in the exponential growth phase by centrifugation (4,000 × g , 10 min), and the cells were completely ground by liquid nitrogen. Total DNA was initially extracted using a Plant Genomic DNA kit, analyzed by electrophoresis in 1% agarose gel to confirm the presence and concentration of products, and then used for PCR amplification (Bio-Rad Thermal Cycler, USA). Amplification reactions used the following universal eukaryotic primers: forward, 5’-TTCGGCTAGGGATAGGCTTG-3’; and reverse, 5’-TTTGAT TTCTCATAAGGTGC-3’. The PCR products were analyzed by electrophoresis in 1% agarose gel and sent to the sequencing facility (WuHan Tingke Biotech Co., Ltd., China) for the corresponding analysis. 18S rDNA sequences of the isolates were searched against GenBank using BLAST for homologous analysis, according to similarities by the Clustal W program. Phylogenetic trees were constructed using the neighbor-joining method from the Molecular Evolutionary Genetics Analysis ver. 5 software after 1,000 bootstrap replicates.
- Microalgal Cultivation
The capacity of growth and lipid production of strains was investigated. Eight microalgae strains were selected and cultivated in a column bioreactor (30 mm × 600 mm), containing 250 ml of BG11 medium with aeration by sterile air, under a 14 h:10 h light:dark photoperiod of white fluorescent light (100 ± 2 µmol m -2 s -1 ) at 25 ± 1℃ for 10 days. The algal density was determined by measuring the OD 680 (the optical density of algal at 680 nm) with TU-1900 UV spectroscopy.
The cells were harvested by centrifugation (8,000 × g , 5 min) and lyophilized using a vacuum freeze dryer (Alpha 1-2 LD plus; Christ). Each experiment was conducted in triplicate.
- Lipid Analysis
The total lipid was extracted from approximately 50 to 100 mg of dried algae (W 0 ) using a Soxhlet apparatus, with chloroform–methanol (1:2 (v/v)) as the solvent at 90℃ for 4 to 6 h [5 , 15] . The total lipid was transferred into a pre-weighed grass dish (V 1 ), dried to a constant weight in an oven at 65℃, and weighed (V 2 ).
The lipid content (LC, %) and the lipid productivity (LP, mg l -1 day -1 ) were determined according to Eqs. (1) to (2):
PPT Slide
Lager Image
PPT Slide
Lager Image
where BP represents the biomass productivity (mg l -1 day -1 ).
- Analysis of Fatty Acid Profiles
The methanolysis of FAs was performed with 1 M H 2 SO 4 -CH 3 OH at 100℃ for 1 h using the modified Davila method [6] . FA components of extracted lipid were analyzed by gas chromatography mass spectrometry (Ultra trace, Thermo Scientific ITQ 700, USA) equipped with a fused silica capillary column (60 mm × 0.25 mm × 0.25 µm; Agilent Technologies, USA) and a flame ionization detector (FID). The initial temperature was maintained at 50℃ for 1 min and then increased to 170℃ at 40℃/min. The oven temperature was raised from 170℃ to 210℃ at 18℃/min after a 1 min hold. The injector temperature was 270℃, and nitrogen was used as a carrier gas with a flow rate of 2.0 ml/min. The detector temperature was 280℃, and air and hydrogen flows were set at 350 and 35 ml/min, respectively. All the parameters of FA methyl ester (FAME) were derived from the calibration curves generated from the FAME standard mix (Supelco 37 Component FAME Mix; Sigma-Aldrich, USA).
- Estimation of Biodiesel Properties
In recent years, studies have focused on estimating the quality parameters of biodiesel according to the FA components [13 , 19] , and the detailed calculation [21] is as follows:
PPT Slide
Lager Image
where ADU is the average degree of unsaturation of microalgal oil; M is the number of carbon–carbon double bonds in each FA component; and Yi is the mass fraction of each FA component.
