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Chapter 13 Algae and their Pigments
CHAPTER 13
ALGAE AND THEIR PIGMENTS 13.1. STRUCTURE AND OCCURRENCE
All algae contain photosynthetic pigments. These are usually an integral part of the structure of the chloroplast lamellae, but sometimes, as in bluegreen algae, they are homogeneously distributed throughout that part of the protoplasm called the “chromatoplasm”. Pigments are molecules which absorb light. The most efficient organic pigments have a molecular absorption which is one or two orders of magnitude greater than that of the inorganic pigments (such as cobalt blue, cinnabar, chrome yellow, etc.), which are used as paints. Many pigments have a characteristic molecular structure of long carbon chains or closed rings linked by so-called “conjugated” double bonds. These bonds are particularly stable because they involve “resonance”, a situation where two or more molecular configurations can exist simultaneously. Benzene for example “resonates” between the following classical structures: c
+ Addition of oxygen or nitrogen t o such a ring system increases the number of possible resonance positions. The simple heterocyclic pyrrole ring:
is one of the basic building units of many organic and biochemical pigments. Chains or rings with few conjugated bonds have strong absorption at relatively short wavelengths (in the far UV region). Molecules with a larger number of conjugated bonds absorb strongly in the near-UV or in the visible region of the spectrum. Thus in benzene the first absorption band lies below 250 nm, while that of anthracene lies in the yellow region. Strong light absorption by a molecule may be caused by its resonant structure. Owing to the occurrence of the different resonating configurations, several closely spaced energy levels are available. These lie between the ground state and the first excited state. The first absorption band then moves towards longer wavelengths (Fig. 13.1).
234
Fig. 13.1. Splitting of excited energy levels by resonance.
In photosynthetic cells the most commonly occurring pigment is chlorophyll a. The only exceptions are the photosynthetic bacteria which have bacteriochlorophyll (BChl). Chlorophyll a is present in brown, green and bluegreen algae and in higher plants. Its yellow-green colour may be masked by colours of other pigments. It seems probable that chlorophyll complexes RI
H
CH3
RrC
CH
I
t
CHz Rz
I
I
-
-
Chlorophyll b
as chlorophyll a, but with
Chlorophyll c
structure unknown: phytol free?
Chlorophyll d
2 divinyl-2 formyl chlorophyllo? (Holt, AS., 1961. Con. d. Bol., 39: 327)
Bacteriochlorophyll b Bacteriochlorophyll c
R3
phytyleater <-C~dC20H390-)
-H
-H
phytylester
dihydro structure unknown
-CG3 ‘H
Bacteriachlorophyll d
0
RI
34
-C=CHz
Bacteriochlorophyll a
CH2 HC-C=O
Rz
RI Chlorophyll a
-C$&
H ‘
-
-H -H
farnesylester (Cl+ZS*) farnesylester
--CHI
Rs Fig. 13.2. Basic structure of chlorophyll and relationship between chlorophylls and bacteriochlorophylls (Pfennig, 1967).
-H
235 function as the photoenzyme, although final proof is lacking. All other pigments (and a large part of BChl and of chlorophyll a itself) seem to serve solely as physical-energy suppliers. Pigments other than chlorophyll a are called accessory pigments. Green algae contain, in addition to chlorophyll a, the blue-green chlorophyll b. Diatoms and brown algae contain chlorophyll c, a partially oxidised derivate of chlorophyll a , but without the phytol chain. The structure of the chlorophyll molecule is shown in Fig. 13.2, and the occurrence and main absorption peaks of the different types are given in Table 13.1. The chlorophylls a and b have a common basic strxture consisting of four pyrrole rings, joined into a single master ring by CH bridges. The porphyrin structure (see Fig. 13.3) is related to that of the bilin pigments. Bacteriochlorophyll is related to tetrahydroporphyrin, having two fewer bonds and thus four more hydrogen atoms than porphyrin. The centre of the molecule is a magnesium atom, similar to the ferrous iron in the haem molecule. The function of magnesium is probably different: the ferrous haem molecule transports oxygen, whereas the chlorophyll molecule transports energy. It may be that the presence of a suitable central metal atom serves to stabilise TABLE 13.1 Characteristic absorption peaks and occurrence of the chlorophylls (after Rabinowitch and Govindjee, 1969) Type of chlorophyll
Characteristic absorption peaks
Occurrence
In organic
In cells (nm)
chlorophyll a
420,660
435,670-680 (several forms)
all photosynthesising plants (except bacteria)
chlorophyll b
453,643
480,650
higher plants and green algae
chlorophyll c
445,625
red band at 645
diatoms and brown algae
chlorophyll d
450,690
red band at 740
reported in some red algae
chlorobium chlorophyll (also called “bacterioviridin” )
two forms: (1)425,650 (2)432, 660
red band at 750 (or 760)
green bacteria
bacteriochlorophyll a (BChla)
365, 605,770
purple and green bacteria
bacteriochlorophyll b (BChlb)
368,582,795
red bands at 800, 850,and 890 red band at 1 017
solvents (nm)
found in a strain of Rhodopseudomonas, (a purple bacterium)
236
H
H
=o
o=
M
Fig. 13.3. Structure of bilin pigments: (a) bilan; (b) phycoerythrobilin. All unmarked corners are occupied by carbon atoms. E , ethyl; M , methyl; P, propionyl groups. (From Rabinowitch and Govindjee, 1969.)
the molecule. Following algal death, the magnesium is removed from the molecule probably during autolysis. In living cells the chlorophylls are bound to protein-lipid complexes just as most enzymes are. Extraction of the pigments by organic solvents such as acetone separates the chlorophylls from the proteins and has a considerable influence on their structure. Bacteriochlorophyll for example, which has three absorption bands at 800, 850 and 890 nm in vivo, shows only one band at 770 nm after it has been extracted by organic solvents. It may be that in vivo three separate complexes are formed with three proteins or that three different aggregation stages exist. Chlorophyll a in green algae shows a similar but less conspicuous polymorphism All photosynthetic cells contain, in addition to one or more chlorophyll pigments, an assortment of carotenoids. These are pigments related to that found in the root of the carrot plant. Carotenoids are either hydrocarbons
-
237 H
CH<,CH3
H
‘
H
H
H
H
H
H
H
H
H
H
I
l
l
I
I
I
I
I
l
l
H
CH3
H CH3
~ ~ ~ - ~ ~ ~ - c ~ ~ - c ~ c - c ~ c - c = c - c = c - c ~ c - c ~ c - c = c H H2
2
R Hz
‘ CH3
CH3 I
I
CH3
I
CH3
I
CH3
I
1%11
H3C
Hz
Fig. 13.4. Structure of p carotene (aand p forms are stereoisomers). All corners on the rings at the two ends are occupied by carbon atoms. (From Rabinowitch and Govindjee, 1969.)
