Response to nutrient variation on lipid productivity in green microalgae captured using second derivative FTIR and Raman spectroscopy

M. contortum contained a relatively higher proportion of CH, TAGs, SFA, and UFA compared to Chlamydomonas sp.

“M. contortum was found to contain a relatively higher proportion of CH, TAGs, SFA, and UFA compared to Chlamydomonas sp.”

CH band intensities of NP1 are relatively higher than the other NP treatments.

“The CH band intensities of NP1 (Fig. 1B and D) are relatively higher than the other NP treatments.”

In all four NP treatments, distinct peaks at ~2924 cm-1 and ~2846 cm-1 from CH2 asymmetric and symmetric stretches of hydrocarbons (CH) were observed in FTIR spectra.

“It can be seen in the overlay of FTIR spectra (Fig.1A) that in all four NP treatments, distinct peaks at ~2924 cm -1 and ~2846 cm -1 from the CH2 asymmetric and symmetric stretches of hydrocarbons (CH) were observed.”

Intensity variation between the NP treatments for UFA peaks is not discernible.

“Intensity variation between the NP treatments for UFA peaks is not discernible.”

There is intensity variation in biomolecular bands (mainly lipids, proteins, carbohydrates, and carotenoids) between the four NP treatments.

“It can be seen in figures 1 to 4 that there is intensity variation in the biomolecular bands (mainly lipids, proteins, carbohydrates, and carotenoids) between the four NP treatments.”

Peak areas from the negative SD spectra, informed by PCA analysis, enabled capturing quantifiable changes in a manner that is consistent with known microalgal physiology.

“Peak areas from the negative SD spectra, informed by PCA analysis, enabled capturing quantifiable changes in a manner that is consistent with known microalgal physiology.”

For M. contortum on its 20th day of growth, similar results were observed except for a reduction in the intensity of the peak at 1380 cm-1 compared to the 10th day.

“Similar results were observed for M.contortum on its 20 th day of growth (Fig.2), except for a reduction in the intensity of the peak 1380cm -1 than on its 10 th day of growth (Table 2).”

The intensity of the ν(C=C) stretches from carotenoids (~1515 cm-1) is high for the replete (NP4) condition in Raman and SD-Raman spectra of both species.

“The Raman and SD-Raman spectra of the two species M.contortum (Fig.1C and D) and Chlamydomonas sp. (Fig.3C and D) showed that the intensity of the ν(C=C) stretches from carotenoids (~1515 cm -1 ) is high for the replete (NP4) condition.”

SD-FTIR and SD-Raman spectra revealed the presence of all four characteristic stretches of hydrocarbons (νasCH3, νasCH2, νsCH3, νsCH2) in the expected region 3000-2800 cm-1.

“As SD spectra enhance the spectral resolution, the presence of all the four characteristic stretches of hydrocarbons ( ν asCH3 , ν asCH2 , ν sCH3 , ν sCH2) was revealed by the SD-FTIR (Fig. 1B) and SD-Raman spectra (Fig. 1D) in the expected region 3000 -2800 cm -1 [23,52,54].”

• cites [23] [23] C. Esther Elizabeth Grace, P. Kiruthika Lakshmi, S. Meenakshi, S. Vaidyanathan, S. Srisudha, M. Briget Mary, Biomolecular transitions and lipid accumulation in green microalgae monitored by FTIR and Raman analysis, Spectrochim. Acta -Part A Mol. Biomol. Spectrosc. 224 (2020). https://doi.org/10.1016/j.saa.2019.117382. - sourceNot checked yet

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• cites [52] [52] B.H. Stuart, Infrared Spectroscopy: Fundamentals and Applications, John Wiley & Sons, Ltd, Chichester, UK, 2004. https://doi.org/10.1002/0470011149. - sourceNot checked yet

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• cites [54] [54] P. Larkin, Infrared and Raman spectroscopy: Principles and Spectral Interpretation, 1st Editio, Elsevier, 2011. - http://www.chemistry.uoc.gr/lapkin/Infrared\_and\_Raman\_Spectroscopy\_\_Principles\_and\_Spectral\_Inte rpretation.pdf. - sourceNot checked yet

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The increase in CH intensity for NP4 was not as prominent in Chlamydomonas sp. as in M. contortum.

“However, an increase in CH intensity for NP4 was not as prominent as in M.contortum (Fig.3B, 3D, 4B, and 4D).”

The normal FTIR and SD-FTIR spectra of the two species on their 10th and 20th days of growth show a gradual decrease in the amide I (~1654 cm-1) and amide II (~1540 cm-1) band intensities of proteins from the replete (NP4) to deplete condition (NP1).

“The normal FTIR (Fig. 1A, 2A, 3A, and 4A) and SD-FTIR (Fig. 1B, 2B, 3B, and 4B) spectra of the two species on their 10 th and 20 th days of growth clearly show a gradual decrease in the amide I (~1654cm -1 ) and amide II (~1540 cm -1 ) band intensities of proteins from the replete (NP4) to deplete condition (NP1).”

Biomolecules present in M. contortum were identified from FTIR, SD-FTIR, Raman, and SD-Raman spectra.

“Biomolecules present in M.contortum (Table 2) were identified from the FTIR (Fig. 1A, 2A), SD-FTIR (Fig.1B, 2B), Raman (Fig. 1C, 2C) and SD-Raman spectra (Fig.1D, 2D).”

There was no increase in intensity of CH deformation bands at ~1460 cm-1 (δasCH3) and ~1380 cm-1 (δsCH3) with NP1 treatment.

“Although these bands represent both lipids and proteins, there was no increase in their intensity with NP1 treatment [26,53].”

• cites [26] [26] M. Kansiz, P. Heraud, B. Wood, F. Burden, J. Beardall, D. McNaughton, Fourier transform infrared microspectroscopy and chemometrics as a tool for the discrimination of cyanobacterial strains, Phytochemistry. 52 (1999) 407 -417. https://doi.org/10.1016/S0031-9422(99)00212-5. - sourceNot checked yet

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• cites [53] [53] J.N. Murdock, D.L. Wetzel, FT-IR Microspectroscopy Enhances Biological and Ecological Analysis of Algae, Appl. Spectrosc. Rev. 44 (2009) 335 -361. https://doi.org/10.1080/05704920902907440. -Not checked yet

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A decrease in carotenoid pigments from the NP4 to NP1 treatments was revealed in the Raman and SD-Raman spectra.

“Furthermore, the decrease in carotenoid pigments from the NP4 to NP1 treatments was revealed in the Raman and SD-Raman spectra.”

An increase in the intensity of the CH band for NP4 (replete condition) was also observed.

“On the contrary, an increase in the intensity of the CH band for NP4 (a replete condition with surplus nutrients) was also observed (Fig.1B and D).”

M. contortum has a higher amount of carotenoids than Chlamydomonas sp.

“The increased amount of carotenoids in M.contortum than Chlamydomonas sp. can also be seen in the SD-FTIR at ~1510 cm -1 (IR) (Fig.1B and 3B) and SD-Raman at ~1515 cm -1 (Raman) (Fig.1D and 3D).”

