Accepted Manuscript

Title: Phycobiliprotein: potential microalgae derived

pharmaceutical and biological reagent

Author: Emmanuel Manirafasha Theoneste Ndikubwimana

Xianhai Zeng Yinghua Lu Keju Jing

PII: S1369-703X(16)30026-2
DOI: http://dx.doi.org/doi:10.1016/j.bej.2016.01.025
Reference: BEJ 6397

To appear in: Biochemical Engineering Journal

Received date: 13-10-2015

Revised date: 12-1-2016
Accepted date: 29-1-2016

Please cite this article as: Emmanuel Manirafasha, Theoneste Ndikubwimana,

Xianhai Zeng, Yinghua Lu, Keju Jing, Phycobiliprotein: potential microalgae
derived pharmaceutical and biological reagent, Biochemical Engineering Journal
http://dx.doi.org/10.1016/j.bej.2016.01.025

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Phycobiliprotein: potential microalgae derived pharmaceutical and biological reagent

Emmanuel Manirafasha1, Theoneste Ndikubwimana1, Xianhai Zeng2,3, Yinghua Lu1,3, Keju Jing1,3*

jkj@xmu.edu.cn
1
Department of Chemical and Biochemical Engineering, College of Chemistry and Chemical

Engineering, Xiamen University, Xiamen 361005, China

2
College of Energy, Xiamen University, Xiamen 361005, China
3
The Key Lab for Synthetic Biotechnology of Xiamen City, Xiamen University, Xiamen 361005,

China
*
Corresponding author. Tel.: +86 592 2186038.

1
Highlights

 Phycobiliproteins have high potential pharmaceutical and biological properties.

 Classification, structure, and stability of phycobiliproteins from microalgae.

 Mechanism governing phycobiliproteins biosynthesis in microalgae.

 Effect of strains, bioreactor design, cultivation on phycobiliproteins production.

 Biomass harvesting, and phycobiliproteins extraction, purification and character.

2
Abstract

Microalgae are considered as a great potential for reliable and sustainable feedstock for the

production of biofuels and biochemically active compounds such as phycobiliproteins.

Phycobiliproteins are a group of colored proteins present commonly not only in cyanobacteria

(blue-green algae) but also in red algae, cryptomonads, etc. They are extensively commercially used

in foods, cosmetics, biotechnology, pharmacology and medicine. Phycobiliproteins have high

potential pharmaceutical and biological properties but their application is confronted with two major

obstacles: (i) the upstream and downstream processes still have some hindrances such as selection of

suitable microalgae strains, bioreactor design, culture conditions, etc.; (ii) the purification of

phycobiliproteins from microalgae is still low. Biosynthesis of higher purity phycobiliproteins from

microalgae is a multiple concomitant steps for which the main criteria and factors were discussed in

this paper. This review highlights an overview on the phycobiliproteins application fields such as

pharmaceutical, medicine, biological and diagnostic reagents, etc., and meanwhile provides an

insight for further research in development, production, and application of phycobiliproteins.

Keywords: Microalgae; Biosynthesis; Downstream Processing; Phycobiliprotein; Purification;

Biological reagent.

3
1. Introduction

Global growth in population has increased the high demand on energy, food, drugs and other

resources. Microalgae are one of the most underutilized resources among water environment

organisms that are highly attracted the attention worldwide [1]. Several microalgae species have been

grown commercially to produce various metabolites including proteins, polysaccharides, lipids,

pigments, minerals, etc. with several attractive properties for various industries [2-4]. For example,

green microalgae for several decades have been utilized for commercial exploitation, with

applications ranging from health food for human consumption, aquaculture and animal feed, coloring

agents, cosmetics and others [5, 6]. The main advantage associated with the use of microalgae is that

their cultivation requires simple growing conditions (such as light, carbon source considered as sugar,

CO2, N, P, and K) and produces various metabolites in large amounts over short periods of time.