(called a , /3 and y carotenes; C,H,,) or oxygen-containing compounds (called carotenols or xanthophylls). The structure of 0 carotene is shown in Fig. 13.4 ( a and p carotene are “stereoisomers” distinguished only by their spatial configuration; y carotene has one open ring structure at the end of the molecule). The xanthophylls are composed mainly of varying amounts of five compounds: lutein (or luteol), violaxanthin, fucoxanthin, spirilloxanthin and neoxanthin. Of these there seems to be most lutein. I t contains a -CHOHgroup in a position occupied by a -CH2 group in carotenoid. It is the major carotenoid of green algae. Fucoxanthin occurs in diatoms (Bacillariophyceae) and brown algae (Phaeophyceae, of which the genus FUCUS,from which the name derives, is found mainly on oceanic beaches). Fucoxanthin may serve t o transfer light energy t o chlorophyll. In addition diadinoxanthin also occurs in diatoms, but this pigment cannot transfer light energy. The possible function of diadinoxanthin has been mentioned in section 7.3. Zeaxanthin contains two, whilst violaxanthin and neoxanthin each have four oxygen atoms in the form of hydroxyl, carbonyl or carboxyl groups attached to the “ionone” ring. In a series of papers, Hertzberg and Liaaen-Jensen (cited in Hertzberg et al., 1971) reported a study of the carotenoid composition of blue-green algae, including Oscillatoria (several species) Anabaena flos aquae, Aphanizomenon flos aquae and Phormidium (several species). /3 carotene was the major carotenoid, and several less well-known xanthophylls occurred. The distribution patterns of the different pigments were compared and some correlations with the taxonomic status of the various algae were discussed. Thus zeaxanthin occurred in three species of Phormidium in amounts nearly equal to 0 carotene. In some species of Anabaena and Nostoc echinenone (= myxoxanthin) is more abundant than 0 carotene. A third important group of algal pigments are the phycobilins (phycos = alga, bilin = bile pigment). The basic molecular structure is rather like that of chlorophyll. Phycoerythrin gives the red algae (Rhodophyceae) their typical brick red colour, while the bluish colour of blue-green algae (Cyanophyceae) is due to phycocyanin, which may be present in amounts equal t o or greater than those of chlorophyll a. The pigments are not distributed homogeneously over the photosystems
I and 11. Chlorophyll a has been found in both systems, except in brown algae, where it is found only in system 11. Fucoxanthin, phycobilin, and chlorophyll b and c are only found in system 11, but /3 carotene and xanthophyll only in system I. The chemical properties of the pigments determine the methods used to extract them. Phycobilins have no phytol chains and are covalently bound to water-soluble proteins. They are therefore easily extractable with pure water. Chlorophyll extractions require organic solvents such as methanol, ethanol or acetone. Most carotenoids are soluble in organic solvents such as petroleum, and they also dissolve in fats and oils. 13.2. ABSORPTION SPECTRA AND FUNCTIONS
Chlorophyll has one strong absorption band in the blue-violet - the so-called Soret band - and one in the red region of the spectrum. The Soret band is characteristic of the porphyrin derivatives. In ether the maximum absorbance is found at 430 nm but in living cells these bands lie at about 440 nm and 675 nm. At wavelengths between these two regions the absorption is weak, and this causes most land vegetation to appear green. Bacteriochlorophyll a also has two absorption bands, but they lie further apart (365nm and 770 nm in methanol). Bacteriochlorophyll b has an infrared absorption at 1014 nm . Chlorobium chlorophyll (also called bacterioviridin) absorbs at 750 or 760 nm. The auxiliary or accessory (non-chlorophyll) pigments absorb blue to green light. Red algae contain phycoerythrin with absorption bands in the middle of the visible spectrum (500,545,570 nm). Light energy absorbed by these pigments can be used for photosynthesis. Light of these wavelengths penetrates water further than does that of other useful wavelengths. It is often suggested that this enables red algae to live in deeper parts of the aquatic environment than can other light-dependent organisms. Marine red sea weeds are often found in lower zones of the intertidal shore region than are the green and brown ones. This ability to grow in deeper water is probably also attributable, at least partly, to their possession of the pigment phycoerythrin. Phycocyanin has its main absorption band at 630 nm. It does not completely bridge the absorption gap in the green part of the chlorophyll spectrum, but it narrows it, as does chlorophyll b . The carotenoids absorb in the blue-violet part of the spectrum; /3 carotene for example has bands at 430,450 and 480 nm. Fucoxanthin has a much broader absorption band than have other carotenoids. The absorption spectrum does not fall off sharply at about 500 nm but declines slowly, filling in the gap left by chlorophyll. Phycobilin shows the same pattern. Some of those pigments which absorb light at wavelengths which are not absorbed by chlorophyll seem to be active in light-energy transport. Thus, 80-90% of the quanta absorbed by phycoerythrin and fucoxanthin may be transferred to chlorophyll a. It has been shown that ener'
239 gy transfer from accessory pigments to chlorophyll is possible only for the pigments found in photosystem 11. Much less efficient is the transfer from the carotenoids (ca. 20%). Transfer from p carotene and xanthophyll (both found in system I) seems to be impossible. The main function of these pigments remains-obscure at present. It has been suggested that they have a protective role. In the presence of light and oxygen many photo-oxidations could occur and chlorophyll in particular can easily be destroyed. Experimental evidence for such a protective screening role of carotenoids is scarce. Sistrom et al. (1956) showed that a Rhodopseudomonas spheroides mutant which lacks the coloured carotenoids was rapidly killed in the air in the presence of light, while cells of the normal carotenoid-containing type thrived. A colourless mutant of Halobacterium sulinarium grew far more slowly than did its parent strain so that in mixtures of mutant and wild-type parent the latter always became dominant in effect displacing its light-sensitive daughter population. Dundas and Larsen (1962), and Mathews and Krinsky (1965) also showed that carotenoids shield Micrococcus against injury by light. The carotenoids are not essential t o these bacteria: the colourless mutant of Rhodopseudom o m s spheroides functions normally as long as no oxygen is present. It has been mentioned in Chapter 7 that diatoms, especially when silicastarved, are much more sensitive t o light than are other algae. Diadinoxanthin synthesis in some diatom species decreases in darkness, but is maintained in the light, even during SiOp depletion. Moderate levels of light become then detrimental or lethal. The synthesis of the energy-transporting pigments chlorophyll and fucoxanthin ceased during this depletion. Diatoms, which are amongst the most successful plants on earth (they occur in the upper layers of most oceans and may account for as much photosynthesis
Anthemxanthln
t
Photosensltized oxidation
Fig. 13.5. Oxidation of zeaxanthin as a protective mechanism against photo-oxidation.
240 on earth as the green land plants), may have adapted themselves to lower irradiance during evolution. This is because they are able t o grow only during the period that silica is present, i.e. the very early spring, during which light levels are low. The ability to use carotenoids to fill the gap in the absorption spectrum left by chlorophyll would then be of great value. A similar adaptation is found in green and purple sulphur bacteria (see subsection 16.4.1) and in blue-greens, which owing to their phycocyanin are able to grow under a layer of green algae. It has been suggested that the protective mechanism acts via a reversible oxidation of carotenoids (Fig. 13.5).It has indeed been shown ;Chat at light levels high enough t o inhibit photosynthesis, O2 production was more inhibited than was C 0 2 uptake. 13.3. RATIO BETWEEN CAROTENOIDS AND CHLOROPHYLL
If one extracts the pigments from algal populations of most temperate lakes, the extinction value in the 400-nm region of the spectrum is most likely t o be 2-2.5 times greater than that at 665 nm. As the blue maximum of chlorophyll is roughly equal t o that at 660 nm, the extra light absorption at 400-430 nm can be used t o estimate the concentration of the carotenoids. Spectral diversity of the pigments (Fig. 13.6)is reflected in the ratio of the absorbances in the 400-430 nm region to that near 665 nm. The ratio between chlorophyll and carotenoids depends greatly on the physiological condition of the cells. Stress conditions also influence this ratio. Yentsch and Vaccaro (1958)compared chlorophyll and nitrogen concentrations in cultures of marine phytoplankton and found that when nitrogen was deficient so was chlorophyll. As soon as nitrogen was added, chlorophyll synthesis was resumed. During nitrogen starvation chlorophyll breakdown prevailed but carotenoid synthesis continued, even after the synthesis of chlorophyll ceased. The pigment ratio therefore changed. Only in conditions of extreme nitrogen deficiency did carotenoid synthesis decline. Yentsch and Vaccaro suggested therefore that this physiological measure could be used to estimate the rate of protein synthesis and cell division. Actual measurements based on the ratio of chlorophyll to carotenoids in natural populations gave disappointing results however. Theoretical ratios were calculated but these were not those obtained either in healthy or unhealthy cultures. The inconsistency was most probably attributable t o the great diversity in natural populations. Margalef (1965)used the ratios of pigments as an index to species diversity and tried t o relate primary production to community structure. Diversity, D , was expressed by Margalef as bits per individual or:
where p i denotes probability of occurrence of each species. He found a strong positive correlation between A,,/A6,, (absorption at
E L;d 0.3
0.2
0.1 0.0 lbo
45U
350
6%
7u)
241
u 350
45U
%Q
65U
7%
nm
Fig. 13.6. Absorption curves for somt chlorophylls and the corresponding phaeophytins and carotenoids in 85%acetone. A. Chlorophyll a found in all photosynthetic plants. B. Chlorophyll b found in higher green plants and green algae. C. Chlorophyll c found in higher brown algae, diatoms, dinoflagellates and coccolithophores. D. Fucoxanthin, the principal xanthophyll of brown algae and diatoms. E. Lutein, the principal xanthophyll of green plants. F. Carotene. (From Yentsch, 1967.)