Chlamydomonas sp. exhibited an increase in CH intensities (3000-2800 cm-1) with nutrient depletion (NP1) on the 10th and 20th days of growth.

“Chlamydomonas sp. also exhibited an increase in CH intensities (3000-2800 cm -1 ) with nutrient depletion (NP1) on the 10 th (Fig.3) and 20 th (Fig.4) days of growth, in accordance with previous studies [9,13,58].”

• cites [9] [9] A.P. Dean, D.C. Sigee, B. Estrada, J.K. Pittman, Using FTIR spectroscopy for rapid determination of lipid accumulation in response to nitrogen limitation in freshwater microalgae, Bioresour. Technol. 101 (2010) 4499 -4507. https://doi.org/10.1016/j.biortech.2010.01.065. - sourceNot checked yet

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• cites [13] [13] A.K. Bajhaiya, A.P. Dean, T. Driver, D.K. Trivedi, N.J.W. Rattray, J.W. Allwood, R. Goodacre, J.K. Pittman, High-throughput metabolic screening of microalgae genetic variation in response to nutrient - limitation, Metabolomics. 12 (2016) 1 -14. https://doi.org/10.1007/s11306-015-0878-4. - sourceNot checked yet

  Not checked in the free run.

• cites [58] [58] A.P. Dean, J.M. Nicholson, D.C. Sigee, Impact of phosphorus quota and growth phase on carbon allocation in Chlamydomonas reinhardtii: An FTIR microspectroscopy study, Eur. J. Phycol. 43 (2008) 345 -354. https://doi.org/10.1080/09670260801979287. - sourceNot checked yet

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In NP2 to NP4 treatments, the TAG peak at ~1740 cm-1 was observed as shoulder peaks.

“In NP2 to NP4 treatments, it was observed as shoulder peaks.”

All NP treatments showed CH3 asymmetric and symmetric stretches at ~2950 cm-1 and ~2862 cm-1 in Raman spectra.

“Similarly, all of the NP treatments showed CH3 asymmetric and symmetric stretches at ~ 2950 cm -1 and ~ 2862 cm -1 in Raman spectra (Fig.1C).”

The FTIR spectra of M. contortum and Chlamydomonas sp. on both 10th and 20th days for the four NP treatments show that the spectra of the limited NP concentration (NP1) were distinct from the rest by the presence of a sharp peak at ~1740 cm-1 that corresponds to TAGs.

“FTIR spectra of M.contortum (Fig. 1A and 2A) and Chlamydomonas sp. (Fig. 3A and 4A) on both 10 th and 20 th days of growth for the four NP treatments show that the spectra of the limited NP concentration (NP1) were distinct from the rest of the NP treatments by the presence of a sharp peak at ~1740cm -1 that corresponds to TAGs.”

SD-FTIR spectra showed a gradual decrease in the content of proteins from replete (NP4) to deplete (NP1) condition.

“SD-FTIR spectra (Fig.11A, and B) showed a gradual decrease in the content of proteins, from replete (NP4) to deplete (NP1) condition.”

The negative peaks at ~1515, ~2820, and ~2860 cm-1 indicate that carotenoids are higher in Chlamydomonas sp. for NP1 treatment.

“While, the negative peaks at ~1515, ~2820, and ~2860 cm -1 , indicate that carotenoids are higher in Chlamydomonas sp. for NP1 treatment.”

The reduction in carotenoid for NP1 is obvious in SD-Raman.

“Although a gradual decrease as in the case of proteins, was not observed, the reduction in carotenoid for NP1 is obvious, as discussed in Section 3.1.2.”

A fourfold increase in CH was observed for both species for the NP1 treatment from the CH:carotenoid ratio.

“Similarly, a fourfold increase in CH was observed for both the species for the NP1 treatment from the CH:carotenoid ratio (Fig.9B).”

The region corresponding to hydrocarbons (~2924, ~2846 cm-1), TAGs (~1740 cm-1), and carbohydrates (~1153, ~1079, ~1058, and ~1030 cm-1) are more in NP1 compared to NP4.

“Interpreting the corresponding loadings plot (Fig. 5B) shows that the region corresponding to hydrocarbons (~2924, ~2846 cm -1 ), TAGs (~1740 cm -1 ), and carbohydrates (~1153,~1079,~1058, and ~ 1030 cm -1 ) are more in NP1 compared to NP4.”

The response of M. contortum to NP1 is more drastic than to NP2-NP4 on the 20th day of its growth.

“It can be seen that the response of M.contortum to NP1 is drastic than for NP2 - NP4 on the 20 th day of its growth.”

Chlamydomonas sp. showed nearly a fourfold increase in TAGs on both days of growth.

“While, Chlamydomonas sp., showed nearly a fourfold increase on both days of growth.”

Both species tend to accumulate more carbohydrates on their 20th day of growth.

“Both species tend to accumulate more carbohydrates on their 20 th day of growth.”

The production of carbohydrates in the two species is higher for nutrient depletion (NP1) than for other treatments.

“The production of carbohydrates in the two species is higher for nutrient depletion (NP1) than for other treatments.”

The accumulation of TAGs and saturated lipids (SFA) for the two species was evident in both species in response to NP1 treatment.

“The accumulation of TAGs (Fig.11A and B) and saturated lipids (SFA) (Fig.11C and D) for the two species was evident in both species in response to NP1 treatment.”

PCA indicated a higher accumulation of carbohydrates in M. contortum compared to Chlamydomonas sp.

“PCA indicated a higher accumulation of carbohydrates in M.contortum compared to Chlamydomonas sp.”

Both M. contortum and Chlamydomonas sp. respond to nutrient deplete condition (NP1) with higher accumulation of lipids in the form of hydrocarbons (CH), TAGs, saturated lipids (SFA), and unsaturated lipids.

“Both M.contortum and Chlamydomonas sp., have been found to respond to the nutrient deplete condition (NP1) with higher accumulation of lipids in the form of hydrocarbons (CH), TAGs, saturated lipids (SFA), and unsaturated lipids (Fig.11A-D).”

• Also stated in: § 4. Conclusion · p. 27 · plain

  “Both microalgae species are shown to respond to nutritional stress (NP1) by a higher accumulation of lipids, particularly neutral lipids which indicate the suitability of these species for biodiesel application, as well as lipid production, in general.”

The production of lipids is higher on the 20th day than the 10th day of growth for both species.

“The production of lipids is higher on the 20 th day than the 10 th day of growth for both species.”

Almost a threefold increase in the carbohydrate:protein ratio was observed for both species on both days of growth.

“Almost a threefold increase in the carbohydrate: protein ratio was observed for both species on both days of growth (Fig.10A).”

The difference in PC2 loadings is clear in M. contortum than in Chlamydomonas sp.

“The difference is clear in M. contortum than in Chlamydomonas sp.”

