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Using DMSO for chlorophyll spectroscopy

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  • Prince of Songkla University - Phuket
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Abstract and Figures

DMSO (dimethyl sulphoxide, (CH3)2SO) is an alternative solvent for spectroscopic assay of chlorophylls (Chls) but has mainly been used on Chl a & b organisms. Here, we develop algorithms for the estimation of Chl a, b & c1c2, and d in DMSO solvent. The common unicellular green alga Chlorella sp. (Chl a & b) is used as an example of an oxygenic photo-organism with Chl a as the primary photosynthetic pigment and Chl b as the accessory Chl. The cyanobacterium Synechococcus is used as an example of organisms containing only Chl a. The diatom Chaetoceros sp. is used as the representative Chl a & c1c2 organism. The unusual chlorobacterium Acaryochloris marina has Chl d & a. Algorithms for use on mixed phytoplankton and algal mats (Chl a, b & c) were also developed. The algorithms for DMSO solvent are compared to those developed for 90% acetone as the benchmark solvent. DMSO solvent offers the advantages of low volatility, low toxicity, low flammability, biodegradability, and ease of transport and is an effective extractant of chlorophylls. However, there appear to be significant storage problems with DMSO extracts of chlorophylls because of its high freezing point (+18.4°C) and probable breakdown in chlorophylls in DMSO over several days.
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Using DMSO for chlorophyll spectroscopy
Raymond J. Ritchie
1,2
&Suhailar Sma-Air
1
&Sornsiri Phongphattarawat
1
Received: 20 January 2021 /Revised and accepted: 24 February 2021
#The Author(s), under exclusive licence to Springer Nature B.V. 2021
Abstract
DMSO (dimethyl sulphoxide, (CH
3
)
2
SO) is an alternative solvent for spectroscopic assay of chlorophylls (Chls) but has mainly
been used on Chl a&borganisms. Here, we develop algorithms for the estimation of Chl a, b &c
1
c
2
,anddin DMSO solvent.
The common unicellular green alga Chlorella sp.(Chl a&b) is used as an example of an oxygenic photo-organism with Chl aas
the primary photosynthetic pigment and Chl bas the accessory Chl. The cyanobacterium Synechococcus is used as an example of
organisms containing only Chl a. The diatom Chaetoceros sp. is used as the representative Chl a&c
1
c
2
organism. The unusual
chlorobacterium Acaryochloris marina has Chl d&a. Algorithms for use on mixed phytoplankton and algal mats (Chl a, b &c)
were also developed. The algorithms for DMSO solvent are compared to those developed for 90% acetone as the benchmark
solvent. DMSO solvent offers the advantages of low volatility, low toxicity, low flammability, biodegradability, and ease of
transport and is an effective extractant of chlorophylls. However, there appear to be significant storage problems with DMSO
extracts of chlorophylls because of its high freezing point (+18.4°C) and probable breakdown in chlorophylls in DMSO over
several days.
Keywords Spectroscopy .Absorbance (Abs) .Spectrophotometric equations .Chlorophyll, Oxygenic photo-organisms .Algae
Introduction
Chlorophyll (Chl) contents of oxygenic algae and cyanobacteria
(Chl aonly) and chlorobacteria (prokaryotic oxygenic organisms
with Chls in addition to Chl a) are routinely calculated from
spectrophotometric measurements of absorbance (Abs) at the
red peaks of the various chlorophylls using acetone, ethanol,
and methanol solvents. Many such algorithms have been pub-
lished for solvents such as 100, 90, and 80% acetone, ethanol,
methanol, and acetone/ethanol mixtures (Arnon 1949; Jeffrey
and Humphrey 1975; Lichtenthaler 1987; Porra et al. 1989;
Scheer 1991;Barnesetal.1992; Wellburn 1994; García and
Nicholás 1997;TaitandHik2003;Porra2006,2011;Ritchie
2006,2008,2018; Scheer 2006), but most of these algorithms
have been only for Chl a&bthat is for terrestrial plants and
green algae. Porra (2002) pointed out that although convenient to
use, such algorithms have tended to become entrenched in the
literature because they involve the use of the measurements of
the extinction coefficients for Chls that were available at the time
andthesolventsroutinelyusedat the time. Algorithms for pho-
tosynthetic organisms with other types of chlorophyll are not as
common (Jeffrey and Humphrey 1975; the trichromatic SCOR-
UNESCO algorithms, Humphrey and Jeffrey 1997; Jeffrey and
Welschmeyer 1997;Ritchie2006,2008).
Correct estimations of Chl contents are a key aspect of algal
research because Chl content is used to monitor abundances of
phytoplankton and algal blooms and photosynthetic
performance is typically quoted on a chlorophyll basis.
Hence, correct estimations of chlorophyll are of critical
importance to marine and aquatic sciences as well as
terrestrial plant research. Unfortunately, Chl estimation in
photosynthetic organisms has a chequered history with
historical inertia playing a role in the persistent use of
obsolete methods of assay of chlorophylls. Arnon (1949)de-
veloped the current method of estimating Chl a&bin plants
and algae using simultaneous equations based on the extinc-
tion coefficients of pure Chl aand pure Chl bat their red
spectral peaks and the peak of the other chlorophyll present:
thus, the extinction of Chl awas measured at its red peak
(663 nm, in 90% acetone) and also at the peak of Chl b
(663 nm, in 90% acetone) and similarly Chl bwas measured
at its red peak (647 nm, in 90% acetone) and at 663 nm.