wavelengths 430 and 665 nm, respectively) and diversity. Using data from man-made lakes and laboratory cultures, Margalef demonstrated that productivity per unit biomass was negatively correlated with the pigme& ratio A430/A665 (r = -0.588) and with diversity. Margalef found that production per unit biomass, P, could be expressed as: log P = 1.047 i0.728 log C - 0.615 log (A430/A665) where C = concentration of chlorophyll. Another form of this relation is:
P
= 11.1 Co.728/(A430/A665)0.615
and since C is approximately proportional to 4 6 5 , this equation may be writ-
242 ten as:
P = 67.7(A665)1'343/(A430)0'615
.
From the data presented by Margalef one cannot tell whether this relationship is better than the classical hypothesis that production is related linearly to chlorophyll concentration or not. Margalef's fig. 2 shows two groups of data: one group has a A430/A665 ratio of about 10 with a low productivity (10mg C per g C per h) and the other has aA430/A665ratio of about 5 with high productivity values (> 20 mg C per g C per h). In an earlier paper Margalef (1964)had computed the community diversity using the following definition:
1 N
d = - log
N!
N,!Nb!N,!...Ns! where N,,Nb, ...,Ns= numbers of cells of species a, b, ..., s, and N = total number of cells per sample. C02 uptake was found to be positively correlated
with the amount of chlorophyll and negatively with pigment ratio. Changes in the ratio A430/A665 reflect changes not only in species composition of the community but also in the physiological state of single species populations. The sensitive response of the pigment ratio to nutrient depletion and supply makes it a useful parameter in the assessment of growth-limiting factors. High values of A430/A665 may thus reflect high species diversity or stress due to nutrient depletion. In alcohol extracts of green algae values between 1.8and 2.0 are commonly found. These values result from a mixture of chlorophyll a, which itself has a A,0/A665 ratio of 1.25 (Fig. 13.6),with yellow pigments. Relatively high values can also be found in unialgal populations in nature. We found a ratio O f A430/A665 of about 4 in a population of Oscillatoriu sp. in crater lake in Uganda (Fig. 13.7).This blue-green alga gave the water a rather pink appearance and caused us to suppose that the lake contained purple bacteria. The presence of such an impressive quantity of carotenoids might be due to a combination of conditions; the lake received full sun, had a temperature of about 35"C,and contained 60 g 1-1 of Cland the same amount of sulphate. Two or three subcultures from the lake population retained the pink-orange colour. The organism showed 14C02uptake in the presence of H,S (see section 16.4).The lakewater itself contained no detectable 02. Margalef (1965)found surprisingly high values of A430/&65 : between 5 and 20 in his cultures and values of 4-7 in some Spanish reservoirs (Margalef, 1964). These values seem rather high but may be due to the extremely low chlorophyll concentrations in the reservoirs (ca. 1-2 mg rn-' ) or to difficulties in the extraction. During the evolutionary process diversity might be better achieved by proliferation of different species than by wide variation of pigments in one species.
243 0.8' 0.7.
0.6. 0.5.
es 0.4'
.-+C
i
W
0.3
0.2'
0.1
'
I
300
LOO
500
600
700
800
900
1OOOnm
Fig. 13.7. Absorption spectrum of Oscillatoria sp. from a crater lake in Uganda.
In addition to the pigments described earlier, degradation products of chlorophyll are usually present. Yentsch and Ryther (1959)filtered large volumes of seawater from more than 100 m depth. In extracts of the filtered particles they found almost no chlorophyll but they did find decomposition products of chlorophyll. Two degradation pathways for chlorophyll breakdown are possible (Yentsch, 1967): chlorophyll =% phaeophytin - P ~ Y ~ O chlorophyllide I ~
-phytol --Mg
> phaeophorbide
p
The loss of phytol results in little or no change in absorption in the visible part of the spectrum, so that chlorophyllide will be indistinguishable from chlorophyll. In dead cells however, Mg will be quickly removed from the chlorophyll or chlorophyllide molecule, resulting in a shift of the blue band towards the ultraviolet while the absorption of the red light is shifted slightly towards longer wavelengths and decreases by almost 50%.Mg removal can be effected by mineral acids as well, so that solutions of chlorophyll may be distinguished from those of chlorophyllide by noting the changes in absorption spectrum following the addition of a (mineral) acid ( F o / F a ) .Yentsch (1967)showed an increase in proportion of phaeophytin with increasing depth, both in the Indian and Atlantic Oceans; at depths greater than 100 m most of the pigment was phaeophytin. The conversion of chlorophyll to phaeopigment may take place
244
‘0
5
10
15
DAYS
20
25
30
Fig. 13.8. Cell numbers, potential photosynthesis and pigments of the diatom Skeletonema costaturn kept in darkness at 20°C. (Absorbance at 750 nm indicates cell number. Potential carbon fixation (14C) is determined by removing a portion of the darkened culture and exposing it to light in the presence of radioactive carbonate.) (From Yentsch, 1967.)
