Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophylls a and b extracted with four different solvents: verification of the concentration of chlorophyll standards by atomic absorption spectroscopy

A fluorometric method is described which provides sensitive measurements of extracted chlorophyll a free from the errors associated with conventional acidification techniques. Fluorometric optical configurations were optimized to produce maximum sensitivity to Chl a while maintaining desensitized responses from both Chl b and pheopigments. Under the most extreme Chl b:Chl a ratio likely to occur in nature (1 : 1 molar), the new method results in only a 10% overestimate of the true Chl a value, while estimates from older acidification methods are 2.5-fold low. Under conditions of high pheopigment concentrations (pheo a: Chl a = 1 : 1 molar), the new method provides Chl a estimates that are equivalent to those determined from the acidification technique. The new simple method requires a single fluorescence determination and provides adequate sensitivity for small sample sizes (<200 ml) even in the most oligotrophic marine and freshwater environments.

https://aslopubs.onlinelibrary.wiley.com/doi/epdf/10.4319/lo.1994.39.8.1985

Fluorometric analysis of chlorophyll a in the presence of chlorophyll b and pheopigments

Fatty acid and lipid composition of 10 species of microalgae used in mariculture

A set of equations for determining chlorophyll a (Chl a) and accessory chlorophylls b, c
 2
 , c
 1
  + c
 2 and the special case of Acaryochloris marina, which uses Chl d as its primary photosynthetic pigment and also has Chl a, have been developed for 90% acetone, methanol and ethanol solvents. These equations for different solvents give chlorophyll assays that are consistent with each other. No algorithms for Chl c compounds (c
 2
 , c
 1
  + c
 2) in the presence of Chl a have previously been published for methanol or ethanol. The limits of detection (and inherent error, ± 95% confidence limit), for chlorophylls in all organisms tested, was generally less than 0.1 µg/ml. The Chl a and b algorithms for green algae and land plants have very small inherent errors (< 0.01 µg/ml). Chl a and d algorithms for Acaryochloris
 marina are consistent with each other, giving estimates of Chl d/a ratios which are consistent with previously published estimates using HPLC and a rarely used algorithm originally published for diethyl ether in 1955. The statistical error structure of chlorophyll algorithms is discussed. The relative error of measurements of chlorophylls increases hyperbolically in diluted chlorophyll extracts because the inherent errors of the chlorophyll algorithms are constants independent of the magnitude of absorbance readings. For safety reasons, efficient extraction of chlorophylls and the convenience of being able to use polystyrene cuvettes, the algorithms for ethanol are recommended for routine assays of chlorophylls. The methanol algorithms would be convenient for assays associated with HPLC work.

https://www.researchgate.net/profile/Raymond_J_Ritchie/publication/7019194_Consistent_Sets_of_Spectrophotometric_Chlorophyll_Equations_for_Acetone_Methanol_and_Ethanol_Solvents/links/0d1c84f9233a3012fd000000.pdf

Consistent sets of spectrophotometric chlorophyll equations for acetone, methanol and ethanol solvents.

The microphytobenthos consists of unicellular eukaryotic algae and cyanobacteria that grow within the upper several millimeters of illuminated sediments, typically appearing only as a subtle brownish or greenish shading. The surficial layer of the sediment is a zone of intense microbial and geochemical activity and of considerable physical reworking. In many shallow ecosystems, the biomass of benthic microalgae often exceeds that of the phytoplankton in the overlying waters. Direct comparison of the abundance of benthic and suspended microalgae is complicated by the means used to measure biomass and by the vertical and horizontal distribution of the microphytobenthos in the sediment. Where biomass has been estimated as chlorophyll a, there may be negligible to large (40%) error due to interference by degradation products, except where chlorophyll is measured by high-performance liquid chromatography. The vertical distribution of microphytobenthos, aside from mat-forming species, is determined by the opposing effects of their vertical migration, which tends to concentrate them near the surface, and physical mixing by overlying currents, which tends to cause an even vertical distribution through the mixed layer of sediment. Uncertainties in vertical distribution are compounded by frequently patchy horizontal distribution. Under-sampling on small (<1 m) scales can lead to errors in the estimate that are comparable to the ranges of seasonal and geographic variation. These uncertainties are compounded by biases in the techniques used to estimate production by the microphytobenthos. In most environments studied, biomass (as chlorophyll a) and light availability appear to be the principal determinants of benthic primary production. The effect of variable light intensities on integral production can be described by a functional response curve. When normalized to the chlorophyll content of the surficial sediment, the residual variation in the data described by the functional response curve is due to changes in the chlorophyll-specific response to irradiance. Production by the benthos is often a significant fraction of production in the water column and microphytobenthos may contribute directly to water column production when they are resuspended. Thus on both the basis of biomass and biogeochemical reactivity, benthic microalgae play significant roles in system productivity and trophic dynamics, as well as such habitat characteristics as sediment stability. *** DIRECT SUPPORT *** A01BY074 00003

https://www.jstor.org/stable/info/1352224

Microphytobenthos: The ecological role of the “secret garden” of unvegetated, shallow-water marine habitats. I. Distribution, abundance and primary production

G.F. Humphrey

Papers

New spectrophotometric equations for determining chlorophylls a, b, c1 and c2 in higher plants, algae and natural phytoplankton

The metabolism of oyster spermatozoa. 2. The effect of malonic acid and esters of substrates.