Metabolic and physiological regulation of Chlorella sp.(Trebouxiophyceae, Chlorophyta) under nitrogen deprivation
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摘要
A freshwater green microalgae Chlorella sp., UMACC344 was shown to produce high lipid content and has the potential to be used as feedstock for biofuel production. In this study, photosynthetic effciency, biochemical pro?les and non-targeted metabolic pro?ling were studied to compare between the nitrogen-replete and deplete conditions. Slowed growth, change in photosynthetic pigments and lowered photosynthetic effciency were observed in response to nitrogen deprivation. Biochemical pro?les of the cultures showed an increased level of carbohydrate, lipids and total fatty acids, while the total soluble protein content was lowered. A trend of fatty acid saturation was observed in the nitrogen-deplete culture with an increase in the level of saturated fatty acids especially C16:0 and C18:0, accompanied by a decrease in proportions of monounsaturated and polyunsaturated fatty acids. Fifty-nine metabolites, including amino acids, lipids, phytochemical compounds, vitamins and cofactors were signi?cantly dysregulated and annotated in this study. Pathway mapping analysis revealed a rewiring of metabolic pathways in the cells, particularly purine, carotenoid, nicotinate and nicotinamide, and amino acid metabolisms. Within the treatment period of nitrogen deprivation, the key processes involved were reshu ? ing of nitrogen from proteins and photosynthetic machinery, together with carbon repartitioning in carbohydrates and lipids.
A freshwater green microalgae Chlorella sp., UMACC344 was shown to produce high lipid content and has the potential to be used as feedstock for biofuel production. In this study, photosynthetic effciency, biochemical pro?les and non-targeted metabolic pro?ling were studied to compare between the nitrogen-replete and deplete conditions. Slowed growth, change in photosynthetic pigments and lowered photosynthetic effciency were observed in response to nitrogen deprivation. Biochemical pro?les of the cultures showed an increased level of carbohydrate, lipids and total fatty acids, while the total soluble protein content was lowered. A trend of fatty acid saturation was observed in the nitrogen-deplete culture with an increase in the level of saturated fatty acids especially C16:0 and C18:0, accompanied by a decrease in proportions of monounsaturated and polyunsaturated fatty acids. Fifty-nine metabolites, including amino acids, lipids, phytochemical compounds, vitamins and cofactors were signi?cantly dysregulated and annotated in this study. Pathway mapping analysis revealed a rewiring of metabolic pathways in the cells, particularly purine, carotenoid, nicotinate and nicotinamide, and amino acid metabolisms. Within the treatment period of nitrogen deprivation, the key processes involved were reshu ? ing of nitrogen from proteins and photosynthetic machinery, together with carbon repartitioning in carbohydrates and lipids.
A freshwater green microalgae Chlorella sp., UMACC344 was shown to produce high lipid content and has the potential to be used as feedstock for biofuel production. In this study, photosynthetic effciency, biochemical pro?les and non-targeted metabolic pro?ling were studied to compare between the nitrogen-replete and deplete conditions. Slowed growth, change in photosynthetic pigments and lowered photosynthetic effciency were observed in response to nitrogen deprivation. Biochemical pro?les of the cultures showed an increased level of carbohydrate, lipids and total fatty acids, while the total soluble protein content was lowered. A trend of fatty acid saturation was observed in the nitrogen-deplete culture with an increase in the level of saturated fatty acids especially C16:0 and C18:0, accompanied by a decrease in proportions of monounsaturated and polyunsaturated fatty acids. Fifty-nine metabolites, including amino acids, lipids, phytochemical compounds, vitamins and cofactors were signi?cantly dysregulated and annotated in this study. Pathway mapping analysis revealed a rewiring of metabolic pathways in the cells, particularly purine, carotenoid, nicotinate and nicotinamide, and amino acid metabolisms. Within the treatment period of nitrogen deprivation, the key processes involved were reshu ? ing of nitrogen from proteins and photosynthetic machinery, together with carbon repartitioning in carbohydrates and lipids.
引文
Allen J W,DiRusso C C,Black P N.2015.Triacylglycerol synthesis during nitrogen stress involves the prokaryotic lipid synthesis pathway and acyl chain remodeling in the microalgae Coccomyxa subellipsoidea.Algal Res.,10:110-120,https://doi.org/10.1016/j.algal.2015.04.019.