as the result of grazing; the acidity of the gut of the animal seems to be sufficient to effect the change, but autolytic or bacterial processes may be just as important. Yentsch (1965,1967) noted that prolonged periods of darkness (> 100 h) induced formation of phaeopigment in the diatom Skeletonemu costaturn (Fig. 13.8). During a 30-day period of continuous darkness, there was an initial increase of cell numbers and photosynthetic capacity, but later both decreased rapidly. Total chlorophyll and the ratio F,/F, declined at a slower rate during the first 15 days. Yentsch found that both Mg and phytol were lost from the pigments in deeper water layers. He suggested that the loss of Mg is an “apparently reversible process” which can be explained as a rapid photo-oxidation of phaeopigment in light and a de novo synthesis of chlorophyll from some intact cells. Yentsch also showed, by thin layer chromatography, that there are a variety of different pigments at different depths. Phaeophorbide appeared to be predominant in the deep waters. Although Yentsch studied these processes in deep oceans, the same processes may operate in deep, stratifying lakes. Diatoms, as soon as they are trapped in deep dark layers, will rapidly lose their photosynthetic pigments and thus their capacity for photosynthesis. ,
13.4. THE PHOTOSYNTHETIC UNIT
Not all chlorophyll molecules are active in light capture. Emerson and
245 Arnold (1932)made experiments in which the yield per light flash (using flashes lasting only a few micro seconds) appeared to be extremely low. Flash saturation was found to occur in normal cells when only one out of 2 500 chlorophyll molecules had received sufficient energy to reduce one molecule of C 0 2 during the flash. This suggested the occurrence of a photosynthetic unit of 2 500 chlorophyll molecules, of which one is active as a photosynthetic rate-limitingenzyme (Gaffron and Wohl, 1936). A structure which may correspond with such a unit, the quantosome, is distinguishable in electron micrographs of chloroplasts. Evidence indicating the existence of such a unit is the fact that a dense suspension of ChZoreZZu will start to evolve oxygen immediately after light is switched on, although kinetic reasoning would lead one to suppose that each chlorophyll molecule in these conditions would have to wait an average of an hour to collect the quanta necessary for the reduction of one molecule of C02. The supposition of a transfer of energy from many pigment molecules to one active enzyme molecule provides an explanation for the absence of any lag in O2 evolution. The transfer can be sufficiently rapid for this: one estimate is that 10 000 transfers can take place during sec, which is the duration of the excited state of the chlorophyll molecule. The active centre where the absorbed energy is collected is probably concerned with the transfer of an electron and not directly with the reduction of the C02 molecule. Since the reduction of a single CO, molecule is estimated to require a minimum of 8 quanta it seems likely that the photosynthetic unit contains at least 300 (200-400) chlorophyll molecules. If isolated chloroplasts are broken down into progressively smaller units, these lose their characteristic photochemical activity when the fragments become smaller than about 200 chlorophyll molecules. Furthermore, it is found that one molecule of DCMU (see page 64)is sufficient to inactivate 200 chlorophyll molecules. Chlorophyll is not the only pigment which is able to transfer energy to the photosynthetic centre: fucoxanthin and phycocyanin may also do so. This has been demonstrated by measuring the quantum yield of photosynthesis at different wavelengths (Fig. 13.9; Tanada, 1951). Quantum yield was found to be nearly constant between 520 and 680 nm, falling abruptly at longer wavelengths and showing a slight depression in the blue-green between 430 and 520 nm. It is clear that at 550 nm,”where fucoxanthin is the pigment mainly responsible for the light absorption, the quantum yield is the same as at 600 nm, where absorption is entirely by chlorophyll. In a similaf way the phycocyanin can be shown to be capable of transferring energy. The physical basis for the energy transfer is still unclear. Resonance may be important if absorption bands overlap, and the occurrence of crystal structures such as those in metals where electron orbits are shared is also possible. These facts may have ecological significance. The high efficiency of the energy transfer from pigments absorbing light between the two chlorophyll peaks
246
I u
E
400 4 4 0 480 520 560 600 640 680 720 Wavelength (nm)
Fig. 13.9. Quantum yield of photosynthesis of the diatom Navicula minima as a function of wavelength (a), and the estimated distribution of light absorption among pigments in living cells of Navicula minima as a function of wavelength (b). A : chlorophylls a and c ; B : fucoxanthin; C : other carotenoids. (From Fogg, 1968.)
raises the photosynthetic efficiency of diatoms (fucoxanthin) and blue-green algae (phycocyanin) above that of other competitors (such as green algae) lacking such pigments. One can also understand why change of photosynthesis with depth does not always parallel the most penetrating light component (see subsection 4.3.1) When calculating primary production per unit of photosynthetic machinery (mg of O2 per mg of chlorophyll) a correction for the presence of energytransferring accessory pigments should be made, and when correlating primary production with diversity, a distinction must be made between energytransferring and non-energy-transferringaccessory pigments. Photosynthesising bacteria also contain photosynthetic units, but the number of pigment molecules per unit is smaller than in green plants: about 50 instead of 300. This may be related to the smaller amount of energy needed to split H2S (see subsection 16.4.2).The number of pigment molecules per photosynthetic unit is a species characteristic for normal, healthy cells. It seems likely that the adaptation of algal cells to low irradiance by increasing the chlorophyll content (subsection 4.3.2)is related to the size of the photosynthetic unit, although the ecological significance of this has not yet been studied. More details about the physiology of photosynthesis are given by Forti (1965),Fogg (1968),and Rabinowitch and Govindjee (1969).
247 REFERENCES Dundas, I.D. and Larsen, H., 1962. The physiological role of the carotenoid pigment of Halobacterium salinarium. Arch. Mikrobiol., 44: 233-239. Emerson, R. and Arnold, W.,1932. Photosynthesis in flashing light. J. Gen. Physiol., 16: 191. Fogg, G.E., 1968.Photosynthesis. English University Press, London, 116 pp. Forti, G., 1965. Light energy utilization in photosynthesis. Mem. Ist. Ital. Idrobiol., 18 (Suppl.): 17-35. Gaffron, H. and Wohl, K., 1936.Zur Theorie der Assimilation. Naturwissenschaften, 24: 81-90; 103-107. Hertzberg, S., Liaaen-Jensen, S. and Siegelmann, H.W., 1971.The carotenoids of bluegreen algae. Phytochemistry, 10: 3121-3127. Holt, A.S., 1961. Further evidence of the relation between 2-desvinyl-2-formyl-chlorophyll-a and chlorophyll-d. Can. J. Bot., 39: 327-331. Margalef, R., 1964.Correspondence between the classic types of lakes and the structural and dynamic properties of their populations. Verh. Int. Ver. Theor. Angew. Limnol., 15: 169-175. Margaief, R., 1965. Ecological correlations and the relationship between primary productivity and community structure. Mem. Ist. Ital. Idrobiol., 18 (Suppl.): 355-364. Mathews, M.M. and Krinsky, N.I., 1965. The relationships between carotenoid pigments and resistance to radiation in non-photosynthetic bacteria. Photochem. Photobiol., 4: 813. Pfennig, N., 1967.Photosynthetic bacteria. Ann. Rev. Microbiol., 21 : 285-324. Rabinowitch, E. and Govindjee, 1969.Photosynthesis. John Wiley, New York, 273 pp. Sistrom, W.R., Griffiths, M. and Stanier, R.Y., 1956.The biology of a photosynthetic bacterium which lacks colored carotenoids. J. Cell Comp. Physiol.. 48: 459-515. Tanada, T., 1951.The photosynthetic efficiency of carotenoid pigments in Navicula minima. A m . J. Bot., 38: 276-283. Yentsch, Ch.S., 1965. The relationship between chlorophyll and photosynthetic carbon production with reference t o the measurement of decomposition products of chloroplastic pigments. Mem. Ist. Ztal. Idrobiol., 18 (Suppl.): 323-346. Yentsch, Ch.S., 1967. The measurements of chloroplastic pigments - thirty years of progress? In: Proc. IBP-Symp. Amsterdam-Nieuwersluis, 1966, pp. 255-270. Yentsch, Ch.S. and Menzel, D.W., 1963.A method for the determination of phytoplankton chlorophyll and phaeophytin by fluorescence. Deep-sea Res., 10: 221-231. Yentsch, Ch.S. and Ryther, J.H., 1959. Absorption curves of acetone extracts ofdeep water particulate matter. Deep-sea Res., 6: 72-74. Yentsch, Ch.S. and Vaccaro, R.F., 1958. Phytoplankton nitrogen in the oceans. Limnol. Oceanogr., 3: 445-448.