Moreover, with their adaptation strategies, with rapid growth potential and high productivity without

interfering with arable land usage; cultivation of microalgae does not have any negative implications

on food security but is considered as a solution. Furthermore, the microalgae can be cultivated within

the whole year even though the yield is not the same depending on the climatological conditions

variability due to their ability to survive harsh environmental conditions as well as two culturing

modes (open and closed systems) can supplement each other for whole year production; Therefore,

they can be used for industrial purpose where they are considered as ubiquitous raw materials [5, 7].

Also considering their capability of integrating environmental bioremediation such as biological

sequestration of CO2 emission, microalgae biomass production may be used as one compatible

mitigation strategy to reduce the excess CO2 in the atmosphere and other greenhouse gases [8-10].

Phycobiliproteins are a group of colored proteins with linear tetrapyrrole prosthetic groups (bilins),

which in their functional state, are covalently linked to specific cysteine residues of the proteins [11].

Phycobiliproteins are water-soluble, brilliantly colored and highly fluorescent protein-pigment

components of the photosynthetic light-harvesting antenna complexes, commonly presented in

cyanobacteria (blue-green algae), red algae, some cryptomonads etc., and possess a spectrum of

4
applications [12-15]. Commercially, Phycobiliproteins are high-value natural products with actual

and/or potential biotechnological applications in nutraceuticals and pharmaceuticals, food and

cosmetic industries as well as in biomedical research and clinical diagnostics. The use of

phycobiliproteins as non-toxic and non-carcinogenic natural food colorants is gaining importance

worldwide in the view of the potential toxicity and carcinogenicity of the synthetic food colorants,

moreover their therapeutic value has also been demonstrated [12-14].

Production of high purity phycobiliproteins is a multiple concomitant steps involving several criteria

and factors. Therefore, it is imperative to pay more attention on each and every step involved in the

phycobiliproteins production process. Microorganism used to produce a given phycobiliprotein

affects its properties and consequently the application of the desired product is affected. For example,

Synechococcus lividus has been reported to be a strain that grows at highest temperature of any

known cyanobacterium, as results the purified C-phycocyanin from S. lividus denatures at 90 o C; and

it has a uniquely blue-shifted absorption maximum at 608 nm compared with normal C-phycocyanin

maxima between 615 and 622 nm [16]. Therefore, more works should be done in the biomass

production and phycobiliproteins accumulation by selecting adequate microorganisms; optimization

of culture media, and optimization of production conditions. Moreover, harvesting /dewatering

methods and downstream processing (such as extraction and purification techniques) should be

improved in order to get high purity phycobiliprotein for desired applications.

2. Phycobiliprotein distinctness: definition, types, structure and stability

Phycobiliproteins are a family of colored proteins that commonly presented in algae including

cyanobacteria, rhodophytes, cryptomonads, cyanelles, etc [14, 17]. These molecules are water

soluble, natively highly fluorescent proteins with linear prosthetic groups (bilins) that are linked to

specific cysteine residues [7, 18]. Generally, they are categorized into three main types namely

phycoerythrin (PE), phycocyanin (PC), and allophycocyanin (APC) which differ in their spectral

properties [19, 20]; and same classification according to their structure. Phycobiliproteins also are the

5
main light-harvesting chromoproteins in a certain type of marine algae [12, 13, 21-25]. These

compounds are classified according to different criteria (such as structure, spectra of absorption, color,

etc.). Moreover, based on absorbance wavelength, the phycobiliproteins existing in cyanobacteria and

red algae are commonly categorized into four groups known as, phycoerythrin (PE; λmax= 490-570

nm), phycoerythrocyanin (PEC; λmax= 560-600 nm), (3) phycocyanin (PC; λmax= 610-625 nm) and

allophycocyanin (AP; λmax= 650-660 nm) while in the cryptomonads, exist two classes of

phycobiliproteins: phycoerythrin (PE; λmax= 540 - 570 nm) and phycocyanin (PC; λmax= 610-650 nm)

[26-29]. Also based on their colors, phycobiliproteins are classified into two large groups namely:

phycoerythrin (red) and phycocyanin (blue); furthermore, phycocyanins are subdivided into

C-phycocyanin (C-PC), R-phycocyanin (R-PC) and allophycocyanin (APC) [11, 13, 23, 30].

