Light-harvesting and light-protecting pigments in simple life forms

Authors


* Email: timldawson@aol.com

Abstract

Photosynthesis is the basis of life on the earth and the development of the vast range of simple organisms existing today can often be traced back to the earliest geological times. Most life forms depend directly or indirectly on the synthetic processes which harvest the sun's energy, utilising a range of pigments such as the chlorophylls and carotenoids, which not only determine the colour of each organism, but often also serve a protective role against the adverse effects of ultraviolet radiation. Recent research has elucidated the detailed photochemical mechanisms and the complex nature of the light-harvesting pigment–protein complexes. This review covers algae and fungi and their symbiotic forms in lichens and corals, particularly with respect to the pigments they synthesise and the commercial uses to which these and other metabolites have been (and may in the future be) put.

Introduction

Some 3 billion years ago, the earliest forms of bacterial life on earth developed in shallow waters warmed by both volcanic activity and the sun's rays. In the more aggressive environments of high temperature and salinity, bacteria, now classed as thermophiles and halophiles, developed while other extremophiles existed at pH levels as low as zero [1]. Only a decade ago bacteria living inside rock some 500 m from the surface were discovered [2] while in other sedimentary rocks there is evidence of many early life forms such as filamentous microfossils closely resembling those seen inside the stony, pillar-like, stromalite formations still found today in warm, shallow seas [3]. Over the passage of time other simple unicellular taxa appeared which are classed as algae, fungi and the symbiotic lichens and corals. These, in common with the abundant plant life that developed subsequently, evolved efficient systems for harvesting the energy of sunlight to drive complex metabolic reactions leading to the fixation of carbon from atmospheric carbon dioxide.

The earliest photosynthetic organisms were anoxygenic (i.e. grew in an oxygen-deficient atmosphere) and would have had to withstand the damaging effects of intense shortwave ultraviolet (UV) radiation but by 2.5 billion years ago forms were beginning to develop with the ability to generate oxygen that in due course enabled the evolution of more complex marine and later terrestrial animals [4]. Oxygen levels and surface temperatures on the primordial earth underwent a number of extreme fluctuations, following major atmospheric and geological changes that resulted in at least five extinctions of many life forms [5,6]. In addition, there is evidence that some 55 million years ago the atmosphere contained high concentrations of carbon dioxide (causing extreme global warming) due to the release of clathrates (methane–ice complexes) from the seabed. It is believed that the excess carbon dioxide was eventually absorbed by surface blooms of plankton [7]. Eventually in the upper atmosphere ozone was formed, thus affording protection to all forms of life from harmful UVB (280–320 nm) and particularly UVC (200–280 nm) solar radiation [8].

Photosynthetic processes

The basic overall chemistry of photosynthesis is that of trapping energy from sunlight which, by a complex chain of energy transfers, splits water molecules into electrons and protons while releasing oxygen (Eqn 1):

image(1)

The released electrons provide a driving force for pumping the protons within the cell membrane structure of the organism while the protons release the energy needed to produce adenosine triphosphate (ATP), a process known as photophosphorylation. Both protons and electrons participate in the production of nicotinamide adenine dinucleotide phosphate (NADPH, reduced dihydride form). These light-dependent reactions are followed by light-independent ones, which result in the ultimate fixation of atmospheric carbon dioxide, initially as glucose, the overall reaction being as shown in Eqn 2:

image(2)

Adenosine triphosphate and NADPH are recycled as adenosine dinucleotide phosphate (ADP) and nicotinamide adenine dinucleotide phosphate (NADP+), respectively. The overall system is summarised in Figure 1. There are two basic photosystems, named PS I and PS II in the chronological order in which they were discovered, the former involved in the production of NADPH, the latter with that of oxygen, and both PS I and II with the synthesis of ATP [9].

Figure 1.

 Basic photosynthetic cycle

The light energy for photosynthesis is collected by ‘antenna systems’ composed of light-harvesting complexes (LHCs), which are protein/pigment macromolecules. In green plants, the main pigment is of course chlorophyll, but although various chlorophylls are present in many simpler organisms also, a wide range of other coloured pigments such as the carotenoids is to be found associated with the protein complexes. Many years of research have been spent in elucidating their mode of action and detailed structures of several LHCs have been finally elucidated using in particular sophisticated, high resolution, X-ray diffraction equipment [10].

When photons strike at the surface of simple living cells that consist essentially of many double membrane sacs within which are stacks (grana) of thylakoid cells which are surrounded by a fluid medium (the stroma), the light is absorbed by various pigment molecules, such as the chlorophylls, of the antenna system lying within the disc-like thylakoid membrane system of the cell. The absorbed energy raises orbital electrons to an excited state. As the molecule rapidly (in nanoseconds) returns to its normal ground state some energy may be lost as fluorescent radiation; although it is characteristic of photosynthetic systems such losses are minimal. Most of the energy is then passed via a series of extremely fast (in a few femtoseconds to picoseconds) radiation-less transfers, by an intermolecular, inductive dipole resonance process, until it reaches the ‘reaction centre’ molecule, which becomes ionised and finally passes on its electron to an acceptor within the photosynthetic chemical reaction chain. These processes are summarised in Figure 1 and Eqns 1 and 2.

Light-harvesting pigment–protein complexes

The best known and the most common photosynthetic pigment is chlorophyll which imparts its characteristic green colour to many life forms ranging from bacteria to the higher plants. The basic structure of the pigment is that of a magnesium complex, substituted porphyrin (1). There are, however, several structural forms that differ in both their carbon–carbon bonding and the side-chain substituents, these variations being shown in Table 1 [11].