ALGAE AND THEIR PIGMENTS 13.1. STRUCTURE AND OCCURRENCE
All algae contain photosynthetic pigments. These are usually an integral part of the structure of the chloroplast lamellae, but sometimes, as in bluegreen algae, they are homogeneously distributed throughout that part of the protoplasm called the “chromatoplasm”. Pigments are molecules which absorb light. The most efficient organic pigments have a molecular absorption which is one or two orders of magnitude greater than that of the inorganic pigments (such as cobalt blue, cinnabar, chrome yellow, etc.), which are used as paints. Many pigments have a characteristic molecular structure of long carbon chains or closed rings linked by so-called “conjugated” double bonds. These bonds are particularly stable because they involve “resonance”, a situation where two or more molecular configurations can exist simultaneously. Benzene for example “resonates” between the following classical structures: c
+ Addition of oxygen or nitrogen t o such a ring system increases the number of possible resonance positions. The simple heterocyclic pyrrole ring:
is one of the basic building units of many organic and biochemical pigments. Chains or rings with few conjugated bonds have strong absorption at relatively short wavelengths (in the far UV region). Molecules with a larger number of conjugated bonds absorb strongly in the near-UV or in the visible region of the spectrum. Thus in benzene the first absorption band lies below 250 nm, while that of anthracene lies in the yellow region. Strong light absorption by a molecule may be caused by its resonant structure. Owing to the occurrence of the different resonating configurations, several closely spaced energy levels are available. These lie between the ground state and the first excited state. The first absorption band then moves towards longer wavelengths (Fig. 13.1).
234
Fig. 13.1. Splitting of excited energy levels by resonance.
In photosynthetic cells the most commonly occurring pigment is chlorophyll a. The only exceptions are the photosynthetic bacteria which have bacteriochlorophyll (BChl). Chlorophyll a is present in brown, green and bluegreen algae and in higher plants. Its yellow-green colour may be masked by colours of other pigments. It seems probable that chlorophyll complexes RI
H
CH3
RrC
CH
I
t
CHz Rz
I
I
-
-
Chlorophyll b
as chlorophyll a, but with
Chlorophyll c
structure unknown: phytol free?
Chlorophyll d
2 divinyl-2 formyl chlorophyllo? (Holt, AS., 1961. Con. d. Bol., 39: 327)
Bacteriochlorophyll b Bacteriochlorophyll c
R3
phytyleater <-C~dC20H390-)
-H
-H
phytylester
dihydro structure unknown
-CG3 ‘H
Bacteriachlorophyll d
0
RI
34
-C=CHz
Bacteriochlorophyll a
CH2 HC-C=O
Rz
RI Chlorophyll a
-C$&
H ‘
-
-H -H
farnesylester (Cl+ZS*) farnesylester
--CHI
Rs Fig. 13.2. Basic structure of chlorophyll and relationship between chlorophylls and bacteriochlorophylls (Pfennig, 1967).
-H
235 function as the photoenzyme, although final proof is lacking. All other pigments (and a large part of BChl and of chlorophyll a itself) seem to serve solely as physical-energy suppliers. Pigments other than chlorophyll a are called accessory pigments. Green algae contain, in addition to chlorophyll a, the blue-green chlorophyll b. Diatoms and brown algae contain chlorophyll c, a partially oxidised derivate of chlorophyll a , but without the phytol chain. The structure of the chlorophyll molecule is shown in Fig. 13.2, and the occurrence and main absorption peaks of the different types are given in Table 13.1. The chlorophylls a and b have a common basic strxture consisting of four pyrrole rings, joined into a single master ring by CH bridges. The porphyrin structure (see Fig. 13.3) is related to that of the bilin pigments. Bacteriochlorophyll is related to tetrahydroporphyrin, having two fewer bonds and thus four more hydrogen atoms than porphyrin. The centre of the molecule is a magnesium atom, similar to the ferrous iron in the haem molecule. The function of magnesium is probably different: the ferrous haem molecule transports oxygen, whereas the chlorophyll molecule transports energy. It may be that the presence of a suitable central metal atom serves to stabilise TABLE 13.1 Characteristic absorption peaks and occurrence of the chlorophylls (after Rabinowitch and Govindjee, 1969) Type of chlorophyll
Characteristic absorption peaks
Occurrence
In organic
In cells (nm)
chlorophyll a
420,660
435,670-680 (several forms)
all photosynthesising plants (except bacteria)
chlorophyll b
453,643
480,650
higher plants and green algae
chlorophyll c
445,625
red band at 645
diatoms and brown algae
chlorophyll d
450,690
red band at 740
reported in some red algae
chlorobium chlorophyll (also called “bacterioviridin” )
two forms: (1)425,650 (2)432, 660
red band at 750 (or 760)
green bacteria
bacteriochlorophyll a (BChla)
365, 605,770
purple and green bacteria
bacteriochlorophyll b (BChlb)
368,582,795
red bands at 800, 850,and 890 red band at 1 017
solvents (nm)
found in a strain of Rhodopseudomonas, (a purple bacterium)
236
H
H
=o
o=
M
Fig. 13.3. Structure of bilin pigments: (a) bilan; (b) phycoerythrobilin. All unmarked corners are occupied by carbon atoms. E , ethyl; M , methyl; P, propionyl groups. (From Rabinowitch and Govindjee, 1969.)
the molecule. Following algal death, the magnesium is removed from the molecule probably during autolysis. In living cells the chlorophylls are bound to protein-lipid complexes just as most enzymes are. Extraction of the pigments by organic solvents such as acetone separates the chlorophylls from the proteins and has a considerable influence on their structure. Bacteriochlorophyll for example, which has three absorption bands at 800, 850 and 890 nm in vivo, shows only one band at 770 nm after it has been extracted by organic solvents. It may be that in vivo three separate complexes are formed with three proteins or that three different aggregation stages exist. Chlorophyll a in green algae shows a similar but less conspicuous polymorphism All photosynthetic cells contain, in addition to one or more chlorophyll pigments, an assortment of carotenoids. These are pigments related to that found in the root of the carrot plant. Carotenoids are either hydrocarbons
-
237 H
CH<,CH3
H
‘
H
H
H
H
H
H
H
H
H
H
I
l
l
I
I
I
I
I
l
l
H
CH3
H CH3
~ ~ ~ - ~ ~ ~ - c ~ ~ - c ~ c - c ~ c - c = c - c = c - c ~ c - c ~ c - c = c H H2
2
R Hz
‘ CH3
CH3 I
I
CH3
I
CH3
I
CH3
I
1%11
H3C
Hz
Fig. 13.4. Structure of p carotene (aand p forms are stereoisomers). All corners on the rings at the two ends are occupied by carbon atoms. (From Rabinowitch and Govindjee, 1969.)
