Restricted accessResearch articleFirst published online 2021-12
Revealing Lipid Body Formation and its Subcellular Reorganization in Oleaginous Microalgae Using Correlative Optical Microscopy and Infrared Nanospectroscopy
The purpose of this work is to develop an integrated imaging approach to characterize without labeling at the sub-cellular level the formation of lipid body droplets (LBs) in microalgae undergoing nitrogen starvation. First conventional optical microscopy approaches, gas chromatography, and turbidimetry measurements allowed to monitor the biomass and the total lipid content in the oleaginous microalgae Parachlorella kesslerii during the starvation process. Then a local analysis of the LBs was proposed using an innovative infrared nanospectroscopy technique called atomic force microscopy-based infrared spectroscopy (AFM-IR). This label-free technique assessed the formation of LBs and allowed to look into the LB composition thanks to the acquisition of local infrared spectra. Last correlative measurements using fluorescence microscopy and AFM-IR were performed to investigate the subcellular reorganization of LB and the chloroplasts.
T. Taparia, M. Mvss, R. Mehrotra, P. Shukla, S. Mehrotra. “Developments and Challenges in Biodiesel Production from Microalgae: A Review”. Biotechnol. Appl. Biochem. 2016. 715–726. doi: 10.1002/Bab.1412.
2.
ChewK.W.YapJ.Y.ShowP.L.SuanN.H., et al.“Microalgae Biorefinery: High Value Products Perspectives”. Bioresour. Technol. 2017; 229: 53–62. doi: 10.1016/J.Biortech.2017.01.006.
3.
OdjadjareE.C.MutandaT.OlaniranA.O.“Potential Biotechnological Application of Microalgae: A Critical Review”. Crit. Rev. Biotechnol. 2017; 37(1): 37–52. doi: 10.3109/07388551.2015.1108956.
4.
MallickN.BagchiS.K.KoleyS.SinghA.K.“Progress and Challenges in Microalgal Biodiesel Production”. Front. Microbiol. 2016; 7: 1019doi: 10.3389/fmicb.2016.01019.
5.
R. Razeghifard. “Algal Biofuels”. Photosynth. Res. 2013. 207–219. doi: 10.1007/s11120-013-9828-Z.
6.
ShowP.L.TangM.S.Y.NagarajanD.LingT.C., et al.“A Holistic Approach to Managing Microalgae for Biofuel Applications”. Int. J. Mol. Sci. 2017; 18(1): 215doi: 10.3390/Ijms18010215.
7.
L.D. Zhu, Z.H. Li, E. Hiltunen. “Strategies for Lipid Production Improvement in Microalgae as a Biodiesel Feedstock”. Biomed. Res. Int. 2016. Article ID 8792548. doi: 10.1155/2016/8792548.
8.
WobbeL.BassiR.KruseO.“Multi-Level Light Capture Control in Plants and Green Algae”. Trends Plant Sci. 2016; 21(1): 55–68. doi: 10.1016/j.tplants.2015.10.004.
9.
MinhasA.K.HodgsonP.BarrowC.J.AdholeyaA.“A Review on the Assessment of Stress Conditions for Simultaneous Production of Microalgal Lipids and Carotenoids”. Front. Microbiol. 2016; 7: 546doi: 10.3389/fmicb.2016.00546.
10.
TanK.W.M.LeeY.K.“The Dilemma for Lipid Productivity in Green Microalgae: Importance of Substrate Provision in Improving Oil Yield Without Sacrificing Growth”. Biotechnol. Biofuels. 2016; 9: 255.doi: 10.1186/S13068-016-0671-2.
11.
BanerjeeA.MaitiS.K.GuriaC.BanerjeeC.“Metabolic Pathways for Lipid Synthesis Under Nitrogen Stress in Chlamydomonas and Nannochloropsis”. Biotechnol. Lett. 2017; 39(1): 1–11. doi: 10.1007/S10529-016-2216-Y.
12.
GomaaM.A.Al-HajL.AbedR.M.M.“Metabolic Engineering of Cyanobacteria and Microalgae for Enhanced Production of Biofuels and High-Value Products”. J. Appl. Microbiol. 2016; 121(4): 919–931. doi: 10.1111/Jam.13232.
