Mixotrophic cultivation, a preferable microalgae cultivation mode for biomass/bioenergy production, and bioremediation, advances and prospect

Abstract Microalgae have received much attention in recent years as a feedstock for producing renewable fuels. Microalgae cultivation technology is one of the main factors restricting biomass production as well as energy fuel production and bioremediation. There are four types of cultivation conditions for microalgae: photoautotrophic, heterotrophic, mixotrophic and photoheterotrophic cultivation. Though photoautotrophic and heterotrophic cultivation are two common growth modes of microalgae, some microalgae can also grow better under mixotrophic condition, which may combine the advantages of autotrophic and heterotrophic and overcome the disadvantages. This review compared these growth modes of microalgae and discussed the advantages of mixotrophic mode in bioenergy production by considering the difference in growth, photosynthesis characteristic and bioenergy production. Also, the influence factors of mixotrophic cultivation and the application of mixotrophic microalgae in bioremediation are discussed, laying theoretical foundation for large scale microalgae cultivating for biomass production, bioenergy production and environmental protection.

[1]  Si-yuan Guo,et al.  Growth and phycocyanin formation of Spirulina platensis in photoheterotrophic culture , 1996, Biotechnology Letters.

[2]  M. Mimuro,et al.  Photosynthetic hydrogen production , 2010 .

[3]  B. Rym,et al.  Modeling growth and photosynthetic response in Arthrospira platensis as function of light intensity and glucose concentration using factorial design , 2010, Journal of Applied Phycology.

[4]  C. Picioreanu,et al.  A mass-spring model unveils the morphogenesis of phototrophic Diatoma biofilms , 2014, Scientific Reports.

[5]  M. Ghirardi,et al.  A comparison of hydrogen photoproduction by sulfur-deprived Chlamydomonas reinhardtii under different growth conditions. , 2007, Journal of biotechnology.

[6]  M. Ghirardi,et al.  Maximizing the Hydrogen Photoproduction Yields in Chlamydomonas Reinhardtii Cultures: The Effect of the H2 Partial Pressure , 2012 .

[7]  C. Popovich,et al.  Photosynthetic aspects and lipid profiles in the mixotrophic alga Neochloris oleoabundans as useful parameters for biodiesel production , 2016 .

[8]  Guangce Wang,et al.  Immobilization of Chlorella sorokiniana GXNN 01 in alginate for removal of N and P from synthetic wastewater. , 2012, Bioresource technology.

[9]  Paul Chen,et al.  Integration of algae cultivation as biodiesel production feedstock with municipal wastewater treatment: strains screening and significance evaluation of environmental factors. , 2011, Bioresource technology.

[10]  F. Kargı,et al.  Bio-hydrogen production from waste materials , 2006 .

[11]  Paul Chen,et al.  Influence of Exogenous CO2 on Biomass and Lipid Accumulation of Microalgae Auxenochlorella protothecoides Cultivated in Concentrated Municipal Wastewater , 2012, Applied Biochemistry and Biotechnology.

[12]  Juan Chen,et al.  The reduced state of the plastoquinone pool is required for chloroplast-mediated stomatal closure in response to calcium stimulation. , 2016, The Plant journal : for cell and molecular biology.

[13]  Jane-Yii Wu,et al.  Effect of carbon sources on growth and lipid accumulation of newly isolated microalgae cultured under mixotrophic condition. , 2015, Bioresource technology.

[14]  H. Endo,et al.  Studies on Chlorella regularis, heterotrophic fast-growing strain II. Mixotrophic growth in relation to light intensity and acetate concentration , 1977 .

[15]  M. Ghirardi,et al.  Microalgae: a green source of renewable H(2). , 2000, Trends in biotechnology.

[16]  Jack Legrand,et al.  Investigation of H2 production using the green microalga Chlamydomonas reinhardtii in a fully controlled photobioreactor fitted with on-line gas analysis , 2008 .

[17]  M. Kawachi,et al.  Effects of carbon source on growth and morphology of Botryococcus braunii , 2011, Journal of Applied Phycology.

[18]  K. Chojnacka,et al.  Trace element removal by Spirulina sp. from copper smelter and refinery effluents , 2004 .

[19]  Y. Chisti Biodiesel from microalgae. , 2007, Biotechnology advances.

