Sustainable Production of Pigments from Cyanobacteria

Abstract

Pigments are intensely coloured compounds used in many industries to colour other materials. The demand for naturally synthesised pigments is increasing and their production can be incorporated into circular bioeconomy approaches. Natural pigments are produced by bacteria, cyanobacteria, microalgae, macroalgae, plants and animals. There is a huge unexplored biodiversity of prokaryotic cyanobacteria which are microscopic phototrophic microorganisms that have the ability to capture solar energy and CO2 and use it to synthesise a diverse range of sugars, lipids, amino acids and biochemicals including pigments. This makes them attractive for the sustainable production of a wide range of high-value products including industrial chemicals, pharmaceuticals, nutraceuticals and animal-feed supplements. The advantages of cyanobacteria production platforms include comparatively high growth rates, their ability to use freshwater, seawater or brackish water and the ability to cultivate them on non-arable land. The pigments derived from cyanobacteria and microalgae include chlorophylls, carotenoids and phycobiliproteins that have useful properties for advanced technical and commercial products. Development and optimisation of strain-specific pigment-based cultivation strategies support the development of economically feasible pigment biorefinery scenarios with enhanced pigment yields, quality and price. Thus, this chapter discusses the origin, properties, strain selection, production techniques and market opportunities of cyanobacterial pigments.Graphical AbstractKeywordsAstaxanthinChlorophyllFucoxanthinLuteinPhycocyanin
Spirulina

Full text

Adv Biochem Eng Biotechnol
https://doi.org/10.1007/10_2022_211
©The Author(s), under exclusive license to Springer Nature Switzerland AG 2022
Sustainable Production of Pigments from
Cyanobacteria
Charu Deepika, Juliane Wolf, John Roles, Ian Ross, and Ben Hankamer
Contents
1 Introduction
2 Cyanobacterial Pigments
2.1 Phycobiliproteins
2.2 Chlorophylls
2.3 Carotenoids
2.4 Scytonemin
3 Applications
3.1 Food and Nutraceuticals
3.2 Cosmetics
3.3 Pharmaceuticals and Diagnostics
4 Pigment Production in Cyanobacteria
4.1 Cultivation Parameters and Their Impact on Biomass and Pigment Yields
4.2 Mass Cultivation Systems and Process Management
5 Downstream Processing
5.1 Biomass Harvesting
5.2 Product Release via Cell Disruption or Pre-Treatment
5.3 Product Recovery via Pigment Extraction
5.4 Pigment Purification
6 Pigment Bioprocessing Challenges
7 Commercial Pigment Production Technologies
7.1 Patents and Technology Transfer
7.2 Techno-Economic Analysis and Life-Cycle Analysis: CAPEX/OPEX and Price
Points
8 Global Pigment Market Analysis: Opportunities and Challenges
9 Future Perspectives
References
C. Deepika, J. Wolf, J. Roles, I. Ross, and B. Hankamer (*)
Institute of Molecular Bioscience, The University of Queensland, Brisbane, QLD, Australia
e-mail: b.hankamer@uq.edu.au

Abstract Pigments are intensely coloured compounds used in many industries to
colour other materials. The demand for naturally synthesised pigments is increasing
and their production can be incorporated into circular bioeconomy approaches.
Natural pigments are produced by bacteria, cyanobacteria, microalgae, macroalgae,
plants and animals. There is a huge unexplored biodiversity of prokaryotic
cyanobacteria which are microscopic phototrophic microorganisms that have the
ability to capture solar energy and CO
2
and use it to synthesise a diverse range of
sugars, lipids, amino acids and biochemicals including pigments. This makes them
attractive for the sustainable production of a wide range of high-value products
including industrial chemicals, pharmaceuticals, nutraceuticals and animal-feed
supplements. The advantages of cyanobacteria production platforms include com-
paratively high growth rates, their ability to use freshwater, seawater or brackish
water and the ability to cultivate them on non-arable land. The pigments derived
from cyanobacteria and microalgae include chlorophylls, carotenoids and
phycobiliproteins that have useful properties for advanced technical and commercial
products. Development and optimisation of strain-specific pigment-based cultivation
strategies support the development of economically feasible pigment biorefinery
scenarios with enhanced pigment yields, quality and price. Thus, this chapter
discusses the origin, properties, strain selection, production techniques and market
opportunities of cyanobacterial pigments.
Graphical Abstract
Keywords Astaxanthin, Chlorophyll, Fucoxanthin, Lutein, Phycocyanin, Spirulina
Abbreviations
ASE Accelerated solvent extraction
ATP Adenosine triphosphate
BDW Biomass dry weight
CAGR Compound annual growth rate
Chl Chlorophyll
Cytb6 Cytochrome b6
EFSA European Food Safety Authority
ETC Electron transport chain
Fd Ferredoxin
FDA Food and Drug Administration
C. Deepika et al.

FNR Ferredoxin NADP
+
reductase
FRP Fluorescence recovery protein
HPH High-pressure homogenisation
HRP High-rate pond
LCA Life-cycle assessment
L
CM
Linker (protein) core membrane
MEP Methylerythritol phosphate
NADPH Nicotinamide adenine dinucleotide phosphate
NPQ Non-photochemical quenching
OCP Orange carotenoid protein
PAR Photosynthetically active radiation
PBP Phycobiliproteins
PBR Photobioreactor
PC Phycocyanin
PCB Phycocyanobilin
PE Phycoerythrin
PEB Phycoerythrobilin
PEF Pulsed-electric field
PLE Pressurised liquid extraction
PQ Plastoquinone
PS Photosystem
PUB Phycourobilin
PVB Phycoviolobilin
RC Reaction centre
SCCO
2
Super critical carbon dioxide
TEA Techno-economic assessment
1 Introduction
Earth formed around 4.6 billion years ago [1] and the Sun remains its largest energy
source, delivering 3,020 ZJ year
-1
to the Earth’s surface. The massive scale of this
energy supply is highlighted by the fact that every 2 h Earth receives more energy
than we need to power our total global economy for an entire year (~0.56 ZJ year
-1
)
[2]. Geological records indicate that around 3.4 billion years ago, early anoxygenic
photosynthetic organisms evolved [3] using light absorbing pigments, today typified
by chlorophylls and carotenoids bound as cofactors to proteins. These organisms
were not yet able to catalyse the highly oxidising photosynthetic water splitting
reaction of oxygenic photosynthesis. As a result, instead of water, purple bacteria,
green sulphur bacteria, acidobacteria and heliobacteria used a range of alternative,
available and more energetically accessible substrates as electron donors. These
included hydrogen sulphide, dihydrogen, thiosulphate, elemental sulphur and fer-
rous iron [4]. Of these, early cyanobacteria evolved to use sulphides [5]. About 2.4
billion years ago, a genetic fusion event is thought to have taken place between two
bacteria, one with a pheophytin-quinone reaction centre (Type II –an archetypal
Sustainable Production of Pigments from Cyanobacteria

form of Photosystem II; Q-type) and the other with an iron-sulphur reaction centre
(Type I –an archetypal form of Photosystem I; FeS-type) to produce a chimeric
photosynthetic organism with two unlinked photosystems [3]. Subsequently, these
two archetypal photosystems evolved further and were linked into one operational
photosynthetic electron transport chain. Development of the oxygen evolving com-
plex of PSII [6,7] enabled it to catalyse the most oxidising reaction in biology (water
photolysis). This photosynthetic electron transport chain enabled cyanobacteria to
use the huge energy resource of the Sun to split water into protons, electrons and
oxygen to provide ATP and reducing equivalents such as NADPH [7]. Cyanobacteria
remained the principal oxygenic photosynthetic organisms throughout the Protero-
zoic Eon (2,500 to 541 mya) and are thought to be responsible for the Great
Oxidation Event (i.e. the rise of the oxygen concentrations in the atmosphere and
oceans [8]). Later, capture of cyanobacteria by eukaryotes expanded oxygenic
photosynthesis into a range of other organisms, including red algae, glaucophyta,
green algae and higher plants, capable of producing and coordinating a range of
pigments involved in photosynthesis to provide the food, fuel, biomaterials and
atmospheric oxygen that support aerobic life on Earth [8]. This chapter elaborates on
the many pigments coordinated within these intricate cyanobacterial cells and partic-
ularly their role in photosynthesis and the economic opportunities that these provide
for commercial scale sustainable production platforms across the food, pharmaceutical,
biomaterials and primary production (aquaculture and livestock feed) sectors.
Cyanobacteria are commonly referred to as blue-green algae but are strictly
speaking microscopic prokaryotic photosynthetic bacteria. They exist as single
cells, filaments, sheets or spherical clusters of cells and are found in diverse habitats
including fresh, brackish and salt water. Under favourable environmental conditions,
cyanobacteria can exhibit high growth rates but can also resist harsh environments
through dormancy [9]. Cyanobacteria contain a range of pigments including chlo-
rophylls (green), carotenoids (red, orange and yellow), phycobiliproteins (red and
blue) and scytonemin (yellow-brown). These pigments function largely in photo-
synthesis and photoprotection and have useful properties that can be translated into
advanced technical and commercial products [10,11] and in certain cases
(e.g. phycocyanin which has been explored to treat autoimmune encephalomyelitis
[12]) are potentially beneficial to human health [13–15] and the environment
(through biodegradability) [16].
Pigments are intensely coloured compounds that are used in a broad range of
industries to colour other materials. They are extensively used to enhance the
attractiveness of industrial products and are usually termed ‘pigments’in the phar-
maceutical, ink and cosmetic industries and ‘dyes’in the food and textile industries
[17]. They are broadly classified into organic vs. inorganic as well as
natural vs. synthetic categories [17]. Organic pigments are carbon-based compounds
with conjugated chains and rings, either synthetic or natural. Inorganic pigments are
usually metals and metallic salts that are typically insoluble, heat stable opaque
oxides such as Prussian blue (Iron (III) ferrocyanide, produced by the oxidation of
ferrous ferrocyanide salts), cobalt blue, cadmium yellow, lead oxide and titanium
yellow. Natural pigments are mainly organic and include chlorophyll, lutein,
C. Deepika et al.