Table 1.   The chlorophylls – structural variationsa
Ring carbonabc1c2d
  1. a In this table, x = phytyl

Chlorophyll type
 C3–CH=CH2–CH=CH2–CH=CH2–CH=CH2–CHO
 C7–CH3–CHO–CH3–CH3–CH3
 C8–CH2CH3–CH2CH3–CH2CH3–CH=CH2–CH2CH3
 C17–C2H4COOx–C2H4COOx–CH=CHCOOH–CH=CHCOOH–CH=CHCOOH
 C17/18 bondSingleSingleDoubleDoubleSingle

inline image

Chlorophyll a is the most widely distributed form but is usually accompanied by either the b or c forms, depending on the particular organism. When isolated, most forms of chlorophyll have a characteristic absorption spectrum exhibiting two peaks (around 450 and 650 nm) which is why they appear green in reflected light, but the associated protein structures forming the LHCs shift the peak absorption to longer wavelengths. In many bacterial chlorophyll complexes, chlorophyll d is, however, present for which the peak absorption is above 700 nm which assists the capture of light under conditions where shorter wavelengths of incident radiation have already been absorbed externally (e.g. on the underside of floating microbial ‘mats’) [12].

Chlorophyll d is the least commonly occurring form and was originally isolated from red algae [13] but it has been suggested that its true source was in the cyanobacteria with which the seaweed was associated [14]. More recently, chlorophyll d has been shown by Mizoguchi et al. to be the main pigment in a unique photosynthetic prokaryote, Acaryochloris marina. The work confirmed the presence of a characteristic formyl substituent at the 3-position and determined the complete stereochemistry [15]. It has also been shown that if the 3-formyl group is reduced to hydroxymethyl, the pigment will form stable, aggregated suspensions in non-polar solvents which the natural forms will not. It is thought that these self-aggregating suspensions could be valuable in the construction of novel photoactive nano-devices [16].

In LHCs there are other pigments, particularly carotenoids (carotenes and xanthophylls), which may not only act to increase light absorption in the middle wavebands where chlorophyll does not, but also serve other essential functions including a photoprotective action against damage from UV radiation by dissipation of excess excitation energy [17]. The evolution and widespread existence of UV-protective compounds have been well reviewed [18]. Apart from the carotenoids, there are many other naturally occurring products, which have been shown to exert a photoprotective role and these often contain basic cyclohexanone or cyclohexenimine structures, which tend to have absorption maxima in the UVB (280–320 nm) and UVA (320–400 nm) wavebands, respectively. Such compounds are exemplified by the mycosporine-amino acids, which were first identified in fungi, but later found in cyanobacteria, algae, lichens and corals.

Many decades of research have been spent in determining the detailed structures of LHCs such as investigations led by Blankenship [19] and Cogdell et al. [20]. These studies have been assisted by developments in multidimensional nuclear magnetic resonance (NMR) [21], time lapse, fluorescence spectrographic examination [22,23], high-performance liquid chromatography (HPLC) separation of pigments [24,25] and high-resolution X-ray diffraction crystallography [10,20] which has revealed the 3D details of these macromolecular structures. Thus, the purple bacteria Rhodospirillium rubrum produce a primary (LH1) LHC, which has a peak absorption wavelength somewhat shorter than that of the peripheral complexes (LH2 and sometimes an LH3 complex) [26]. There is therefore a downward gradient of energy levels from the LH3, via LH2 to the LH1 antennas.

The form of the LH2/LH3 protein structures varies from one organism to another. An LH2 from purple bacteria has been shown to be a macrocyclic nonamer with ninefold symmetry, each subunit of which binds three bacterial chlorophyll a and one lycopene carotenoid molecules, while LH2 from the Rhodospirilliumacidophila bacterium has an eightfold symmetry [27,28]. A more complex structure was found for the LH2 complex in green plants [29], which contained both chlorophyll a and b, and three carotenoid pigments. An LHC from dinoflagellates has been shown to be a trimeric complex but with a much higher proportion of carotenoid to chlorophyll content compared with that of other LHCs [30].

A further type of LHC are the phycobilisomes (PBSs), which occur in cyanobacteria and in some red algae. PBSs harvest light in the blue–green, yellow and orange wavebands (450–650 nm), which is advantageous for organisms existing in low light, aquatic habitats. The PBSs have been actively studied since the mid-nineteenth century [31] and being water soluble can be obtained as crystals suitable for X-ray diffraction examination from which their structures can be interpolated. The elucidation of their three-dimensional structure was finally resolved in the 1980s [32]. The pigments such as phycoerythrin (red) and phycocyanin (blue) themselves are covalently attached via thiol linkages to the associated protein in the PBS [33]. The phycobilin pigments all have tetrapyrrole structures, varying in the positions of the carbon–carbon double bonds and in their stereochemistry, the two most important being phycocyanobilin and phycoerythrobilin (2) [34].

inline image

Environmental changes, such as the quality of ambient light can also have a profound effect on the colour of some algae, which adapt the proportions of the different pigments bound in their PBSs to optimise energy transfer. For example, a cyanobacterium Fremyella diplosiphon, when illuminated with green light, produces red cells and vice versa. This process is described as (biological) ‘complementary chromatic adaptation’ [35]. More familiar are the changes we see in many green plants in which the presence of pigments other than the chlorophylls becomes visually evident as a result of seasonal changes. Thus, the autumn colour display of trees and the ripening of many fruits results from a reduction in the synthesis of chlorophyll, which degrades rapidly leaving the colours of the anthocyanins (red) and carotenoids (yellow) that are also present to predominate [36].