[20]  S. Yoshikawa,et al.  Spatiotemporal distribution of bacteriochlorophylls in the meromictic Lake Suigetsu, Japan , 2013, Limnology.

[21]  Jo-Shu Chang,et al.  Effects of cultivation conditions and media composition on cell growth and lipid productivity of indigenous microalga Chlorella vulgaris ESP-31. , 2012, Bioresource technology.

[22]  O. Kruse,et al.  Microalgae as substrates for fermentative biogas production in a combined biorefinery concept. , 2010, Journal of biotechnology.

[23]  A. Scoma,et al.  Interplay between light intensity, chlorophyll concentration and culture mixing on the hydrogen production in sulfur‐deprived Chlamydomonas reinhardtii cultures grown in laboratory photobioreactors , 2009, Biotechnology and bioengineering.

[24]  Joel L. Cuello,et al.  Effects of implementing PSI-light on hydrogen production via biophotolysis in Chlamydomonas reinhardtii mutant strains , 2013 .

[25]  J. Doucha,et al.  Utilization of flue gas for cultivation of microalgae Chlorella sp.) in an outdoor open thin-layer photobioreactor , 2005, Journal of Applied Phycology.

[26]  M. Mandal,et al.  Mixotrophic cultivation of Chlorella sp. BTA 9031 and Chlamydomonas sp. BTA 9032 isolated from coal field using various carbon sources for biodiesel production , 2016 .

[27]  Ben Hankamer,et al.  Challenges and opportunities for hydrogen production from microalgae , 2016, Plant biotechnology journal.

[28]  Matthew R Melnicki,et al.  Integrated biological hydrogen production , 2006 .

[29]  Qiang Wang,et al.  Current Status and Outlook in the Application of Microalgae in Biodiesel Production and Environmental Protection , 2014, Front. Energy Res..

[30]  J. Cárdenas,et al.  The nitrate-reducing enzyme system of Chlamydomonas reinhardii , 1975, Archives of Microbiology.

[31]  K. Abe,et al.  Removal of nitrate, nitrite, ammonium and phosphate ions from water by the aerial microalga Trentepohlia aurea , 2002, Journal of Applied Phycology.

[32]  Olaf Kruse,et al.  RNAi Knock-Down of LHCBM1, 2 and 3 Increases Photosynthetic H2 Production Efficiency of the Green Alga Chlamydomonas reinhardtii , 2013, PloS one.

[33]  Rao Y. Surampalli,et al.  Biodiesel production from heterotrophic microalgae through transesterification and nanotechnology application in the production , 2013 .

[34]  Ken Sasaki,et al.  Growth characteristics of Spirulina platensis in mixotrophic and heterotrophic conditions , 1993 .

[35]  S. Oncel,et al.  “Effect of light intensity and the light: dark cycles on the long term hydrogen production of Chlamydomonas reinhardtii by batch cultures” , 2011 .

[36]  A. Tsygankov,et al.  The effect of light intensity on hydrogen production by sulfur-deprived Chlamydomonas reinhardtii. , 2004, Journal of biotechnology.

[37]  Philip Owende,et al.  Biofuels from microalgae—A review of technologies for production, processing, and extractions of biofuels and co-products , 2010 .

[38]  M. Gibbs,et al.  Fermentative Metabolism of Chlamydomonas reinhardtii: I. Analysis of Fermentative Products from Starch in Dark and Light. , 1984, Plant physiology.

[39]  Lipid production for biofuels from hydrolyzate of waste activated sludge by heterotrophic Chlorella protothecoides. , 2013, Bioresource technology.

[40]  Seeram Ramakrishna,et al.  Hydrogen photoproduction by use of photosynthetic organisms and biomimetic systems , 2009, Photochemical & photobiological sciences : Official journal of the European Photochemistry Association and the European Society for Photobiology.

[41]  W. Cong,et al.  Growth and Physiological Features of Cyanobacterium Anabaena sp. Strain PCC 7120 in a Glucose-Mixotrophic Culture , 2011 .

[42]  Michael Seibert,et al.  Effects of extracellular pH on the metabolic pathways in sulfur-deprived, H2-producing Chlamydomonas reinhardtii cultures. , 2003, Plant & cell physiology.

[43]  T. Keskin,et al.  Photofermentative hydrogen production from wastes. , 2011, Bioresource technology.