β-carotene, astaxanthin, indole based dyes and anthocyanins and are widely used as
food colourants (e.g. chlorophyll derivatives) and nutraceuticals (e.g. lutein from
marigold flowers used in functional foods) for human consumption [18]. Synthetic
pigments are usually carbon-based molecules chemically derived from petrochem-
ical products, acids and other chemicals. Even when synthetic pigments are copies of
natural products, their activity may not be the same. This is because natural products
are often chiral in nature while their synthetic counterparts may be racemic. For
example, synthetic astaxanthin produced from petrochemical products (e.g. the
Wittig reaction) is reported to provide less antioxidative activity than natural
astaxanthin (55x less singlet oxygen quenching capacity and 20x less free radical
elimination [19]). Some synthetic pigments (e.g. citrus red II, metanil yellow and
rhodamine B) are reported to have various toxicological effects, including carcino-
genesis, oestrogenic activity and neurotoxicity [20] which has increased the desir-
ability of natural pigments. Pigments in the food sector are strictly regulated due to
health and safety concerns [21,22]. Synthetic pigments are inexpensive and typi-
cally stable, but increasing health and environmental awareness has led to market-
driven expansion of the naturally derived pigment sector as part of an expanding
circular bioeconomy [23,24]. In terms of industrial-scale pigment production it is
important to note that pigments can be produced as isolated coloured chromophores
such as chlorophylls, carotenoids and pheophytin (Fig. 1b), phycoerythrobilin (PEB)
and phycocyanobilin (PCB; Fig. 1c), or as the coloured proteins that coordinate
them (e.g. phycoerythrin, phycocyanin and allophycocyanin). To avoid confusion,
isolated chromophores are here referred to as chromophores and chromophore
binding proteins as coloured proteins. Collectively, along with other coloured
molecules, they are referred to as pigments.
The global pigment market including both natural and synthetic pigments was
estimated to be USD $36.4 billion in 2020 and based on a 5.1% Compound Annual
Growth Rate (CAGR) between 2021–2028 is forecast to expand to USD $51.7
billion in 2028 [25]. Different market sectors comprising textiles (62%), leather
(10%), printing inks (10%) and others (food, nutraceuticals, pharmaceuticals and
cosmetics, 18%) provide significant opportunities for high quality natural pigments.
Compared to plant and animal sources, microbial pigment production is more
sustainable [26], providing opportunities for the production of biodegradable
colourants (e.g. phycocyanin from Arthrospira platensis (Spirulina)). For large-
scale production, cyanobacteria offer specific advantages for pigments unique to
cyanobacteria (e.g. phycocyanin and scytonemin) or that they can deliver higher
yields (e.g. lutein yields are reported to be three- to sixfold higher than in marigold).
Other potential benefits of cyanobacterial systems include lower cultivation time
(compared to plants; days/weeks vs season), lower cultivation cost [27], less arable
land (ability to use non-arable land and floating systems), low freshwater demand
(ability to grow in closed systems using recycled freshwater/seawater/brackish
water) and labour requirements [28–30]. Furthermore, cyanobacteria are amenable
to genetic engineering to support further improvement.
This chapter focusses specifically on natural pigment production from
cyanobacteria –their properties, applications, current extraction technologies and
market trends.
Sustainable Production of Pigments from Cyanobacteria

2 Cyanobacterial Pigments
The first step of photosynthesis is light capture, which is mediated by the light
harvesting antenna proteins of photosystems I (PSI) and II (PSII). These light
harvesting antenna systems are designed to capture Photosynthetically Active Radi-
ation (PAR) in the visible spectrum (400–700 nm). In cyanobacteria, these antenna
systems consist of pigment-protein complexes located on and in the thylakoid
Fig. 1 Cyanobacterial light harvesting antenna and pigment organisation. (a) Cyanobacterial
photosynthetic electron transport chain including the dynamic extrinsic antenna system consisting
of phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC) is connected to the stromal
surface of the PSI and PSII core complexes via the Core-Membrane Linker (L
CM
). (b) Example
of pigment coordination within the PSII monomer. (c) Four major chromophores in cyanobacteria.
The chromophores Phycocyanobilin (PCB; C
33
H
40
N
4
O
6
), Phycoerythrobilin (PEB; C
33
H
38
N
4
O
6
),
Phycourobilin (PUB; C
33
H
42
N
4
O
6
) and Phycoviolobilin (PVB; C
33
H
34
N
4
O
6
). (d) Typical
phycobilisome (PBS) organisation: rod-shaped, bundle-shaped, hemi-discoidal and hemi-
ellipsoidal. In most cyanobacteria the hemi-discoidal organisation occurs but the pigment compo-
sition within these rods is species-specific
C. Deepika et al.

membranes, which lie under the cell membrane (see Fig. 1), typically in a dense
multilayered wrapping (Fig. 6, Sect. 5.2). The extrinsic and intrinsic antenna pro-
teins have evolved to provide a dynamic scaffold that coordinates an intricate and
excitonically coupled network of chromophores including phycoerythrobilin (PEB;
Fig. 1c), phycocyanobilin (PCB; Fig. 1c), phycourobilin (PUB; Fig. 1c),
phycoviolobilin (PVB; Fig. 1c), chlorophylls, pheophytins and carotenoids that
collectively support the dual function of PSI and PSII light-driven charge separation
and photoprotection. The extrinsic antenna systems include the light harvesting
protein complexes (phycoerythrin, phycocyanin and allophycocyanin) which usu-
ally coordinate the chromophores phycoerythrobilin and phycocyanobilin within
them and connect them into the excitonically coupled chromophore network coor-
dinated by the PSI and PSII core complexes [31].
The cyanobacterial PSII core complex is composed of around 20 subunits
(Fig. 1a). In 2001 a 3.8 Å resolution PSII core complex structure from
Synechococcus elongatus was described [32]. Each 350 kDa PSII monomer
(Fig. 1b) is reported to contain 17 membrane spanning protein subunits as well,
three extrinsic proteins, 99 cofactors, 35 chlorophyll a, 12 β-carotene, 2 pheophytin,
2 plastoquinone and 2 heme molecules, the water splitting Mn
4
CaO
5
cluster and one
non-heme Fe
2+
[33]. The electrons extracted from water by PSII are passed, via the
cytochrome b
6
fcomplex (a dimer which includes one chlorophyll and one caroten-
oid per monomer) to PSI, contributing to the generation of an electrochemical
gradient across the membrane that drives ATP production [34]. At PSI, photons
harvested by its phycoerythrin, phycocyanin and allophycocyanin antenna system
are passed on to the PSI core complex to drive charge separation and raise the redox
potential of the donated electrons [35]. Specifically, PSI catalyses the light-induced
electron transfer from plastocyanin or cytochrome c6 to ferredoxin or flavodoxin via
its chain of electron carriers [36,37]. The first crystal structure (2.5 Å resolution) of
the cyanobacterial Synechococcus elongatus PSI complex was also reported in 2001
[38]. Cyanobacterial PSI core complexes are typically trimeric with each monomer
core consisting of 12 subunits and 127 cofactors which include 96 chlorophylls,
22 carotenoids, two phylloquinones and three iron-sulphur (4Fe4S) clusters
[36,37]. The subunits collectively stabilise the core-antenna system and help them
interconnect with peripheral antenna systems. Within the PSI core is the redox active
PSI reaction complex which consists of PsaA and PsaB which coordinate the key
intrinsic redox active cofactors in the membrane [37]. Plastocyanin/cytochrome c6
are soluble electron carrier proteins that donate electrons at the luminal surface of
PSI. Cytochrome c6 is likely the evolutionary older electron donor as it can be found
in most cyanobacteria [39,40]. Excitation energy transfer from the antenna chloro-
phylls leads to excitation of P
700
to the excited state P
700
*, which catalyses the
primary charge separation [41]. Upon illumination, electrons are transferred from
plastocyanin/cytochrome c6 at the luminal surface of the PSI reaction centre to
ferredoxin/flavodoxin at the PSI stromal surface.
Sustainable Production of Pigments from Cyanobacteria