The relationships between ADU and other biodiesel properties, namely, kinematic viscosity (KV), specific gravity (SG), cloud point (CP), cetane number (CN), iodine value (IV), and higher heating value (HHV) as well as the cold filter plug point (CFPP) and long-chain saturated factor (LCSF) were all determined by empirical equations from FA composition as described previously [9 , 19] .
- Statistical Analysis
The data were analyzed by one-way ANOVA and cluster analysis using SPSS statistical software (ver. 19.0). P < 0.05 denotes a statistically significant difference. The values were expressed as the mean ± standard deviation.
Results and Discussion
- Isolation and Identification
Separation of algal strains is a necessary prerequisite for screening [8] . A total of 646 algal strains, including green algae and diatoms, were initially isolated from 220 samples in Northern Xinjiang and then cultivated in 100 ml flasks. Eight cultured microalgae with LP higher than 40 mg l -1 day -1 were selected for further study ( Table 1 ).
Habitat information of eight microalgal strains.
PPT Slide
Lager Image
“E” and “N” represent east longitude and north latitude, respectively.
After the morphological analysis, phylogenetic trees were created according to the BLAST analysis of corresponding sequences ( Fig. 1 ). The eight microalgae strains were respectively named as Desmodesmus intermedius XJ-498, D. intermedius XJ-145, D. intermedius XJ-99, Monoraphidium pusillum XJ-489, M. dybowskii XJ-435, M. dybowskii XJ-151, Mychonastes homosphaera XJ-488, and Podohedriella falcata XJ-176.
PPT Slide
Lager Image
Phylogenetic tree of the 18S rDNA of eight microalgal strains. The topology of this tree was constructed using the neighbor-joining method after 1,000 rounds of bootstrap resampling. The tested algae are indicated in bold font.
- Growth Characteristics
The eight strains were cultured in a column photobioreactor for 10 days. Comparison between the specific growth rates ( Table 2 ) showed that two strains from saline environment were lower than the freshwater strains. The specific growth rate (day -1 ) of M. pusillum XJ- 489 was the highest (1.14 ± 0.06), followed by P. falcata XJ-176 (1.08 ± 0.16), which was higher in previous reports [11 , 21] . The specific growth rate of M. dybowskii XJ-435 was the lowest and significantly lower than the other strains ( p < 0.05). The biological growth potential of M. pusillum XJ-489 and P. falcata XJ-176 was great, whereas the growth of the two algae strains from saline environment was unsatisfactory. This result suggests that the medium conditions were inappropriate and need further study.
Comparison of biomass and lipid productivity of some microalgae in different cultivated conditions.
PPT Slide
Lager Image
µ, the special growth rate; BC, biomass concentration; BP, biomass productivity; LC, lipid content; LP, lipid productivity.
D. intermedius XJ-99 showed the highest biomass concentration (2.84 ± 0.3 g/l) ( Table 2 ), which was higher than the reported Desmodesmus sp. WC08 [23] ( Table 2 ); except for M. dybowskii XJ-435, the biomass concentrations of other strains were over 2 g/l. This result indicates that the algae from the common fresh water grew faster than those from the stress environment when cultured under the present conditions.
- Lipid Content and Lipid Productivity
After being cultured, the total lipid contents of the strains ranged from 22% to 33% ( Table 2 ). The LC (%) of M. dybowskii XJ-151 was the highest (33.5 ± 4.38), followed by M. dybowskii XJ-435 (33.2 ± 0.57), which was higher than the literature data for M. dybowskii [3] ( Table 2 ).