There are two types of Phycoerythrin (PE) namely R-PE and B-PE, which were named after the taxa

of the organism form which they were isolated. R-PE was isolated first from Rhodophyta and B-PE

from Bangiales order (red seaweed) of Rhodophyta [29]. Different classes/types of phycobiliproteins

have different properties that have to be taken into consideration during their study and applications.

For example, the spectral characteristics of classes/types of phycobiliproteins (see Table 1). Briefly,

phycobiliproteins have wide variety of applications that is used in the food, biotechnology and

cosmetic industry because of their color, fluorescent and antioxidant properties [31].

The phycobiliproteins in many algae are arranged in subcellular structures called phycobilisomes.

These structures allow the pigments to be arranged geometrically in a manner which help to optimize

the capture of light and transfer of energy. All of the phycobiliproteins absorb incident light directly,

but in addition they participate in an energy transfer chain within the phycobilisome [32]. Whereby

the transfer of energy occurs from phycoerythrin →phycocyanin →allophycocyanin→chlorophyll A

[32, 33]. Phycobiliproteins function as predominant light-harvesting complexes to absorb the sunlight

in the region range from 470 to 660 nm, and efficiently transfer the energy to chlorophyll A [7, 32].

The phycobiliproteins are composed of a number of subunits, each having a protein backbone to

which linear tetrapyrrole chromophores are covalently bound. All phycobiliproteins contain either

6
phycocyanobilin or phycoerythrobilin chromophores, and may also contain one of three minor bilins;

Each bilin has unique spectral characteristics which may be further modified by interactions of the

subunits and of the chromophore with the apoprotein [34], means that the specific spectral

characteristics, of each type of phycobiliproteins, are a results of the composition of its subunits [34].

They are all composed of hetero-subunits α and β and commonly exist in trimer (αβ)3 or hexamer

(αβ)6 made up of equimolar monomer (αβ) [19, 27], where (αβ) is traditionally designated as the

monomer of phycobiliproteins. Phycoerythrins, especially from red algae, have another

chromophore-carrying subunit named  [27]. One monomer contains two to five chromophore

phycobilins, the subunit is considered to be a connecter by which two (αβ)3 trimers (about 120–30
kDa) are combined to form a more stable hexamer ((αβ)-  - (αβ)3) (about 240–260 kDa) [19].

Among different phycobiliproteins, phycocyanin is one of greater importance compound because of

its various biological and pharmacological properties [35](see Table2), its production and stability as

a promising and efficient pharmaceutical and biological reagent due to its activities such as

neuroprotection [36], antioxidant, and anti-inflammatory [11, 35].

Phycocyanin (PC) is a natural blue pigment that belongs to the class of phycobiliproteins and also

considered as one of the key pigments of Arthrospira (Spirulina), a microalgae used in many

countries as a dietary supplement [30]. Phycocyanin is mostly present in the cyanobacteria (also

known as blue green algae (BGA), which are considered as the oldest form of life on the earth)

especially Arthrospira species [30, 37]. Arthrospira species mostly used to produce phycocyanin are

Arthrospira (Spirulina) plantesis [10, 38, 39], Arthrospira (Spirulina) maxima [40], etc. Apart from

Arthrospira species, there are other microalgae that are used to produce the phycocyanin such as

Pyrophyridium sp. [41], Synechocystis sp. [42], Phormidium ceylanicum [43], Limnothrix sp [44],

Synechococcus lividus [16], Nostoc species [45], etc. The Arthrospira is the more preferred

microalgae because it is well known and widely commercialized in different countries for its

applications in different domains such as food additives, health food, cosmetic, pharmaceutical and

medicine [2, 46]. Moreover, it has clinical potentials in pharmaceutical, physiological and medicine

7
[47], because of its activities such as anticancer [48], neuroprotective [49], antioxidant,

anti-inflammatory, hypolipidermic [50], etc.