Bligh E G,Dyer W J.1959.A rapid method of total lipid extraction and puri?cation.Can.J.Biochem.Physiol.,37(8):911-917,https://doi.org/10.1139/o59-099.
Chu W L,Phang S M,Goh S H.1994.Studies on the production of useful chemicals,especially fatty acids in the marine diatom Nitzschia conspicua Grunow.Hydrobiologia,285(1-3):33-40,https://doi.org/10.1007/BF00005651.
Dubois M,Gilles K A,Hamilton J K,Rebers P A,Smith F.1956.Colorimetric method for determination of sugars and related substances.Anal.Chem.,28(3):350-356,https://doi.org/10.1021/ac60111a017.
Gao B Y,Yang J,Lei X Q,Xia S,Li A F,Zhang C W.2016.Characterization of cell structural change,growth,lipid accumulation,and pigment pro?le of a novel oleaginous microalga,Vischeria stellata(Eustigmatophyceae),cultured with dif ferent initial nitrate supplies.J.Appl.Phycol.,28(2):821-830,https://doi.org/10.1007/s10811-015-0626-1.
Gao C F,Wang Y,Shen Y,Yan D,He X,Dai J B,Wu Q Y.2014.Oil accumulation mechanisms of the oleaginous microalga Chlorella protothecoides revealed through its genome,transcriptomes,and proteomes.BMC Genomics,15:582,https://doi.org/10.1186/1471-2164-15-582.
Gargouri M,Park J J,Holguin F O,Kim M J,Wang H X,Deshpande R R,Shachar-Hill Y,Hicks L M,Gang D R.2015.Identi?cation of regulatory network hubs that control lipid metabolism in Chlamydomonas reinhardtii.J.Exp.Bot.,66(15):4 551-4 566,https://doi.org/10.1093/jxb/erv217.
Gowda H,Ivanisevic J,Johnson C H,Kurczy M E,Benton HP,Rinehart D,Nguyen T,Ray J,Kuehl J,Arevalo B,Westenskow P D,Wang J H,Arkin A P,Deutschbauer AM,Patti G J,Siuzdak G.2014.Interactive XCMS Online:simplifying advanced metabolomic data processing and subsequent statistical analyses.Anal.Chem.,86(14):6 931-6 939,https://doi.org/10.1021/ac500734c.
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(4):621-639,https://doi.org/10.1111/j.1365-313X.2008.03492.x.
Ito T,Tanaka M,Shinkawa H,Nakada T,Ano Y,Kurano N,Soga T,Tomita M.2013.Metabolic and morphological changes of an oil accumulating trebouxiophycean alga in nitrogen-de?cient conditions.Metabolomics,9(S1):178-187,https://doi.org/10.1007/s11306-012-0463-z.
Juneja A,Ceballos R M,Murthy G S.2013.Ef fects of environmental factors and nutrient availability on the biochemical composition of algae for biofuels production:a review.Energies,6(9):4 607-4 638,https://doi.org/10.3390/en6094607.
Levitan O,Dinamarca J,Zelzion E,Lun D S,Guerra L T,Kim M K,Kim J,Van Mooy B A S,Bhattacharya D,Falkowski P G.2015.Remodeling of intermediate metabolism in the diatom Phaeodactylum tricornutum under nitrogen stress.Proc.Natl.Acad.Sci.U.S.A.,112(2):412-417,https://doi.org/10.1073/pnas.1419818112.
Li T T,Gargouri M,Feng J,Park J J,Gao D F,Miao C,Dong T,Gang D R,Chen SL.2015.Regulation of starch and lipid accumulation in a microalga Chlorella sorokiniana.Bioresour.Technol.,180:250-257,https://doi.org/10.1016/j.biortech.2015.01.005.
Li Y Q,Han F X,Xu H,Mu J X,Chen D,Feng B,Zeng H Y.2014.Potential lipid accumulation and growth characteristic of the green alga Chlorella with combination cultivation mode of nitrogen(N)and phosphorus(P).Bioresour.Technol.,174:24-32,https://doi.org/10.1016/j.biortech.2014.09.142.
Li Y T,Han D X,Yoon K,Zhu S N,Sommerfeld M,Hu Q.2013.Molecular and cellular mechanisms for lipid synthesis and accumulation in microalgae:Biotechnological implications.In:Richmond A,Hu Qeds.Handbook of Microalgal Culture:Applied Phycology and Biotechnology.2 nd edn.John Wiley&Sons,Ltd,Oxford,UK.p.545-565.https://doi.org/10.1002/9781118567166.ch28.