Biological classification of simple life forms

Classical phylogeny is based on five ‘kingdoms’ (Animals, Plants, Fungi, Protista and Monera), with three ‘domains’ (eucarya, bacteria and archaea). There is, however, considerable ambiguity within some of these classifications, which has become particularly evident the more their genetic make up and probable evolutionary lineage is discovered. For example, algae were classed in the plant kingdom but many are now considered to belong with the Protista (a wide-ranging kingdom including many microscopic plant-, algae- and fungus-like organisms) with the exception of certain multicellular phyla remaining as plants. As a result of many such difficulties there have been major revisions in classification and the five kingdoms have been expanded to eight with Monera (simple bacteria) being divided into Eubacteria (primordial life forms still found toady in extremely harsh environments) and Archaebacteria (the more familiar complex, single cell organisms), with the result that what were previously known as blue–green algae which may be regarded as cyanobacteria. Furthermore, coloured bacteria were once all classed as chromobacteria but not all are photosynthetic and some have been assigned to two other genera [37]. With the exception of the cyanobacterial pigments already mentioned, the many other colours of bacterial origin are not covered by the present review. The eighth ‘new’ proposed kingdom is Chromista, a group comprising many seaweeds. All these changes demand the use of new phylogenic descriptions [38]. Even now, as a result of increasing knowledge, there are constant changes in the categories into which individual species are placed. In this review, the relevant simpler photosynthetic species will be grouped as either algae or fungi or as their symbiotic associations existing as lichens and corals. The constantly changing situation is well illustrated by the algae where three new phyla, 10 new classes and four new orders have been proposed in a classification system which once more includes the cyanophyta [39]. In this review, the terms cyanophyta, blue–green algae or cyanobacteria are used depending on the classification given in a particular reference.

Algal pigments

The algae are one of the most diverse forms of life and comprise some 7500 species [40]. They are simple organisms, which evolved ca. 2 billion years ago and in contrast to members of the plant kingdom have no true root/stem/leaf system although their morphology is very varied as they may form filaments, flat sheets or take on spherical shapes. They also vary considerably in size from microscopic phytoplankton to giant seaweeds. All contain chlorophylls and usually two or three other pigments which affect their overall colour. Most algae store their food as starch or other polysaccharides and have cellulosic cell walls while others known as coralline algae acquire an outer shell of calcium carbonate. Other exceptions are some brown algae (Chrysophyta) and diatoms, which have a silaceous cell wall and store their food as oils, thus millions of years ago laying down today's oil deposits. In this respect the production of bio-diesel fuel from oil extracted from certain cultivated brown, red or green algae may one day compensate for the earth's diminishing fuel resources.

Algae may be grouped into several phyla, all of which are to be found in marine environments and several in freshwater also [41] (Table 2).

Table 2.   Some varieties of photosynthetic algae
PhylumOccurrenceFormsColours (chromophores)
CyanophytaWidespreadMostly unicellularBlue–green
Cyanobacteria Early life form
May be colonial
 (chlorophyll a/d, phycocyanin and phycoerythrin, zeaxanthin and fucoxanthin, echinenone)
BacillariophytaFreshwater and marineDiatoms/phytoplanktonYellow–green/brown (chlorophyll a/c, fucoxanthin and diadinoxanthin, α- and β-carotene)
ChlorophytaFreshwater and marineUnicellular – sometimes colonial, e.g. Volvox, SpirogyraGreen or orange (chlorophyll a/b, lutein, violaxanthin and neoxanthin)
ChrysophytaFreshwater and marineUnicellular, colonial flagellatesGolden brown (chlorophyll a/c, fucoxanthin and neoxanthin and echinenone)
PhaeophytaMarineMulticellular seaweedsOlive green to brown (chlorophyll a/c, lutein, fucoxanthin and violaxanthin)
RhodophytaMarineMainly seaweeds
May be coralline
Red (chlorophyll a/d, α- and β-carotene, phycobilins and xanthophylls)
XanthophytaFresh and brackishMostly unicellular and waters colonial microalgaeYellow–green (chlorophyll c, β-carotene, violaxanthin and neoxanthin)
Pyrrhophyta/ DinophytaMarine and freshwaterMostly dinoflagellates (symbiotic in corals)Red or brown (chlorophyll a/c, peridinin, dinoxanthin and diadinoxanthin)

Table 3 illustrates the variety of chlorophylls and the carotenoid pigments that give each species its characteristic colour, together with references to their individual chemical structures. The first structure to be elucidated over 50 years ago was that of β-carotene and since then almost the full range of natural carotenoid pigments have been synthesised and their stereochemistry determined, particularly through the work of Weedon and colleagues at Nottingham University [42]. As well as chlorophyll, other pigments and in particular α-carotene (a structural isomer of the β-form), peridinin and fucoxanthin are directly involved in the photosynthetic process while others, including β-carotene, lutein, zeaxanthin and diadinoxanthin play a mainly photoprotective role [43]. Photoprotection may take the form of either preferential absorption of damaging UV radiation or by a quenching of free energy from molecules that have been raised to higher energy levels and which might lead to the formation of free radicals or oxidising species.

Table 3.   Some pigments found in coloured algae
ClassNameStructureColourλmaxa [25]
  1. a Main absorption maxima of the isolated pigment to nearest 5 nm

ChlorophyllChlorophyll a 1 (see Table 1)Mid green430, 665
Chlorophyll b Yellowish green460, 645
Chlorophyll c1 Yellowish green445, 630
Chlorophyll c2 Yellowish green445, 630
Chlorophyll d Olive green400, 445, 700
Carotenoid/caroteneβ-Carotene3Reddish yellow455, 480
Lutein4Yellow450, 480
Carotenoid/xanthophyllsFucoxanthin5Orange530
Neoxanthin6Greenish yellow440, 465
Zeaxanthin7Orange455, 485
Diatoxanthin8Orange455, 480
Diadinoxanthin9Greenish yellow450, 475
Violaxanthin10Yellow440, 470
Peridinin11Orange/red480, 520
Dinoxanthin12Yellow445, 475
Echinenone13Orange460
PhycobilinsPhycoerythrin 2 (R = vinyl)Orange/red540
Phycocyanin 2 (R = ethyl)Greenish blue620

Structurally, diatoxanthin and diadinoxanthin are particularly unusual molecules in that they possess acetylenic groups, while fucoxanthin, neoxanthin and dinoxanthin and peridinin have allenic linkages. Using C-13- and H-3-labelled model compounds a mechanism for the formation of these groups has been postulated [44]. Peridinin also differs from other xanthophylls in that the molecule includes a lactone ring structure. The proportions of diadinoxanthin and diatoxanthin in an organism can change reversibly with the strength of incident light, high levels of which convert the epoxy groups to ethylene bonds; the reverse reaction occurring in the dark. A similar ‘xanthophyll cycle’ has been observed with the two-stage de-epoxidation of violaxanthin to zeaxanthin [45].