13.
CourantF.MartzolffA.RabinG.AntignacJ.P., et al.“How Metabolomics Can Contribute to Bio-Processes: A Proof of Concept Study for Biomarkers Discovery in the Context of Nitrogen-Starved Microalgae Grown in Photobioreactors”. Metabolomics. 2013; 9(6): 1286–1300. doi: 10.1007/S11306-013-0532-Y.
14.
CoatR.MontalescotV.LeónE.S.KucmaD., et al.“Unravelling the Matrix Effect of Fresh Sampled Cells for In Vivo Unbiased FTIR Determination of the Absolute Concentration of Total Lipid Content of Microalgae”. Bioprocess Biosyst. Eng. 2014; 37(11): 2175–2187. doi: 10.1007/S00449-014-1194-5.
15.
MoutelB.AndréM.KucmaD.LegrandJ., et al.“Assessing the Biofuel Production Potential of Botryococcus braunii Strains by Sensitive Rapid Qualitative Chemotyping Using Chemometrically Assisted Gas Chromatography-Mass Spectrometry”. Algal Res. 2015; 11: 33–42. doi: 10.1016/J.Algal.2015.05.001.
16.
MoutelB.GonçalvesO.Le GrandF.LongM., et al.“Development of a Screening Procedure for the Characterization of Botryococcus Braunii Strains for Biofuel Application”. Process Biochem. 2016; 51(11): 1855–1865. doi: 10.1016/J.Procbio.2016.05.002.
17.
Serrano LeónE.CoatR.MoutelB.PruvostJ., et al.“Influence of Physical and Chemical Properties of HTSXT-FTIR Samples on the Quality of Prediction Models Developed to Determine Absolute Concentrations of Total Proteins, Carbohydrates, and Triglycerides: A Preliminary Study on the Determination of Their Ab”. Bioprocess Biosyst. Eng. 2014; 37(11): 2371–2380. doi: 10.1007/S00449-014-1215-4.
18.
S. Ota, K. Oshima, T. Yamazaki, S. Kim, et al. “Highly Efficient Lipid Production in the Green Alga Parachlorella kessleri: Draft Genome and Transcriptome Endorsed by Whole-Cell 3D Ultrastructure”. Biotechnol. Biofuels. 2016. 913. doi: 10.1186/S13068-016-0424-2.
19.
RhoadsA.AuK.F.“PacBio Sequencing and Its Applications”. Genomics, Proteomics Bioinf. 2015; 13(5): 278–289. doi: 10.1016/J.Gpb.2015.08.002.
FuD.LuF.K.ZhangX.FreudigerC., et al.“Quantitative Chemical Imaging with Multiplex Stimulated Raman Scattering Microscopy”. J. Am. Chem. Soc. 2012; 134(8): 3623–3626. doi: 10.1021/Ja210081h.
22.
TanS.T.BalasubramanianR.K.DasP.ObbardJ.P.ChewW.“Application of Mid-Infrared Chemical Imaging and Multivariate Chemometrics Analyses to Characterise a Population of Microalgae Cells”. Bioresour. Technol. 2013; 134: 316–323. doi: 10.1016/J.Biortech.2013.01.060.
23.
WongD.M.FranzA.K.“A Comparison of Lipid Storage in Phaeodactylum tricornutum and Tetraselmis suecica Using Laser Scanning Confocal Microscopy”. J. Microbiol. Methods. 2013; 95(2): 122–128. doi: 10.1016/J.Mimet.2013.07.026.
24.
YanC.FanJ.XuC.“Analysis of Oil Droplets in Microalgae”. Methods Cell. Biol. 2013; 116: 71–82. doi: 10.1016/B978-0-12-408051-5.00005-X.
25.
HosokawaM.AndoM.MukaiS.OsadaK., et al.“In Vivo Live Cell Imaging for the Quantitative Monitoring of Lipids by Using Raman Microspectroscopy”. Anal. Chem. 2014; 86(16): 8224–8230. doi: 10.1021/Ac501591d.
26.
CavoniusL.FinkH.KiskisJ.AlbersE., et al.“Imaging of Lipids in Microalgae with Coherent Anti-Stokes Raman Scattering Microscopy”. Plant Physiol. 2015; 167(3): 603–616. doi: 10.1104/Pp.114.252197.