2.1 Phycobiliproteins
Definition: Cyanobacterial phycobilisomes (PBS) (Fig. 1a) are large organised
complexes of water-soluble phycobiliproteins (PBPs), phycoerythrin (PE), phyco-
cyanin (PC), allophycocyanin (APC) and their chromophores [42,43]. Their chro-
mophores (phycocyanobilin and phycoerythrobilin) are synthesised from glutamic
acid, which is converted to aminolevulinic acid (ALA), two molecules of which
form porphobilinogen and ultimately protoporphyrin IX by the action of three
enzymes (Fig. 2a). The enzyme Fe-chelatase catalyses the formation of protoheme
from protoporphyrin IX. Subsequently, this protoheme is converted to biliverdin IX,
from which phycocyanobilin and phycoerythrobilin are produced.
Classes: The 3 major PBPs (PE, PC and APC) [35] have been further classified
into six groups based on their light absorption and fluorescence properties:
phycoerythrocyanin, C-phycoerythrin (C-PE) and R-phycoerythrin (R-PE),
C-phycocyanin (C-PC), allophycocyanin (APC) and allophycocyanin-B (AP-B)
[35] (Table 1).
Sources: Phycobilisomes (PBS) are unique to cyanobacteria and some red
macroalgae [45]. In green microalgae and higher plants they were replaced by
transmembrane chlorophyll a/b binding proteins [46]. In cyanobacteria,
phycobiliproteins make up a large proportion of soluble proteins; e.g. Nostoc com-
mune (54%), Scytonema sp. (37%), Lyngbya sp. (32%) and Anabaena sp. (8%) [47].
Structures & Properties: The PBS consist of water-soluble phycobiliproteins
(PBPs) and hydrophobic linker peptides and are classified into 4 structural types
which are both species and light-dependent: rod-shaped, hemi-ellipsoidal, hemi-
discoidal and bundle-shaped (Fig. 1b). The most common and stable type of PBS
organisation is reported to be the hemi-discoidal form (4.5–15 MDa) [48]. It is
thought to accommodate a maximum of 800 chromophores per PSII dimer [49]. The
bundle-shaped PBS was found in Gloeobacter violaceus and reported to support
among the fastest energy transfer rates [49]. The rod-shaped PBS was found in
Acaryochloris marina and the excitation energy transfer is reported to be unidirec-
tional and faster in PS II (compared to hemi-discoidal form) because of its differen-
tial organisation of APC and PC [50].
PC is ubiquitous in cyanobacteria and present at high intracellular levels. It
consists of two subunits: α-PC (15 kDa) and β-PC (19 kDa). These subunits
coordinate three PCBs via thioether bonds within each αβ PC monomer
[51]. These αβ PC monomers can in turn form PC trimers (αβ)
3
and hexamers
(αβ)
6
. The fluorescence of PC has been attributed to the covalent linkage of
phycocyanobilin to cysteine-84 of α-subunits as well as cysteine-82 and cysteine-
153 residues of β-subunits [51]. These coordinated phycocyanobilins collectively
contribute to the high Stokes shift of PC (i.e. the difference between the band
maxima of the absorption and emission spectra [51]) and its high quantum yield,
with maximum fluorescence emission at ~640 nm, and the molar extinction coeffi-
cient at ε
620
is 1.54 ×10
6
M
-1
cm
-1
for a 242 kDa C-PC hexamer [52].
C. Deepika et al.

Fig. 2 Cyanobacterial pigments –biosynthesis and absorption spectra. (a) Phycobiliprotein and
Chlorophyll biosynthesis. The enzymes Fe-chelatase, Mg-chelatase and Heme oxygenase play
important regulatory roles in chlorophyll and bilin synthesis. The enzymes PebS synthase and
PcyA synthase catalyse key steps in phycoerythrobilin and phycocyanobilin synthesis, respectively,
and are either NAD(P)H- or ferredoxin-dependent bilin reductases. During chlorophyll biosynthe-
sis, Mg-chelatase catalyses the insertion of Mg
2+
into protoporphyrin IX at the branch point
between bilin synthesis and chlorophyll biosynthesis [35]. (b) Carotenoid biosynthetic pathway
via the Methyl-Erythritol 4-Phosphate (MEP) pathway [44]. Phytoene synthase and phytoene
desaturase (red dotted boxes) are both important enzymes in carotenoid biosynthesis. The carotenes
and xanthophyll pathways are highlighted by the orange and yellow boxes, respectively. (c)
Absorption spectra of major cyanobacterial pigments of commercial interest –Chlorophyll (Chlo-
rophyll a), Carotenoids (β-carotene, lutein, fucoxanthin, astaxanthin) and Phycobiliproteins
(phycocyanin)
Sustainable Production of Pigments from Cyanobacteria

APC consists of the two subunits α-APC (15 kDa) and β-APC (17 kDa). They
coordinate 2 PCB per αβ-APC monomer via thioether bonds [42,53]. These αβ PC
monomers usually form trimeric APC ((αβ)
3
). As for PC, the fluorescence of APC
has been attributed to the covalent linkage of phycocyanobilin to cysteine-84 of the
α-subunit as well as to cysteine-84 and cysteine-155 residues of β-subunit. The APC
core (Fig. 1a) is formed by four APC trimers in Synechocystis sp. PCC6803 [54] and
has a maximum fluorescence emission at ~660 nm, and the molar extinction
coefficient at ε
650
is 0.7 ×10
6
M
-1
cm
-1
for the 104 kDa APC trimer [55].
The two subunits of PE named α-PE (20 kDa) and β-PE (22 kDa) are reported to
coordinate from 2–6 chromophores via thioether bonds (i.e. 2–6. PEB, PUB or PVB
or a combination thereof; Fig. 1) per αβ monomer (αβ)
1
[56]. These αβ-PE mono-
mers are generally organised into disc-shaped trimers (αβ)
3
or hexamers (αβ)
6
.Asan
example, PE in Gloeobacter violaceus (PDB: 2VJH) is reported to form hexamers
Table 1 Phycobiliproteins structure (PDB; scale bar 10 nm) and spectral properties (λ
exc
–
excitation wavelength)
PBP pigments Structure Colour
Absorption
maxima (nm)
Fluorescence emission
maxima (nm)
Allophycocyanin
(4RMP)
Bright
blue
652 657
(λ
exc
=633)
C-phycocyanin
(1HA7)
Dark
blue
621 642
(λ
exc
=620)
R-phycocyanin
(1F99)
Blue 533,544 636
(λ
exc
=580)
C-phycoerythrin
(5FVB)
Reddish
pink
565 573
(λ
exc
=560)
R-phycoerythrin
(1B8D)
Red 566 578
(λ
exc
=561)
B-phycoerythrin
(3 V58)
Orange 545 572
(λ
exc
=545)
C. Deepika et al.