inline image

inline image

inline image

inline image

inline image

inline image

inline image

inline image

inline image

inline image

inline image

Conventional spectroscopy may be used to determine the amounts of each pigment component in an algal solvent extract by applying empirical weighting factors to the absorption values at selective wavelengths, sometimes in conjunction with certain simultaneous equations. More specific pigment identification can be achieved using chromatographic separation techniques followed by conventional spectrographic analysis [46,47]. When illuminated with light at 400–500 nm, each of the algal pigments exhibits a characteristic fluorescence which may be used both for identification and for quantitative measurements. A simple fluorimeter instrument is marketed using filters and a diode-illuminant and diode-photodetector to determine concentrations of chlorophyll, phycoerythrin and phycocyanin for in vivo monitoring of algal blooms in reservoirs [48]. Recently, there has been considerable interest in tracking changes in the overall biomass in the oceans, the relative quantities of chlorophyll being indicative of phytoplankton concentrations which may be determined fluorimetrically [49]. Spectra which characterise each specific pigment better are obtained with fluorimeters employing monochromators for both illuminant and detector which yields excitation-emission matrix (EEM) spectra. Fluorescence emission takes place after a very short time interval following a flash pulse of exciting radiation and the emission then quickly decays. This is the basis for techniques which can examine more complex systems, using either a sequence of isolated flash pulses (fast repetition rate or FRR fluorimetry), or the pulse amplitude modulated (PAM) fluorescence method commonly used to measure the photosynthetic efficiency of an organism [50]. Fluorimeters can be fitted with fibre optic probes which allow the investigation of fluorescent responses from a very small organism such as an individual coral polyp [51]. In contrast, much larger scale spectral monitoring of both land and water areas can be carried out from aircraft or even satellites using so-called hyperspectral reflectance devices [52].

Algal blooms

Algae colonise open water very readily and usually the warmer and more sunlit the situation the faster they grow so long as there are adequate supplies of trace nutrients such as nitrogen and phosphorus. Under certain conditions their presence is made very evident by the body of water becoming strongly coloured. Such is the case with certain species of green algae, which although common in tropical countries (where they are often not regarded as hazardous and even used as fertiliser and for food) are here treated as a potential public health risk. They can give drinking water an unpleasant taste due to the presence of 2-methylisoborneol and block filters in water treatment plant. Blue–green algae in particular can produce hepato- (liver), neuro- (nerve), endo- (internal) and cyto- (organ cell) toxins which can be fatal to cattle but only rarely to humans [53,54].

In the sea, an excessive bloom of algal dinoflagellates of the Gymnodinium or Peridinium genus can give rise to an effect known as the ‘red tide’ [55]. These events, described generally as harmful algal blooms (HABs) appear to be becoming more prevalent worldwide, with increasing reports of contamination of fish and especially shellfish by the associated toxins, particularly where the water contains raised levels of nitrogen and phosphorus [56].

Marine dinoflagellates belong to the phylum Dinophyta or Pyrrophyta, so named because they are responsible for green luminescent effects sometimes seen in darkness when walking over seashore mud or agitating the water surface such as when rowing. The effect is due to bioluminescence involving a luciferin having an open chain tetrapyrrole structure contained in specialised algal vesicles called scintillons, which become activated after darkness falls when there is a drop in pH in these vessels [57].

Commercial uses for algal products

Cyanophyta

Phycobilins have considerable commercial value not only as natural pigments for colouring foodstuffs and cosmetics [58], but also have become well established for fluorimetric immunoassay [59] a very sensitive method of analysing drugs, hormones, proteins, antigens, etc. [60]. The phycobilins are particularly suitable for use as fluorescent markers in immunoassay techniques because these natural pigments fluoresce much brighter and have higher quantum efficiencies (ca. 90%) for incident light absorption than synthetic fluorescent dyes such as the fluoresceins and rhodamines. Further improved performance is said to be possible by using a related, novel polypeptide marker called a phytofluor [61].

Flow cytometers usually employ one or two laser excitation beams and from two to four fluorescent markers, typically phycoerythrin and phycocyanin (which bond via thiol linkages) and fluorescein isothiocyanate (FITC) which couples with protein amino groups. A common light source for flow cytometry [62] is a pulsed argon ion laser radiating at 488 nm which is appropriate for excitation of FITC and phycoerythrin and phycocyanin which fluoresce at 520 nm (yellow), 575 nm (orange) and 640 nm (red), respectively. These three responses are separated by the dichroic mirrors and band-pass filters fitted within the cytometer [63].

Phaeophyta and Rhodophyta

For many centuries, certain varieties of seaweed have been used as food by local communities and this tradition continues today particularly in the Far East [64,65]. Typical examples are the red seaweed Porphyra (nori), used to wrap Japanese sushi and the fried brown seaweed Undaria (wakame) used in Chinese cooking, although the dark green ‘seaweed’ served in most restaurants in the UK is actually a cabbage, bok choy (Brassica chinensis). In China alone, some 10 million tonnes per annum of Laminaria seaweed is cultivated for food. These types of seafood were originally all collected from the wild but present day demand can only be met by large-scale cultivation methods. In the UK, the seaweed Porphyra dioica is eaten as Welsh laverbread and Palmaria palmata is the basis of dulse in Scotland. Extracts from both red and brown seaweed are also approved as flavour enhancers.