27.
ParkJ.W.NaS.C.NguyenT.Q.PaikS.M., et al.“Live Cell Imaging Compatible Immobilization of Chlamydomonas reinhardtii in Microfluidic Platform for Biodiesel Research”. Biotechnol. Bioeng. 2015; 112(3): 494–501. doi: 10.1002/Bit.25453.
28.
JaegerD.PilgerC.HachmeisterH.OberländerE., et al.“Label-Free In Vivo Analysis of Intracellular Lipid Droplets in the Oleaginous Microalga Monoraphidium neglectum by Coherent Raman Scattering Microscopy”. Sci. Rep. 2016; 6(1): 1–9. doi: 10.1038/Srep35340.
29.
HuangY.Y.BealC.M.CaiW.W.RuoffR.S.TerentjevE.M.“Micro-Raman Spectroscopy of Algae: Composition Analysis and Fluorescence Background Behavior”. Biotechnol. Bioeng. 2010; 105(5): 889–898. doi: 10.1002/bit.22617.
30.
WengY.M.WengR.H.TzengC.Y.ChenW.“Structural Analysis of Triacylglycerols and Edible Oils by Near-Infrared Fourier Transform Raman Spectroscopy”. Appl. Spectrosc. 2003; 57(4): 413–418. doi: 10.1366/00037020360625952.
31.
LahrechA.BachelotR.GleyzesP.BoccaraA.C.“Infrared-Reflection-Mode Near-Field Microscopy Using an Apertureless Probe with a Resolution of Λ/600”. Opt. Lett. 1996; 21(17): 1315doi: 10.1364/Ol.21.001315.
32.
KnollB.KeilmannF.“Near-Field Probing of Vibrational Absorption for Chemical Microscopy”. Nature. 1999; 399(6732): 134–137. doi: 10.1038/20154.
33.
DazziA.PrazeresR.GlotinF.OrtegaJ.M.“Local Infrared Microspectroscopy with Subwavelength Spatial Resolution with an Atomic Force Microscope Tip Used as a Photothermal Sensor”. Opt. Lett. 2005; 30(18): 2388doi: 10.1364/Ol.30.002388.
34.
DazziA.PraterC.B.“AFM-IR: Technology and Applications in Nanoscale Infrared Spectroscopy and Chemical Imaging”. Chem. Rev. 2017; 117(7): 5146–5173. doi: 10.1021/Acs.Chemrev.6b00448.
35.
CentroneA.“Infrared Imaging and Spectroscopy Beyond the Diffraction Limit”. Annu. Rev. Anal. Chem. 2015; 8(1): 101–126. doi: 10.1146/Annurev-Anchem-071114-040435.
36.
Clavijo RiveraE.MontalescotV.ViauM.DrouinD., et al.“Mechanical Cell Disruption of Parachlorella kessleri Microalgae: Impact on Lipid Fraction Composition”. Bioresour. Technol. 2018; 256: 77–85. doi: 10.1016/J.Biortech.2018.01.148.
37.
PiaseckaA.CieślaJ.KoczańskaM.KrzemińskaI.“Effectiveness of Parachlorella kessleri Cell Disruption Evaluated with the Use of Laser Light Scattering Methods”. J. Appl. Phycol. 2019; 31(1): 97–107. doi: 10.1007/S10811-018-1583-2.
38.
FernandesB.TeixeiraJ.DragoneG.VicenteA.A., et al.“Relationship Between Starch and Lipid Accumulation Induced by Nutrient Depletion and Replenishment in the Microalga Parachlorella kessleri”. Bioresour. Technol. 2013; 144: 268–274. doi: 10.1016/j.biortech.2013.06.096.
39.
LiX.PřibylP.BišováK.KawanoS., et al.“The Microalga Parachlorella kessleri: A Novel Highly Efficient Lipid Producer”. Biotechnol. Bioeng. 2013; 110(1): 97–107. doi: 10.1002/bit.24595.
40.
MizunoY.SatoA.WatanabeK.HirataA.TakeshitaT.OtaS., et al.“Sequential Accumulation of Starch and Lipid Induced by Sulfur Deficiency in Chlorella and Parachlorella Species”. Bioresour. Technol. 2013; 129: 150–155. doi: 10.1016/j.biortech.2012.11.030.