coordinating 4 PEB and 1 PUB per αβ monomer. The maximum fluorescence
emission occurs at ~578 nm and the molar extinction coefficient at ε
578
is
2×10
6
M
-1
cm
-1
for a 240 kDa R-PE hexamer [52].
PBPs emit an intense autofluorescence which results from their strong light
absorption and intense fluorescence emission within the visible spectrum when not
coupled into the photosystems [57]. Wynam et al. (1985) [57] reported that a
proportion of the light energy is absorbed by PE in PBS of Synechococcus
sp. DC2 when cultivated under excess nitrate. As a result the cells exhibited high
autofluorescence as the PE granules accumulated (as a form of stored nitrogen) and
were uncoupled from PBS in the photosystems. Efficient excitation energy coupling
among the chromophores in the PBP trimers and hexamers in the PBS contributes to
high autofluorescence.
Biological functions: PE, PC and APC absorb radiation in regions of the visible
spectrum in which Chl has a low absorptivity (Fig. 2, 470–620 nm). Photosynthetic
organisms typically have antenna systems that are tuned to their environmental
conditions to best capture the light energy that they require. For example at the
illuminated surface of a water column (euphotic zone) PAR in the 400–700 nm range
is abundant, while below this (disphotic zone) less red, yellow and green light is
available, resulting in dim blue illumination [58]. Consequently, organisms have
evolved antenna systems best adapted to capture differing wavelengths of light under
a range of light intensities to support optimal light to chemical energy conversion
[35,59]. Phycoerythrin is adapted to capture high energy wavelengths
(λ
max
~ 565 nm), phycocyanin intermediate energy wavelengths (λ
max
~ 620 nm)
and allophycocyanin low energy wavelengths (λ
max
~ 650 nm) [60]. Their major
biological function is to increase the energy absorbed from light and its transfer to
the redox active reaction centres and the special pair chlorophylls (i.e. P
680
in PSII
and P
700
in PSI). In cyanobacteria, they also offer protection against
photodamage [61].
2.2 Chlorophylls
Definition: Chlorophylls are tetrapyrrole based chromophores that are generally
green in colour.
Classes: Chlorophylls are classified as Chl a,b, c
1
,c
2
,c
3
,dand fin the order that
they were discovered [62] (Table 2).
Sources: Chlorophylls are abundant in the photosynthetic machinery of
cyanobacteria, algae and plants where they are coordinated within specific light
harvesting antenna proteins and the redox active reaction centres of PSI and PSII.
In cyanobacteria, green plants and green microalgae, Chl ais the predominant form
of chlorophyll with other chlorophylls usually considered to be accessory chloro-
phylls. Chl bis common in land plants and microalgae while Chl chas been
reported in marine algae including diatoms, brown algae and dinoflagellates [63].
Sustainable Production of Pigments from Cyanobacteria

Table 2 Chlorophyll structure (ChemDraw 20.1.0) and spectral properties. (λ
exc
–excitation wavelength; NA –Not available)
Chlorophyll pigments Chemical structure Chemical formula Colour Absorption maxima (nm) Fluorescence emission maxima (nm)
Chl aC
55
H
72
O
5
N
4
Mg Blue/green 430,664 668
(λ
exc
=430)
Chl bC
55
H
70
MgN
4
O
6
Green/yellow 460,647 652
(λ
exc
=453)
Chl c1 C
35
H
28
MgN
4
O
5
Green/
yellow
442,630 633,694
(λ
exc
=450)
Chl c2 C
35
H
28
MgN
4
O
5
Green/yellow 444,630 635,696
(λ
exc
=453)
Chl c3 C
35
H
28
MgN
4
O
5
Green/yellow 452,627 635,690
(λ
exc
=452)
Chl dC
54
H
70
MgO
6
N
4
Green 401,696 NA
Chl fC
55
H
70
MgO
6
N
4
Green/yellow 700 720
(λ
exc
=425)
C. Deepika et al.

Chl dhas been reported in certain cyanobacteria, for example in the cyanobacte-
rium Acaryochloris marina it makes up 99% of the chlorophyll [64]. Chl fwas
found in extracts from stromatolytes, layered sedimentary formations which are
rich in cyanobacteria [65].
Chlorophyll synthesis (Fig. 2a) involves the reduction of protochlorophyllide.
Two pathways exist for chlorophyll biosynthesis, one taking place in darkness
(using the enzyme dark-operative protochlorophyllide oxidoreductase) and the
other requiring continuous light (light-dependent protochlorophyllide
oxidoreductase).
Structures & Properties: Chlorophylls a, b, c
1
,c
2
,c
3
,dand fconsist of a large
aromatic tetrapyrrole macrocycle with a fifth modified cyclopentane, responsible for
their light absorption and redox chemistry [66,67]. A central Mg ion maximises
excited state lifetime and the interactions of Chls with their proteins, and in many
cases a hydrophobic phytyl tail is present (Chl a, b, d & f) although this tail is absent
in Chl c1, c2 and c3 [68]. Chlorophylls differ in their chemical formulae at their C2,
C3, C7, C8, C17 positions and in their C17-C18 bonds (Table 2). The only
difference between Chl aand Chl bis that at the C-7 position on the pyrrole
ring B, there is a methyl group (–CH
3
) in Chl a, while in Chl bthere is a formyl
group (–CHO) at the same position.In Chl da formyl group (–CHO) replaces the
vinyl group (–CH=CH
2
) at the C-3 position of the pyrrole ring A of Chl a(Table 2).
In Chl fa formyl group (–CHO) instead replaces the methyl group (–CH
3
) at the C-2
position of the pyrrole ring A of Chl a(Table 2).
Although most chlorophylls absorb in the red (660–665 nm) and blue (~430 nm)
regions of the spectrum, these structural differences result in subtle shifts in their
respective absorption and fluorescence spectra. Consequently, chlorophylls differ
somewhat in their colour: Chl ais blue-green (absorbs predominantly violet-blue
and orange-red light), Chl bis yellow-green, Chl c’s are blue-green, Chl d is green
and absorbs in the far-red region of the spectrum (710 nm, outside of the visible
range) as does Chl f(yellow-green). The phytyl chains of Chl a, b, d and fmake these
chlorophylls oil soluble and give them a wax like consistency as solids [69].
Biological functions: Collectively chlorophylls have four major biological func-
tions including light capture, excitation energy transfer, acting as electron donors,
and energy dissipation (Fig. 1a).
Light capture: The first function is to capture light. Different chlorophylls have
different absorption spectra. Consequently, by coordinating different combinations
of chlorophylls within the antenna systems (e.g. Chl aand bin the light harvesting
systems of microalgae and higher plants) photosynthetic organisms can use chloro-
phylls to optimise their absorption spectra to capture the light that they require. The
broader the absorption spectra and the larger the cross-sectional area of a given
antenna, the more light can theoretically be captured [37]. Interestingly in Chl dand f
the typical red peaks of Chl aand bare shifted towards the far red (which enables
capture of the infra-red portion of the spectrum). Consistent with this it was recently
suggested that Chl fmay function solely as an antenna chromophore [70], but in
Acaryochloris marina, Chl dmakes up 99% of the chlorophyll (~80% of total lipid
soluble pigment and >2% cell dry weight) suggesting that it also has a role in
Sustainable Production of Pigments from Cyanobacteria

primary light harvesting in certain organisms [64,71]. Chl d assists in the capture of
far-red light (FRL) and is thus thought to be responsible for remodelling PSI under
FRL-induced photoacclimation (FaRLiP) [64].
Excitation energy transfer: The second function of chlorophylls is to support the
transfer of excitation energy from the antenna to the redox active Chl adimer (P
680
and P
700
) in PSII and PSI reaction centres, respectively. Chlorophylls can support
long-lived excited states, making them powerful photosensitisers that play an impor-
tant role in excitation energy transfer. The safe transduction of this excited state into
chemical energy is the basis of photosynthesis. Typically, the absorption spectra
shift from blue (shorter/higher energy wavelength) towards the red (longer/lower
energy wavelength) towards the reaction centres to facilitate energy transfer.
Electron donor: The third biological function of chlorophylls is to drive P
680
and
P
700
-mediated redox chemistry. Chlorophylls and chlorophyll derivatives
(e.g. pheophytin) can act as primary electron donors and acceptors, transporting
electrons within a few picoseconds across half the thylakoid membrane [72]. Here
again the ability to support long-lived excited states is important.
Energy dissipation: The fourth function of chlorophylls is photoprotection.
Under conditions of excess light, the photosystems and particularly PSII are subject
to photodamage due to the formation of reactive oxygen species. To prevent this,
certain photosynthetic organisms including higher plants and microalgae have
evolved mechanisms to dissipate excess light (up to 85–90%) derived energy from
chlorophyll-containing proteins [73].
2.3 Carotenoids
Definition: Carotenoids are lipophilic tetraterpene derivatives which consist of eight
isoprene molecules and typically contain 40 carbon atoms [74,75].
Classes: Approximately 1,100 carotenoids [76] have been reported and these
have been categorised into carotenes (hydrocarbons) and xanthophylls, which addi-
tionally contain oxygen. The structure and properties of some of the most industri-
ally relevant carotenoids are summarised in Table 3. Of these, the carotenes include
α-carotene, β-carotene, γ-carotene and lycopene. The xanthophylls include lutein,
zeaxanthin, neoxanthin, violaxanthin, canthaxanthin, fucoxanthin, antheraxanthin,
myxoxanthophyll, β-cryptoxanthin and echinenone.
Sources: Carotenoids are produced by bacteria, fungi, cyanobacteria, algae,
plants and animals, where they fulfil a plethora of different roles, but they are
most abundant in photosynthetic organisms. Of these 1,100 carotenoids about
30 are reported to have a function in photosynthesis [77]. Consequently, in photo-
synthetic organisms, these hydrophobic molecules are often enriched in the thyla-
koid membrane [74]. In higher plants certain xanthophylls (i.e. zeaxanthin,
antheraxanthin and violaxanthin) that are involved in the photoprotective xantho-
phyll cycle and so are located in the light harvesting complexes in the thylakoid
membranes. In cyanobacteria, xanthophylls have been reported to be located in the
C. Deepika et al.