Appropriate control of the pH, ionic strength and temperature during extraction, separation and

purification processes are crucial for the stability of phycocyanin molecules [51]. Moreover,

temperature and pH play an important role in the stability of phycocyanin [52]. Systematic

investigations revealed that the maximum stability of phycocyanin was in the pH range of 5.5–6.0

[51]. The pH of the buffer to be used for phycocyanin extraction should be well controlled because it

may denature it. Sekar and Chandramohan (2008) [14] reported that phycocyanin was found to be

stable over a pH range of 5.0 to 7.5 at 9 °C, whereas temperatures above 40 °C led to instability.

The cyanobacteria strains used to produce phycocyanins also influence their stability. For example,

the stability of C-phycocyanin from cyanobacteria able to grow at high temperatures is different to

that of C-phycocyanin from cyanobacteria of usual environments [53]; for example, S. lividus is able

to grow at the highest temperature thus the purified C-phycocyanin from this strain denatures at 90
o
C, and it has a uniquely blue-shifted maximum absorption at 608 nm compared with normal

C-phycocyanin maxima between 615 and 622 nm [16]. The degradation of phycocyanin depends on

the aggregation state of the protein, which is influenced by different factors such as light, temperature,

pH and protein concentration [51, 52].

3. Technology of higher production of phycobiliprotein

The production of microalgae metabolites, including phycobiliproteins, requires two main

consecutive processes as indicate in Figure1: upstream process including microalgae biomass

production/metabolites accumulation and downstream processing (including harvesting, extraction

and biorefinery methods). The downstream processing, i.e. biomass recovery and metabolites

recovery, of microalgae-derived products, represents important cost components of the overall

process [54, 55]. Even though the harvesting step is included in the process of downstream (in this

review) but normally, is commonly considered as intermediate step between up-stream process and

downstream process. The achievement of the high quality phycobiliproteins production from
8
microalgae needs the optimum production conditions including effective microalgal biomass

production and stimulation of phycobiliproteins accumulation that leads to the high content of

phycobiliproteins in the biomass. Most microalgae accumulate phycobiliproteins under specific

environmental stress conditions especially the light. The light strongly stimulates the synthesis of

Phycobiliproteins while the availability of carbon source (such as glucose) strongly presses their

synthesis by inhibiting the synthesis of other proteins thus favoring phycobiliproteins synthesis [56].

The cells grown without light and glucose can produce other proteins than phycobiliprotiens under

heterotrophic conditions [56]. The nitrogen source contributes in cell growth, the phycocyanin

accumulation, and nitrate-sufficient culture can help to maintain a high phycocyanin content in the

cell [57, 58]. Therefore, the management of the environmental conditions is a common approach used

for improving phycobiliproteins accumulation in the microalgal cells.

Concisely, the process of phycoblilprotein production is a multiple steps process. The phycocyanin

production is a good example to summarize the upstream and downsteam of biosynthesis and

separation of phycobiliprotein. Those steps include selection of efficient microorganism strain and

culture medium; microalgae biomass production and phycocyanin accumulation,

harvesting/dewatering, extraction and isolation of phycocyanin, and finally, purification and

stabilization (see Figure2) [55]. The downstream procedures for phycocyanin from microalgae

biomass, generally combine different techniques such as ammonium sulfate precipitation,

ion-exchange chromatography, and gel filtration chromatography to get pure phycocyanin [30].

Mechanisms governing phycobiliprotein production in microalgae

Microalgae metabolism, the same as other plants in general, is regulated to a large extent by

environmental factors such as nutrient availability, temperature and lights, and then microalgae

produce the metabolites [59, 60]. Biosynthesis of phycobiliproteins in microalgae cells is carried out

through the transcription, translational and posttranslational pathways through which there are

synthesis of amino acids, proteins and phycobilins, and finally, the ligation of phycobilins

synthesized to apoproteins to form the phycobiliproteins during the posttranslational phase [61, 62].

9
This review summarizes the mechanisms of phycobilins (i.e. chromophores of phycobiliproteins)

biosynthesis that are ligated to the apoproteins to form the phycobiliproteins as depicted in Figure 3.