Longworth J,Wu D Y,Huete-Ortega M,Wright P C,Vaidyanathan S.2016.Proteome response of Phaeodactylum tricornutum,during lipid accumulation induced by nitrogen depletion.Algal Res.,18:213-224,https://doi.org/10.1016/j.algal.2016.06.015.
Lu N,Wei D,Chen F,Yang S T.2013.Lipidomic pro?ling reveals lipid regulation in the snow alga Chlamydomonas nivalis in response to nitrate or phosphate deprivation.Process Biochem.,48(4):605-613,https://doi.org/10.1016/j.procbio.2013.02.028.
Martin G J O,Hill D R A,Olmstead I L D,Bergamin A,Shears M J,Dias D A,Kentish S E,Scales P J,BottéC Y,Callahan D L.2014.Lipid pro?le remodeling in response to nitrogen deprivation in the microalgae Chlorella sp.(Trebouxiophyceae)and Nannochloropsis sp.(Eustigmatophyceae).PLoS One,9(8):e103389,https://doi.org/10.1371/journal.pone.0103389.
Millán-Oropeza A,Fernández-Linares L.2017.Biomass and lipid production from Nannochloropsis oculata growth in raceway ponds operated in sequential batch mode under greenhouse conditions.Environ.Sci.Pollut.Res.Int.,24(33):25 618-25 626,https://doi.org/10.1007/s11356-016-7013-6.
Msanne J,Xu D,Konda A R,Casas-Mollano J A,Awada T,Cahoon E B,Cerutti H.2012.Metabolic and gene expression changes triggered by nitrogen deprivation in the photoautotrophically grown microalgae Chlamydomonas reinhardtii and Coccomyxa sp.C-169.Phytochemistry,75:50-59,https://doi.org/10.1016/j.phytochem.2011.12.007.
Ng F L,Phang S M,Periasamy V,Yunus K,Fisher A C.2014.Evaluation of algal bio?lms on indium tin oxide(ITO)for use in biophotovoltaic platforms based on photosynthetic performance.PLoS One,9(5):e97643,https://doi.org/10.1371/journal.pone.0097643.
Nichols H W,Bold H C.1965.Trichosarcina polymorpha gen.et sp.nov.J.Phycol.,1(1):34-38,https://doi.org/10.1111/j.1529-8817.1965.tb04552.x.
Park J J,Wang H X,Gargouri M,Deshpande R R,Skepper J N,Holguin F O,Juergens M T,Shachar-Hill Y,Hicks L M,Gang D R.2015.The response of Chlamydomonas reinhardtii to nitrogen deprivation:a systems biology analysis.Plant J.,81(4):611-624,https://doi.org/10.1111/tpj.12747.
Recht L,T?pfer N,Batushansky A,Sikron N,Gibon Y,Fait A,Nikoloski Z,Boussiba S,Zarka A.2014.Metabolite pro?ling and integrative modeling reveal metabolic constraints for carbon partitioning under nitrogen starvation in the green algae Haematococcus pluvialis.J.Biol.Chem.,289(44):30 387-30 403,https://doi.org/10.1074/jbc.M114.555144.
Salomon E,Bar-Eyal L,Sharon S,Keren N.2013.Balancing photosynthetic electron?ow is critical for cyanobacterial acclimation to nitrogen limitation.Biochim.Biophys.Acta,1827(3):340-347,https://doi.org/10.1016/j.bbabio.2012.11.010.
Schmollinger S,Mühlhaus T,Boyle N R,Blaby I K,Casero D,Mettler T,Moseley J L,Kropat J,Sommer F,Strenkert D,Hemme D,Pellegrini M,Grossman A R,Stitt M,Schroda M,Merchant S S.2014.Nitrogen-sparing mechanisms in Chlamydomonas af fect the transcriptome,the proteome,and photosynthetic metabolism.Plant Cell,26(4):1 410-1 435,https://doi.org/10.1105/tpc.113.122523.
Sharma K K,Schuhmann H,Schenk P M.2012.High lipid induction in microalgae for biodiesel production.Energies,5(5):1 532-1 553,https://doi.org/10.3390/en5051532.