41.
NozueS.MukunoA.TsudaY.ShiinaT., et al.“Characterization of Thylakoid Membrane in a Heterocystous Cyanobacterium and Green Alga with Dual-Detector Fluorescence Lifetime Imaging Microscopy with a Systematic Change of Incident Laser Power”. Biochim. Biophys. Acta, Bioenerg. 2016; 1857(1): 46–59. doi: 10.1016/j.bbabio.2015.10.003.
42.
OtaS.YoshiharaM.YamazakiT.TakeshitaT., et al.“Deciphering the Relationship Among Phosphate Dynamics, Electron-Dense Body and Lipid Accumulation in the Green Alga Parachlorella kessleri”. Sci. Rep. 2016; 6: 25731doi: 10.1038/srep25731.
43.
TalebA.KandilianR.TouchardR.MontalescotV., et al.“Screening of Freshwater and Seawater Microalgae Strains in Fully Controlled Photobioreactors for Biodiesel Production”. Bioresour. Technol. 2016; 218: 480–490. doi: 10.1016/j.biortech.2016.06.086.
44.
LatourG.RobinetL.DazziA.PortierF., et al.“Correlative Nonlinear Optical Microscopy and Infrared Nanoscopy Reveals Collagen Degradation in Altered Parchments”. Sci. Rep. 2016; 6(1): 26344doi: 10.1038/Srep26344.
45.
GoodsonC.RothR.WangZ.T.GoodenoughU.“Structural Correlates of Cytoplasmic and Chloroplast Lipid Body Synthesis in Chlamydomonas reinhardtii and Stimulation of Lipid Body Production with Acetate Boost”. Eukaryotic Cell. 2011; 10(12): 1592–1606. doi: 10.1128/EC.05242-11.
46.
WangZ.T.UllrichN.JooS.WaffenschmidtS.GoodenoughU.“Algal Lipid Bodies: Stress Induction, Purification, and Biochemical Characterization in Wild-Type and Starchless Chlamydomonas reinhardtii”. Eukaryotic Cell. 2009; 8(12): 1856–1868. doi: 10.1128/EC.00272-09.
47.
NguyenH.M.BaudetM.CuinéS.AdrianoJ.-M., et al.“Proteomic Profiling of Oil Bodies Isolated from the Unicellular Green Microalga Chlamydomonas reinhardtii: With Focus on Proteins Involved in Lipid Metabolism”. Proteonomics. 2011; 11(21): 4266–4273. doi: 10.1002/pmic.201100114.
48.
H. Goold, F. Beisson, G. Peltier, Y. Li-Beisson. “Microalgal Lipid Droplets: Composition, Diversity, Biogenesis, and Functions”. Plant Cell Rep. 2015. 545–555. doi: 10.1007/S00299-014-1711-7.
49.
YonedaK.YoshidaM.SuzukiI.WatanabeM.M.“Identification of a Major Lipid Droplet Protein in a Marine Diatom Phaeodactylum tricornutum”. Plant Cell Physiol. 2016; 57(2): 397–406. doi: 10.1093/Pcp/Pcv204.
50.
MoelleringE.R.BenningC.“RNA Interference Silencing of a Major Lipid Droplet Protein Affects Lipid Droplet Size in Chlamydomonas reinhardtii”. Eukaryotic Cell. 2010; 9(1): 97–106. doi: 10.1128/EC.00203-09.
51.
VielerA.BrubakerS.B.VickB.BenningC.“A Lipid Droplet Protein of Nannochloropsis with Functions Partially Analogous to Plant Oleosins”. Plant Physiol. 2012; 158(4): 1562–1569. doi: 10.1104/Pp.111.193029.
52.
Deniset-BesseauA.PraterC.B.VirolleM.-J.DazziA.“Monitoring Triacylglycerols Accumulation by Atomic Force Microscopy Based Infrared Spectroscopy in Streptomyces Species for Biodiesel Applications”. J. Phys. Chem. Lett. 2014; 5(4): 654–658. doi: 10.1021/jz402393a.
53.