For Chlamydomonas sp. on its 10th day of growth, the accumulation of carbohydrates is higher than M. contortum.

“On the other hand, it can be seen that for Chlamydomonas sp. on its 10 th day of growth, the accumulation of carbohydrates is higher than M.contortum (Fig.11B).”

The relative content of carbohydrates was higher in the two species for nutrient deplete conditions (NP1).

“Similar to lipids, the relative content of carbohydrates (Fig.10) was higher in the two species for nutrient deplete conditions (NP1).”

The amount of proteins in the cells on the 20th day of growth was higher than on the 10th day.

“The amount of proteins in the cells on the 20 th day of growth was higher than on the 10 th day, which was evident on the PC3 loadings (Fig.7B) in the amide regions (~1653, 1540cm -1 ).”

The integrated band area of proteins and carotenoids used to normalize lipid and carbohydrate bands changed in response to the various nutrient treatments.

“the integrated band area of proteins and carotenoids that were used to normalize the lipids and carbohydrate bands, also changed in response to the various nutrient treatments (Fig.1, 2, 3, and 4).”

The indication that unsaturated lipid is higher in NP1 compared to NP4 is not evident in the Chlamydomonas response.

“This is not evident in the Chlamydomonas response.”

The accumulation of TAGs was higher for M. contortum than Chlamydomonas sp.

“The accumulation of TAGs was high for M.contortum than Chlamydomonas sp. (Fig.9C).”

The regions corresponding to carbohydrates (~1153, ~1079, ~1058, and ~1030 cm-1) are higher in Chlamydomonas sp. compared to M. contortum.

“While the regions corresponding to carbohydrates (~1153, ~1079,~1058, and ~ 1030 cm -1 ) are higher in Chlamydomonas sp . compared to M. contortum .”

Only the first three PCs were considered, as this explained 94% of the total variance for both M. contortum and Chlamydomonas sp.

“Only the first three PCs were considered, as this explained 94 % of the total variance for both M. contortum and Chlamydomonas sp.”

For M. contortum, the response to NP1 for both days clusters in the positive PC2 and PC3 scores plot.

“Similar results were observed for M. contortum (Fig. 5C and D), where the response to NP1 for both days clusters in the positive PC2 and PC3 scores plot.”

An increase in CH intensity was accompanied by an increase in TAGs:protein ratio and SFA:carotenoid ratio for the nutrient deplete condition (NP1) for both species.

“An increase in CH intensity was accompanied by an increase in TAGs: protein ratio (Fig. 9C) and SFA:carotenoid ratio (Fig. 9D) for the nutrient deplete condition (NP1) for both the species.”

For NP1 treatment, the peak at ~1515 cm-1 was very weak for M. contortum than for Chlamydomonas sp.

“This was also evident from the overlay of SD-Raman spectra (Fig.1C and 3C), where for NP1 treatment, the peak at ~1515 was very weak for M.contortum (Fig.1C and Table 2) than for Chlamydomonas sp.(Fig.3C and Table 3).”

It is possible to differentiate the contributions attributed to the species along the PC3 direction, which explains 14% of the variance.

“It is also possible to differentiate the contributions that can be attributed to the species, along the PC3 direction, which explains 14% of the variance.”

The scores plot indicates the response separated on PC2 for the two species on both days for NP1 treatment.

“The scores plot (Fig. 7A) indicates the response separated on PC2 for the two species on both days.”

The signal at ~1440 cm-1 corresponding to SFA is higher in M. contortum than Chlamydomonas sp. in the PCA on SD-Raman for NP1 treatment.

“This was evident from the PCA on SD-Raman for NP1 treatment (Fig.8B), where the signal at ~1440 cm -1 that corresponds to SFA is noted to be higher in M.contortum than Chlamydomonas sp.”

NP1/NP4 from the CH:protein ratio showed a threefold increase of CH from replete to deplete condition for M. contortum on its 20th day of growth.

“Computing NP1/NP4 from CH:protein ratio (Fig.9A) showed a threefold increase of CH from replete to deplete condition for M.contortum on its 20 th day of growth”

Maximum peak area for accumulation of CH about 0.06 (SD-FTIR) and 90 (SD-Raman) was observed for M. contortum on its 20th day of growth.

“Maximum peak area (sq. units) for the accumulation of CH about 0.06 (SD-FTIR) (Fig.11A) and 90 (SD-Raman) (Fig.11C) was observed for M.contortum on its 20 th day of growth.”

Maximum peak area for UFA of 20 (SD-Raman) is observed for M. contortum.

“Maximum peak area for UFA of 20(SDRaman) is observed for M.contortum .”

The responses of the two microalgae to the various NP treatments can also be seen for other biomolecules such as proteins, carotenoids, and carbohydrates.

“The responses of the two microalgae to the various NP treatments can also be seen for other biomolecules such as proteins (Fig.11A and B), carotenoids (Fig.11C and D), and carbohydrates (Fig.11A-D).”

The variation in the accumulation of carotenoids was noticed in SD-Raman.

“Similarly, the variation in the accumulation of carotenoids was noticed in SD-Raman (Fig.11C and D).”

The difference in the accumulation of lipids between the two microalgae cells is clear.

“The difference in the accumulation of lipids between the two microalgae cells is also clear.”

An increase in CH, TAGs, SFA, and UFA for M. contortum was evident from the PCA.

“An increase in CH, TAGs, SFA, and UFA for M.contortum was also evident from the PCA (Fig.7 and 8).”

The responses of the two species to the NP1 treatment are distinct from the other NP treatments.

“The accumulation of lipids relative to proteins (Fig.9A, C, and E) and carotenoids (Fig.9B, D, and F) clearly show that the responses of the two species to the NP1 treatment are distinct from the other NP treatments.”

The difference in the responses between the two species is clear in the carbohydrate:carotenoid ratio.

“The difference in the responses between the two species is clear in carbohydrate:carotenoid ratio (Fig.10B).”

PCA showed spectral variance between species and days for NP1 treatment.

“This was also observed in the PCA that showed spectral variance between species and days for NP1 treatment (Fig.7B).”

The reason to choose the carotenoid band to normalize lipid and carbohydrate bands in Raman spectra was the characteristic reduction in intensity of its peak at ~1515 cm-1 with nutrient depletion (NP1), similar to proteins in FTIR spectra.

“The reason to choose the carotenoid band to normalize the lipid and carbohydrate bands in the Raman spectra was due to the characteristic reduction in the intensity of its characteristic peak at ~1515 cm -1 ( ν C=C) with nutrient depletion (NP1), similar to proteins in FTIR spectra.”

The quantity of unsaturated lipids (UFA) is clearer in SD-Raman than in SD-FTIR.

“The quantity of unsaturated lipids (UFA) is clear in SD-Raman (Fig.11C and D) than in SD-FTIR.”

Monoraphidium contortum and Chlamydomonas sp. were identified to accumulate lipids.