Solovchenko A E,Khozin-Goldberg I,Cohen Z,Merzlyak MN.2009.Carotenoid-to-chlorophyll ratio as a proxy for assay of total fatty acids and arachidonic acid content in the green microalga Parietochloris incisa.J.Appl.Phycol.,21(3):361-366,https://doi.org/10.1007/s10811-008-9377-6.
Stansell G R,Gray V M,Sym S D.2012.Microalgal fatty acid composition:Implications for biodiesel quality.J.Appl.Phycol.,24(4):791-801,https://doi.org/10.1007/s10811-011-9696-x.
Stasolla C,Katahira R,Thorpe T A,Ashihara H.2003.Purine and pyrimidine nucleotide metabolism in higher plants.J.Plant Physiol.,160(11):1 271-1 295,https://doi.org/10.1078/0176-1617-01169.
Strickland J D H,Parsons T R.1972.A Practical Handbook of Seawater Analysis.2 nd edn.Fisheries Research Board of Canada,Ottawa,Canada.310p.
Wase N,Black P N,Stanley B A,DiRusso C C.2014.Integrated quantitative analysis of nitrogen stress response in Chlamydomonas reinhardtii using metabolite and protein pro?ling.J.Proteome Res.,13(3):1 373-1 396,https://doi.org/10.1021/pr400952z.
Worley B,Powers R.2013.Multivariate analysis in metabolomics.Curr.Metabolomics,1(1):92-107,https://doi.org/10.2174/2213235X11301010092.
Yang D W,Zhang Y T,Barupal D K,Fan X L,Gustafson R,Guo R B,Fiehn O.2014.Metabolomics of photobiological hydrogen production induced by CCCP in Chlamydomonas reinhardtii.Int.J.Hydrogen Energy,39(1):150-158,https://doi.org/10.1016/j.ijhydene.2013.09.116.
Yang Z K,Niu Y F,Ma Y H,Xue J,Zhang M H,Yang W D,Liu J S,Lu S H,Guan Y F,Li H Y.2013.Molecular and cellular mechanisms of neutral lipid accumulation in diatom following nitrogen deprivation.Biotechnol.Biofuels,6(1):67,https://doi.org/10.1186/1754-6834-6-67.
Bligh E G,Dyer W J.1959.A rapid method of total lipid extraction and puri?cation.Can.J.Biochem.Physiol.,37(8):911-917,https://doi.org/10.1139/o59-099.
Chu W L,Phang S M,Goh S H.1994.Studies on the production of useful chemicals,especially fatty acids in the marine diatom Nitzschia conspicua Grunow.Hydrobiologia,285(1-3):33-40,https://doi.org/10.1007/BF00005651.
Dubois M,Gilles K A,Hamilton J K,Rebers P A,Smith F.1956.Colorimetric method for determination of sugars and related substances.Anal.Chem.,28(3):350-356,https://doi.org/10.1021/ac60111a017.
Gao B Y,Yang J,Lei X Q,Xia S,Li A F,Zhang C W.2016.Characterization of cell structural change,growth,lipid accumulation,and pigment pro?le of a novel oleaginous microalga,Vischeria stellata(Eustigmatophyceae),cultured with dif ferent initial nitrate supplies.J.Appl.Phycol.,28(2):821-830,https://doi.org/10.1007/s10811-015-0626-1.
Gao C F,Wang Y,Shen Y,Yan D,He X,Dai J B,Wu Q Y.2014.Oil accumulation mechanisms of the oleaginous microalga Chlorella protothecoides revealed through its genome,transcriptomes,and proteomes.BMC Genomics,15:582,https://doi.org/10.1186/1471-2164-15-582.
Gargouri M,Park J J,Holguin F O,Kim M J,Wang H X,Deshpande R R,Shachar-Hill Y,Hicks L M,Gang D R.2015.Identi?cation of regulatory network hubs that control lipid metabolism in Chlamydomonas reinhardtii.J.Exp.Bot.,66(15):4 551-4 566,https://doi.org/10.1093/jxb/erv217.
Gowda H,Ivanisevic J,Johnson C H,Kurczy M E,Benton HP,Rinehart D,Nguyen T,Ray J,Kuehl J,Arevalo B,Westenskow P D,Wang J H,Arkin A P,Deutschbauer AM,Patti G J,Siuzdak G.2014.Interactive XCMS Online:simplifying advanced metabolomic data processing and subsequent statistical analyses.Anal.Chem.,86(14):6 931-6 939,https://doi.org/10.1021/ac500734c.