[44]  N. T. Eriksen,et al.  Mixotrophic continuous flow cultivation of Chlorella protothecoides for lipids. , 2013, Bioresource technology.

[45]  V. Himabindu,et al.  Mixotrophic Cultivation of Botryococcus Braunii for Biomass and Lipid Yields with Simultaneous CO2 Sequestration , 2014 .

[46]  H. Kaltwasser,et al.  Photoproduction of hydrogen by photosystem I of Scenedesmus , 2004, Planta.

[47]  Lu Zhang,et al.  Sustained photobiological hydrogen gas production upon reversible inactivation of oxygen evolution in the green alga Chlamydomonas reinhardtii. , 2000, Plant physiology.

[48]  M. Seibert,et al.  Hydrogen photoproduction by nutrient‐deprived Chlamydomonas reinhardtii cells immobilized within thin alginate films under aerobic and anaerobic conditions , 2009, Biotechnology and bioengineering.

[49]  Jack Legrand,et al.  Autotrophic and Mixotrophic Hydrogen Photoproduction in Sulfur-Deprived Chlamydomonas Cells , 2005, Applied and Environmental Microbiology.

[50]  Hui Chen,et al.  Nitrogen Starvation Induced Oxidative Stress in an Oil-Producing Green Alga Chlorella sorokiniana C3 , 2013, PloS one.

[51]  Arthur R. Grossman,et al.  Anaerobic Acclimation in Chlamydomonas reinhardtii , 2007, Journal of Biological Chemistry.

[52]  R. Jennings,et al.  Biogenesis of chloroplast membranes: XIV. Inhomogeneity of membrane protein distribution in photosystem particles obtained from Chlamydomonas reinhardi. Y-l , 1973 .

[53]  B. Cheirsilp,et al.  Enhanced growth and lipid production of microalgae under mixotrophic culture condition: effect of light intensity, glucose concentration and fed-batch cultivation. , 2012, Bioresource technology.

[54]  K. C. Das,et al.  Biomass and bioenergy production potential of microalgae consortium in open and closed bioreactors using untreated carpet industry effluent as growth medium. , 2010, Bioresource technology.

[55]  Li-Hua Cheng,et al.  Enhanced lipid production of Chlorella vulgaris by adjustment of cultivation conditions. , 2010, Bioresource technology.

[56]  Jo‐Shu Chang,et al.  pH-stat photoheterotrophic cultivation of indigenous Chlorella vulgaris ESP-31 for biomass and lipid production using acetic acid as the carbon source , 2012 .

[57]  C. Anbazhagan,et al.  Microalgae: a sustainable feed source for aquaculture , 2011 .

[58]  J. Day,et al.  An investigation of the heterotrophic culture of the green algaTetraselmis , 2004, Journal of Applied Phycology.

[59]  J. Pekkoh,et al.  Effects of organic carbon source and light-dark period on growth and lipid accumulation of Scenedesmus sp. AARL G022 , 2014 .

[60]  L. T. Angenent,et al.  Production of bioenergy and biochemicals from industrial and agricultural wastewater. , 2004, Trends in biotechnology.

[61]  Ganti S. Murthy,et al.  Effects of Environmental Factors and Nutrient Availability on the Biochemical Composition of Algae for Biofuels Production: A Review , 2013 .

[62]  S. Jia,et al.  Growth characteristics of the cyanobacterium Nostoc flagelliforme in photoautotrophic, mixotrophic and heterotrophic cultivation , 2009, Journal of Applied Phycology.

[63]  Haizhen Yang,et al.  Mixotrophic Cultivation of Microalgae for Biodiesel Production: Status and Prospects , 2014, Applied Biochemistry and Biotechnology.

[64]  Y. Chisti,et al.  Comparative evaluation of compact photobioreactors for large-scale monoculture of microalgae , 1999 .

[65]  A. Maietti,et al.  Morphophysiological analyses of Neochloris oleoabundans (Chlorophyta) grown mixotrophically in a carbon-rich waste product , 2012, Protoplasma.

[66]  R. Kang,et al.  Interactions between organic and inorganic carbon sources during mixotrophic cultivation of Synechococcus sp. , 2004, Biotechnology Letters.

[67]  J. K. Kim,et al.  Comparison of biomass production and total lipid content of freshwater green microalgae cultivated under various culture conditions , 2014, Bioprocess and Biosystems Engineering.