Comparison of the lipid productivity (mg l -1 day -1 ) of the eight tested strains ( Table 2 ) showed that D. intermedius XJ-99 was the highest (86.41 ± 9.04), followed by My. homosphaera XJ-488 (85.58 ± 6.24). The LP (mg l -1 day -1 ) of D. intermedius XJ-498 (52.56 ± 13.34) and D. intermedius XJ-145 (52.35 ± 4.88) was the lowest. Compared with previous reports, the strains were higher than Scenedesmus sp. [1 , 19] , but lower than Desmodesmus sp. WC08 [23] ( Table 2 ). Consistent with previous reports, several strains with high LC presented low biomass concentration. Although no necessary connection was found between biomass concentration and LC [12 , 19] , this phenomenon still affected the application prospect of these strains. In terms of LP, My. homosphaera XJ-488 and D. intermedius XJ-99 were the more promising strains for lipid production. In terms of resisting to pollution from the outdoor culture, D. intermedius XJ-498 and M. dybowskii XJ-435 may have potential, but these saline strains still need further study.
- Fatty Acid Profiles and Biodiesel Properties
Screening of better oleaginous microalgae should not only consider the LP, but also the composition and quality attribute of FAs for biodiesel production [14] . The main components of biodiesel are saturated fatty acids (SFA) and monounsaturated fatty acids (MUFA) [4] . The major FA compositions of green algae are C16 to C18, which are suitable for biodiesel production [16] . The results ( Table 3 ) showed that the overall FA compositions were similar, where the main components of FA were C16 and C18. The contents of C16 to C18 were 86% to 98%, whereas the longchain FAs above C20 exhibited low quantities. By comparison, SFAs of M. dybowskii XJ-435 and D. intermedius XJ-99 showed the highest proportion (41.09% and 41.94%, respectively). Polyunsaturated fatty acid (PUFA) contents among them were different; the highest was 37.95% ( D. intermedius XJ-498) and the lowest was only 9.81% ( D. intermedius XJ-99). D. intermedius XJ-498 exhibited the lowest C18:1 content (18.15%), but had the highest C18:3 content (18.18%). However, C18 FA contents composed of PUFAs in algal oils were much less than in vegetable oils [18] . The aforementioned results showed that the FA compositions of the eight strains were suitable for biodiesel production.
Fatty acid profiles of eight microalgal strains (% of total FAMEs).
PPT Slide
Lager Image
XJ-498, Desmodesmus intermedius XJ-498; XJ-145, Desmodesmus intermedius XJ-145; XJ-99, Desmodesmus intermedius XJ-99; XJ-435, Monoraphidium dybowskii XJ-435; XJ-151, Monoraphidium dybowskii XJ-151; XJ-489, Monoraphidium pusillum XJ-489; XJ-488, Mychonastes homosphaera XJ-488; XJ-176, Podohedriella falcata XJ-176.
Based on FA compositions, this study also estimated several important qualities of the strains as biodiesel ( Table 4 ). According to the present knowledge, with increasing values of ADU and CN, its oxidation stability is lower; and the high IV will be easily oxidized to form sediments, affecting its lubrication [19] . Thus, these properties are particularly important. In terms of the two common quality standards for biodiesel, namely, ASTM D6751 in the US and EN 14214 in Europe, all eight strains satisfied the standards. Given that their CFPP was below -15℃, the strains have better low-temperature fluidity [17] . Futhermore, the SFAs and MUFAs of these strains accounted for a high proportion, resulting in better diesel oxidation stability.
Comparison of biodiesel properties between the selected strains, and ASTM biodiesel and EN biodiesel standards.
PPT Slide
Lager Image
ADU, average degree of unsaturation; KV, kinematic viscosity; SG, specific gravity; CP, cloud point; CN, cetane number; IV, iodine value; HHV, higher heating value; CFPP, the cold filter plug point; LCSF, long-chain saturated factor.
This research combined LP and biodiesel properties by cluster analysis to comprehensively evaluate the potential of the eight strains for biodiesel production [9 , 19] . The results showed that My. homosphaera XJ-488, D. intermedius XJ-99, and M. dybowskii XJ-151 could be classified into one category ( Fig. 2 ), because of their high LPs and similar biodiesel qualities. Therefore, these strains are the most promising for further study.
PPT Slide
Lager Image
Cluster analysis comparing the variation of microalgae lipid productivity and biodiesel properties.