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(4):621-639,https://doi.org/10.1111/j.1365-313X.2008.03492.x.
Ito T,Tanaka M,Shinkawa H,Nakada T,Ano Y,Kurano N,Soga T,Tomita M.2013.Metabolic and morphological changes of an oil accumulating trebouxiophycean alga in nitrogen-de?cient conditions.Metabolomics,9(S1):178-187,https://doi.org/10.1007/s11306-012-0463-z.
Juneja A,Ceballos R M,Murthy G S.2013.Ef fects of environmental factors and nutrient availability on the biochemical composition of algae for biofuels production:a review.Energies,6(9):4 607-4 638,https://doi.org/10.3390/en6094607.
Levitan O,Dinamarca J,Zelzion E,Lun D S,Guerra L T,Kim M K,Kim J,Van Mooy B A S,Bhattacharya D,Falkowski P G.2015.Remodeling of intermediate metabolism in the diatom Phaeodactylum tricornutum under nitrogen stress.Proc.Natl.Acad.Sci.U.S.A.,112(2):412-417,https://doi.org/10.1073/pnas.1419818112.
Li T T,Gargouri M,Feng J,Park J J,Gao D F,Miao C,Dong T,Gang D R,Chen SL.2015.Regulation of starch and lipid accumulation in a microalga Chlorella sorokiniana.Bioresour.Technol.,180:250-257,https://doi.org/10.1016/j.biortech.2015.01.005.
Li Y Q,Han F X,Xu H,Mu J X,Chen D,Feng B,Zeng H Y.2014.Potential lipid accumulation and growth characteristic of the green alga Chlorella with combination cultivation mode of nitrogen(N)and phosphorus(P).Bioresour.Technol.,174:24-32,https://doi.org/10.1016/j.biortech.2014.09.142.
Li Y T,Han D X,Yoon K,Zhu S N,Sommerfeld M,Hu Q.2013.Molecular and cellular mechanisms for lipid synthesis and accumulation in microalgae:Biotechnological implications.In:Richmond A,Hu Qeds.Handbook of Microalgal Culture:Applied Phycology and Biotechnology.2 nd edn.John Wiley&Sons,Ltd,Oxford,UK.p.545-565.https://doi.org/10.1002/9781118567166.ch28.
Longworth J,Wu D Y,Huete-Ortega M,Wright P C,Vaidyanathan S.2016.Proteome response of Phaeodactylum tricornutum,during lipid accumulation induced by nitrogen depletion.Algal Res.,18:213-224,https://doi.org/10.1016/j.algal.2016.06.015.
Lu N,Wei D,Chen F,Yang S T.2013.Lipidomic pro?ling reveals lipid regulation in the snow alga Chlamydomonas nivalis in response to nitrate or phosphate deprivation.Process Biochem.,48(4):605-613,https://doi.org/10.1016/j.procbio.2013.02.028.
Martin G J O,Hill D R A,Olmstead I L D,Bergamin A,Shears M J,Dias D A,Kentish S E,Scales P J,BottéC Y,Callahan D L.2014.Lipid pro?le remodeling in response to nitrogen deprivation in the microalgae Chlorella sp.(Trebouxiophyceae)and Nannochloropsis sp.(Eustigmatophyceae).PLoS One,9(8):e103389,https://doi.org/10.1371/journal.pone.0103389.
Millán-Oropeza A,Fernández-Linares L.2017.Biomass and lipid production from Nannochloropsis oculata growth in raceway ponds operated in sequential batch mode under greenhouse conditions.Environ.Sci.Pollut.Res.Int.,24(33):25 618-25 626,https://doi.org/10.1007/s11356-016-7013-6.
Msanne J,Xu D,Konda A R,Casas-Mollano J A,Awada T,Cahoon E B,Cerutti H.2012.Metabolic and gene expression changes triggered by nitrogen deprivation in the photoautotrophically grown microalgae Chlamydomonas reinhardtii and Coccomyxa sp.C-169.Phytochemistry,75:50-59,https://doi.org/10.1016/j.phytochem.2011.12.007.
Ng F L,Phang S M,Periasamy V,Yunus K,Fisher A C.2014.Evaluation of algal bio?lms on indium tin oxide(ITO)for use in biophotovoltaic platforms based on photosynthetic performance.PLoS One,9(5):e97643,https://doi.org/10.1371/journal.pone.0097643.