“Two green microalgae species Monoraphidium contortum (M. contortum) and Chlamydomonas sp. that were identified to accumulate lipids were subjected to four different nutrient treatments (NP1-NP4)”

• Also stated in: § 2.1. Microalgal culture and growth conditions · p. 5 · plain

  “Two freshwater green microalgae isolates, Monoraphidium contortum (M. contortum) and Chlamydomonas sp., that were identified to accumulate lipids [23]were subjected to four different treatments”

• cites [23] [23] C. Esther Elizabeth Grace, P. Kiruthika Lakshmi, S. Meenakshi, S. Vaidyanathan, S. Srisudha, M. Briget Mary, Biomolecular transitions and lipid accumulation in green microalgae monitored by FTIR and Raman analysis, Spectrochim. Acta -Part A Mol. Biomol. Spectrosc. 224 (2020). https://doi.org/10.1016/j.saa.2019.117382. - sourceNot checked yet

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Nutritional variability causes changes in the macromolecular composition of microalgae cells.

“Several studies to improve lipid production in microalgae have shown that nutritional variability causes changes in the macromolecular composition of microalgae cells [5,6].”

• cites [5] [5] K. Stehfest, J. Toepel, C. Wilhelm, The application of micro-FTIR spectroscopy to analyze nutrient stressrelated changes in biomass composition of phytoplankton algae, Plant Physiol. Biochem. 43 (2005) 717 -726. https://doi.org/10.1016/j.plaphy.2005.07.001. - sourceNot checked yet

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• cites [6] [6] M. Palmucci, S. Ratti, M. Giordano, Ecological and evolutionary implications of carbon allocation in marine phytoplankton as a function of nitrogen availability: A fourier transform infrared spectroscopy approach, J. Phycol. 47 (2011) 313 -323. https://doi.org/10.1111/j.1529-8817.2011.00963.x. - sourceNot checked yet

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Not all microalgae species respond to nutrient scarcity by accumulating carbohydrates or lipids.

“Although nutritional scarcity has caused metabolic changes in microalgae, such as the accumulation of carbohydrates and lipids, not all species have responded to nutrient scarcity by accumulating carbohydrates or lipids [6,13,14].”

• cites [6] [6] M. Palmucci, S. Ratti, M. Giordano, Ecological and evolutionary implications of carbon allocation in marine phytoplankton as a function of nitrogen availability: A fourier transform infrared spectroscopy approach, J. Phycol. 47 (2011) 313 -323. https://doi.org/10.1111/j.1529-8817.2011.00963.x. - sourceNot checked yet

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• cites [13] [13] A.K. Bajhaiya, A.P. Dean, T. Driver, D.K. Trivedi, N.J.W. Rattray, J.W. Allwood, R. Goodacre, J.K. Pittman, High-throughput metabolic screening of microalgae genetic variation in response to nutrient - limitation, Metabolomics. 12 (2016) 1 -14. https://doi.org/10.1007/s11306-015-0878-4. - sourceNot checked yet

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• cites [14] [14] T. Driver, A.K. Bajhaiya, J.W. Allwood, R. Goodacre, J.K. Pittman, A.P. Dean, Metabolic responses of eukaryotic microalgae to environmental stress limit the ability of FT-IR spectroscopy for species identi fi cation, 11 (2015) 148 -155. https://doi.org/10.1016/j.algal.2015.06.009. - sourceNot checked yet

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Nitrogen and phosphorus limitation causes changes in photosynthetic activity, lipid synthesis, and biomass composition in freshwater and marine microalgae.

“Studies have indicated changes in photosynthetic activity, lipid synthesis, and biomass composition in freshwater and marine microalgae as a result of nitrogen and phosphorus limitation [8 -11].”

• cites [8] [8] Y. Jiang, T. Yoshida, A. Quigg, Photosynthetic performance, lipid production and biomass composition in response to nitrogen limitation in marine microalgae, Plant Physiol. Biochem. 54 (2012) 70 -77. https://doi.org/10.1016/j.plaphy.2012.02.012. - sourceNot checked yet

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• cites [9] [9] A.P. Dean, D.C. Sigee, B. Estrada, J.K. Pittman, Using FTIR spectroscopy for rapid determination of lipid accumulation in response to nitrogen limitation in freshwater microalgae, Bioresour. Technol. 101 (2010) 4499 -4507. https://doi.org/10.1016/j.biortech.2010.01.065. - sourceNot checked yet

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• cites [10] [10] A.M. Illman, A.H. Scragg, ScienceDirect Enzyme and Microbial Technology : Increase in Chlorella strains calorific values when grown in low nitrogen medium, Enzyme Microb. Technol. 27 (2000) 631 -635. 11. http://www.sciencedirect.com/science/article/pii/S0141022900002660%0Apapers2://publication/uuid/B 7537DAC-F40B-4395-9341-739FEEA48F97. - sourceNot checked yet

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• cites [11] [11] A.P. Dean, J.M. Nicholson, D.C. Sigee, Impact of phosphorus quota and growth phase on carbon allocation in Chlamydomonas reinhardtii: An FTIR microspectroscopy study, Eur. J. Phycol. 43 (2008) 345 -354. https://doi.org/10.1080/09670260801979287. - sourceNot checked yet

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Second derivative spectroscopy enables accurate measurements of wavelength position by identifying small and overlapping peaks that are not resolved in the original spectra.

“SD spectroscopy enables accurate measurements of wavelength position by identifying small and overlapping peaks that are not resolved in the original spectra. [23,46,47].”

• cites [23] [23] C. Esther Elizabeth Grace, P. Kiruthika Lakshmi, S. Meenakshi, S. Vaidyanathan, S. Srisudha, M. Briget Mary, Biomolecular transitions and lipid accumulation in green microalgae monitored by FTIR and Raman analysis, Spectrochim. Acta -Part A Mol. Biomol. Spectrosc. 224 (2020). https://doi.org/10.1016/j.saa.2019.117382. - sourceNot checked yet

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• cites [46] [46] L. Rieppo, S. Saarakkala, T. Närhi, H.J. Helminen, J.S. Jurvelin, J. Rieppo, Application of second derivative spectroscopy for increasing molecular specificity of fourier transform infrared spectroscopic imaging of articular cartilage, Osteoarthr. Cartil. 20 (2012) 451 -459. https://doi.org/10.1016/j.joca.2012.01.010. - sourceNot checked yet

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• cites [47] [47] D. Ami, R. Posteri, P. Mereghetti, D. Porro, S.M. Doglia, P. Branduardi, Fourier transform infrared spectroscopy as a method to study lipid accumulation in oleaginous yeasts, Biotechnol. Biofuels. 7 (2014) 1 -14. https://doi.org/10.1186/1754-6834-7-12. - sourceNot checked yet

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The N:P ratio approximating 18 is in a physiologically acceptable range for assessing nutrient stress.

“at an N:P ratio approximating 18, which is in a physiologically acceptable range for assessing nutrient stress [48 -51].”

