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Cited by 104Spectroscopy Techniques in Biomedical and Chemical ResearchAlgal biology and biofuel productionSpectroscopy and Chemometric Analyses

Biomolecular transitions and lipid accumulation in green microalgae monitored by FTIR and Raman analysis

Open access
C. Esther Elizabeth Grace, P. Kiruthika Lakshmi, S. Meenakshi, Vaidyanathan, Seetharaman, S. Srisudha, M. Briget Mary·2020·Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy
DOI 10.1016/j.saa.2019.117382
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Abstract

Fourier transform infrared (FTIR) and Raman spectroscopic techniques were employed to analyze the biomolecular transitions and lipid accumulation in three freshwater green microalgal species, Monoraphidium contortum (M. contortum), Pseudomuriella sp. and Chlamydomonas sp. during various phases of their growth. Biomolecular transitions and lipid [hydrocarbons, triacylglycerides (TAGs)] accumulation within the microalgal cells were identified using second derivatives of the FTIR absorption spectroscopy. Second derivative analysis normalized and resolved the original spectra and led to the identification of smaller, overlapping bands. Both relative and absolute content of lipids were determined using the integrated band area. M. contortum exhibited higher accumulation of lipids than the other two species. The integrated band area of the vibrations from saturated (SFA) and unsaturated lipids (UFA) enabled quantification of fatty acids. The percentage of SFA and UFA was determined using GC, FTIR and Raman spectroscopy. From the spectral data, the order of increasing concentration of SFA among the three microalgal species was M. contortum > Chlamydomonas sp. >Pseudomuriella sp. The spectral results on fatty acids were consistent with the separation of lipids by gas chromatography. The results emphasized the significance of FTIR and Raman spectroscopic methods in monitoring the biomolecular transitions and rapid quantification of lipids, without the need for extraction of lipids.

AI snapshot

Written from the paper’s full text.

TL;DR

FTIR and Raman spectroscopy with second-derivative analysis tracked biomolecular transitions and lipid accumulation in three freshwater green microalgae across growth phases, with *M. contortum* accumulating the most lipids/TAGs. Spectral SFA estimates were consistent with GC separation, supporting rapid, extraction-free lipid quantification.

Key findings

  • *M. contortum* showed higher lipid accumulation (hydrocarbons and TAGs) than *Pseudomuriella* sp. and *Chlamydomonas* sp.; its absolute content increased nearly 6-fold for hydrocarbons (0.7 to 3.9) and 13-fold for TAGs (0.2 to 2.6).
  • Relative TAGs:Amide I ratio rose from ~0.5 to 4.3 for *M. contortum* and 3.8 for *Chlamydomonas* sp., a seven- to eight-fold increase during the declining phase compared with *Pseudomuriella* sp.; relative hydrocarbon content increased 2–3 fold by day 40 for all three species.
  • SFA order by FTIR/Raman was *M. contortum* > *Chlamydomonas* sp. > *Pseudomuriella* sp.; FTIR SFA percentages were 93, 83, and 74, and Raman SFA percentages were 80, 75, and 71, respectively. GC showed maximum SFA for *Chlamydomonas* sp., but total SFA+MUFA was high for *M. contortum*; spectral results were consistent with GC separation.

Methods

  • Three freshwater green microalgal isolates were cultured in modified BG11, 300 mL, in triplicates at 27 µmol m−2 s−1, 25±2°C, and 12:12 h light/dark; cells were harvested on days 10, 20, 30, and 40 and freeze-dried.
  • FTIR used KBr pellets (1:100), a Thermo Nicolet 380 spectrometer, 32 scans, 4 cm−1 resolution, 4000–500 cm−1, DTGS detector, yielding 9 spectra per species per phase (108 total). Raman used a Modular 1064 Raman system, 1064 nm laser at 149 mW, 200–3200 cm−1, 10–15 cm−1 resolution, 5 s/10 scans, InGaAs detector (36 spectra total).
  • Spectra were smoothed (Savitzky–Golay), second derivatives were obtained, and integrated band areas were used. Relative lipids were normalized to amide I; absolute lipids were normalized to a zero-intensity spectral region. GC-FID after modified Bligh–Dyer extraction and FAME preparation validated fatty acid percentages.