In conclusion, among the 646 strains (543 strains from water samples and 103 strains from soil samples), eight strains with high LP were selected from the aquatic environment. After being cultured in a photobioreactor, M. pusillum XJ-489 reached the highest specific growth rate (1.14 ± 0.06 day -1 ), D. intermedius XJ-99 reached the highest biomass concerntration (2.84 ± 0.3 g/l), M. dybowskii XJ-151 reached the highest LC (33.5 ± 4.38%), and My. homosphaera XJ-488 reached the highest LP (86.41 ± 9.04 mg l -1 day -1 ). The C16 to C18 contents of the eight strains were over 86%, and the biodiesel qualities of the selected algae corresponded to international standards. Considering their LP and biodiesel quality, this study suggests that My. homosphaera XJ-488, D. intermedius XJ-99, and M. dybowskii XJ-151 are the most potential strains for biodiesel production.
Prior to large-scale application, finding more effective ways of cultivation and evaluating the ability of the strains to resist pollution are also necessary. D. intermedius XJ-498 from a saline environment and M. dybowskii XJ-435 with better growth potential need to be further investigated as promising strains.
Acknowledgements
This work benefited from the support from the Ministry of Science and Technology of China (2013AA065804, 2012FY112900), and we thank Dr. Shubin Lan, Dr. Qingyi Zhang, and Dr. Hailong Ouyang for their sampling work support in the field.
References
Abou-Shanab RAI , Matter IA , Kim S-N , Oh Y-K , Choi J , Jeon B-H 2011 Characterization and identification of lipidproducing microalgae species isolated from a freshwater lake. Biomass Bioenergy 35 3079 - 3085    DOI : 10.1016/j.biombioe.2011.04.021
Ahmad AL , Yasin NHM , Derek CJC , Lim JK 2011 Microalgae as a sustainable energy source for biodiesel production: a review. Renew. Sust. Energy Rev. 15 584 - 593    DOI : 10.1016/j.rser.2010.09.018
Bogen C , Klassen V , Wichmann J , La Russa M , Doebbe A , Grundmann M 2013 Identification ofMonoraphidium contortumas a promising species for liquid biofuel production. Bioresour. Technol. 133 622 - 626    DOI : 10.1016/j.biortech.2013.01.164
Borges L , Morón-Villarreyes JA , D’Oca MGM , Abreu PC 2011 Effects of flocculants on lipid extraction and fatty acid composition of the microalgaeNannochloropsis oculataandThalassiosira weissflogii. Biomass Bioenergy 35 4449 - 4454    DOI : 10.1016/j.biombioe.2011.09.003
Cheung PCK , Leung AYH , Ang PO 1998 Comparison of supercritical carbon dioxide and soxhlet extraction of lipids from a brown seaweed,Sargassum hemiphyllum(Turn.) C. Ag. J. Agric. Food Chem. 46 4228 - 4232    DOI : 10.1021/jf980346h
Davila AM , Marchal R , Monin N , Vandecasteele JP 1993 Identification and determination of individual sophorolipids in fermentation products by gradient elution high-performance liquid-chromatography with evaporative light-scattering detection. J. Chromatogr. 648 139 - 149    DOI : 10.1016/0021-9673(93)83295-4
Doan TTY , Sivaloganathan B , Obbard JP 2011 Screening of marine microalgae for biodiesel feedstock. Biomass Bioenergy 35 2534 - 2544    DOI : 10.1016/j.biombioe.2011.02.021
Duong VT , Li Y , Nowak E , Schenk PM 2012 Microalgae isolation and selection for prospective biodiesel production. Energies 5 1835 - 1849    DOI : 10.3390/en5061835