hydrophobic part of the cytoplasmic membranes [78] but they may also be present in
the thylakoids [79].
The carotenoids are typically synthesised from isopentenyl pyrophosphate (IPP)
via the methylerythritol-4-phosphate (MEP) pathway in cyanobacteria and in chlo-
roplasts of microalgae and higher plants (Fig. 2a) and via the mevalonic acid (MVA)
Table 3 Major carotenoid structures (ChemDraw 20.1.0) and spectral properties
Carotenoid
pigments Chemical structure
Chemical
formula Colour
Absorption
maxima (nm)
Carotenes
α-Carotene C
40
H
56
Light-
yellow
378, 400 and
425
β-Carotene C
40
H
56
Orange 425, 450 and
480
γ-Carotene C
40
H
56
Yellowish-
orange
437, 462 and
492
Lycopene C
40
H
56
Red 443, 471 and
502
Xanthophylls
Astaxanthin C
40
H
52
O
24
Red 482
Lutein C
40
H
56
O
2
Yellowish-
red
425, 448 and
476
Zeaxanthin C
40
H
56
O
2
Yellow 428, 454 and
481
Neoxanthin C
40
H
56
O
5
Yellow 486,495
Violaxanthin C
40
H
56
O
5
Orange 417, 440 and
470
Canthaxanthin C
40
H
56
O
2
Yellowish-
orange
450, 475 and
506
Fucoxanthin C
40
H
56
O
6
Orange 423 and 445
Myxoxanthophyll C
46
H
66
O
8
Bright red 450, 475 and
506
β-Cryptoxanthin C
40
H
56
O Yellowish-
orange
425, 449 and
476
Echinenone C
40
H
54
O Brownish-
red
452
Sustainable Production of Pigments from Cyanobacteria

pathway in the cytosol of bacteria and fungi [77]. Two important enzymes which
regulate the first committed steps towards carotene biosynthesis are phytoene
synthase and phytoene desaturase. Silencing the genes encoding these enzymes is
reported to completely eliminate carotenoid production [80,81].
Structures & Properties: Carotenoids are unsaturated hydrocarbons with
extended conjugated double bond networks that are an essential component of
their light absorbing (chromophore) [82] and antioxidant properties [77]. Caroten-
oids generally absorb light in the violet to green (400–550 nm) region of the
spectrum and so tend to be yellow, orange and red in colour [83]. Carotenoids
which capture light from shorter wavelengths (e.g. 400 nm) are redder. Their
individual colours depend on the length of the polyene component (3–13 conjugate
double bond systems) which influences the delocalisation of electrons along the
entire length of the polyene chain [72,77]. The longer the conjugated bond system,
the more delocalised the electrons within and the lower the energy required to
change state. The range of the light energy captured reduces as the length of the
conjugated bond system increases [72,77]. Xanthophylls, which additionally con-
tain oxygen, may possess hydroxyl groups (e.g. hydroxycarotenoids such as zea-
xanthin and lutein), keto groups (canthaxanthin and echinenone) and epoxy groups
(violaxanthin and diadinoxanthin) [77]. The structures of some xanthophylls are
even more complex, combining several functional groups, for example astaxanthin
(keto-hydroxy groups), dinoxanthin and fucoxanthin (epoxy-acetylated groups and
allene linkages) and monadoxanthin (acetylene linkages) [21].
Biological functions: Carotenoids are indispensable components of chlorophyll/
carotenoid binding photosystems (Fig. 2a) of photoautotrophs (e.g. cyanobacteria,
eukaryotic algae and plants) but also have other roles including the protection of
membranes from oxidation [79,84]. In photosynthesis carotenoids have three key
roles: Structural stabilisation of the photosystems [85], regulation of light capture
[86] and supporting energy dissipation and photoprotection, for example through the
process of Non-Photochemical Quenching (NPQ) which dissipates excess energy as
heat [86].
Structural stabilisation:β-carotene is the only carotenoid reported in the atomic
resolution structure of the cyanobacterial PSII complex [84]. For example,
Synechococcus sp. PCC7335 was reported to have 11–12 β-carotene molecules
[87,88] in PSI (19 β-carotene molecules per monomer of the PSI trimer) when
cultivated under far-red light [89]. Carotenoids are reported to assist in maintaining
the stability of the PSII structure [90]. For example, the Synechocystis sp. PCC 6803,
the △crtB mutant (deletion of the crtB gene coding for phytoene synthase) exhibited
limited carotenoid biosynthesis and the absence of xanthophylls. Yet although
cyanobacterial phycobilisomes, PSII and PSI reportedly lack xanthophyll, these
mutants produced intact phycobilisomes while displaying reduced PSI and PSII
oligomerisation. Interestingly, xanthophylls reportedly rigidify the fluid phase of
the membranes and limit oxygen penetration to the hydrophobic membrane core
(susceptible to oxidative degradation) [78]. This is due to the presence of lipid acyl
chains in xanthophyll molecules that are responsible for van-der-Waals interactions
[78]. In thylakoids, therefore, this may be important for the correct assembly of PSI,
C. Deepika et al.

PSII and their antenna systems [79]. It may also be important for the protection of
other membranes against oxidative damage.
Light capture: Carotenoids can capture violet-green light. Excited β-carotene
molecules that are excitonically coupled to chlorophylls within a light harvesting
antenna system can transfer the derived excitation energy to a neighbouring chloro-
phyll molecule (usually Chl a), thereby broadening the absorption spectrum or
antenna size of the photosystem [75]. Carotenoids can account for ~20–30% of all
light harvested [4,91].
Energy dissipation and photoprotection: In cyanobacteria, the water-soluble
Orange Carotenoid Proteins (OCP) which bind a single carotenoid (3′-hydroxy-
echinenone; chromophore) can act as photosensors that can trigger light-activation
[92,93] and quenching of excess light energy in the PBS through the release of
excess heat. This can prevent oxidative damage to proteins, DNA and lipids
[94]. Absorption of blue-green light induces structural changes in both the protein
and carotenoid, which triggers NPQ induction, although the NPQ mechanism is still
under active investigation [93]. Under low light or in darkness, OCP converts back
to the inactive state. This process has been shown to be mediated by another protein
called the Fluorescence Recovery Protein (FRP) that interacts with the active form of
OCP and accelerates the reconversion of active OCP to the inactive form [95]. Carot-
enoids also serve as sacrificial molecules to neutralise reactive species (e.g. oxygen
free radicals) [4,96,97]. Here, β-carotene helps to quench excess light in the
chlorophyll triplet state by releasing it as heat [77]. It is the only carotenoid bound
to the core reaction centre complex of photosystem II and offers protection against
UV radiation [4,98]. Zeaxanthin and echinenone are reported to protect the repair
stage of the PSII recovery cycle from photoinhibition in cyanobacteria by decreasing
the level of singlet oxygen that inhibits protein synthesis [99].
2.4 Scytonemin
Definition: Scytonemin is an aromatic indole alkaloid (Table 4).
Sources: Scytonemin has been reported to accumulate in the extracellular matrix
of a broad range of cyanobacteria [100] including species of the genera Scytonema,
Aulosira (A. fertilissima), Nostoc (N. linckia,N. spongiaeforme, N. punctiforme),
Schizothrix (S. coriacea), Lyngbya (L. majuscule, L. aestuarii), Leptolyngbya
(L. boryana), Laspinema (L. thermale) and Chlorogloeopsis (C. fritschii). It has
been reported that an 18-gene cluster responsible for scytonemin synthesis in
N. punctiforme is upregulated upon exposure to UV-A radiation and
co-transcribed as a single operon [101].
Structures & Properties: Scytonemin is a secondary metabolite that absorbs
UV-C (100–280 nm), UV-B (280–315 nm) and UV-A (315–400 nm) radiation but
has a low absorbance in the PAR (400–700 nm) range. It is generally insoluble in
water and moderately soluble in organic solvents. Derivatives of scytonemin include
scytonine, dimethoxy-scytonemin, tetramethoxy-scytonemin and scytonemin-imine
(Table 4)[101,102].
Sustainable Production of Pigments from Cyanobacteria