Limitations

  • No explicit limitations section; the paper does not state limitations.
  • Evident from the text: only three freshwater green microalgal species and one cultivation/growth series were examined; Raman fatty acid quantification and the GC/FTIR/Raman comparison were recorded on day 30 (stationary phase), so broader species or condition generalization is not tested.
  • Evident from the text: relative lipid content depends on amide I normalization, which the authors note changes with growth phase; they address this for absolute content by normalizing to a zero-intensity spectral region.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1016/j.saa.2019.117382

Cites

51 of 51 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 45 cited sources checked · 0 supported · 0 flagged

  • Findingcitation

    Bands at ~3008 cm−1 ν(=C-H) and ~1270 cm−1 δ(=C-H) are due to stretching and bending vibrations of unsaturated fatty acids in algal lipids.

    “The smaller bands at ~3008 cm -1 ν (= C-H) and ~ 1270 cm -1 (= C-H) were due to the stretching and bending vibrations of unsaturated fatty acids in the algal lipids.[21,26,50].”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [21]

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    • cites [26] source

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  • Findingown data

    The ~3008 cm−1 band was very weak in Pseudomuriella sp. and Chlamydomonas sp.

    “However, this band was observed as a very weak band in Pseudomuriella sp., and Chlamydomonas sp. (Fig. 3).”
    Quote in the paperNot a substantial claim
  • Findingown data

    Amide I and amide II protein bands were observed near 1653 and 1550 cm−1.

    “Two characteristic bands of the amide groups of protein chains, amide I and amide II were observed at ~1653cm -1 and ~1550 cm -1 respectively.”
    Quote in the paperNot a substantial claim
  • Findingown data

    From day 20 to day 40, drastic changes in spectral intensity of the three species were observed.

    “However, from the 20 th day to the 40th day of growth, drastic changes in the spectral intensity of the three species were observed (Fig. 4b).”
    Quote in the paperSubstantial claim
  • Findingown data

    FTIR bands in the 3050-2800 cm−1 region correspond to CH3 and CH2 asymmetric and symmetric stretches of lipid hydrocarbons.

    “The bands observed in the region 3050 to 2800 cm -1 corresponds to ν(CH 3 ) and ν(CH 2 ) asymmetric and symmetric stretches of hydrocarbons from lipids”
    Quote in the paperSubstantial claim
  • May combine two claims. 22
  • The quote only partly states this claim. 1
  • Classified as probably not a substantial claim. 13

  • May combine two claims.

    Findingown data

    Protein amide I/II and carbohydrate bands increased until stationary phase and decreased during declining phase.

    “While the intensity of the amide I (~ 1653 cm -1 ) and amide II (~ 1550 cm -1 ) bands of proteins and the carbohydrate bands increased till the stationary phase and decreased in the declining phase.”
    Quote in the paperSubstantial claim

  • The quote only partly states this claim.

    Findingown data

    Raw FTIR absorption spectra of the three species showed distinct bands for carbohydrates, proteins, and lipids across 4000-500 cm−1.

    “The raw absorption spectra showed distinct absorption bands corresponding to different biomolecules such as carbohydrates, proteins and lipids over the wave number range 4000 -500 cm -1 .”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Biomolecular band positions were nearly the same at all growth phases, but band intensities varied markedly.

    “the position of the bands corresponding to the biomolecules was observed to be almost the same (i.e. in the expected wavenumber range) at every phase of growth, but their intensity varied remarkably.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingcitation

    Second derivative analysis of FTIR absorption spectra resolved overlapping bands, removed baseline errors, identified small adjacent bands, and enabled quantitative analysis.

    “Second derivatives resolved the overlapping bands in the raw spectra, removed the baseline errors and lead to the identification of small and adjacent absorption bands and enabled quantitative analysis [26,49].”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [26] source

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  • May combine two claims.

    Findingown data

    Lipid bands at ~1745, ~1462, ~1380 cm−1 and protein bands at ~1658 and ~1547 cm−1 were identified.

    “Bands due to lipids were ~1745cm -1 (C=O), ~1462cm -1 ( as CH 3 ), ~1380cm -1 ( s CH 3 ) and of proteins were ~1658cm -1 (amide I mainly C=O), ~ 1547 cm -1 (amide II N-H, C-N).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    M. contortum exhibited nearly a 6-fold increase in hydrocarbons (0.7 to 3.9) and a 13-fold increase in TAGs (0.2 to 2.6).