Phycobilins are linear tetrapyrrole pigments that function as light harvesting entities in

photosynthetic mechanisms of microlagae [63, 64]. Phycobilins are biosynthesized from heme by the

action of heme oxygenase, which converts heme to biliverdin, followed by the action of other

enzymes that convert biliverdin to the phycobilins [65] and finally, phycobilins are covalently linked

to different specific cysteine residues via thioether bonds by the bilin lyases; the latter process is

considered as last step of phycobiliprotein biosynthesis.

Heme s a cofactor with diverse biological functions including modulation of enzymes activities etc.

[66]. Heme molecule is believed as the suitable metabolic pathway of all biosynthetic pathways of all

billins [67]. The heme synthesis has the core center on aminolevulinic acid (ALA) synthesis from

either on glutamate or pyruvate or succinyl CoA as immediate precursor of heme but the succinyl

Coenzyme A (CoA) is more immediate precursor as shown in figure 3. Heme synthesis is carried out

in the cytoplasm and mitochondria through eight main steps in case the succinyl Coenzyme A is

immediate precursor [68]. The heme mechanism starts from the condensation of succinyl CoA and

glycine to form ALA [68, 69], the latter condensation is followed by other enzymatic reactions that

lead to different intermediate molecules such as porphobilinogen (PGB) containing pyrrole ring and

two side genes, acetate group A and propionate group P [70], uroporphyrinogen III,

corproporphyrinogen III [71], protoporphyrinogen, Protoporphyrin IX, the heme synthesis is

accomplished when Ferrochelatase intervene in addition of the ferrous to Protoporphyrin IX to form

heme.

The heme undergoes an oxidative cleavage by heme oxygenase to form biliverdin IXα (BV) [67].

The biliverdin is an intermediate, i.e. common biosynthetic precursor molecule, in the biosynthesis of

phycobilins; but there some cases where the biliverdin can be or not the intermediate molecule for the

phycocyanobilin biosynthesis. If heme cleavage is directly to bile pigment before reduction, then

10
biliverdin would be an intermediate in phycocyanin synthesis. In case one or both of the reduction

step(s) occur(s) before heme cleavages then biliverdin would not be an intermediate [72]. Three main

phycobilins are directly formed from the biliverdin: Phycocyanobilin synthase (PcyA) catalyzes the

biliverdin to form phycocyanobilin, biliverdin reduction catalyzed by ferrodoxin oxidoreductase

(PebA) lead to 15, 16-dihydrobiliverdin, which undergoes further reduction by ferrodoxin

oxidoreductase (PebB) catalyst to form phycoerythrobilin; the third phycobilin formed from the

biliverdin is phytochromobilin through the Ferrodoxin oxidoreductase (H2Y) catalysis.

Apart of this first group of phycobilins produced from heme via biliverdin (i.e. common known ones:

phycocyanobilin, phycoerythrobilin and photochromobilin), there is another second group of

phycobilin which includes phycoviolobilin (PVB) and phycourobilin (PUB). The second group of

phycobilins containing a vinyl group at C3 and they cannot be cleaved directly from the

phycobiliprotein but they should be produced by other mechanisms such as isomerization [67].

During the posttranslational phase, the phycobilins tetrapyrrole formed are usually attached to

specific cysteine residues within the alpha and beta subunits via thioether linkages by bilin lyases [73,

74].

Improving the phycobiliprotein production in microalgae

Differences levels of bioactivity may be a result of environmental influences (biotic and abiotic

conditions) which cause microalgae cell content [4]. Therefore, the phycobiliproteins productivity is

affected by various factors such as microalgae strains (i.e. microalgae species), photobioreactors (i.e.

photobioreactor types and design), cultivation parameters (i.e. culture media and light, aeration, pH)

[41, 53, 75, 76].