Biological functions: The location of scytonemin in the extracellular matrix and
its UV absorbing and PAR light transmitting properties likely provide cyanobacterial
cells with UV protection while allowing PAR light (400–700 nm) into the cell to
drive photosynthesis. The energy captured in the UV range is thought to be released
as heat [103]. Scytonemin synthesis is induced by high irradiance and most effec-
tively by UV-A and UV-B radiation (~85%) [104]. Cells surrounded by a
scytonemin containing sheath [105] exhibited resistance to UV-A induced
photobleaching of Chl a.InChlorogloeopsis sp., photosynthesis was inhibited and
growth delayed until substantial amounts of scytonemin had been deposited in the
sheaths [105].
Table 4 Scytonemin-derivatives structure (ChemDraw 20.1.0) and spectral properties
Scytonemin
derivatives Chemical structure
Chemical
formula Colour
Absorption
maxima (nm)
Scytonemin C
36
H
20
N
2
O
4
Yellowish
brown
252,278,300,386
Reduced
scytonemin
C
36
H
24
N
2
O
4
Bright red 246,276,314,378
Dimethoxy
scytonemin
C
38
H
28
N
2
O
6
Red 215,316,422
Tetramethoxy
scytonemin
C
40
H
36
N
2
O
8
Purple 212,562
Scytonine C
31
H
22
N
2
O
6
Reddish
pink
207,224,270
Scytonemin-3a-
imine
C
38
H
25
N
3
O
4
Reddish
brown
237, 366, 437,
564
C. Deepika et al.

3 Applications
This diverse array of pigments derived from cyanobacteria, i.e. phycobiliproteins
(blue and red, Table 1), chlorophylls (green, Table 2), carotenoids (red, orange and
yellow, Table 3) and scytonemin (Table 4), can be translated into advanced technical
and commercial products [9,10]. Indeed, cyanobacterial pigments already have a
wide range of industrial applications (Fig. 3) especially in the food, cosmetics,
nutraceutical and pharmaceutical sectors [17,106]. Besides their use as colourants
and dyes, they are used as food additives, nutraceuticals, putative pharmaceuticals,
cosmetics, molecular assays, aquaculture feeds and textiles. One of the first potential
Fig. 3 Applications of cyanobacterial pigments. Cyanobacterial pigments have been reportedly
used as fluorescence probes (Single-Cell Imaging –e.g. Supernova 428 dye), food colourants, food
additives, nutraceuticals, putative pharmaceuticals, cosmetics, molecular assays, aquaculture feed
and textiles
Sustainable Production of Pigments from Cyanobacteria

industrial uses for chlorophyll was during experiments in early colour photography
by Becquerel (1874) [107] by employing chlorophyll as a photosensitiser of collo-
dion (a flammable, viscous solution of nitrocellulose in ether and alcohol) and silver
bromide. Chlorophylls were also used in surgical dressings and as chelators (carriers
of micronutrients like cobalt, zinc, manganese, iron and molybdenum) in hydropon-
ics [11,16,21].
3.1 Food and Nutraceuticals
Commercially, phycobiliproteins (PBP) are broadly classified into two categories –
phycocyanin and phycoerythrin, based on their colour. Phycocyanin has a bright
blue colour and is considered versatile, although it is heat and light sensitive.
Phycoerythrin is a bright red water-soluble pigment used as a natural food colourant.
Both are non-toxic and have been reported to provide antioxidant [108], anti-cancer
[109], anti-inflammatory [110], anti-obesity [111], anti-angiogenic [112],
neuroprotective [113] and anti-ageing properties [51,114], though in many cases
this may require further study to verify these claims. Phycocyanin is widely used as a
natural colourant in ice cream, soft drinks, candies, chewing gum, desserts, cake
decorations, icings and frostings, milk shakes as well as lipsticks and eyeliners
[51]. Although PBP-rich Spirulina extracts are FDA approved (2013) food
colourants and additives, they are susceptible to heavy metal contamination and
therefore, human use is tightly regulated [115]. Stable isotope labelled metabolites
with phycoerythrin have gained attention as fluorescent probes for cytometry and
immunodiagnostics [116,117].
Cyanobacteria can be produced to contain high levels of carotenoids [118]. The
global carotenoid market in 2016 was valued at approximately USD 1.24 billion and
forecast to increase to USD 1.74 billion by 2025 at a 4.3% CAGR [119]. The market
share of the major carotenoids in this sector, anticipated in 2021 is in the order of
β-carotene (26%), astaxanthin (25%), lutein (18%), fucoxanthin (15%), canthaxan-
thin (10%) and lycopene (6%) [120]. The global chlorophyll market was valued to be
USD 279.5 million in 2018 and is anticipated to reach USD 463.7 million by 2025
with a 7.5% CAGR from 2018 to 2025 [121]. In Europe, both carotenoids (yellow,
orange and red colour) and chlorophyllins (90% of green colour in food) are widely
used as food-colouring agents (approved as Group II food additives; authorised by
the European Commission).
Carotenoids play an important role in the global food industry as food additives.
Of the many known carotenoids, only ~40 are produced commercially. These
include β-carotene and astaxanthin, and, to a lesser extent, lutein, zeaxanthin and
lycopene. The major carotenoids produced commercially today are β-carotene and
astaxanthin, which are currently produced from the commercial strains Dunaliella
salina (14% β-carotene of dry weight) [122]andHaematococcus pluvialis (3%
astaxanthin of dry weight), respectively [123]. The largest astaxanthin consumer is
the salmon feed industry (FDA approved in 1987) [124]. Astaxanthin is widely used
C. Deepika et al.

in aquaculture feeds [106] as a colourant for fish and shrimp; the reddish pink
pigmentation of salmon is considered an important consumer criterion of quality
[125]. The annual aquaculture market of this pigment is estimated at USD 200 mil-
lion, with an average price of USD 2,500 kg
-1
[123]. Astaxanthin is also known as
‘super vitamin E’as it exhibits the highest antioxidant property (500×more potent
than α-tocopherol). Natural carotenoids from cyanobacteria have potential to replace
commonly used synthetic colourants such as Erythrosine (pinkish red; E127), Sunset
Yellow FCF (yellowish orange; E110), Tartrazine (lemon yellow; E102) and Allura
red (red; E129). β-Carotene is used as a food-colouring agent with the E number
E160. Lutein (bright yellow) cannot be synthesised by humans and has a protective
role against macular degeneration of the eye. It is therefore an important dietary
supplement (E161b in the European Union) [126,127]. Hammond et al. (2014)
studied the effect of daily uptake of lutein (10 mg) and zeaxanthin (2 mg) supple-
ment in 100 healthy adults over a period of 1 year and regularly recorded their
contrast sensitivity and glare tolerance. The study concluded good improvement in
both the parameters and thus suggested lutein and zeaxanthin good for ocular health.
Carotenoids are also used in nutraceuticals (e.g. astaxanthin approved by FDA as a
human nutraceutical ingredient in 2004 [128]). Carotenoids extracted from Spirulina
sp. are used to treat vitamin A deficiency, β-carotene and cryptoxanthin being
precursors of vitamin A [30,129].
3.2 Cosmetics
The global pigment-based cosmetic market was valued at USD $10 billion in 2020
and is anticipated to increase to USD $17 billion by 2028 at a ~7% CAGR [130]. The
demand for natural pigments in the cosmetic industry has significant traction due to
the increasing safety concerns associated with synthetic sunscreen compounds that
exhibit cytotoxicity [20,131]. The interest in cyanobacterial pigments in cosmetics
(e.g. sunscreens, creams, lotions) is mainly due to their reported photoprotective
property (see biological functions in Sect. 2.4) that prevents skin cancer and sup-
presses ageing-related skin issues (demonstrated through increased cell viability in
keratinocyte cell line HaCat, fibroblast cell line 3T3L1 and endothelial cell line
hCMEC/D3 exposed to 10 μgmL
-1
aqueous cyanobacterial extract containing high
levels of phycocyanin) [132]. Scytonemin is a yellow to brown lipophilic pigment
that is exclusively found in cyanobacteria and is employed in sunscreens due to their
promising effect on protection from UV radiation [104,105]. Scytonemin is
extracted from the cell wall of cyanobacteria cultivated under harsh conditions
(e.g. exposure to high solar radiation; desiccation). The UV radiation trigger for
natural scytonemin production prevented ~92% of radiation from entering the cell,
making it a promising ingredient for cosmetics [110,133]. Further, the
cyanobacterial carotenoids, including β-carotene, fucoxanthin, zeaxanthin, lutein,
echinenone, astaxanthin and canthaxanthin also exhibit strong antioxidative proper-
ties which help in the reduction of UV-induced oxidative damage [123,134]. Darvin
Sustainable Production of Pigments from Cyanobacteria