ClèdeS.LambertF.SandtC.KascakovaS., et al.“Detection of an Estrogen Derivative in Two Breast Cancer Cell Lines Using a Single Core Multimodal Probe for Imaging (SCoMPI) Imaged by a Panel of Luminescent and Vibrational Techniques”. Analyst. 2013; 138(19): 5627doi: 10.1039/C3an00807j.
54.
MayetC.Deniset-BesseauA.PrazeresR.OrtegaJ.-M.DazziA.“Analysis of Bacterial Polyhydroxybutyrate Production by Multimodal Nanoimaging”. Biotechnol. Adv. 2013; 31(3): 369–374. doi: 10.1016/J.Biotechadv.2012.05.003.
55.
PancaniE.MathurinJ.BilentS.Bernet-CamardM.-F., et al.“Biocompatible Nanoparticles: High-Resolution Label-Free Detection of Biocompatible Polymeric Nanoparticles in Cells”. Part. Part. Syst. Charact. 2018; 35(3): 1870008doi: 10.1002/ppsc.201870008.
56.
ReboisR.OnidasD.MarcottC.NodaI.DazziA.“Chloroform Induces Outstanding Crystallization of Poly(hydroxybutyrate) (PHB) Vesicles Within Bacteria”. Anal. Bioanal. Chem. 2017; 409(9): 2353–2361. doi: 10.1007/S00216-017-0181-5.
57.
MiglioR.PalmeryS.SalvalaggioM.CarnelliL., et al.“Microalgae Triacylglycerols Content by FT-IR Spectroscopy”. J. Appl. Phycol. 2013; 25(6): 1621–1631. doi: 10.1007/S10811-013-0007-6.
58.
MengY.YaoC.XueS.YangH.“Application of Fourier Transform Infrared (FT-IR) Spectroscopy in Determination of Microalgal Compositions”. Bioresour. Technol. 2014; 151: 347–354. doi: 10.1016/j.biortech.2013.10.064.
59.
FreemanN.K.“Applications of Infrared Absorption Spectroscopy in the Analysis of Lipids”. J. Am. Oil Chem. Soc. 1968; 45(11): 798–809. doi: 10.1007/bf02631958.
60.
IsmailA.A.Van de VoortF.R.EmoG.SedmanJ.“Rapid Quantitative Determination of Free Fatty Acids in Fats and Oils by Fourier Transform Infrared Spectroscopy”. J. Am. Oil Chem. Soc. 1993; 70(4): 335–341. doi: 10.1007/BF02552703.
61.
LupetteJ.JaussaudA.SeddikiK.MorabitoC., et al.“The Architecture of Lipid Droplets in the Diatom Phaeodactylum tricornutum”. Algal Res. 2019; 38: 101415doi: 10.1016/J.Algal.2019.101415.
62.
López-GarcíaF.MicolV.VillalaínJ.Gómez-FernándezJ.C.“Infrared Spectroscopic Study of the Interaction of Diacylglycerol with Phosphatidylserine in the Presence of Calcium”. Biochim. Biophys. Acta, Lipids Lipid Metab. 1993; 1169(3): 264–272. doi: 10.1016/0005-27609390250-D.
63.
OikawaK.KasaharaM.KiyosueT.KagawaT., et al.“Chloroplast Unusual Positioning1 is Essential for Proper Chloroplast Positioning”. Plant Cell. 2003; 15(12): 2805–2815. doi: 10.1105/Tpc.016428.
64.
OikawaK.YamasatoA.KongS.G.KasaharaM., et al.“Chloroplast Outer Envelope Protein Chup1 is Essential for Chloroplast Anchorage to the Plasma Membrane and Chloroplast Movement”. Plant Physiol. 2008; 148(2): 829–842. doi: 10.1104/Pp.108.123075.
65.
A. Reis, C.M. Spickett. “Chemistry of Phospholipid Oxidation”. Biochim. Biophys. Acta, Biomembr. 2012. 2374–2387. doi: 10.1016/J.Bbamem.2012.02.002.
66.
GrayJ.I.“Measurement of Lipid Oxidation: A Review”. J. Am. Oil Chem. Soc. 1978; 55(6): 539–546. doi: 10.1007/BF02668066.
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