• cites [48] [48] R.W. Sterner, Nutrient Stoichiometry in Aquatic Ecosystems, in: Encycl. Inl. Waters, Elsevier, 2009: pp. 820 -831. https://doi.org/10.1016/B978-012370626-3.00113-7. - sourceNot checked yet

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• cites [49] [49] P.G. Falkowski, Rationalizing elemental ratios in unicellular algae, J. Phycol. 36 (2000) 3 -6. https://doi.org/10.1046/j.1529-8817.2000.99161.x. - sourceNot checked yet

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• cites [50] [50] A.C. Redfield, Redfield\_AmSci\_1958.pdf, Am. Sci. (1958). -Not matched

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• cites [51] [51] R.J. Geider, J. La Roche, Redfield revisited: Variability of C:N:P in marine microalgae and its biochemical basis, Eur. J. Phycol. 37 (2002) 1 -17. https://doi.org/10.1017/S0967026201003456. - sourceNot checked yet

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Primary carotenoids associated with growth are degraded under stress.

“Although studies have shown that nutrient deprivation can increase carotenoid production [66,67], it is also known that the primary carotenoids which are associated with growth are degraded under stress [68,69].”

• cites [68] [68] T.Q. Shi, L.R. Wang, Z.X. Zhang, X.M. Sun, H. Huang, Stresses as First-Line Tools for Enhancing Lipid and Carotenoid Production in Microalgae, Front. Bioeng. Biotechnol. 8 (2020) 1 -9. https://doi.org/10.3389/fbioe.2020.00610. - sourceNot checked yet

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• cites [69] [69] A.C. Guedes, H.M. Amaro, F.X. Malcata, Microalgae as sources of carotenoids, Mar. Drugs. 9 (2011) 625 -644. https://doi.org/10.3390/md9040625. - sourceNot checked yet

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Changes in signals attributed to lipids in proportion to other biochemical components were consistent with physiological changes expected from nutrient depletion.

“Changes in signals attributed to lipids in proportion to other biochemical components were consistent with physiological changes expected from nutrient depletion.”

The reduction in intensity of the 1380 cm-1 peak may indicate reduction of the symmetric CH3 deformation either from lipids or proteins.

“This may indicate the reduction of the symmetric CH3 deformation either from lipids or proteins.”

SD-FTIR and SD-Raman spectroscopy possess superior potential to capture relevant microalgal physiological changes.

“SD-FTIR and SD-Raman spectroscopy have been shown to possess superior potential to capture relevant microalgal physiological changes.”

An increase in the intensity of CH along with other indicators of lipids such as TAGs (IR) and fatty acids (Raman) can confirm the enhancement of lipid accumulation in the microalgae cells in response to NP1 treatment.

“Therefore, an increase in the intensity of CH along with the other indicators of lipids such as TAGs (IR), and fatty acids (Raman) can confirm the enhancement of lipid accumulation in the microalgae cells in response to NP1 treatment.”

The nutritional surplus is impacting carotenoid accumulation in microalgae cells.

“This indicates that the nutritional surplus is impacting carotenoid accumulation in microalgae cells.”

The weak intensity of the UFA bands in comparison to the SFA band (~1440 cm-1) indicates that these two microalgae are rich in saturated fatty acids.

“The weak intensity of these bands in comparison to the SFA band (~1440 cm -1 ) indicates that these two microalgae are rich in saturated fatty acids [36,38,60].”

• cites [36] [36] O. Samek, P. Zemánek, A. Jonáš, H.H. Telle, Characterization of oil -producing microalgae using Raman spectroscopy, Laser Phys. Lett. 8 (2011) 701 -709. https://doi.org/10.1002/lapl.201110060. - sourceNot checked yet

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• cites [38] [38] S.K. Sharma, D.R. Nelson, R. Abdrabu, B. Khraiwesh, K. Jij akli, M. Arnoux, M.J. O'Connor, T. Bahmani, H. Cai, S. Khapli, R. Jagannathan, K. Salehi-Ashtiani, An integrative Raman microscopy-based workflow for rapid in situ analysis of microalgal lipid bodies, Biotechnol. Biofuels. 8 (2015) 1 -14. https://doi.org/10.1186/s13068-015-0349-1. - sourceNot checked yet

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• cites [60] [60] J. De Gelder, K. De Gussem, P. Vandenabeele, L. Moens, Reference database of Raman spectra of biological molecules, J. Raman Spectrosc. 38 (2007) 1133 -1147. https://doi.org/10.1002/jrs.1734. -Not checked yet

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In addition to proteins, other factors may have contributed to the increase in CH intensities for M. contortum in NP4 that is not seen to the same extent in Chlamydomonas sp.

“This suggests that in addition to proteins, other factors may have contributed to the increase in CH intensities for M.contortum in NP4 that is not seen to the same extent in Chlamydomonas sp.”

The two species M. contortum and Chlamydomonas sp. have produced more neutral lipids (TAGs) with nutritional depletion.

“This indicates that the two species M.contortum and Chlamydomonas sp. have produced more neutral lipids (TAGs) with nutritional depletion [9,20,61 -63] and it is a positive indication that these microalgae species can be tapped for biodiesel production.”

• cites [9] [9] A.P. Dean, D.C. Sigee, B. Estrada, J.K. Pittman, Using FTIR spectroscopy for rapid determination of lipid accumulation in response to nitrogen limitation in freshwater microalgae, Bioresour. Technol. 101 (2010) 4499 -4507. https://doi.org/10.1016/j.biortech.2010.01.065. - sourceNot checked yet

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• cites [20] [20] K. Stehfest, J. Toepel, C. Wilhelm, The application of micro-FTIR spectroscopy to analyze nutrient stressrelated changes in biomass composition of phytoplankton algae, 43 (2005) 717 -726. https://doi.org/10.1016/j.plaphy.2005.07.001. - sourceNot checked yet

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• cites [61] [61] R. Miglio, S. Palmery, M. Salvalaggio, L. Carnelli, F. Capuano, R. Borrelli, Microalgae triacylglycerols content by FT-IR spectroscopy, (2013) 1621 -1631. https://doi.org/10.1007/s10811-013-0007-6. - sourceNot checked yet

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• cites [62] [62] K. Zienkiewicz, Z.Y. Du, W. Ma, K. Vollheyde, C. Benning, Stress-induced neutral lipid biosynthesis in microalgae -Molecular, cellular and physiological insights, Biochim. Biophys. Acta - Mol. Cell Biol. Lipids. 1861 (2016). https://doi.org/10.1016/j.bbalip.2016.02.008. - sourceNot checked yet

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• cites [63] [63] P.-L. Shen, H.-T. Wang, Y.-F. Pan, Y.-Y. Meng, P.-C. Wu, S. Xue, Identification of Characteristic Fatty Acids to Quantify Triacylglycerols in Microalgae, Front. Plant Sci. 7 (2016). https://doi.org/10.3389/fpls.2016.00162. - sourceNot checked yet

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These spectral variations reflect the cell metabolism of the two microalgae species in response to the nutrient variations.