et al. [135] performed in-vivo carotenoid assays on human skin from healthy normal
skin volunteers (20–70 years old) at multiple points over a year and also studied
differences in absorption capacity based on the application. They concluded that
carotenoids are crucial components of the antioxidative protective system of the
human skin and ideally supplied as a topical application. Scarmo et al. [136]
demonstrated the effect of carotenoids on skin health by performing dermal biopsies
and analysing blood samples to generate a correlation of individual and total
carotenoid content in human skin. Carotenoids absorbed in the gut are transported
to the epidermis and the two abundant carotenoids found in skin were beta-carotene
and lycopene which suggested their role in photoprotection. Lutein and zeaxanthin
are marketed as nutraceutical tablets to be ingested and then deposited in lipophilic
tissues in humans. Phycobiliproteins have an already established market in the
cosmetic sector and are mainly derived from Arthrospira platensis (commonly
known as Spirulina platensis)[51,137]. Similarly, phycocyanin and phycoerythrin
are widely incorporated into hair conditioners, anti-ageing, skin-whitening and anti-
wrinkle skin creams and moisturisers, colourant in eye shadow, eye liners, soaps,
nail polish and lipsticks [138]. Given the potential of scytonemin in UV screening
and free radical scavenging, together with its non-toxic properties [139], this highly
stable pigment [133] offers biotechnological opportunities for exploitation by the
cosmetics industry [104]. Examples of companies that use cyanobacterial pigments
in their cosmetic products today include Lush Cosmetics Pty. Ltd., L’Oreal Pty. Ltd.
and Aubrey Organics Inc.
3.3 Pharmaceuticals and Diagnostics
PC is commonly used in immunoassays such as flow cytometry and high-throughput
screening [35,51,59]. PE is considered one of the world’s brightest fluorophores
and is widely employed in Time Resolved Laser Induced Fluorescence (TR-LIF),
flow cytometry and immunofluorescent staining [140]. Similarly, fluorescent
phycobiliproteins are used in fluorescent microscopy, flow cytometry,
fluorescence-activated cell sorting, diagnostics, immunolabelling, Fluorescence Res-
onance Energy Transfer (FRET) assays and immunohistochemistry [59,60,
137]. Phycobiliproteins are also reported to possess therapeutic properties such as
anti-inflammatory and anti-tumour activities [138,141]. Czerwonka et al. 2018
[142] demonstrated anti-tumour activity of phycocyanin extracts from Spirulina
sp. Using A549 lung adenocarcinoma cells, and recording cell viability, proliferation
and morphology, the cell viability and proliferation of A549 tumour cells were found
to be significantly reduced (cell cycle inhibited in G1 phase). The tumour cells were
also much more sensitive to PC than the normal skin fibroblasts. Lopes et al. [118]
reported the effective treatment of psoriasis using carotenoid extracts from five
different cyanobacterial strains from the genera Alkalinema,Cyanobium,
Nodosilinea,Cuspidothrix and Leptolyngbya. HPLC analysis of acetone carotenoid
extracts showed high levels of β-carotene, zeaxanthin, echinenone and lutein.
C. Deepika et al.

Lutein also has applications in maintaining ocular health, reportedly acting as a
photoprotective agent for macular cells [126]. Reynoso-Camacho et al. [15] dem-
onstrated the efficacy of lutein to treat colon cancer in rat models, by investigating
the protein expression levels of K-ras (coded by Kirsten rat sarcoma virus gene,
responsible for delivering signals to the cell’s nucleus), PKB (Protein Kinase-B,
regulates cell survival and apoptosis), and β-catenin (regulates cell–cell adhesion and
signal transduction) in rats. Lutein treatment reduced these levels by 25%, 32% and
28% in the prevention phase and by 39%, 26% and 26% in the treatment phase. In
another study, FloraGLO
®
Lutein was found to increase the sensitivity/response of
transformed and tumour cells to chemotherapy agents, inducing apoptosis in MCF-7
tumour cells [143]. Scytonemin has antioxidant activity and functions as a radical
scavenger to prevent cellular damage resulting from reactive oxygen species pro-
duced upon UV radiation exposure and thus has potential applications in biomedical
products [104]. Scytonemin is reported to repress proliferation of T-cell leukaemia
Jurkat cells (IC
50
=7.8 μM) in humans [61] and to act as an inhibitor of human polo-
like kinase 1 (PLK1), the enzyme involved in regulating the G2/M transition in the
cell cycle. Zhang et al. (2013) [144] demonstrated the antiproliferative activity of
scytonemin (3–4μmol/l) against multiple myeloma (anti-tumour activity) targeting
PLK1 on three different myeloma cell lines (U266, RPMI8226 and NCI-H929). The
study concluded that scytonemin significantly decreased cell proliferation. Thus
scytonemin could be used as a therapeutic agent for the management of chronic
disorders involving inflammation and proliferation (such as Alzheimer’s, arthritis
and cystic fibrosis) [145]. Consequently, cyanobacterial pigments offer a broad array
of opportunities for further evaluation and industrial scale-up to supply existing
markets and realise new opportunities.
4 Pigment Production in Cyanobacteria
Cyanobacteria can be used as renewable microbial cell factories [146]. Their opti-
misation for pigment production requires augmentation of both biomass productivity
and pigment yield [11,17,147]. The interdependence of these two variables depends
on pigment type, and whether the pigments are primary or secondary metabolites.
Understanding pigment synthesis pathways and the growth characteristics of pro-
duction strains are therefore both important.
Cyanobacterial biomass and pigment yields rely on strain-specific characteristics
and their alignment with cultivation parameters, such as light intensity and spectral
quality [34], the availability of macro and micronutrients [148–150], CO
2
supply
[150,151], temperature [152,153] and mixing rates [151,154].
Sustainable Production of Pigments from Cyanobacteria

4.1 Cultivation Parameters and Their Impact on Biomass
and Pigment Yields
4.1.1 Carbon and Energy Supply
The industrial production modes for microbes differ in their supply strategy for
carbon (e.g. hetero- and mixotrophic) and energy (e.g. photo-, chemotrophic).
Chemo-heterotrophic organisms have a metabolic strategy that derives both energy
and carbon from organic compounds (chemosynthesis) to enable growth. Thus, the
production processes applying chemo-heterotrophs are essentially depending on the
organic carbon source, typically sugars, which can add cost (both media costs and
the cost of maintaining sterile cultures) and limit viable options for specific-
applications. That said photo-autotrophic cultures have added costs due to the
need for light and CO
2
delivery. Economic and environmental feasibility is thus
product-, process and location-specific and can be assessed using techno-economic
and life-cycle analysis tools [172].
However, many cyanobacteria are neither completely photo-autotrophic nor
completely chemo-heterotrophic; they can perform both photosynthesis and chemo-
synthesis in a mixed mode of growth called mixotrophy, which has advantages for
commercial production. Photo-heterotrophic growth is a specific type of
mixotrophy, where light is an essential energy source for the cells but can be
supplemented with energy derived from the metabolisation of organic carbon com-
pounds, e.g. when growing under light limiting conditions. Under facultative
mixotrophic growth light is not essential anymore and the organisms can be grown
either heterotrophically or autotrophically, and modes can be changed throughout
the production process [173]. Under obligate mixotrophic growth, the organism
utilises both, organic and inorganic carbon (CO
2
), simultaneously to support growth
and maintenance.
Several studies found that mixotrophic and particularly photo-heterotrophic cul-
tivation modes resulted in higher biomass yields compared to chemo-heterotrophic
cultivation [174–178] (Table 5). Schwarz et al. (2020) [179] studied the influence of
different growth modes (using different carbon sources; mixotrophic and heterotro-
phic) on two xenic cyanobacterial strains –Trichocoleus sociatus and Nostoc
muscorum. Mixotrophic cultivation at a light intensity of 100 μmol photons m
-
2
s
-1
led to the highest biomass concentrations. Glucose was identified as the best
organic carbon source for N. muscorum (2.46 g L
-1
) while raffinose was best for
T. sociatus (3.77 g L
-1
)[179]. The uptake of complex sugars such as raffinose in
cyanobacteria is believed to be mediated through sugar transporters such as the GlcP
transporter (fructose/glucose transport system) which was identified in the model
organism Synechocystis sp. PCC6803 [180] and the ABC fructose transporter which
was identified in Nostoc punctiforme [181]. Synechococcus elongatus PCC7942 was
identified to have three different sugar transporters, including galP (glucose), cscB
C. Deepika et al.