Francisco EC , Neves DB , Jacob-Lopes E , Franco TT 2010 Microalgae as feedstock for biodiesel production: carbon dioxide sequestration, lipid production and biofuel quality. J. Chem. Technol. Biotechnol. 85 395 - 403    DOI : 10.1002/jctb.2338
Griffiths MJ , Harrison STL 2009 Lipid productivity as a key characteristic for choosing algal species for biodiesel production. J. Appl. Phycol. 21 493 - 507    DOI : 10.1007/s10811-008-9392-7
Griffiths MJ , Hille RP , Harrison STL 2011 Lipid productivity, settling potential and fatty acid profile of 11 microalgal species grown under nitrogen replete and limited conditions. J. Appl. Phycol. 24 989 - 1001    DOI : 10.1007/s10811-011-9723-y
Hempel N , Petrick I , Behrendt F 2012 Biomass productivity and productivity of fatty acids and amino acids of microalgae strains as key characteristics of suitability for biodiesel production. J. Appl. Phycol. 24 1407 - 1418    DOI : 10.1007/s10811-012-9795-3
Hoekman SK , Broch A , Robbins C , Ceniceros E , Natarajan M 2012 Review of biodiesel composition, properties, and specifications. Renew. Sust. Energy Rev. 16 143 - 169    DOI : 10.1016/j.rser.2011.07.143
Hong JW 2013 Isolation of a Korean domestic microalga,Chlamydomonas reinhardtiiKNUA021, and analysis of its biotechnological potential. J. Microbiol. Biotechnol. 23 375 - 381    DOI : 10.4014/jmb.1111.11073
Hsieh CH , Wu WT 2009 Cultivation of microalgae for oil production with a cultivation strategy of urea limitation. Bioresour. Technol. 100 3921 - 3926    DOI : 10.1016/j.biortech.2009.03.019
Knothe G 2008 “Designer” biodiesel: optimizing fatty ester composition to improve fuel properties. Energy Fuels 22 1358 - 1364    DOI : 10.1021/ef700639e
Knothe G 2009 Improving biodiesel fuel properties by modifying fatty ester composition. Energy Environ. Sci. 2 759 - 766    DOI : 10.1039/b903941d
Knothe G 2011 A technical evaluation of biodiesel from vegetable oils vs. algae. Will algae-derived biodiesel perform? Green Chem. 13 3048 - 3065    DOI : 10.1039/c0gc00946f
Nascimento IA , Marques SSI , Cabanelas ITD , Pereira SA , Druzian JI , de Souza CO 2013 Screening microalgae strains for biodiesel production: lipid productivity and estimation of fuel quality based on fatty acids profiles as selective criteria. Bioenerg. Res. 6 1 - 13    DOI : 10.1007/s12155-012-9222-2
Rodolfi L , Chini Zittelli G , Bassi N , Padovani G , Biondi N , Bonini G , Tredici MR 2009 Microalgae for oil: strain selection, induction of lipid synthesis and outdoor mass cultivation in a low-cost photobioreactor. Biotechnol. Bioeng. 102 100 - 112    DOI : 10.1002/bit.22033
Song M , Pei H , Hu W , Ma G 2013 Evaluation of the potential of 10 microalgal strains for biodiesel production. Bioresour. Technol. 141 245 - 251    DOI : 10.1016/j.biortech.2013.02.024
Xie T , Liu J , Du K , Liang B , Zhang Y 2013 Enhanced biofuel production from high-concentration bioethanol wastewater by a newly isolated heterotrophic microalga,Chlorella vulgarisLAM-Q. J. Microbiol. Biotechnol. 23 1460 - 1471    DOI : 10.4014/jmb.1301.01046
Zhang S , Liu P-H , Yang X , Hao Z-D , Zhang L , Luo N , Shi J 2014 Isolation and identification by 18S rDNA sequence of high lipid potential microalgal species for fuel production in Hainan Dao. Biomass Bioenergy 66 197 - 203    DOI : 10.1016/j.biombioe.2014.01.015
Zheng Y , Yuan C , Liu J , Hu G , Li F 2014 Lipid production by a CO2-tolerant green microalga,Chlorellasp sp. MRA-1. J. Microbiol. Biotechnol. 24 683 - 689    DOI : 10.4014/jmb.1308.08050