Other varieties of seaweed provide valuable polysaccharide thickening agents, such as carrageenan and agar, and especially the alginates, without which the introduction of reactive dyes for textile printing would have had serious technical problems. Reactive dyes react with primary hydroxy groups in other polysaccharide thickening agents such as locust bean and guar gums that do not posses carboxylic groups, producing a fabric with a harsh handle and prints with inferior wash fastness. Carrageenans and agars are derived from red algae and algins from brown algae (Ascophyllum nodosum and Laminaria hyperborea in the UK). Both types of product are used as stabilisers and binding agents in formulating foodstuffs and medicines. A proprietary mixture of sodium bicarbonate and alginate is widely used as a simple remedy to overcome the digestive discomfort caused by gastric ‘oesophageal refluxing’. Another unusual use for alginates is that of wound dressings made from extruded and entangled fibres of calcium and sodium alginate which form a highly absorbent, breathable and non-adhesive dressing [66].

Dietary uses for carotenoid pigments

Just as the carotenoid pigments have a photoprotective action when present in simple organisms this can also be their function in higher animals. Lutein and its structural isomer zeaxanthin are the yellow pigments present in the retina of the human eye, mainly in the foveal region, where they act as anti-oxidants. Although we should acquire these products in our normal diet they are marketed as dietary supplements, ostensibly as a protection against age-related macular pigmentation disease (AMD). AMD is the leading cause of blindness in people over 65 but although many epidemiological studies and experimental dietary trials do tend to support the benefit of such supplements, other factors (such as gender, lens density, the colour of the iris and obesity), which also appear to influence the incidence of AMD prevent any hard and fast conclusions [67,68]. Carotene and lutein are also permitted food colours (E160a and E161b) used, for example, for colouring margarine and as dietary supplements for poultry feed, as a means of controlling the colour of both egg yolks and chicken flesh.

Fungi

Fungi belong to an individual taxonomic kingdom and range from large and often colourful mushrooms down to the microscopic yeast and moulds [69]. Selected natural yeast and moulds have of course been used for thousands of years for fermentation, baking bread and cheese making but the fossil record shows that fungi were developing and land plants were already forming symbiotic relationships some 400 million years ago. Fungi fulfil an essential ecological role in breaking down dead organic matter. Unlike the algae, fungi do not fix carbon by photosynthesis but absorb nutrients produced by other photosynthetic organisms, with which they often coexist symbiotically, for example, as a mycorrhizal partnership with the root systems of plants or with cyanophytes or other algae as in lichens, or they may be parasitic as infectious fungi on living creatures. Their most colourful forms are, however, as mushrooms, many of which range from the brightest yellows, oranges and reds to the more common brown shades.

Fungal pigments cover an astonishing range of chemical structures, many of which are cyclopentanone and quinone structures, and progress in their identification has been regularly reviewed [70]. There is, at present, no systematic method of grouping this array of natural products other than by their perceived biosynthetic pathways [71]. Some examples of the range of their structural complexity are given in Table 4.

Table 4.   Examples of fungal pigmentation
Source (common name)PigmentColourStructureReference
Stephanospora caroticolor (Carrot truffle)StephanosporinOrange overall2-Chloro-4-nitrophenol[70]
Hapalopilus nidulans (Cinnamon bracket)Polyporic acidOrange brown14[72]
Echodontium tinctorium (Indian paint fungus)EchinotinctoneReddish orange15[73]
Chamonixia caespitosa (none)ChamonixinBright blue spores16[74]
Amanita muscaria (Fly agaric)MuscarufinRed to orange cap17[75]
Tricholoma aurantium (Orange knight)AurantricholoneScarlet cap18[76]
Scleroderma citrinum (common puff- or earth-ball)SclerocitrinYellow spores19[77]
Chalciporus piperatus (Peppery bolete)ChalcitrinBright yellow stalk20[77]

inline image

inline image

inline image

inline image

inline image

inline image

inline image

Potential uses for fungal products

Certain fungi have been used since prehistoric times for many purposes such as spark-tinder for lighting fires or as a source of traditional medicine. For example, the skin of the birch polypore (Piptoporus betulinus), a common bracket fungus, has had uses ranging from tinder to wound dressings to razor strops and was even found in a bag hanging round the neck of the late Neolithic iceman ‘Oetzi’ whose mummified body was discovered in the alps in 1992 [78]. The names ‘Indian paint fungus’ and Echodontium tinctorium (see Table 4) indicate that certain fungi have also been traditionally used for coloration (in this case for making red paint by American native Indians) and in the past 25 years such arts have seen something of a renaissance for craft dyeing [79,80]. Certain fungi are a potentially rich natural source of anthraquinone derivatives which they produce as secondary metabolites. It has been shown, for example, that a strain of Curvulata lunata produces just three anthraquinones with the blue cynodontin (1,4,5,8-tetrahydroxy-3-methylanthraquinone) making up 70% of the extract from which it proved possible to produce CI Disperse Blue 7 and CI Acid Green 28 [81]. Cheaper and more ecologically friendly alternatives to present processes for manufacturing anthraquinone dyes are still being sought and bio-production might one day prove feasible, although any novel structures would still require the extensive toxicological testing required for all new dyes [82].

Fungi have a further potential role to play, not as a source of dyes but in wastewater treatment, as a means of removing them. One possibility is to use dead fungi and bacteria as a ‘microbial biomass’ [83,84]. It has also been shown that, using a suitable continuous bioreactor containing an immobilised live fungus (Phanerochaete chrysosporum), even anthraquinone dyes can be decolorised, the main effect believed to be due to the presence of a manganese peroxidase [85]. Enzymes present in a newly developed strain of the fungus Tamates modesta are also effective decolorisers [86]. In fact, many of the enzymes produced by fungi are now commercially available and used by both the foodstuffs and textile industries. For example, cultures of the black mould Aspergillus niger are a source of asparaginase, amylase, catalase, cellulase, glucanase, glucoamylase and hemicellulase. Some of these enzymes are used for the desizing of woven cotton textiles, producing stone-washed effects on denims and for smooth ‘biopolishing’ finishes [87]. Fungal enzymes have been found which can degrade even nylon fibre [88], while the enzymes of white-rot fungi are capable of degrading a wide range of ‘difficult’ pollutants such as polychlorinated biphenyls, polycyclic hydrocarbons, bleach plant effluent and many types of dye [89]. Aspergillus niger is also used for the commercial production of citric acid from corn starch-derived glucose, replacing lemons as the former source of manufacture, chiefly in Italy [90].