“These spectral variations reflect the cell metabolism of the two microalgae species in response to the nutrient variations.”

SD-FTIR and SD-Raman spectroscopy have greater potential as superior screening methods for enhancing the resolution and easy quantification of key biochemical composition in algae, relevant to its usage in developing environmentally sustainable manufacturing solutions.

“Thus SD-FTIR and SD-Raman spectroscopy have greater potential as superior screening methods for enhancing the resolution and easy quantification of key biochemical composition in algae, relevant to its usage in developing environmentally sustainable manufacturing solutions.”

According to Beer Lambert's law, absorbance (FTIR and its derivatives SD-FTIR) is proportional to the analyte concentration.

“According to Beer L ambert's law, absorbance [(FTIR) and its derivatives (SD-FTIR)] is proportional to the analyte concentration [45,46].”

• cites [45] [45] H. Mark, J.W. Jr, J.W. Jr, Derivatives in Spectroscopy, 18 (2003). - sourceNot checked yet

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• cites [46] [46] L. Rieppo, S. Saarakkala, T. Närhi, H.J. Helminen, J.S. Jurvelin, J. Rieppo, Application of second derivative spectroscopy for increasing molecular specificity of fourier transform infrared spectroscopic imaging of articular cartilage, Osteoarthr. Cartil. 20 (2012) 451 -459. https://doi.org/10.1016/j.joca.2012.01.010. - sourceNot checked yet

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SD spectroscopy has captured the effect of nutrient variation on lipid production and other biomolecules in the two species.

“Thus SD spectroscopy has captured the effect of nutrient variation on lipid production and the other biomolecules in the two species.”

• Also stated in: § 4. Conclusion · p. 27 · boosted

  “Second derivative (SD) FTIR and Raman spectroscopy has effectively captured the responses of the two microalgae species M.contortum and Chlamydomonas sp. to the four different nutrient treatments.”

The negative peaks in the corresponding loadings plots can be associated more with NP1 than with NP4.

“Therefore, the negative peaks in the corresponding loadings plots can be associated more with this set of samples (NP1) than with the diagonally opposite set (NP4).”

In the NP1 treatment, M. contortum is likely to have more hydrocarbons and unsaturated lipids than Chlamydomonas sp.

“This suggests that in the NP1 treatment, M. contortum is likely to have more hydrocarbons and unsaturated lipids than Chlamydomonas sp.”

SD spectroscopy, in particular the negative regions, combined with PCA has considerably improved the quality of assessment of lipids and other biomolecular compositions compared to normal (zero-order) FTIR and Raman spectroscopy.

“SD spectroscopy, in particular, the negative regions, combined with PCA has considerably improved the quality of assessment of lipids and other biomolecular compositions which was not possible with the normal (Zero-order) FTIR and Raman spectroscopy.”

The usage of the negative region of the SD spectra is of value in enabling consistent and quantifiable monitoring of changes relevant to the biochemical composition of microalgal cells that is consistent with known physiology.

“In particular, the usage of the negative region of the SD spectra has been shown to be of value in enabling consistent and quantifiable monitoring of changes relevant to the biochemical composition of microalgal cells that is consistent with known physiology.”

These latter differences in PC3 are distanced further in the nutrient rich conditions (NP3 and 4), suggesting that the species level differences are evident better in these conditions.

“It can also be noted that these latter differences in PC3 are distanced further in the nutrient rich conditions (NP3 and 4), suggesting that the species level differences are evident better in these conditions.”

This investigation provides evidence for the use of the negative regions of both SD-FTIR and SD-Raman to be of considerable value in assessing quantitative changes consistent with known physiology of the microalgae examined.

“This investigation thus provides evidence for the use of the negative regions of both the SD-FTIR and SD-Raman to be of considerable value in assessing quantitative changes that are consistent with the known physiology of the microalgae examined.”

For the peak area to be applied as absolute content, it is important to have the sample quantity and analysis conditions the same for all analyses, to prevent alteration in the spectral information.

“However, for this to be applied, it is important to have the sample quantity and analysis conditions the same for all analyses, to prevent alteration in the spectral information [43].”

• cites [43] [43] M.J. Pelletier, Quantitative Analysis Using Raman Spectrometry, Appl. Spectrosc. 57 (2003) 20A-42A. https://doi.org/10.1366/000370203321165133. - sourceNot checked yet

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The ~2850 cm−1 IR band is assigned to νs(CH2) symmetric stretches.

“~ 2846 | ~ 2820 | s + | m + | s + | w + | s + | m + | vs + |”

• cites [9] [9] A.P. Dean, D.C. Sigee, B. Estrada, J.K. Pittman, Using FTIR spectroscopy for rapid determination of lipid accumulation in response to nitrogen limitation in freshwater microalgae, Bioresour. Technol. 101 (2010) 4499 -4507. https://doi.org/10.1016/j.biortech.2010.01.065. - sourceNot checked yet

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• cites [20] [20] K. Stehfest, J. Toepel, C. Wilhelm, The application of micro-FTIR spectroscopy to analyze nutrient stressrelated changes in biomass composition of phytoplankton algae, 43 (2005) 717 -726. https://doi.org/10.1016/j.plaphy.2005.07.001. - sourceNot checked yet

  Not checked in the free run.

• cites [52] [52] B.H. Stuart, Infrared Spectroscopy: Fundamentals and Applications, John Wiley & Sons, Ltd, Chichester, UK, 2004. https://doi.org/10.1002/0470011149. - sourceNot checked yet

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• cites [53] [53] J.N. Murdock, D.L. Wetzel, FT-IR Microspectroscopy Enhances Biological and Ecological Analysis of Algae, Appl. Spectrosc. Rev. 44 (2009) 335 -361. https://doi.org/10.1080/05704920902907440. - sourceNot checked yet

  Not checked in the free run.

• cites [54] [54] P. Larkin, Infrared and Raman spectroscopy: Principles and Spectral Interpretation, 1st Editio, Elsevier, 2011. - http://www.chemistry.uoc.gr/lapkin/Infrared\_and\_Raman\_Spectroscopy\_\_Principles\_and\_Spectral\_Inte rpretation.pdf. - sourceNot checked yet

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The main advantage of using second derivative spectroscopy is that it allows for quantitative analysis.

“The main advantage of using second derivative spectroscopy is that it allows for quantitative analysis.”

The ~1740 cm−1 IR band is assigned to ν(C=O) stretches of esters from lipids - Triacylglycerides (TAGs).

“~ 1740 | | m + | | m + | | m + | | s + |”

• cites [9] [9] A.P. Dean, D.C. Sigee, B. Estrada, J.K. Pittman, Using FTIR spectroscopy for rapid determination of lipid accumulation in response to nitrogen limitation in freshwater microalgae, Bioresour. Technol. 101 (2010) 4499 -4507. https://doi.org/10.1016/j.biortech.2010.01.065. - sourceNot checked yet

  Not checked in the free run.