[68]  A. Melis,et al.  Microalgal hydrogen production research , 2016 .

[69]  Biotransformations of carbon dioxide in photobioreactors , 2010 .

[70]  Dong-Woo Lee,et al.  Biohydrogen Production: Strategies to Improve Process Efficiency through Microbial Routes , 2015, International journal of molecular sciences.

[71]  E. Fernández,et al.  Low oxygen levels contribute to improve photohydrogen production in mixotrophic non-stressed Chlamydomonas cultures , 2015, Biotechnology for Biofuels.

[72]  W. J. Dyer,et al.  A rapid method of total lipid extraction and purification. , 1959, Canadian journal of biochemistry and physiology.

[73]  Haiying Wang,et al.  A study on lipid production of the mixotrophic microalgae Phaeodactylum tricornutum on various carbon sources , 2012 .

[74]  Jana Stöckel,et al.  High rates of photobiological H2 production by a cyanobacterium under aerobic conditions. , 2010, Nature communications.

[75]  Y. Bashan,et al.  Heterotrophic cultures of microalgae: metabolism and potential products. , 2011, Water research.

[76]  Giuseppe Torzillo,et al.  Hydrogen production with the microalga Chlamydomonas reinhardtii grown in a compact tubular photobioreactor immersed in a scattering light nanoparticle suspension , 2012 .

[77]  Y. Alkhamis,et al.  Comparison of pigment and proximate compositions of Tisochrysis lutea in phototrophic and mixotrophic cultures , 2015, Journal of Applied Phycology.

[78]  Jean-François Cornet,et al.  Investigation of the combined effects of acetate and photobioreactor illuminated fraction in the induction of anoxia for hydrogen production by Chlamydomonas reinhardtii , 2010 .

[79]  S. Chinnasamy,et al.  Chlorella minutissima—A Promising Fuel Alga for Cultivation in Municipal Wastewaters , 2010, Applied biochemistry and biotechnology.

[80]  S. Mohan,et al.  CO2 supplementation to domestic wastewater enhances microalgae lipid accumulation under mixotrophic microenvironment: effect of sparging period and interval. , 2012, Bioresource technology.

[81]  Michael Seibert,et al.  Hydrogen production by sulfur-deprived Chlamydomonas reinhardtii under photoautotrophic conditions , 2006 .

[82]  Y. Bashan,et al.  ORGANIC CARBON SUPPLEMENTATION OF STERILIZED MUNICIPAL WASTEWATER IS ESSENTIAL FOR HETEROTROPHIC GROWTH AND REMOVING AMMONIUM BY THE MICROALGA CHLORELLA VULGARIS 1 , 2011, Journal of phycology.

[83]  R. Banerjee,et al.  Comparison of biohydrogen production processes , 2008 .

[84]  Wei Zhang,et al.  Two-stage photo-biological production of hydrogen by marine green alga Platymonas subcordiformis , 2004 .

[85]  Yeoung-Sang Yun,et al.  Carbon Dioxide Fixation by Algal Cultivation Using Wastewater Nutrients , 1997 .

[86]  S. Venkata Mohan,et al.  Heterotrophic microalgae cultivation to synergize biodiesel production with waste remediation: progress and perspectives. , 2015, Bioresource technology.

[87]  R. Ruan,et al.  Mixotrophic cultivation of Chlorella vulgaris and its potential application for the oil accumulation from non-sugar materials , 2011 .

[88]  Jingli Xie,et al.  Mixotrophic cultivation of Platymonas subcordiformis , 2001, Journal of Applied Phycology.

[89]  K. Miyamoto,et al.  Hydrogen Production by a Mixed Culture of a Green Alga, Chlamydomonas reinhardtii and a Photosynthetic Bacterium, Rhodospirillum rubrum , 1987 .

[90]  Jun Zhu,et al.  Anaerobic digested dairy manure as a nutrient supplement for cultivation of oil-rich green microalgae Chlorella sp. , 2010, Bioresource technology.

[91]  C. Faraloni,et al.  Enhanced hydrogen production by means of sulfur-deprived Chlamydomonas reinhardtii cultures grown in pretreated olive mill wastewater , 2011 .

[92]  José G García-Cerdán,et al.  Truncated Photosystem Chlorophyll Antenna Size in the Green Microalga Chlamydomonas reinhardtii upon Deletion of the TLA3-CpSRP43 Gene1[C][W][OA] , 2012, Plant Physiology.