The best known commercial use of fungi in the food industry has been the manufacture of mycoprotein products as vegetarian meat substitutes, such as Quorn (originally developed by Marlow Foods in the 1980s). This was produced in biofermentation tanks using natural, locally occurring soil fungi, Fusareum venenatum or Fusareum graminearum. This mycoprotein was initially produced in response to a predicted global famine which did not develop and it took a further 20 years for Quorn-based foods to become widely accepted by the UK public [91]. Although covered by patent in the USA, there is still resistance to Quorn's acceptance and it might be thought to pose a threat to the large soya-product industry there, where Quorn was advertised somewhat misleadingly as ‘derived from mushrooms’ [92].

Algaecides and fungicides

There are of course circumstances where the occurrence of fungal or algal growths is very undesirable and it is therefore necessary to apply preventative chemicals. On textiles, fungal growths are to be avoided as they can lead to staining which is very difficult to remove. Pentachlorphenol was used for many years until a ban was imposed with very stringent maximum levels of dioxin-related chemicals in cloth being permitted. This created quite a problem for some time because imported cloth free from this contaminant was difficult to obtain. A number of alternative treatments such as the use of 2-(thiocyanomethylthio)benzothiazole (TCMTB) are now available. In other fields, there is an extensive range of fungicides which are used to prevent damage to agricultural crops by fungal spores (‘smut’ or ‘rust’). Traditional non-selective fungicides included copper-, sulphur- and mercury-based compositions (such as Bordeaux mixture, Flowers of sulphur and Calomel) but it is mostly crop/disease-specific organic chemicals which are used today. With these synthetic fungicides, if genetic mutations occur, resistant strains of disease-causing fungi can develop, for these agents act by inhibiting a particular part of the biochemistry of the target fungus. Fortunately, the wide range and differing modes of action of modern fungicides available usually allow an alternative, more effective product (or mixture of products) to be found [93].

Undesirable algal blooms on water can also be killed by treatment with copper compounds or chlorine but where this is unacceptable (aquaria and some swimming pools), proprietary products based on polyhexamethylene biguanide hydrochloride are available. This product is also used as a bacteriocide for cellulosic fibres while Triclosan [5-chloro-2-(2,4-dichlorophenoxy)phenol] can be incorporated as a sanitiser into synthetic fibres at the spinning stage [94]. Where extensive blue–green algal growth occurs on a reservoir it has been shown that killing the algae can actually exacerbate the possible effects of cyanotoxins [95].

Textile thickeners when dissolved and exposed to the atmosphere may trap fungal spores which, particularly in hot weather, can degrade the polymer, cause bad odours and reduce printpaste viscosity. To prevent this, it is usual for the thickener manufacturer to include a fungicide but otherwise a small addition of formaldehyde or other aldehyde precursor such as paraldehyde to the stock thickener is very effective.

Lichen

Lichens are an example of mutual symbiosis, the body of the fungus forming a protective framework around a green algal partner which is photosynthetic and provides the host with nutrients. In turn, the fungus protects the algae so well that lichens are often very resistant to extreme climatic conditions, being found in both arid and cold locations. They are classified taxonomically according to the fungus involved (mainly Ascomycota) and also according to the appearance of the main body (thallus) of the lichen, being described as foliose (leaf-like), crustose (crust-like), fruticose (bushy) or squamulose (scaly) [96,97].

There are probably ca. 15 000 lichen species with representatives to be found clinging to a variety of surfaces be they bare rocks in sunbaked deserts or on frozen tundra soils where they may, for example, provide a vital food for caribou and reindeer. Many lichens are greenish grey from the greyish colour of the fungus host and the chlorophyll contained in the green alga within, but some are coloured bright yellow, orange or red. Of particular interest are the lichens Leconora tartarla/Ochrolechia tartarea and Rocella tinctoria, from which the natural dyes orchil and cudbear have long been obtained [98]. These lichens contain orcinol depsides such as lecanoric acid (21) and extraction followed by a slow anaerobic fermentation process in aqueous ammonia, yields the red to purple orcein dye or, if reacted under more alkaline conditions, the acid/base indicator, litmus, which is a trimer of orcein. These colours are classed as Natural Red 28 (red at low pH and blue when alkaline) but are complex mixtures derived from 7-amino-2-phenoxazone (hydroxy- and amino-orcein are illustrated in structure 22) [99].

Other yellow lichen pigments, which act as UV-absorbers, are usnic acid (23) and parietin (1,8-dihydroxy-3-methyl-6-methoxyanthraquinone). Usnic acid, which is the most abundant secondary metabolite characteristic of lichens, is extracted from Usnea barbarta. It has long been used in folk medicine and more recently for antibiotic and personal hygiene products as well as in health food supplements as an aid to weight loss [100,101]. It has been shown that the formation of parietin in some lichens can be induced by exposure to light in the visible wavebands alone, but its formation is much greater when the light includes UVB radiation (280–320 nm) [102,103]. Thus, the biosynthesis of this UV-screening pigment responds to the intensity of incident UVB radiation, which, for example, increases in Antarctica each spring due to ozone depletion in the upper atmosphere [104]. This response by an organism in increasing the production of protective pigment with increasing levels of ambient UV radiation is reported to be characteristic and these chemicals often possess long chain, or polycyclic, conjugated double bond structures forming π-orbitals [18].

