Abstract:BackgroundLipid/carbohydrate content and ratio are extremely important when engineering algal cells for liquid biofuel production. However, conventional methods for such determination and quantification are not only destructive and tedious, but also energy consuming and environment unfriendly. In this study, we first demonstrate that Raman spectroscopy is a clean, fast, and accurate method to simultaneously quantify the lipid/carbohydrate content and ratio in living microalgal cells.ResultsThe quantification r… Show more
“…A strong case can be made that substrate characterization in biorefinery has emerged as a research field in its own right. Characterization of biorefinery resources has been examined by several state-of-the-art analytical techniques including Fourier transform infrared spectroscopy (FTIR) 2 , fluorescence spectroscopy 3 , HPLC 4 , gas chromatography-mass spectrometry (GC-MS) 5 , NMR spectroscopy 6 , gel permeation chromatography (GPC) 7 , scanning electron microscopy 8 , tunneling electron microscopy 9 , atomic force microscopy 10 , Raman spectrometry 11 , time-of-flight secondary ion mass spectrometry (ToF-SIMS) 12 , and small-angle neutron scattering 13 along with a host of wet-chemistry and biological assays.…”
The analysis of chemical structural characteristics of biorefinery product streams (such as lignin and tannin) has advanced substantially over the past decade, with traditional wet-chemical techniques being replaced or supplemented by NMR methodologies. Quantitative 31 P NMR spectroscopy is a promising technique for the analysis of hydroxyl groups because of its unique characterization capability and broad potential applicability across the biorefinery research community. This protocol describes procedures for (i) the preparation/solubilization of lignin and tannin, (ii) the phosphitylation of their hydroxyl groups, (iii) NMR acquisition details, and (iv) the ensuing data analyses and means to precisely calculate the content of the different types of hydroxyl groups. Compared with traditional wet-chemical techniques, the technique of quantitative 31 P NMR spectroscopy offers unique advantages in measuring hydroxyl groups in a single spectrum with high signal resolution. The method provides complete quantitative information about the hydroxyl groups with small amounts of sample (~30 mg) within a relatively short experimental time (~30-120 min).
“…A strong case can be made that substrate characterization in biorefinery has emerged as a research field in its own right. Characterization of biorefinery resources has been examined by several state-of-the-art analytical techniques including Fourier transform infrared spectroscopy (FTIR) 2 , fluorescence spectroscopy 3 , HPLC 4 , gas chromatography-mass spectrometry (GC-MS) 5 , NMR spectroscopy 6 , gel permeation chromatography (GPC) 7 , scanning electron microscopy 8 , tunneling electron microscopy 9 , atomic force microscopy 10 , Raman spectrometry 11 , time-of-flight secondary ion mass spectrometry (ToF-SIMS) 12 , and small-angle neutron scattering 13 along with a host of wet-chemistry and biological assays.…”
The analysis of chemical structural characteristics of biorefinery product streams (such as lignin and tannin) has advanced substantially over the past decade, with traditional wet-chemical techniques being replaced or supplemented by NMR methodologies. Quantitative 31 P NMR spectroscopy is a promising technique for the analysis of hydroxyl groups because of its unique characterization capability and broad potential applicability across the biorefinery research community. This protocol describes procedures for (i) the preparation/solubilization of lignin and tannin, (ii) the phosphitylation of their hydroxyl groups, (iii) NMR acquisition details, and (iv) the ensuing data analyses and means to precisely calculate the content of the different types of hydroxyl groups. Compared with traditional wet-chemical techniques, the technique of quantitative 31 P NMR spectroscopy offers unique advantages in measuring hydroxyl groups in a single spectrum with high signal resolution. The method provides complete quantitative information about the hydroxyl groups with small amounts of sample (~30 mg) within a relatively short experimental time (~30-120 min).
“…They are considered as a prospective source of biofuel production, and much research has been devoted to engineering novel or enhanced synthesis pathways of energy‐rich metabolites, lipids and carbohydrates in algae . Recently, we have reported the application of Raman spectroscopy to algae characterization for a label‐free and non‐destructive evaluation of biofuel production . In our previous study, high fluorescence background from the photosynthesis system as well as the inhomogeneous and dynamic intracellular structures were major limiting factors which degrade the speed and accuracy of the quantification.…”
A fluorescence background is one of the common interference factors of the Raman spectroscopic analysis in the biology field. Shifted‐excitation Raman difference spectroscopy (SERDS), in which a slow (typically 1 Hz) modulation to excitation wavelength is coupled with a sequential acquisition of alternating shifted‐excitation spectra, has been used to separate Raman scattering from excitation‐shift insensitive background. This sequential method is susceptible to spectral change and thus is limited only to stable samples. We incorporated a fast laser modulation (200 Hz) and a mechanical streak camera into SERDS to effectively parallelize the SERDS measurement in a single exposure. The developed system expands the scope of SERDS to include temporary varying system. The proof of concept is demonstrated using highly fluorescent samples, including living algae. Quantitative performance in fluorescence rejection and the robustness of the method to the dynamic spectral change during the measurement are manifested.
“…GC/MS or LC/MS have excellent sensitivity, molecular specificity, and precision, and represent the current 'gold standard' for the quantification of microalgal cellular composition [101]. However, sample pretreatment makes these methods destructive, time-consuming, environmentally unfriendly, and does not allow for real-time lipid content monitoring.…”
Section: Gas and Liquid Chromatography Coupled To Mass Spectrometrymentioning
confidence: 99%
“…Raman micro-spectroscopy has been applied also to quantify the degree of unsaturation by means of the ratiometric method in six species of Mortierella [144]; in situ analysis of lipids accumulated in Botryococcus sudeticus, Chlamydomonas sp., and Trachydiscus minutus [145]; in vivo lipid and carbohydrate quantification of single Chlamydomonas sp. [101]; TAG accumulation in single Nannochloropsis oceanica cells [146]; and quantitative analysis of lipid unsaturation in immobilized Fistulifera solaris [147]. Urban et al…”
Oleaginous microorganisms are among the most promising feedstocks for the production of lipids for biofuels and oleochemicals. Lipids are synthesized in intracellular compartments in the form of lipid droplets. Therefore, their qualitative and quantitative analysis requires an initial pretreatment step that allows their extraction. Lipid extraction techniques vary with the type of microorganism but, in general, the presence of an outer membrane or cell wall limits their recovery. This review discusses the various types of oleaginous microorganisms, their lipid accumulating capabilities, lipid extraction techniques, and the pretreatment of cellular biomass for enhanced lipid recovery. Conventional methods for lipid quantification include gravimetric and chromatographic approaches; whereas nonconventional methods are based on infrared, Raman, nuclear magnetic resonance, and fluorescence spectroscopic analysis. Recent advances in these methods, their limitations, and fields of application are discussed, with the aim of providing a guide for selecting the best method or combination of methods for lipid quantification.
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