We might measure the absorption spectrum of, say, phytoplankton or a multicellular algal thallus, for a number of different reasons. We might seek information on what pigments are present. We might wish to compare the spectral position and shape of an in vivo absorption peak with those of the same peak in the isolated pigment with a view to assessing the extent to which the absorption properties are modified by binding to protein. We might want to know to what extent an alga is equipped to efficiently harvest light from the underwater radiation field in which it lives.
An absorption spectrum is the variation of some measure of light absorption by a system with wavelength. Light absorption might be expressed in terms of the absorptance, A, the per cent absorption (100 A), the absorption coefficient, a, the absorbance, D (where D = —log10(1 - A)), or some other function such as the first derivative of the absorbance. The particular light absorption parameter chosen will depend on the purpose of the absorption spectrum. If, for example, we wish to be able to estimate the contribution of a planktonic alga to the total absorption coefficient of the aquatic medium at any wavelength, then we would measure the absorbance of a suspension of known concentration of the algae, as a function of wavelength, and (since absorbance is proportional to the absorption coefficient) calculate the specific absorption coefficient per unit algal biomass or pigment at the wavelength of interest. If, on the other hand, we require information on the rate at which an algal thallus is absorbing quanta from a particular incident light field, then the absorp-tance, or per cent absorption, spectrum of that thallus is what is needed.
Before returning to the absorptance spectra of algae we shall in some detail consider absorbance spectra: these are, in effect, absorption coefficient spectra, linked as they are by simple proportionality (see §3.2). At this point it should be noted that since living cells scatter, as well as absorb, light then all measurements, of whatever absorption parameter, must be carried out with procedures that eliminate the effect of scattering on the absorption spectrum, as described in §3.2. Except where otherwise specified, it should be assumed in the remainder of this chapter that all absorption spectra referred to have been corrected for scattering.
We saw in the previous chapter that what actually carries out the light absorption in aquatic plants, the fundamental light-absorbing system, is the thylakoid. The absorbance spectrum of the thylakoid is determined by the particular kind and quantity of chlorophyll/carotenoid-protein and, in some cases, biliprotein complexes present within, or attached to, the membrane. It might therefore be thought that two species of planktonic algae that have the same array of pigment-proteins, and consequently the same absorbance spectrum at the thylakoid level, must also have the same in vivo absorbance spectrum in suspensions of cells or colonies at the same total pigment concentration. This is not the case. While two such algal species would certainly have rather similar spectra with the peaks and the troughs in the same positions, the extent to which the peaks rise above the troughs, and the specific absorption coefficient per unit pigment at any wavelength, can differ markedly between the two species. This is because the in vivo absorbance spectrum is, as we shall see, determined by the size and shape of the chloroplasts, cells or colonies, as well as by the pigment composition.
It is not feasible to determine the absorbance spectrum of a single thylakoid directly. However, in many cases it is possible to disperse the chloroplasts, by physical disruption and/or detergents, into particles so small that the effects of size and shape on the spectrum are eliminated, but with the pigment-protein interaction, so important for spectral
Fig. 9.1 Absorbance spectrum of whole cells of Euglena gracilis compared with that of disrupted cells in which absorption is essentially due to thylakoid fragments (Kirk, unpublished). The spectra of intact cells (-) and of cells fragmented by ultrasonication (------ ) have been corrected for scattering
Fig. 9.1 Absorbance spectrum of whole cells of Euglena gracilis compared with that of disrupted cells in which absorption is essentially due to thylakoid fragments (Kirk, unpublished). The spectra of intact cells (-) and of cells fragmented by ultrasonication (------ ) have been corrected for scattering and in both pathlength.
cases correspond to 12 mg chlorophyll a ml and 1cm characteristics, being unaffected. Spectra of such preparations may be regarded as being reasonable approximations to the true in situ absorbance spectra of the thylakoids. Figure 9.1 shows a spectrum of this type for thylakoid fragments of the green planktonic alga Euglena gracilis.
Was this article helpful?