(called a , /3 and y carotenes; C,H,,) or oxygen-containing compounds (called carotenols or xanthophylls). The structure of 0 carotene is shown in Fig. 13.4 ( a and p carotene are “stereoisomers” distinguished only by their spatial configuration; y carotene has one open ring structure at the end of the molecule). The xanthophylls are composed mainly of varying amounts of five compounds: lutein (or luteol), violaxanthin, fucoxanthin, spirilloxanthin and neoxanthin. Of these there seems to be most lutein. I t contains a -CHOHgroup in a position occupied by a -CH2 group in carotenoid. It is the major carotenoid of green algae. Fucoxanthin occurs in diatoms (Bacillariophyceae) and brown algae (Phaeophyceae, of which the genus FUCUS,from which the name derives, is found mainly on oceanic beaches). Fucoxanthin may serve t o transfer light energy t o chlorophyll. In addition diadinoxanthin also occurs in diatoms, but this pigment cannot transfer light energy. The possible function of diadinoxanthin has been mentioned in section 7.3. Zeaxanthin contains two, whilst violaxanthin and neoxanthin each have four oxygen atoms in the form of hydroxyl, carbonyl or carboxyl groups attached to the “ionone” ring. In a series of papers, Hertzberg and Liaaen-Jensen (cited in Hertzberg et al., 1971) reported a study of the carotenoid composition of blue-green algae, including Oscillatoria (several species) Anabaena flos aquae, Aphanizomenon flos aquae and Phormidium (several species). /3 carotene was the major carotenoid, and several less well-known xanthophylls occurred. The distribution patterns of the different pigments were compared and some correlations with the taxonomic status of the various algae were discussed. Thus zeaxanthin occurred in three species of Phormidium in amounts nearly equal to 0 carotene. In some species of Anabaena and Nostoc echinenone (= myxoxanthin) is more abundant than 0 carotene. A third important group of algal pigments are the phycobilins (phycos = alga, bilin = bile pigment). The basic molecular structure is rather like that of chlorophyll. Phycoerythrin gives the red algae (Rhodophyceae) their typical brick red colour, while the bluish colour of blue-green algae (Cyanophyceae) is due to phycocyanin, which may be present in amounts equal t o or greater than those of chlorophyll a. The pigments are not distributed homogeneously over the photosystems
I and 11. Chlorophyll a has been found in both systems, except in brown algae, where it is found only in system 11. Fucoxanthin, phycobilin, and chlorophyll b and c are only found in system 11, but /3 carotene and xanthophyll only in system I. The chemical properties of the pigments determine the methods used to extract them. Phycobilins have no phytol chains and are covalently bound to water-soluble proteins. They are therefore easily extractable with pure water. Chlorophyll extractions require organic solvents such as methanol, ethanol or acetone. Most carotenoids are soluble in organic solvents such as petroleum, and they also dissolve in fats and oils. 13.2. ABSORPTION SPECTRA AND FUNCTIONS
Chlorophyll has one strong absorption band in the blue-violet - the so-called Soret band - and one in the red region of the spectrum. The Soret band is characteristic of the porphyrin derivatives. In ether the maximum absorbance is found at 430 nm but in living cells these bands lie at about 440 nm and 675 nm. At wavelengths between these two regions the absorption is weak, and this causes most land vegetation to appear green. Bacteriochlorophyll a also has two absorption bands, but they lie further apart (365nm and 770 nm in methanol). Bacteriochlorophyll b has an infrared absorption at 1014 nm . Chlorobium chlorophyll (also called bacterioviridin) absorbs at 750 or 760 nm. The auxiliary or accessory (non-chlorophyll) pigments absorb blue to green light. Red algae contain phycoerythrin with absorption bands in the middle of the visible spectrum (500,545,570 nm). Light energy absorbed by these pigments can be used for photosynthesis. Light of these wavelengths penetrates water further than does that of other useful wavelengths. It is often suggested that this enables red algae to live in deeper parts of the aquatic environment than can other light-dependent organisms. Marine red sea weeds are often found in lower zones of the intertidal shore region than are the green and brown ones. This ability to grow in deeper water is probably also attributable, at least partly, to their possession of the pigment phycoerythrin. Phycocyanin has its main absorption band at 630 nm. It does not completely bridge the absorption gap in the green part of the chlorophyll spectrum, but it narrows it, as does chlorophyll b . The carotenoids absorb in the blue-violet part of the spectrum; /3 carotene for example has bands at 430,450 and 480 nm. Fucoxanthin has a much broader absorption band than have other carotenoids. The absorption spectrum does not fall off sharply at about 500 nm but declines slowly, filling in the gap left by chlorophyll. Phycobilin shows the same pattern. Some of those pigments which absorb light at wavelengths which are not absorbed by chlorophyll seem to be active in light-energy transport. Thus, 80-90% of the quanta absorbed by phycoerythrin and fucoxanthin may be transferred to chlorophyll a. It has been shown that ener'
239 gy transfer from accessory pigments to chlorophyll is possible only for the pigments found in photosystem 11. Much less efficient is the transfer from the carotenoids (ca. 20%). Transfer from p carotene and xanthophyll (both found in system I) seems to be impossible. The main function of these pigments remains-obscure at present. It has been suggested that they have a protective role. In the presence of light and oxygen many photo-oxidations could occur and chlorophyll in particular can easily be destroyed. Experimental evidence for such a protective screening role of carotenoids is scarce. Sistrom et al. (1956) showed that a Rhodopseudomonas spheroides mutant which lacks the coloured carotenoids was rapidly killed in the air in the presence of light, while cells of the normal carotenoid-containing type thrived. A colourless mutant of Halobacterium sulinarium grew far more slowly than did its parent strain so that in mixtures of mutant and wild-type parent the latter always became dominant in effect displacing its light-sensitive daughter population. Dundas and Larsen (1962), and Mathews and Krinsky (1965) also showed that carotenoids shield Micrococcus against injury by light. The carotenoids are not essential t o these bacteria: the colourless mutant of Rhodopseudom o m s spheroides functions normally as long as no oxygen is present. It has been mentioned in Chapter 7 that diatoms, especially when silicastarved, are much more sensitive t o light than are other algae. Diadinoxanthin synthesis in some diatom species decreases in darkness, but is maintained in the light, even during SiOp depletion. Moderate levels of light become then detrimental or lethal. The synthesis of the energy-transporting pigments chlorophyll and fucoxanthin ceased during this depletion. Diatoms, which are amongst the most successful plants on earth (they occur in the upper layers of most oceans and may account for as much photosynthesis
Anthemxanthln
t
Photosensltized oxidation
Fig. 13.5. Oxidation of zeaxanthin as a protective mechanism against photo-oxidation.
240 on earth as the green land plants), may have adapted themselves to lower irradiance during evolution. This is because they are able t o grow only during the period that silica is present, i.e. the very early spring, during which light levels are low. The ability to use carotenoids to fill the gap in the absorption spectrum left by chlorophyll would then be of great value. A similar adaptation is found in green and purple sulphur bacteria (see subsection 16.4.1) and in blue-greens, which owing to their phycocyanin are able to grow under a layer of green algae. It has been suggested that the protective mechanism acts via a reversible oxidation of carotenoids (Fig. 13.5).It has indeed been shown ;Chat at light levels high enough t o inhibit photosynthesis, O2 production was more inhibited than was C 0 2 uptake. 13.3. RATIO BETWEEN CAROTENOIDS AND CHLOROPHYLL
If one extracts the pigments from algal populations of most temperate lakes, the extinction value in the 400-nm region of the spectrum is most likely t o be 2-2.5 times greater than that at 665 nm. As the blue maximum of chlorophyll is roughly equal t o that at 660 nm, the extra light absorption at 400-430 nm can be used t o estimate the concentration of the carotenoids. Spectral diversity of the pigments (Fig. 13.6)is reflected in the ratio of the absorbances in the 400-430 nm region to that near 665 nm. The ratio between chlorophyll and carotenoids depends greatly on the physiological condition of the cells. Stress conditions also influence this ratio. Yentsch and Vaccaro (1958)compared chlorophyll and nitrogen concentrations in cultures of marine phytoplankton and found that when nitrogen was deficient so was chlorophyll. As soon as nitrogen was added, chlorophyll synthesis was resumed. During nitrogen starvation chlorophyll breakdown prevailed but carotenoid synthesis continued, even after the synthesis of chlorophyll ceased. The pigment ratio therefore changed. Only in conditions of extreme nitrogen deficiency did carotenoid synthesis decline. Yentsch and Vaccaro suggested therefore that this physiological measure could be used to estimate the rate of protein synthesis and cell division. Actual measurements based on the ratio of chlorophyll to carotenoids in natural populations gave disappointing results however. Theoretical ratios were calculated but these were not those obtained either in healthy or unhealthy cultures. The inconsistency was most probably attributable t o the great diversity in natural populations. Margalef (1965)used the ratios of pigments as an index to species diversity and tried t o relate primary production to community structure. Diversity, D , was expressed by Margalef as bits per individual or:
where p i denotes probability of occurrence of each species. He found a strong positive correlation between A,,/A6,, (absorption at
E L;d 0.3
0.2
0.1 0.0 lbo
45U
350
6%
7u)
241
u 350
45U
%Q
65U
7%
nm
Fig. 13.6. Absorption curves for somt chlorophylls and the corresponding phaeophytins and carotenoids in 85%acetone. A. Chlorophyll a found in all photosynthetic plants. B. Chlorophyll b found in higher green plants and green algae. C. Chlorophyll c found in higher brown algae, diatoms, dinoflagellates and coccolithophores. D. Fucoxanthin, the principal xanthophyll of brown algae and diatoms. E. Lutein, the principal xanthophyll of green plants. F. Carotene. (From Yentsch, 1967.)