[93]  Jianlong Wang,et al.  Characterization of cell growth and photobiological H2 production of Chlamydomonas reinhardtii in ASSF industry wastewater , 2014 .

[94]  Yan Wang,et al.  Differential fatty acid profiles of Chlorella kessleri grown with organic materials , 2013 .

[95]  K Bala Amutha,et al.  Biological hydrogen production by the algal biomass Chlorella vulgaris MSU 01 strain isolated from pond sediment. , 2011, Bioresource technology.

[96]  Qiang Wang,et al.  An informatics-based analysis of developments to date and prospects for the application of microalgae in the biological sequestration of industrial flue gas , 2016, Applied Microbiology and Biotechnology.

[97]  Jo‐Shu Chang,et al.  Photoheterotrophic growth of Chlorella vulgaris ESP6 on organic acids from dark hydrogen fermentation effluents. , 2013, Bioresource technology.

[98]  Jo-Shu Chang,et al.  Biohydrogen production by a novel integration of dark fermentation and mixotrophic microalgae cultivation , 2013 .

[99]  Alberto Bertucco,et al.  Excess CO2 supply inhibits mixotrophic growth of Chlorella protothecoides and Nannochloropsis salina. , 2012, Bioresource technology.

[100]  L. Qun,et al.  Effects of sodium nitrate and sodium acetate concentrations on the growth and fatty acid composition of Brachiomonas submarina , 2003 .

[101]  Jae-Hoon Hwang,et al.  Photoheterotrophic microalgal hydrogen production using acetate- and butyrate-rich wastewater effluent , 2014 .

[102]  Mark A. White,et al.  Environmental life cycle comparison of algae to other bioenergy feedstocks. , 2010, Environmental science & technology.

[103]  Feng Chen,et al.  Heterotrophic growth of Chlamydomonas reinhardtii on acetate in chemostat culture , 1996 .

[104]  C. Lan,et al.  CO2 bio-mitigation using microalgae , 2008, Applied Microbiology and Biotechnology.

[105]  T. Mock,et al.  Photosynthetic energy conversion under extreme conditions--II: the significance of lipids under light limited growth in Antarctic sea ice diatoms. , 2002, Phytochemistry.

[106]  Sashenka Fierro,et al.  Nitrate and phosphate removal by chitosan immobilized Scenedesmus. , 2008, Bioresource technology.

[107]  Yan Wang,et al.  Effects of iron on fatty acid and astaxanthin accumulation in mixotrophic Chromochloris zofingiensis , 2012, Biotechnology Letters.

[108]  Hideo Tanaka,et al.  Light requirement and photosynthetic cell cultivation – Development of processes for efficient light utilization in photobioreactors , 2000, Journal of Applied Phycology.

[109]  K. Gregory,et al.  Sulfide removal from livestock biogas by Azospirillum-like anaerobic phototrophic bacteria consortium , 2014 .

[110]  S. Venkata Mohan,et al.  Temperature induced stress influence on biodiesel productivity during mixotrophic microalgae cultivation with wastewater. , 2014, Bioresource technology.

[111]  M. Shapira,et al.  A Proposed Mechanism for the Inhibitory Effects of Oxidative Stress on Rubisco Assembly and Its Subunit Expression1 , 2005, Plant Physiology.

[112]  R. Ely,et al.  Photobiological hydrogen production from Synechocystis sp. PCC 6803 encapsulated in silica sol–gel , 2009 .

[113]  Junfeng Rong,et al.  Ca2+-regulated cyclic electron flow supplies ATP for nitrogen starvation-induced lipid biosynthesis in green alga , 2015, Scientific Reports.

[114]  L. Gouveia,et al.  A symbiotic gas exchange between bioreactors enhances microalgal biomass and lipid productivities: taking advantage of complementary nutritional modes , 2011, Journal of Industrial Microbiology & Biotechnology.

[115]  Junfeng Rong,et al.  Effective Biological DeNOx of Industrial Flue Gas by the Mixotrophic Cultivation of an Oil-Producing Green Alga Chlorella sp. C2. , 2016, Environmental science & technology.

[116]  Yanna Liang,et al.  Biomass and lipid productivities of Chlorella vulgaris under autotrophic, heterotrophic and mixotrophic growth conditions , 2009, Biotechnology Letters.