• cites [20] [20] K. Stehfest, J. Toepel, C. Wilhelm, The application of micro-FTIR spectroscopy to analyze nutrient stressrelated changes in biomass composition of phytoplankton algae, 43 (2005) 717 -726. https://doi.org/10.1016/j.plaphy.2005.07.001. - sourceNot checked yet

  Not checked in the free run.

• cites [52] [52] B.H. Stuart, Infrared Spectroscopy: Fundamentals and Applications, John Wiley & Sons, Ltd, Chichester, UK, 2004. https://doi.org/10.1002/0470011149. - sourceNot checked yet

  Not checked in the free run.

• cites [53] [53] J.N. Murdock, D.L. Wetzel, FT-IR Microspectroscopy Enhances Biological and Ecological Analysis of Algae, Appl. Spectrosc. Rev. 44 (2009) 335 -361. https://doi.org/10.1080/05704920902907440. - sourceNot checked yet

  Not checked in the free run.

• cites [54] [54] P. Larkin, Infrared and Raman spectroscopy: Principles and Spectral Interpretation, 1st Editio, Elsevier, 2011. - http://www.chemistry.uoc.gr/lapkin/Infrared\_and\_Raman\_Spectroscopy\_\_Principles\_and\_Spectral\_Inte rpretation.pdf. - sourceNot checked yet

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The ~1658 cm−1 Raman band is assigned to cis ν(C=C) from unsaturated lipids.

“~ 1654 | ~ 1658 | s + | w + | s + | vw + | s + | vw + | m + |”

• cites [9] [9] A.P. Dean, D.C. Sigee, B. Estrada, J.K. Pittman, Using FTIR spectroscopy for rapid determination of lipid accumulation in response to nitrogen limitation in freshwater microalgae, Bioresour. Technol. 101 (2010) 4499 -4507. https://doi.org/10.1016/j.biortech.2010.01.065. - sourceNot checked yet

  Not checked in the free run.

• cites [20] [20] K. Stehfest, J. Toepel, C. Wilhelm, The application of micro-FTIR spectroscopy to analyze nutrient stressrelated changes in biomass composition of phytoplankton algae, 43 (2005) 717 -726. https://doi.org/10.1016/j.plaphy.2005.07.001. - sourceNot checked yet

  Not checked in the free run.

• cites [52] [52] B.H. Stuart, Infrared Spectroscopy: Fundamentals and Applications, John Wiley & Sons, Ltd, Chichester, UK, 2004. https://doi.org/10.1002/0470011149. - sourceNot checked yet

  Not checked in the free run.

• cites [53] [53] J.N. Murdock, D.L. Wetzel, FT-IR Microspectroscopy Enhances Biological and Ecological Analysis of Algae, Appl. Spectrosc. Rev. 44 (2009) 335 -361. https://doi.org/10.1080/05704920902907440. - sourceNot checked yet

  Not checked in the free run.

• cites [54] [54] P. Larkin, Infrared and Raman spectroscopy: Principles and Spectral Interpretation, 1st Editio, Elsevier, 2011. - http://www.chemistry.uoc.gr/lapkin/Infrared\_and\_Raman\_Spectroscopy\_\_Principles\_and\_Spectral\_Inte rpretation.pdf. - sourceNot checked yet

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The ~1654 cm−1 IR band is assigned to ν(C=O), ν(C-N) stretches and δ(N-H) deformations of Amide I from proteins.

“~ 1654 | ~ 1658 | s + | w + | s + | vw + | s + | vw + | m + |”

• cites [9] [9] A.P. Dean, D.C. Sigee, B. Estrada, J.K. Pittman, Using FTIR spectroscopy for rapid determination of lipid accumulation in response to nitrogen limitation in freshwater microalgae, Bioresour. Technol. 101 (2010) 4499 -4507. https://doi.org/10.1016/j.biortech.2010.01.065. - sourceNot checked yet

  Not checked in the free run.

• cites [20] [20] K. Stehfest, J. Toepel, C. Wilhelm, The application of micro-FTIR spectroscopy to analyze nutrient stressrelated changes in biomass composition of phytoplankton algae, 43 (2005) 717 -726. https://doi.org/10.1016/j.plaphy.2005.07.001. - sourceNot checked yet

  Not checked in the free run.

• cites [52] [52] B.H. Stuart, Infrared Spectroscopy: Fundamentals and Applications, John Wiley & Sons, Ltd, Chichester, UK, 2004. https://doi.org/10.1002/0470011149. - sourceNot checked yet

  Not checked in the free run.

• cites [53] [53] J.N. Murdock, D.L. Wetzel, FT-IR Microspectroscopy Enhances Biological and Ecological Analysis of Algae, Appl. Spectrosc. Rev. 44 (2009) 335 -361. https://doi.org/10.1080/05704920902907440. - sourceNot checked yet

  Not checked in the free run.

• cites [54] [54] P. Larkin, Infrared and Raman spectroscopy: Principles and Spectral Interpretation, 1st Editio, Elsevier, 2011. - http://www.chemistry.uoc.gr/lapkin/Infrared\_and\_Raman\_Spectroscopy\_\_Principles\_and\_Spectral\_Inte rpretation.pdf. - sourceNot checked yet

  Not checked in the free run.

Changes in absolute peak areas are likely to reflect the spectral variations more quantitatively.

“Changes in absolute peak areas are also likely to reflect the spectral variations more quantitatively.”

The intensity of Raman scattering and the corresponding derivative (SD-Raman) intensity as a function of Raman shift is proportional to the analyte concentration.

“Similarly, the intensity of Raman scattering and the corresponding derivative (SD-Raman) intensity as a function of Raman shift is proportional to the analyte concentration [43,54].”

• cites [43] [43] M.J. Pelletier, Quantitative Analysis Using Raman Spectrometry, Appl. Spectrosc. 57 (2003) 20A-42A. https://doi.org/10.1366/000370203321165133. - sourceNot checked yet

  Not checked in the free run.

• cites [54] [54] P. Larkin, Infrared and Raman spectroscopy: Principles and Spectral Interpretation, 1st Editio, Elsevier, 2011. - http://www.chemistry.uoc.gr/lapkin/Infrared\_and\_Raman\_Spectroscopy\_\_Principles\_and\_Spectral\_Inte rpretation.pdf. - sourceNot checked yet

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The ~3010 cm−1 IR band is assigned to (=CH) stretches from unsaturated lipids.

“~ 3010 | ~ 3014 | w + | w + | w + | vw + | w + | vw + | w + |”

• cites [9] [9] A.P. Dean, D.C. Sigee, B. Estrada, J.K. Pittman, Using FTIR spectroscopy for rapid determination of lipid accumulation in response to nitrogen limitation in freshwater microalgae, Bioresour. Technol. 101 (2010) 4499 -4507. https://doi.org/10.1016/j.biortech.2010.01.065. - sourceNot checked yet

  Not checked in the free run.