*Raymond J. Ritchie
raymond.r@phuket.psu.ac.th;
Raymond.Ritchie@alumni.sydney.edu.au
1
Faculty of Technology and Environment, Prince of Songkla
University - Phuket, Kathu, Phuket 83120, Thailand
2
Andaman Environment and Natural Disaster Research Centre, Prince
of Songkla University - Phuket, Kathu, Phuket 83120, Thailand
https://doi.org/10.1007/s10811-021-02438-8
/ Published online: 12 March 2021
Journal of Applied Phycology (2021) 33:2047–2055
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
... Acetone is a lousy extractant of chlorophylls from many organisms, and alternative solvents and specialised extraction methods have long been sought (Porra et al. 1989;Porra 2006;2011). We recently published a paper presenting algorithms for DMSO (dimethyl sulfoxide) solvent compared to 90% acetone for Chl a-only organisms, Chl a + b organisms, Chl a + c 1 c 2 organism and organisms with Chl d + a (Ritchie et al. 2021), and a paper on determination of Chl a + c 2 in zooxanthellid dinoflagellates has been published recently (Ritchie et al. 2022). ...
... Ninety percent acetone and 100% DMSO were neutralised with magnesium carbonate. The physical properties of DMSO relevant to its use as a solvent for chlorophylls have been outlined previously (Lee et al. 1999;Spiese et al. 2009;ThermoFisher Scientific Safety Data Sheet 2018;Ritchie et al. 2021Ritchie et al. , 2022. Its melting point is 18.4 °C, meaning that it freezes in a standard refrigerator (4 °C) and freezer (− 20 °C). ...
... The 90% acetone in water was used as the benchmark extraction solvent, and the equations of Ritchie (2006) were used for the benchmark Chl determinations but using 850 nm zero. A scanning range 600 to 850 nm was chosen using 850 nm as the zero because the more conventional 750-nm absorbance blank for chlorophyll solvent extract masks the presence of bacteriochlorophylls (Ritchie 2018;Ritchie et al. 2021Ritchie et al. , 2022. All scans were exported as EXCEL.csv ...
Article
Full-text available
DMSO (dimethyl sulfoxide, (CH3)2SO) is an alternative solvent for the spectroscopic assay of chlorophylls (Chl) but has mainly been used on Chl a and b organisms, but we have previously shown that it is an effective solvent for the spectrophotometric determination of Chl a in Chl a-only organisms (Synechococcus), Chl a + b in Chl a + b organisms (Chlorella), Chl a + c1c2 in the diatom Chaetoceros) and the Chl d organism Acaryochloris marina (Chl d and a). There was some question about the stability of chlorophylls in DMSO solvent compared to the benchmark 90% acetone, and it has an inconvenient freezing point of DMSO (+ 18.4 °C). Here, we show that chlorophyll extracts in DMSO are as stable as those in 90% acetone if stored at − 20 °C for up to 10 days, but if plastic tubes are used, there is often an increase in apparent chlorophyll content by about + 0.2% day⁻¹ due to volatilisation through the plastic. Breakdown rates varied from one species to another. Generally, there was some breakdown of Chl a in DMSO but only a maximum of < 0.5% per day. Unacceptable losses of Chl a (> 5%) occurred in DMSO extracts from Chaetoceros after 10 days but not after 7 days.
... Free-living dinoflagellates are also common in aquatic environments, but some are Chl a + c 1 c 2 organisms, and so it cannot be assumed that all dinoflagellates are necessarily Chl a + c 2 Morden and Sherwood 2002). Although aqueous 90% acetone and 100% acetone are excellent spectroscopic solvents (Jeffrey and Humphrey 1975;Ritchie 2006Ritchie , 2018Ritchie et al. 2021), these solvents are frequently unsatisfactory for quantitative solvent extraction of chlorophylls (Porra 2011). Accurate chlorophyll determinations on photosynthetic organisms is not an arcane problem because photosynthesis is routinely estimated and quoted on a chlorophyll basis, and so correct estimates of chlorophylls have physiological and ecological importance to many branches of marine and aquatic biology. ...
... We were able to culture a symbiotic dinoflagellate (Symbiodinium sp.) from Sarcophyton. Here, we have developed spectrophotometric equations for photosynthetic organisms with Chl a and Chl c 2 (dinoflagellates and some haptophytes) in DMSO solvent using approaches previously described for other chlorophylls in a previous study (Ritchie et al. 2021) using equations in 90% acetone as the benchmark (Jeffrey and Humphrey 1975;Jeffrey and Welschmeyer 1997;Ritchie 2006Ritchie , 2018. Acetone/DMSO extracts from the soft coral and from the cultured symbiont have also been included in the development of the algorithms. ...
... Zooxanthellae cultures were grown in enriched f/2 seawater provided with silicate (McLachlan 1973). The algal cultures were grown in 250 and 500 mL conical flasks in a culture room described previously (Ritchie et al. 2021) and were shaken each day. Chemicals DMSO (CH 3 ) 2 SO) was from WINNEX (Thailand) Co. Ltd, Bangkok, Thailand. ...
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