wavelengths 430 and 665 nm, respectively) and diversity. Using data from man-made lakes and laboratory cultures, Margalef demonstrated that productivity per unit biomass was negatively correlated with the pigme& ratio A430/A665 (r = -0.588) and with diversity. Margalef found that production per unit biomass, P, could be expressed as: log P = 1.047 i0.728 log C - 0.615 log (A430/A665) where C = concentration of chlorophyll. Another form of this relation is:
P
= 11.1 Co.728/(A430/A665)0.615
and since C is approximately proportional to 4 6 5 , this equation may be writ-
242 ten as:
P = 67.7(A665)1'343/(A430)0'615
.
From the data presented by Margalef one cannot tell whether this relationship is better than the classical hypothesis that production is related linearly to chlorophyll concentration or not. Margalef's fig. 2 shows two groups of data: one group has a A430/A665 ratio of about 10 with a low productivity (10mg C per g C per h) and the other has aA430/A665ratio of about 5 with high productivity values (> 20 mg C per g C per h). In an earlier paper Margalef (1964)had computed the community diversity using the following definition:
1 N
d = - log
N!
N,!Nb!N,!...Ns! where N,,Nb, ...,Ns= numbers of cells of species a, b, ..., s, and N = total number of cells per sample. C02 uptake was found to be positively correlated
with the amount of chlorophyll and negatively with pigment ratio. Changes in the ratio A430/A665 reflect changes not only in species composition of the community but also in the physiological state of single species populations. The sensitive response of the pigment ratio to nutrient depletion and supply makes it a useful parameter in the assessment of growth-limiting factors. High values of A430/A665 may thus reflect high species diversity or stress due to nutrient depletion. In alcohol extracts of green algae values between 1.8and 2.0 are commonly found. These values result from a mixture of chlorophyll a, which itself has a A,0/A665 ratio of 1.25 (Fig. 13.6),with yellow pigments. Relatively high values can also be found in unialgal populations in nature. We found a ratio O f A430/A665 of about 4 in a population of Oscillatoriu sp. in crater lake in Uganda (Fig. 13.7).This blue-green alga gave the water a rather pink appearance and caused us to suppose that the lake contained purple bacteria. The presence of such an impressive quantity of carotenoids might be due to a combination of conditions; the lake received full sun, had a temperature of about 35"C,and contained 60 g 1-1 of Cland the same amount of sulphate. Two or three subcultures from the lake population retained the pink-orange colour. The organism showed 14C02uptake in the presence of H,S (see section 16.4).The lakewater itself contained no detectable 02. Margalef (1965)found surprisingly high values of A430/&65 : between 5 and 20 in his cultures and values of 4-7 in some Spanish reservoirs (Margalef, 1964). These values seem rather high but may be due to the extremely low chlorophyll concentrations in the reservoirs (ca. 1-2 mg rn-' ) or to difficulties in the extraction. During the evolutionary process diversity might be better achieved by proliferation of different species than by wide variation of pigments in one species.
243 0.8' 0.7.
0.6. 0.5.
es 0.4'
.-+C
i
W
0.3
0.2'
0.1
'
I
300
LOO
500
600
700
800
900
1OOOnm
Fig. 13.7. Absorption spectrum of Oscillatoria sp. from a crater lake in Uganda.
In addition to the pigments described earlier, degradation products of chlorophyll are usually present. Yentsch and Ryther (1959)filtered large volumes of seawater from more than 100 m depth. In extracts of the filtered particles they found almost no chlorophyll but they did find decomposition products of chlorophyll. Two degradation pathways for chlorophyll breakdown are possible (Yentsch, 1967): chlorophyll =% phaeophytin - P ~ Y ~ O chlorophyllide I ~
-phytol --Mg
> phaeophorbide
p
The loss of phytol results in little or no change in absorption in the visible part of the spectrum, so that chlorophyllide will be indistinguishable from chlorophyll. In dead cells however, Mg will be quickly removed from the chlorophyll or chlorophyllide molecule, resulting in a shift of the blue band towards the ultraviolet while the absorption of the red light is shifted slightly towards longer wavelengths and decreases by almost 50%.Mg removal can be effected by mineral acids as well, so that solutions of chlorophyll may be distinguished from those of chlorophyllide by noting the changes in absorption spectrum following the addition of a (mineral) acid ( F o / F a ) .Yentsch (1967)showed an increase in proportion of phaeophytin with increasing depth, both in the Indian and Atlantic Oceans; at depths greater than 100 m most of the pigment was phaeophytin. The conversion of chlorophyll to phaeopigment may take place
244
‘0
5
10
15
DAYS
20
25
30
Fig. 13.8. Cell numbers, potential photosynthesis and pigments of the diatom Skeletonema costaturn kept in darkness at 20°C. (Absorbance at 750 nm indicates cell number. Potential carbon fixation (14C) is determined by removing a portion of the darkened culture and exposing it to light in the presence of radioactive carbonate.) (From Yentsch, 1967.)