[117]  Abdul Aziz Abdul Raman,et al.  Hydrogen production by Chlamydomonas reinhardtii in a two-stage process with and without illumination at alkaline pH , 2012 .

[118]  Paul J. Harrison,et al.  Effects of temperature on growth rate, cell composition and nitrogen metabolism in the marine diatom Thalassiosira pseudonana (Bacillariophyceae) , 2002 .

[119]  Frank W. R. Chaplen,et al.  Optimization of media nutrient composition for increased photofermentative hydrogen production by Synechocystis sp. PCC 6803 , 2008 .

[120]  Attilio Converti,et al.  Fed-batch mixotrophic cultivation of Arthrospira (Spirulina) platensis (Cyanophycea) with carbon source pulse feeding , 2005 .

[121]  P. Altimari,et al.  Hydrogen Photo-Production by Mixotrophic Cultivation of Chlamydomonas Reinhardtii: Interaction between Organic Carbon and Nitrogen , 2014 .

[122]  Willy Verstraete,et al.  Revival of the biological sunlight‐to‐biogas energy conversion system , 2009, Biotechnology and bioengineering.

[123]  Q. Wang,et al.  Evaluation of an oil-producing green alga Chlorella sp. C2 for biological DeNOx of industrial flue gases. , 2014, Environmental science & technology.

[124]  J. Costa,et al.  Mixotrophic cultivation of microalga Spirulina platensis using molasses as organic substrate , 2007 .

[125]  Otto Pulz,et al.  Photobioreactors: Design and performance with respect to light energy input , 1998 .

[126]  Lawrence Pitt,et al.  Biohydrogen production: prospects and limitations to practical application , 2004 .

[127]  Bo Hu,et al.  Oil Accumulation via Heterotrophic/Mixotrophic Chlorella protothecoides , 2010, Applied biochemistry and biotechnology.

[128]  N. T. Eriksen The technology of microalgal culturing , 2008, Biotechnology Letters.

[129]  J. Qin,et al.  Cultivation of Isochrysis galbana in Phototrophic, Heterotrophic, and Mixotrophic Conditions , 2013, BioMed research international.

[130]  Hui Chen,et al.  Ca2+ signal transduction related to neutral lipid synthesis in an oil-producing green alga Chlorella sp. C2. , 2014, Plant & cell physiology.

[131]  R. Lewin,et al.  The uptake and utilization of organic carbon by algae: an essay in comparative biochemistry* , 1974 .

[132]  G. Murthy,et al.  Life cycle analysis of algae biodiesel , 2010 .

[133]  M. Alvim-Ferraz,et al.  Carbon dioxide capture from flue gases using microalgae: Engineering aspects and biorefinery concept , 2012 .

[134]  K. Shimizu,et al.  Energetics and carbon metabolism during growth of microalgal cells under photoautotrophic, mixotrophic and cyclic light-autotrophic/dark-heterotrophic conditions. , 2000, Biochemical engineering journal.

[135]  H. Xiong,et al.  Mixotrophic cultivation of Chlorella pyrenoidosa with diluted primary piggery wastewater to produce lipids. , 2012, Bioresource technology.

[136]  J. Serra,et al.  Nitrate removal from water by Scenedesmus obliquus immobilized in polymeric foams , 1995 .

[137]  Junzhi Liu,et al.  Enhancement of biomass productivity and nutrients removal from pretreated piggery wastewater by mixotrophic cultivation of Desmodesmus sp. CHX1 , 2013 .

[138]  Keat-Teong Lee,et al.  Microalgae biofuels: A critical review of issues, problems and the way forward. , 2012, Biotechnology advances.

[139]  Jo‐Shu Chang,et al.  Cultivation, photobioreactor design and harvesting of microalgae for biodiesel production: a critical review. , 2011, Bioresource technology.

[140]  Qiang Wang,et al.  Microalgal biofuel revisited: An informatics-based analysis of developments to date and future prospects , 2015 .

[141]  Juergen E. W. Polle,et al.  tla1, a DNA insertional transformant of the green alga Chlamydomonas reinhardtii with a truncated light-harvesting chlorophyll antenna size , 2003, Planta.

[142]  Qingyu Wu,et al.  Application of sweet sorghum for biodiesel production by heterotrophic microalga Chlorella protothecoides , 2010 .