Table 5 Reported biomass and pigment yields achieved in cyanobacteria
Cyanobacteria
strain
Biomass
productivity
(g L
-1
day
-1
)
Pigment
productivity
(mg L
-1
day
-1
)
Reactor type/
scale
Growth condition if different from
BG11 (photo-autotroph)
Illumination intensity
(μmol m
-2
s
-1
) Reference
Phycocyanin (PC)
Spirulina
platensis M2
0.18 15.00 300 L Raceway
Pond
Zarrouk Sunlight (Italy) [155]
Spirulina
platensis
0.32 24.00 282 L Raceway
Pond
Zarrouk Sunlight (Italy) [155]
Spirulina
platensis
0.05 3.00 135000 L Race-
way Pond
Zarrouk Sunlight (Spain) [26]
Anabaena
sp. ATCC 33047
0.24 13.00 Raceway Pond Custom 200 [156]
Spirulina
platensis M2
1.32 92.00 11 L Tubular
PBR
Zarrouk Sunlight (Italy) [157]
Spirulina
platensis
0.12 14.00 500 mL flask Zarrouk 140 [158]
Spirulina
platensis
0.06 10.00 Open tank Zarrouk 30 [159]
Synechocystis
sp.
–12.00 100 mL flask –75 (16 h light) [160]
Spirulina
platensis
0.33 50.00 Tubular PBR Zarrouk 200 (14 h light) [161]
Spirulina
platensis TISTR
8172
0.03 1.3
a
9 L tank Zarrouk Sunlight; white filter [162]
Spirulina
platensis TISTR
8172
0.038 4.3
a
9 L tank Zarrouk +16.8 g L
-1
NaHCO
3
Sunlight; white filter
(continued)
Sustainable Production of Pigments from Cyanobacteria

Table 5 (continued)
Cyanobacteria
strain
Biomass
productivity
(g L
-1
day
-1
)
Pigment
productivity
(mg L
-1
day
-1
)
Reactor type/
scale
Growth condition if different from
BG11 (photo-autotroph)
Illumination intensity
(μmol m
-2
s
-1
) Reference
Spirulina
platensis TISTR
8172
0.034 6.4
a
9 L tank Sunlight; yellow filter
Spirulina
platensis
0.74 13.00 1 L Flat panel
PBR
Spirul 50 [163]
Spirulina
platensis
WH879
0.436 94.00 1 L Flat panel
PBR
Zarrouk 450 [164]
Anabaena
oryzae SOS13
N/A 0.12 250 mL flask BG11-N
0
30 [165]
Nostoc
sp. LAUN0015
0.057 0.01 500 mL flask –156 (12 h light) [166]
Nostoc
sp. UAM206
0.064 0.01
Anabaena sp. 1 0.141 0.08
Anabaena sp. 2 0.115 0.06
Nostoc sp. NK 0.32 57.00 1 L Column
PBR
BG11-N
0
100, Red light [167]
Spirulina sp. S1 0.108 0.07 300 mL flask –100 [168]
Spirulina sp. S2 0.057 0.03 BG11 + 0.3% glucose
Anabaena
sp. C2
0.068 0.02 BG11 + 0.15% glycerol
Anabaena
sp. C5
0.059 0.09 BG11 + 0.3% glucose
Nostoc sp. 2S7B 0.071 0.01 BG11 + 0.3% glycerol
Nostoc sp. 2S9B 0.024 0.03 BG11 + 0.3% glycerol
C. Deepika et al.

Synechocystis
sp. PCC 7338
0.07 0.0006 250 mL flask ASN-III + 1.2 M NaCl 30 [169]
Nostoc sp. NK 0.32 0.057 1 L column PBR BG11-N
0
100 [167]
Synechocystis
salina LEGE
06,155
N/A 7
a
5Lflask Z8 + 25 g L
-1
NaCl 100 (16 h light) [170]
Phycoerythrin (PE)
Anabaena
oryzae SOS15
N/A 0.49 250 mL flask BG11-N
0
30 [165]
Nostoc
sp. LAUN0015
0.057 0.0005 500 mL flask –156 (12 h light) [166]
Nostoc
sp. UAM206
0.064 0.0003
Anabaena sp. 1 0.141 0.10
Anabaena sp. 2 0.115 0.08
Synechocystis
sp. PCC 7338
0.07 0.10 250 mL flask ASN-III + 1.2 M NaCl 30 [169]
Synechocystis
salina LEGE
06,155
N/A 4.3
a
5Lflask Z8 + 25 g L
-1
NaCl 100 (16 h light) [170]
Allophycocyanin (APC)
Anabaena
oryzae SOS14
N/A 0.28 250 mL flask BG11-N
0
30 [165]
Spirulina sp. S1 0.108 0.01 300 mL flask –100 [168]
Spirulina sp. S2 0.057 0.0004 BG11 + 0.3% glucose
Anabaena
sp. C2
0.068 0.0009 BG11 + 0.15% glycerol
Anabaena
sp. C5
0.059 0.05 BG11 + 0.3% glucose
Nostoc sp. 2S7B 0.071 0.03 BG11 + 0.3% glycerol
(continued)
Sustainable Production of Pigments from Cyanobacteria

Table 5 (continued)
Cyanobacteria
strain
Biomass
productivity
(g L
-1
day
-1
)
Pigment
productivity
(mg L
-1
day
-1
)
Reactor type/
scale
Growth condition if different from
BG11 (photo-autotroph)
Illumination intensity
(μmol m
-2
s
-1
) Reference
Nostoc sp. 2S9B 0.024 0.02 BG11 + 0.3% glycerol
Synechocystis
sp. PCC 7338
0.07 0.30 250 mL flask ASN-III + 1.2 M NaCl 30 [169]
Synechocystis
salina LEGE
06,155
N/A 8.7
a
5Lflask Z8 + 25 g L
-1
NaCl 100 (16 h light) [170]
β-Carotene
Synechococcus
elongatus PCC
7942
0.13 0.70 250 mL flask –120 [171]
Synechococcus
elongatus R48
0.12 0.60 –
Synechococcus
elongatus RG48
0.91 0.50 –
Synechocystis
salina LEGE
06,155
N/A 0.11
a
5Lflask Z8 + 25 g L
-1
NaCl 100 (16 h light) [170]
Zeaxanthin
Synechococcus
elongatus PCC
7942
0.13 0.50 250 mL flask –120 [171]
Synechococcus
elongatus R48
0.12 0.80 250 mL flask –
Synechococcus
elongatus RG48
0.91 1.10 250 mL flask –
C. Deepika et al.

Synechocystis
salina LEGE
06,155
N/A 0.05
a
5Lflask Z8 + 25 g L
-1
NaCl 100 (16 h light) [170]
Lutein
Synechocystis
salina LEGE
06,155
N/A 0.14
a
5Lflask Z8 + 25 g L
-1
NaCl 100 (16 h light) [170]
Echinenone
Synechocystis
salina LEGE
06,155
N/A 0.48
a
5Lflask Z8 + 25 g L
-1
NaCl 100 (16 h light) [170]
Chlorophyll a
Spirulina
platensis TISTR
8172
0.031 0.41
a
9 L tank Zarrouk Sunlight; white filter [162]
0.017 0.44
a
Sunlight; blue filter
0.027 0.48
a
Sunlight; red filter
0.038 0.47
a
Zarrouk +16.8 g L
-1
NaHCO
3
Sunlight; white filter
0.024 0.49
a
Sunlight; blue filter
0.031 0.52
a
Sunlight; red filter
Spirulina
platensis
0.12 0.24 500 mL flask Zarrouk 140 [158]
Synechocystis
sp. PCC 7338
0.07 0.30 250 mL flask ASN-III + 1.2 M NaCl 30 [169]
a
Denotes pigment yields in mg g
DW-1
day
-1
Sustainable Production of Pigments from Cyanobacteria

(sucrose) and xylEAB (xylose) [182]. The variability in the carbohydrate uptake
rates between strains were attributed to their metabolic activity and the varying
membrane permeability to different organic substrates [183]. The mixotrophic
cultivation of Spirulina platensis using glucose as a carbon source under continuous
light yielded the highest biomass (2x that obtained in phototrophic and heterotrophic
cultures). This led to the suggestion that photo-driven and oxidative glucose