Nichols H W,Bold H C.1965.Trichosarcina polymorpha gen.et sp.nov.J.Phycol.,1(1):34-38,https://doi.org/10.1111/j.1529-8817.1965.tb04552.x.
Park J J,Wang H X,Gargouri M,Deshpande R R,Skepper J N,Holguin F O,Juergens M T,Shachar-Hill Y,Hicks L M,Gang D R.2015.The response of Chlamydomonas reinhardtii to nitrogen deprivation:a systems biology analysis.Plant J.,81(4):611-624,https://doi.org/10.1111/tpj.12747.
Recht L,T?pfer N,Batushansky A,Sikron N,Gibon Y,Fait A,Nikoloski Z,Boussiba S,Zarka A.2014.Metabolite pro?ling and integrative modeling reveal metabolic constraints for carbon partitioning under nitrogen starvation in the green algae Haematococcus pluvialis.J.Biol.Chem.,289(44):30 387-30 403,https://doi.org/10.1074/jbc.M114.555144.
Salomon E,Bar-Eyal L,Sharon S,Keren N.2013.Balancing photosynthetic electron?ow is critical for cyanobacterial acclimation to nitrogen limitation.Biochim.Biophys.Acta,1827(3):340-347,https://doi.org/10.1016/j.bbabio.2012.11.010.
Schmollinger S,Mühlhaus T,Boyle N R,Blaby I K,Casero D,Mettler T,Moseley J L,Kropat J,Sommer F,Strenkert D,Hemme D,Pellegrini M,Grossman A R,Stitt M,Schroda M,Merchant S S.2014.Nitrogen-sparing mechanisms in Chlamydomonas af fect the transcriptome,the proteome,and photosynthetic metabolism.Plant Cell,26(4):1 410-1 435,https://doi.org/10.1105/tpc.113.122523.
Sharma K K,Schuhmann H,Schenk P M.2012.High lipid induction in microalgae for biodiesel production.Energies,5(5):1 532-1 553,https://doi.org/10.3390/en5051532.
Solovchenko A E,Khozin-Goldberg I,Cohen Z,Merzlyak MN.2009.Carotenoid-to-chlorophyll ratio as a proxy for assay of total fatty acids and arachidonic acid content in the green microalga Parietochloris incisa.J.Appl.Phycol.,21(3):361-366,https://doi.org/10.1007/s10811-008-9377-6.
Stansell G R,Gray V M,Sym S D.2012.Microalgal fatty acid composition:Implications for biodiesel quality.J.Appl.Phycol.,24(4):791-801,https://doi.org/10.1007/s10811-011-9696-x.
Stasolla C,Katahira R,Thorpe T A,Ashihara H.2003.Purine and pyrimidine nucleotide metabolism in higher plants.J.Plant Physiol.,160(11):1 271-1 295,https://doi.org/10.1078/0176-1617-01169.
Strickland J D H,Parsons T R.1972.A Practical Handbook of Seawater Analysis.2 nd edn.Fisheries Research Board of Canada,Ottawa,Canada.310p.
Wase N,Black P N,Stanley B A,DiRusso C C.2014.Integrated quantitative analysis of nitrogen stress response in Chlamydomonas reinhardtii using metabolite and protein pro?ling.J.Proteome Res.,13(3):1 373-1 396,https://doi.org/10.1021/pr400952z.
Worley B,Powers R.2013.Multivariate analysis in metabolomics.Curr.Metabolomics,1(1):92-107,https://doi.org/10.2174/2213235X11301010092.
Yang D W,Zhang Y T,Barupal D K,Fan X L,Gustafson R,Guo R B,Fiehn O.2014.Metabolomics of photobiological hydrogen production induced by CCCP in Chlamydomonas reinhardtii.Int.J.Hydrogen Energy,39(1):150-158,https://doi.org/10.1016/j.ijhydene.2013.09.116.
Yang Z K,Niu Y F,Ma Y H,Xue J,Zhang M H,Yang W D,Liu J S,Lu S H,Guan Y F,Li H Y.2013.Molecular and cellular mechanisms of neutral lipid accumulation in diatom following nitrogen deprivation.Biotechnol.Biofuels,6(1):67,https://doi.org/10.1186/1754-6834-6-67.