• cites [20] [20] K. Stehfest, J. Toepel, C. Wilhelm, The application of micro-FTIR spectroscopy to analyze nutrient stressrelated changes in biomass composition of phytoplankton algae, 43 (2005) 717 -726. https://doi.org/10.1016/j.plaphy.2005.07.001. - sourceNot checked yet

  Not checked in the free run.

• cites [52] [52] B.H. Stuart, Infrared Spectroscopy: Fundamentals and Applications, John Wiley & Sons, Ltd, Chichester, UK, 2004. https://doi.org/10.1002/0470011149. - sourceNot checked yet

  Not checked in the free run.

• cites [53] [53] J.N. Murdock, D.L. Wetzel, FT-IR Microspectroscopy Enhances Biological and Ecological Analysis of Algae, Appl. Spectrosc. Rev. 44 (2009) 335 -361. https://doi.org/10.1080/05704920902907440. - sourceNot checked yet

  Not checked in the free run.

• cites [54] [54] P. Larkin, Infrared and Raman spectroscopy: Principles and Spectral Interpretation, 1st Editio, Elsevier, 2011. - http://www.chemistry.uoc.gr/lapkin/Infrared\_and\_Raman\_Spectroscopy\_\_Principles\_and\_Spectral\_Inte rpretation.pdf. - sourceNot checked yet

  Not checked in the free run.

The ~1440 cm−1 Raman band is assigned to δ(CH2) scissoring from saturated lipids and hydrocarbons.

“| ~ 1440 | | s + | | s + | | s + |”

• cites [9] [9] A.P. Dean, D.C. Sigee, B. Estrada, J.K. Pittman, Using FTIR spectroscopy for rapid determination of lipid accumulation in response to nitrogen limitation in freshwater microalgae, Bioresour. Technol. 101 (2010) 4499 -4507. https://doi.org/10.1016/j.biortech.2010.01.065. - sourceNot checked yet

  Not checked in the free run.

• cites [20] [20] K. Stehfest, J. Toepel, C. Wilhelm, The application of micro-FTIR spectroscopy to analyze nutrient stressrelated changes in biomass composition of phytoplankton algae, 43 (2005) 717 -726. https://doi.org/10.1016/j.plaphy.2005.07.001. - sourceNot checked yet

  Not checked in the free run.

• cites [52] [52] B.H. Stuart, Infrared Spectroscopy: Fundamentals and Applications, John Wiley & Sons, Ltd, Chichester, UK, 2004. https://doi.org/10.1002/0470011149. - sourceNot checked yet

  Not checked in the free run.

• cites [53] [53] J.N. Murdock, D.L. Wetzel, FT-IR Microspectroscopy Enhances Biological and Ecological Analysis of Algae, Appl. Spectrosc. Rev. 44 (2009) 335 -361. https://doi.org/10.1080/05704920902907440. - sourceNot checked yet

  Not checked in the free run.

• cites [54] [54] P. Larkin, Infrared and Raman spectroscopy: Principles and Spectral Interpretation, 1st Editio, Elsevier, 2011. - http://www.chemistry.uoc.gr/lapkin/Infrared\_and\_Raman\_Spectroscopy\_\_Principles\_and\_Spectral\_Inte rpretation.pdf. - sourceNot checked yet

  Not checked in the free run.

The ~1266 cm−1 IR band is assigned to δ(=CH) deformations from unsaturated lipids.

“~ 1266 | ~ 1264 | vw + | w + | vw + | w + | vw + | w + | vw + |”

• cites [9] [9] A.P. Dean, D.C. Sigee, B. Estrada, J.K. Pittman, Using FTIR spectroscopy for rapid determination of lipid accumulation in response to nitrogen limitation in freshwater microalgae, Bioresour. Technol. 101 (2010) 4499 -4507. https://doi.org/10.1016/j.biortech.2010.01.065. - sourceNot checked yet

  Not checked in the free run.

• cites [20] [20] K. Stehfest, J. Toepel, C. Wilhelm, The application of micro-FTIR spectroscopy to analyze nutrient stressrelated changes in biomass composition of phytoplankton algae, 43 (2005) 717 -726. https://doi.org/10.1016/j.plaphy.2005.07.001. - sourceNot checked yet

  Not checked in the free run.

• cites [52] [52] B.H. Stuart, Infrared Spectroscopy: Fundamentals and Applications, John Wiley & Sons, Ltd, Chichester, UK, 2004. https://doi.org/10.1002/0470011149. - sourceNot checked yet

  Not checked in the free run.

• cites [53] [53] J.N. Murdock, D.L. Wetzel, FT-IR Microspectroscopy Enhances Biological and Ecological Analysis of Algae, Appl. Spectrosc. Rev. 44 (2009) 335 -361. https://doi.org/10.1080/05704920902907440. - sourceNot checked yet

  Not checked in the free run.

• cites [54] [54] P. Larkin, Infrared and Raman spectroscopy: Principles and Spectral Interpretation, 1st Editio, Elsevier, 2011. - http://www.chemistry.uoc.gr/lapkin/Infrared\_and\_Raman\_Spectroscopy\_\_Principles\_and\_Spectral\_Inte rpretation.pdf. - sourceNot checked yet

  Not checked in the free run.

The ~1380 cm−1 IR band is assigned to δs(CH3) symmetric bend from lipids and proteins.

“~ 1380 | | vs - | | s - | | m - | | w - |”

• cites [9] [9] A.P. Dean, D.C. Sigee, B. Estrada, J.K. Pittman, Using FTIR spectroscopy for rapid determination of lipid accumulation in response to nitrogen limitation in freshwater microalgae, Bioresour. Technol. 101 (2010) 4499 -4507. https://doi.org/10.1016/j.biortech.2010.01.065. - sourceNot checked yet

  Not checked in the free run.

• cites [20] [20] K. Stehfest, J. Toepel, C. Wilhelm, The application of micro-FTIR spectroscopy to analyze nutrient stressrelated changes in biomass composition of phytoplankton algae, 43 (2005) 717 -726. https://doi.org/10.1016/j.plaphy.2005.07.001. - sourceNot checked yet

  Not checked in the free run.

• cites [52] [52] B.H. Stuart, Infrared Spectroscopy: Fundamentals and Applications, John Wiley & Sons, Ltd, Chichester, UK, 2004. https://doi.org/10.1002/0470011149. - sourceNot checked yet

  Not checked in the free run.

• cites [53] [53] J.N. Murdock, D.L. Wetzel, FT-IR Microspectroscopy Enhances Biological and Ecological Analysis of Algae, Appl. Spectrosc. Rev. 44 (2009) 335 -361. https://doi.org/10.1080/05704920902907440. - sourceNot checked yet

  Not checked in the free run.

• cites [54] [54] P. Larkin, Infrared and Raman spectroscopy: Principles and Spectral Interpretation, 1st Editio, Elsevier, 2011. - http://www.chemistry.uoc.gr/lapkin/Infrared\_and\_Raman\_Spectroscopy\_\_Principles\_and\_Spectral\_Inte rpretation.pdf. - sourceNot checked yet

  Not checked in the free run.