    “M.contortum exhibited nearly 6 fold increase in the hydrocarbons (0.7 to 3.9) and a thirteen fold increase in TAGs (0.2 to 2.6).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The region with almost zero intensity in the second derivative spectra does not change with growth phase or species and has no characteristic bands.

  • May combine two claims.

    Findingown data

    All three species showed the =C-H stretch near 3008 cm-1; it was strongest in M. contortum, a result also seen in the FTIR spectra.

    “The stretching vibration of =C-H at ~3008 cm -1 was observed for all the three species, with maximum intensity for M.contortum, which was also confirmed in the FTIR spectra (Fig. 2 and Fig. 4).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Raman spectra gave SFA percentages of 80 (M. contortum), 75 (Chlamydomonas sp.), and 71 (Pseudomuriella sp.).

    “The percentage of SFA from the Raman spectra was 80 for M.contortum, 75 for Chlamydomonas sp.71 for Colour should be used for this figure in print a) b) c) Fig.10 Fatty acid analysis by a) Gas chromatography b) FTIR and c) Raman Spectroscopy, recorded on the 30 th day growth of the microalgal species.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Integrated band areas in FTIR and Raman spectra are proportional to the amount of the corresponding lipid component.

  • May combine two claims.

    Findingown data

    FTIR spectra gave SFA percentages of 93 (M. contortum), 83 (Chlamydomonas sp.), and 74 (Pseudomuriella sp.).

    “The percentage of SFA from the FTIR spectra was 93 for M.contortum, 83 for Chlamydomonas sp.74 for Pseudomuriella sp.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Integrated band areas in FTIR and Raman spectra are proportional to the amount of the corresponding lipid component.

  • May combine two claims.

    Interpretationreasoning

    During early exponential phase with sufficient nutrients, cells divide; when nutrients become limited in stationary phase, division stops and cells store energy as lipids and carbohydrates.

    “During the early exponential phase, when there is sufficient availability of nutrients, the microalgal cells divide, but as the cells enter the stationary phase, the nutrient availability becomes limited, the cell division stops, the algal cells start to accumulate and stores more energy in the form of lipids and carbohydrates.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    The four characteristic hydrocarbon bands are at ~2960 (as CH3), ~2920 (as CH2), ~2870 (s CH3), and ~2850 cm−1 (s CH2).

    “The characteristic bands of hydrocarbons are the four bands at ~ 2960cm -1 ( as CH 3 ), 2920cm -1 ( as CH 2 ), 2870 cm -1 ( s CH 3 ) and 2850 cm -1 ( s CH 2 ) [22,26].”
    Quote in the paperSubstantial claim0 of 2 sources checked
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  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Previous studies on Chlamydomonas reinhardtii and Scenedesmus subspicatus showed 5- to 6-fold increases in lipid accumulation.

    “Previous studies on green algal species like Chlamydomonas reinhardtii and Scenedesmus subspicatus have exhibited 5 to 6 fold increase in lipid accumulation [8]”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [8] source

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  • May combine two claims.

    Interpretationown data

    FTIR and Raman methods enable monitoring biomolecular transitions and rapid lipid quantification without lipid extraction.

    “The results emphasized the significance of FTIR and Raman spectroscopic methods in monitoring the biomolecular transitions and rapid quantification of lipids, without the need for extraction of lipids.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    FAME were identified using fatty acid standards.

    “the FAME were identified using fatty acid standards (Sigma, Supleco, 37 FAMEs).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    SFA and MUFA in microalgae can be excellent biodiesel source due to high oxidative stability and lower NOx emissions than PUFA, though high SFA melting point can cause cold-weather gelling.

    “SFA and MUFA in microalgae can be an excellent source of biodiesel due to their high oxidative stability and lesser NO X emissions in comparison to polyunsaturated fatty acids (PUFA), except that the high melting point of SFA can cause gelling of fuel in cold weather.[12,13].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
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  • Classified as probably not a substantial claim.

    Premisecitation

    The analysis involves measuring absorption/scattering intensity in relation to molecular vibrational modes due to biomolecules, enabling quantification.

    “The analysis involves the measurement of absorption (FTIR)/scattering (Raman) intensity in relation to a range of molecular vibrational modes due to the presence of biomolecules, enabling quantification.[18,20,21].”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [18]

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  • May combine two claims.

    Premisecitation

    Traditional chromatographic methods require lipid extraction, are destructive, time-consuming, use large biomass and costly reagents, and cannot effectively screen many samples.