A novel pigment pigmentosin A (24), isolated from Hypotrachyna immaculata, is the first lichen metabolite shown to have a bis-naphthopyrone structure [105]. Lichens also synthesise a range of colourless acids and when growing on stone buildings these can, in time, lead to serious, unsightly damage to the surface of the masonry. This arises because of the high water-retention properties of lichens and the presence of metal chelating, carboxylic acids, forming, for example, calcium oxalate dihydrate which builds up as dark, disfiguring encrustation [106]. Lichen acids can also damage trees as a result of chlorophyll degradation leading to defoliation [107].

inline image

inline image

inline image

inline image

Coral

In 1814 Captain Matthew Flinders, the first man to circumnavigate Australia [108], wrote, ‘I went upon the reef with a party of gentlemen and a new creation… was there presented to our view. We had wheat sheaves, mushrooms, stagshorns, cabbage leaves and a variety of other forms glowing under the water with vivid tints of every shade betwixt green, purple, brown and white; equalling in beauty and excelling in grandeur the most favourite parterre of the curious florist’.

Such is the colourful beauty of coral growths (Corallinaceae), which represent another example of a symbiotic relationship involving algae. Coral polyps contain filaments which can capture planktonic food and they gradually acquire a carbonate outer coralline skeleton, which may have either a solid or porous structure. In this association, external protection is afforded by the whole reef structure. The coral polyps are a variety of unicellular, dinoflagellate microalgae called zooanthellae, for example, Symbiodinium microadriaticum [109]. Until relatively recently it was thought that only one species of coral was associated exclusively with one species of zooanthella but it is now evident that there is considerable diversity and interchange [110].

Zooanthellae provide the host coral with additional nutrients, which they produce photosynthetically. Although coral can be found at considerable depths, the most colourful tend to form in clear shallow waters and growth can be seriously affected by silts brought down from river estuaries close to off-shore reefs [111]. On the other hand, it appears that strategies exist to adapt to excessive levels of illumination and protective xanthophyll pigments such as diadinoxanthin are produced to counteract potential damage from shortwave radiation [112].

The tissues of a coral are themselves usually colourless, so it is the coloured pigments of the zooanthelae living within these structures that produce the outward colour displays seen on some reefs. With certain coral species the skeletal material itself is coloured pink, red, black or gold but little has been published as to the precise nature of these pigmented corals, which have long been used for human adornment. In contrast, a range of coloured pigments are associated with the living zooanthellae and although many corals are either brown or golden-yellow, those growing in lagoons tend to be more colourful with pink, yellow and orange varieties. Some of the pigments responsible are listed in Table 5.

Table 5.   Zooanthellae photosynthetically active pigments
PigmentColourStructure
  1. a Green in its oxidised form (25), red when in the reduced state

Chlorophyll aMid green 1 (see Table 1)
Chlorophyll cBright green 1 (see Table 1)
β-CaroteneReddish yellow3
FucoxanthinOrange5
ZeaxanthinGreenish yellow7
DiadinoxanthinGreenish yellow9
DinoxanthinYellow12
PeridininOrange/red11
NeoperidininBrick red 
ScytoneminGreen or reda25

inline image

The pigment scytonemin is a good example of an efficient photoprotective compound, its complex structure resulting in UV absorption over a relatively wide range of wavebands (from 325 to 425 nm). Coral's dynamic adaptation to the intensity of UV radiation has been demonstrated, for the amount of protective pigment produced was found to increase with decreasing depth from which the coral samples were growing [113].

Corals such as the great star coral (Monasteraea cavernosa) also fluoresce due to the presence of certain proteins but the observed effect depends on the nature of the illumination and ambient light at a particular depth. The main contributors to fluorescence are cyan/green fluorescent protein (GFP; also present in some jellyfish), orange/red fluorescent protein and chlorophyll which fluoresce at 515, 585 and 685 nm, respectively [114].

Some doubts have been expressed over Darwinian theories of evolution, with the development of an ‘Intelligent Design’ counter-movement that postulated that any system producing a structure which was ‘irreducibly complex’ could not have evolved from a simpler one [115]. Study of the great star coral's genes, which encode for the different fluorescent proteins, by Ugalde et al. [116] allowed them to prove that this particular irreducibly complex system (requiring the evolution of genes encoding for the cyan/green protein to the more complex, multistage biosynthesis of the red fluorescent protein) can indeed occur by small incremental changes. Furthermore, these authors produced an elegant demonstration in which, using genetically engineered reconstructions of the ancient genes transferred into bacteria, they drew onto the agar gel surface of a Petri dish the reconstructed ‘tree’ of this phylogenetic evolution. The incubated culture was then illuminated with UV light and the resulting traces, each of which fluoresced in the relevant colours (blue, green and red), was photographed.

Coral reef bleaching

It appears that from ca. 1976 there has been a marked increase in the average ocean salinity and temperature which has affected the health of coral reefs worldwide [117] and during the past 20 years there have been an increasing number of reports of coral reef bleaching. Bleaching occurs when, as a result of external stresses, there is a decline either in the pigment concentration or the number of zooanthellae within the coral skeleton. These losses may be as high as 80% [118], but if the stress is not too severe or declines, then the depleted zooanthellae population may recover after a few months. Using computer projections and atmosphere/ocean circulation models it has been predicted that, without an evolutionary increase in tolerance to temperature rises of up to 1 °C per decade, coral bleaching events could eventually be happening every 1 or 2 years [119].

The causes of coral bleaching can vary considerably but that which has received most attention is the incidence of raised sea temperatures, which has a clear connection with the El Niño effect and global warming [120,121]. The temperatures at which coral bleaching occurs seem to lie between 30 and 34 °C [122] but some species of Symbiodinium algae are more resistant than others and may be capable of recolonising a reef [123]. Additional evidence for thermal adaptation has also been reported [124].

In the laboratory, coral bleaching has been studied using chlorophyll fluorescence as a measure of the total photosynthetic capacity of the zooanthellae under different conditions. Depending on the technique used (PAM or saturation pulse fluorescence), it is possible to determine the photosynthetic efficiency of a system under controlled laboratory conditions. A model for coral bleaching was proposed suggesting that the heat-induced bleaching results from impairment of the carbon dioxide fixation mechanisms within the Calvin cycle followed by damage to the PS II reaction centre [125]. More recent investigations reached conflicting conclusions as to whether the PS II system was damaged by heat stress as it has been observed that photosynthetically active zooanthellae can be expelled from the bleaching corals which are first affected [126]. This observation can be explained if a non-photochemical quenching shuts down the PS II reaction centre, but can be explained that when the stress diminishes photosynthesis may recommence [127]. In contrast, reef damage sometimes arises from an unusual cause. As a consequence of the extensive forest fires in Indonesia during 1997–1998, large quantities of iron were carried onto the sea in the smoke, which resulted in a ‘red-tide’ dinoflagellate bloom from which coral toxins were released [128].