as the result of grazing; the acidity of the gut of the animal seems to be sufficient to effect the change, but autolytic or bacterial processes may be just as important. Yentsch (1965,1967) noted that prolonged periods of darkness (> 100 h) induced formation of phaeopigment in the diatom Skeletonemu costaturn (Fig. 13.8). During a 30-day period of continuous darkness, there was an initial increase of cell numbers and photosynthetic capacity, but later both decreased rapidly. Total chlorophyll and the ratio F,/F, declined at a slower rate during the first 15 days. Yentsch found that both Mg and phytol were lost from the pigments in deeper water layers. He suggested that the loss of Mg is an “apparently reversible process” which can be explained as a rapid photo-oxidation of phaeopigment in light and a de novo synthesis of chlorophyll from some intact cells. Yentsch also showed, by thin layer chromatography, that there are a variety of different pigments at different depths. Phaeophorbide appeared to be predominant in the deep waters. Although Yentsch studied these processes in deep oceans, the same processes may operate in deep, stratifying lakes. Diatoms, as soon as they are trapped in deep dark layers, will rapidly lose their photosynthetic pigments and thus their capacity for photosynthesis. ,
13.4. THE PHOTOSYNTHETIC UNIT
Not all chlorophyll molecules are active in light capture. Emerson and
245 Arnold (1932)made experiments in which the yield per light flash (using flashes lasting only a few micro seconds) appeared to be extremely low. Flash saturation was found to occur in normal cells when only one out of 2 500 chlorophyll molecules had received sufficient energy to reduce one molecule of C 0 2 during the flash. This suggested the occurrence of a photosynthetic unit of 2 500 chlorophyll molecules, of which one is active as a photosynthetic rate-limitingenzyme (Gaffron and Wohl, 1936). A structure which may correspond with such a unit, the quantosome, is distinguishable in electron micrographs of chloroplasts. Evidence indicating the existence of such a unit is the fact that a dense suspension of ChZoreZZu will start to evolve oxygen immediately after light is switched on, although kinetic reasoning would lead one to suppose that each chlorophyll molecule in these conditions would have to wait an average of an hour to collect the quanta necessary for the reduction of one molecule of C02. The supposition of a transfer of energy from many pigment molecules to one active enzyme molecule provides an explanation for the absence of any lag in O2 evolution. The transfer can be sufficiently rapid for this: one estimate is that 10 000 transfers can take place during sec, which is the duration of the excited state of the chlorophyll molecule. The active centre where the absorbed energy is collected is probably concerned with the transfer of an electron and not directly with the reduction of the C02 molecule. Since the reduction of a single CO, molecule is estimated to require a minimum of 8 quanta it seems likely that the photosynthetic unit contains at least 300 (200-400) chlorophyll molecules. If isolated chloroplasts are broken down into progressively smaller units, these lose their characteristic photochemical activity when the fragments become smaller than about 200 chlorophyll molecules. Furthermore, it is found that one molecule of DCMU (see page 64)is sufficient to inactivate 200 chlorophyll molecules. Chlorophyll is not the only pigment which is able to transfer energy to the photosynthetic centre: fucoxanthin and phycocyanin may also do so. This has been demonstrated by measuring the quantum yield of photosynthesis at different wavelengths (Fig. 13.9; Tanada, 1951). Quantum yield was found to be nearly constant between 520 and 680 nm, falling abruptly at longer wavelengths and showing a slight depression in the blue-green between 430 and 520 nm. It is clear that at 550 nm,”where fucoxanthin is the pigment mainly responsible for the light absorption, the quantum yield is the same as at 600 nm, where absorption is entirely by chlorophyll. In a similaf way the phycocyanin can be shown to be capable of transferring energy. The physical basis for the energy transfer is still unclear. Resonance may be important if absorption bands overlap, and the occurrence of crystal structures such as those in metals where electron orbits are shared is also possible. These facts may have ecological significance. The high efficiency of the energy transfer from pigments absorbing light between the two chlorophyll peaks
246
I u
E
400 4 4 0 480 520 560 600 640 680 720 Wavelength (nm)
Fig. 13.9. Quantum yield of photosynthesis of the diatom Navicula minima as a function of wavelength (a), and the estimated distribution of light absorption among pigments in living cells of Navicula minima as a function of wavelength (b). A : chlorophylls a and c ; B : fucoxanthin; C : other carotenoids. (From Fogg, 1968.)
raises the photosynthetic efficiency of diatoms (fucoxanthin) and blue-green algae (phycocyanin) above that of other competitors (such as green algae) lacking such pigments. One can also understand why change of photosynthesis with depth does not always parallel the most penetrating light component (see subsection 4.3.1) When calculating primary production per unit of photosynthetic machinery (mg of O2 per mg of chlorophyll) a correction for the presence of energytransferring accessory pigments should be made, and when correlating primary production with diversity, a distinction must be made between energytransferring and non-energy-transferringaccessory pigments. Photosynthesising bacteria also contain photosynthetic units, but the number of pigment molecules per unit is smaller than in green plants: about 50 instead of 300. This may be related to the smaller amount of energy needed to split H2S (see subsection 16.4.2).The number of pigment molecules per photosynthetic unit is a species characteristic for normal, healthy cells. It seems likely that the adaptation of algal cells to low irradiance by increasing the chlorophyll content (subsection 4.3.2)is related to the size of the photosynthetic unit, although the ecological significance of this has not yet been studied. More details about the physiology of photosynthesis are given by Forti (1965),Fogg (1968),and Rabinowitch and Govindjee (1969).
247 REFERENCES Dundas, I.D. and Larsen, H., 1962. The physiological role of the carotenoid pigment of Halobacterium salinarium. Arch. Mikrobiol., 44: 233-239. Emerson, R. and Arnold, W.,1932. Photosynthesis in flashing light. J. Gen. Physiol., 16: 191. Fogg, G.E., 1968.Photosynthesis. English University Press, London, 116 pp. Forti, G., 1965. Light energy utilization in photosynthesis. Mem. Ist. Ital. Idrobiol., 18 (Suppl.): 17-35. Gaffron, H. and Wohl, K., 1936.Zur Theorie der Assimilation. Naturwissenschaften, 24: 81-90; 103-107. Hertzberg, S., Liaaen-Jensen, S. and Siegelmann, H.W., 1971.The carotenoids of bluegreen algae. Phytochemistry, 10: 3121-3127. Holt, A.S., 1961. Further evidence of the relation between 2-desvinyl-2-formyl-chlorophyll-a and chlorophyll-d. Can. J. Bot., 39: 327-331. Margalef, R., 1964.Correspondence between the classic types of lakes and the structural and dynamic properties of their populations. Verh. Int. Ver. Theor. Angew. Limnol., 15: 169-175. Margaief, R., 1965. Ecological correlations and the relationship between primary productivity and community structure. Mem. Ist. Ital. Idrobiol., 18 (Suppl.): 355-364. Mathews, M.M. and Krinsky, N.I., 1965. The relationships between carotenoid pigments and resistance to radiation in non-photosynthetic bacteria. Photochem. Photobiol., 4: 813. Pfennig, N., 1967.Photosynthetic bacteria. Ann. Rev. Microbiol., 21 : 285-324. Rabinowitch, E. and Govindjee, 1969.Photosynthesis. John Wiley, New York, 273 pp. Sistrom, W.R., Griffiths, M. and Stanier, R.Y., 1956.The biology of a photosynthetic bacterium which lacks colored carotenoids. J. Cell Comp. Physiol.. 48: 459-515. Tanada, T., 1951.The photosynthetic efficiency of carotenoid pigments in Navicula minima. A m . J. Bot., 38: 276-283. Yentsch, Ch.S., 1965. The relationship between chlorophyll and photosynthetic carbon production with reference t o the measurement of decomposition products of chloroplastic pigments. Mem. Ist. Ztal. Idrobiol., 18 (Suppl.): 323-346. Yentsch, Ch.S., 1967. The measurements of chloroplastic pigments - thirty years of progress? In: Proc. IBP-Symp. Amsterdam-Nieuwersluis, 1966, pp. 255-270. Yentsch, Ch.S. and Menzel, D.W., 1963.A method for the determination of phytoplankton chlorophyll and phaeophytin by fluorescence. Deep-sea Res., 10: 221-231. Yentsch, Ch.S. and Ryther, J.H., 1959. Absorption curves of acetone extracts ofdeep water particulate matter. Deep-sea Res., 6: 72-74. Yentsch, Ch.S. and Vaccaro, R.F., 1958. Phytoplankton nitrogen in the oceans. Limnol. Oceanogr., 3: 445-448.