    “However, the methods require extraction of lipids from algal biomass prior to analysis and are destructive, time consuming, also involves large amount of biomass, costly chemical reagents for analysis and therefore cannot be effectively used for screening large number of microalgal samples [14][15][16].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [14][15][16]

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  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Several microalgal species, including Chlorella, Haematococcus, Dunaliella, Spirulina, Nannochloropsis, Scenedesmus, Chlamydomonas, and Monoraphidium, have been reported as promising for biodiesel through traditional lipid quantification.

    “Microalgal species like Chlorella, Haemattococcus, Dunaliella , Spirulina , Nannochloropsis, Scenedesmus, Chlamydomonas and Monoraphidium have been reported as promising species for biodiesel applications through traditional lipid quantification methods [10,46,47].”
    Quote in the paperNot a substantial claim0 of 3 sources checked
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  • May combine two claims.

    Interpretationown data

    The three microalgal species accumulate more TAGs than other lipid forms, which is a good indicator for biodiesel production.

    “This confirms that, these microalgal species are accumulating more triacylglycerides (TAGs) than the other forms of lipids, which is a good indicator for biodiesel production.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Study design and conductown data

    Monospecificity of cultures was verified periodically by microscopy and no contamination was found.

    “During incubation, the monospecificity of the cultures were verified periodically by microscopic observation and no evidence of contamination was found.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductown data

    FTIR spectra were recorded for three replicates per sample, yielding 9 spectra per growth phase for all three species and 108 total spectra.

    “Spectra were recorded for three replicates per sample, resulting in 9 spectra at every phase of growth (10 th to 40 th day) for all three species yielding a total of 108 spectra.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductreasoning

    Exposure time was chosen to avoid laser-induced damage to algal biomass.

    “The exposure time was optimally chosen by ensuring that there was no laser induced damage for the algal biomass.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    Spectroscopic techniques FTIR and Raman are non-destructive, fast, versatile tools to analyze algal biomass using minimal volume.

    “Spectroscopic techniques such as Fourier transformed infrared spectroscopy (FTIR) and Raman spectroscopy have been reported to be non-destructive, fast and versatile tools to analyze algal biomass using minimal volume with much ease [17][18][19].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [17][18][19]

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  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    Raman spectra were recorded in triplicate for three species at four growth intervals, total 36 spectra.

    “A total of 36 spectra were recorded for the three species in triplicate at the four intervals of their growth phases.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    TAGs are neutral lipids primarily made up of SFA and MUFA and are preferred compounds for biodiesel.

    “TAGs are neutral lipids primarily made up of saturated (SFA) and monounsaturated fatty acids (MUFA) [10,11] and are the preferred compounds for biodiesel (Section 1).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [10] source

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  • May combine two claims.

    Premisecitation

    Nile Red staining is affected by uneven dye uptake due to cell wall differences, is not quantitative as chromatographic methods, and cannot distinguish neutral from polar lipids.

    “However, it is affected by uneven dye uptake due to differences in cell wall composition among species.Nile red staining is not quantitative as chromatographic methods and cannot distinguish between neutral and polar lipids [5,8].”
    Quote in the paperSubstantial claim0 of 2 sources checked
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  • May combine two claims.

    Interpretationcitation

    Among the three species, increased TAG accumulation was observed for M. contortum, indicating it is rich in saturated and monounsaturated fatty acids.

    “Among the three species, increased accumulation of TAGs (Fig. 5) was observed for M.contortum which indicate that the species is rich with saturated and monounsaturated fatty acids [10,11,47].”
    Quote in the paperSubstantial claim0 of 3 sources checked
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    • cites [11] source

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    • cites [47] source

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  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Chlorella sp. exhibited a 2- to 4-fold increase in lipid content under nitrogen starvation.

    “Among them green algal species, Chlorella sp. has exhibited 2 to 4 fold increase in lipid content, under nitrogen starved condition [48].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [48] source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    FTIR and Raman spectroscopy improved understanding of biomolecular transitions and provided rapid, easy lipid assessment, including fatty acid quantification without extraction.

    “In conclusion, FTIR and Raman spectroscopy enabled better understanding of biomolecular transitions and a rapid and easy assessment of lipid accumulation, especially to quantify fatty acids without the need for the extraction of lipids.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Integrated band areas in FTIR and Raman spectra are proportional to the amount of the corresponding lipid component.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    Every Raman spectrum represented the average of three replicates.