Future developments

Medical products

For centuries, folk medicines have been concocted from many plant and fungal sources, these often being chosen because the material from which they were extracted were supposed to give a visual clue of their efficacy (e.g. yellow lichens for jaundice, Lungwort fungus for bronchial problems), but the most important breakthrough of modern times resulted from Alexander Flemming's discovery of the antibiotic action of the fungus, Penicillium notatum, from which penicillin was subsequently developed [129]. Some 50 years later, another wild fungal spore produced cyclosporin, the anti-immune reaction drug vital to the success of organ transplant surgery [130]. In recent years, interest has increased in finding other drugs which may be discovered in nature, in a similar manner to the discovery of the taxanes (Placlitaxel/taxol), which are particularly effective against stage 2 breast cancer. These products are extracted in rather poor yield from yew tree bark or foliage but it has been shown that they may more easily be produced by culture of the yew tree fungus, Taxomyces andrea [131]. Anticancer activity has also been noted with the carotenoid pigments neoxanthin and fucoxanthin, both being shown to produce apoptosis (cell death) in prostate cancer cells [132,133]. Other metabolites from marine cyanobacteria have also provided novel anticancer drugs such as dolastatin and cryptophycin, derivatives of which are undergoing clinical trials [134]. There is even hope for chronic arthritis sufferers with the recent discovery of powerful COX-2 enzyme inhibitors, related to the polyketide inotilone, isolated from an Inonotus bracket fungus species. Release of the COX-2 enzyme triggers inflammation of joints but blocking its action stops the associated pain [135]. Even natural colours such as monascin and monascorubin [136], which have for centuries been produced in the Far East by fermentation of rice using Monascus yeast, have been found to possess anti-microbial and cholesterol-lowering properties [137,138].

From such promising leads there are hopes of finding entirely new drugs to deal with those diseases that have proved more difficult to treat or where there is presently a drug resistance problem [139].

Fuel technology

With ever-increasing oil prices (and diminishing world reserves) there has long been a potential for alternative ‘greener’ sources of engine fuels generally known as biofuels. The established source of these has been either alcohol, obtained by fermentation processes from beet, wheat or corn sugars, or natural oils, extracted principally from sunflowers. Production from plant sources does, however, demand large areas of land for cultivation. In contrast, certain species of microalgae such as diatoms (Bacillariophyta) and green algae (Chlorophyta) grow extremely rapidly in suitable bioreactors and are rich in natural glycerides, a potential source of biofuels, which are non-toxic and biodegradable. The oils produced can be recovered simply by compressing the dried algal mass, followed by a low loss extraction method with water-miscible solvents or liquid carbon dioxide [140]. Algae need bright sunlight, warmth and large quantities of carbon dioxide (and nitrogen oxides) to grow rapidly which can be assured where the bioreactors are located alongside existing power generating plants, thus offering the double benefits of reducing carbon emissions and the production of a fuel. One such system developed by GreenFuel Technologies has been under test in the USA for the past 3 years [141]. The basic reactor unit comprises a triangular arrangement of three interconnected polycarbonate tubes containing the suspended algae. The exhaust gasses are fed into the vertical riser while solar irradiation takes place in the sloping down-tube, circulation being achieved partly by the air-lift effect but is also driven by density differences in the fluid columns. GreenFuel claims to have attained between 40% and 80% reductions of carbon dioxide from flue gasses, depending on the strength of the sunlight. Installation of some full-scale plant is expected by 2008.

Power generation by the burning of waste organic materials or of crops specially grown for this purpose is largely ‘carbon-neutral’, i.e. the amount of carbon dioxide originally fixed by photosynthesis is simply returned to the atmosphere. There are now an increasing number of sites where power is generated by burning biomass [142,143]. If the above-mentioned biologically generated fuel process becomes a reality, the algal residue left after the extraction of the oils could be used as part of the biomass fuel for steam generation. Going yet one stage, further consideration is now also being given to possibility of building ‘biorefineries’, where biomass forms the feedstock for the production of chemicals [144].

The best basis for pollution-free fuel for vehicles is undoubtedly hydrogen used in a fuel cell, although much remains to be done with respect to the production of the gas itself without using pollutant-producing energy generation and devising a means (e.g. a suitable adsorbent material such as metal hydrides or carbon nanostructures) for storing sufficiently large quantities of the gas on vehicles. In the absence of nuclear-, hydroelectric- or wind-power generation, the production of hydrogen itself results, however, in atmospheric pollution [145]. In the absence of a source of nitrogen, some strains of purple bacteria, when provided with a suitable organic substrate and irradiated, produce hydrogen rather than ammonia, but for a practical system it is better to mix the bacteria with green algae which photosynthesise glucose. Some blue–green algae such as Nostoc muscorum appear to combine these two processes in the one organism. Experiments have been carried out using various bacterial and algal species and with different bioreactor designs, but at the moment, the commercial production of hydrogen by such means still remains a long range target [146].

During our workaday lives, we are only occasionally reminded of the myriad simple life forms surrounding us and when we are made aware, it is usually for negative reasons (disease, decay, toxicity problems, bad smells, etc.). Although much of present day research on the array of metabolites produced by these organisms is still largely academic, the isolation and genetic engineering of new strains will undoubtedly produce commercially and environmentally exciting opportunities for their exploitation. Finally, if ever it does become feasible to produce even a small range of fast dyes using pollution-free biotechnology, then the wheel will certainly have come full circle for those who believe that ‘natural’ colours still have a future.

The author works as an independant consultant in coloration.