    “Each spectrum is an average of 3 replicates.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    FTIR and Raman spectroscopy allowed fast, reliable, and non-destructive fatty acid quantification.

    “Thus quantification of fatty acids by the FTIR and Raman spectroscopic methods was fast, reliable and non-destructive.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    Each data point in the lipid content figures is a mean with standard error from nine replicates.

    “Each point is the mean (±SE) of 9 replicates.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Every FTIR spectrum represented the average of nine replicates.

    “Each spectrum is an average of 9 replicates.”
    Quote in the paperNot a substantial claim
33 of 33

  • The paper relies on this without support.

    The intensity variation in the FTIR spectra reflects absorbance changes proportional to microalgal concentration or cell population, as described by the Beer-Lambert law.

    “The intensity variation in the spectral data represents the change in absorbance which is proportional to the concentration / cell population of the microalgae, according to the Beer Lambert's law.”

  • The paper relies on this without support.

    The region with almost zero intensity in the second derivative spectra does not change with growth phase or species and has no characteristic bands.

    “normalized by a region with almost zero intensity (10 -6 ) in the second derivative spectra, which does not change with the phases of growth as well as with the species and have no characteristic bands”

  • The paper relies on this without support.

    Gas chromatography is a reliable method for fatty acid analysis.

    “Gas chromatography (GC) is one of the conventional methods, widely used as a reliable technique for fatty acid analysis.”

  • The paper relies on this without support.

    Gas chromatography requires extraction of lipids from algal cells.

    “However, the method requires extraction of lipids from algal cells.”

  • The paper relies on this without support.

    Integrated band areas in FTIR and Raman spectra are proportional to the amount of the corresponding lipid component.

    “Relative and absolute lipid contents of the species were calculated using the integrated peak area of the lipid regions.”
5 of 5

Claims other papers make about this one

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Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • GC and spectral analyses identify different species with the most saturated fatty acids

    Major

    GC identifies Chlamydomonas sp. as having the most saturated fatty acids, whereas the stated spectral ranking places M. contortum first. This contradicts the paper's conclusion that the methods give consistent results for saturated fatty acids.

1 of 1

Cites

Flagged first, then by citations

Research works printed but not linked: 2

  1. Stehfest, 2005, vol. 43, 717
  2. Driver, 2015, vol. 11, 148

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Related

Authors
Grace, C. E. E., Lakshmi, P. K., Meenakshi, S., Vaidyanathan, S., Srisudha, S., & Mary, M. B.
Affiliations
  • Lady Doak College (IN)
  • University of Sheffield (GB)
Corresponding author
M. Briget Mary
ORCID
Vaidyanathan, Seetharaman
Venue
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy
Publisher
Elsevier BV
Published
2020-01
Volume
224
Pages
117382
ISSN
1386-1425 (ISSN-L)
DOI
10.1016/j.saa.2019.117382
OpenAlex
W2957070687
Semantic Scholar
9cd4a545f5cae8512e3bba170cd971bc4805224a
Cited by
104
References
50
Citations by year
2026: 9 · 2025: 10 · 2024: 27 · 2023: 14 · 2022: 13 · 2021: 23 · 2020: 7
FWCI
5.17
Type
journal article
Language
en
Open access
Licence
Locations
  • Spectrochimica Acta Part A Molecular and Biomolecular Spectroscopy (publishedVersion)
  • PubMed (publishedVersion)
  • White Rose Research Online (University of Leeds, The University of Sheffield, University of York) (acceptedVersion)
Funders
  • Biotechnology and Biological Sciences Research Council
  • Department of Biotechnology , Ministry of Science and Technology
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Biophysics › Spectroscopy Techniques in Biomedical and Chemical Research
  • Physical Sciences › Energy › Renewable Energy, Sustainability and the Environment › Algal biology and biofuel production
  • Physical Sciences › Chemistry › Analytical Chemistry › Spectroscopy and Chemometric Analyses
Keywords
green microalgae, lipid accumulation, Monoraphidium contortum, Chlamydomonas, triglycerides, fatty acid composition, saturated and unsaturated fatty acids, lipid quantification
MeSH terms
Chlorophyta, Fatty Acids, Lipids, Spectrum Analysis, Raman, Spectroscopy, Fourier Transform Infrared, Biofuels, Microalgae

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