Efficient microalgae strains

There are various microalgal species which can be grown for metabolites synthesis; however, they

are not grown in the same culture conditions, thus different productivity. Selection of efficient

11
microalgal strains is a critical factor that affects the metabolites productivity in a producing organism

[77]. Selection here refers to the isolation and screening of microalgae strains [26]. Those processes

depend on characteristics of process design and the product desired. The isolation process can be

done by enrichment techniques such as nutritional variations (e.g. C and N sources, trace elements,

vitamins, etc.), variations in pH, temperature, aeration, selective inhibitors (e.g. antimetabolites,

antibiotics), etc. [26, 78, 79]. Isolation is mostly used as the first step for the selection of novel

microalgal strains in samples collected from different ecological habitats such as rivers, lake, etc.[80].

The isolation step is followed by the screening which is mainly aimed to improve the producer strains

by eliminating non-producing isolates [26]. The screening technique mostly used, consists of taking

many microalgal strains, expose them to the same environmental stress, and finally choose the most

suitable ones based on the productivity yield [26, 80]. The latter choice is based on adaptability to the

environmental conditions, higher biomass productivity, and phycobiliproteins content [80].

Microalgae strains have to be confirmed as good choice depending on its high metabolites content,

easy cultivation and processing [23]. Different microalgae such as Arthrospira platensis,

Porphyridium sp., Synechococcus sp., etc. have been reported as effective microalgae for produce

phycobiliproteins production [7, 13, 81]. Once the suitable microalgal strain is chosen, cultivation

conditions have to be optimized for further productivity enhancement.

Photobioreactor types and design

Microalgae cultivation can be carried out either in open or closed systems using different trophic

conditions (photoautotrophic, heterotrophic, mixotrophic, etc.) for biomass production and

metabolites accumulation. Open systems can be open ponds which are considered as a solution for

large scale microalgae production, however their applicability is challenged by high risk of

contamination and uncontrollable production conditions [82]. In closed systems, microalgae can be

grown in photobioreactors under photoautotrophic, mixotrophic or heterotrophic conditions [83, 84].

Phycobiliproteins can be production in either open or closed systems at small or large scale. Recently,

photobioreactors have been reported as effective for cultivation of different microalgae species

because of their efficiency; they facilitate productivity, better CO2 consumption, better light transfer

12
to the culture, more effective mixing, less cleaning and maintenance time, etc. [85]. A limited number

of photobioreactors can be practically used for biomass production of microalgae and metabolites

accumulation even though there is a good number of photobioreactors that have been proposed and

designed [85-87]. Furthermore, it has been reported that the tubular and flat panel photobioreactors

are more effective due to the possibility of achieving high volumetric productivity and better biomass

quality reason why they are expected to contribute in the future microalgae industry [87, 88]. When

bioreactors used for microalgae cultivation allow control and monitoring of cultures conditions such

as pH, temperature, air flow rate, light intensity, etc., the optimation of those conditions become more

effective.

Bioreactors that provide data on mixing, gas exchange, energy consumption, biomass productivity

are effective for microalgae production. Moreover, they should reduce the power consumption by

providing simultaneous culture circulation and gas exchange with airlift alone and no centrifugal

recirculating pump, they should also provide sufficient mass transfer (mainly for the exchange of

oxygen and carbon dioxide) [88]. Effective bioreactors design contributes greatly in minimizing

energy consumption, reducing total cost of the production process, and increasing productivity.

Different Microalgae strains have been evaluated in different culture conditions such as heterotrophic,

photoautotrophic and mixotrophic for biomass production and phycobiliprotein accumulation.

Different Microalgae strains have been evaluated in different culture conditions such as heterotrophic,

photoautotrophic and mixotrophic for biomass production and phycobiliprotein accumulation. The

results shown that although most microalgae grow phototrophically, some are able to grow

mixotrophically and heterotrophically [89]. The specific growth rate of mixotrophic cultures grown

on glucose are much more than those of photoautotrophic and heterotrophic in case of

phycobiliproteins synthesis because the energy density of carbon source is higher in comparison with

carbon dioxide [89-91], suggesting that mixotrophy facilitated faster growth, increased maximal

biomass concentrations and accumulation of phycobiliproteins accumulation compared to

13
photoautotrophic and heterotrophic cultures [90]. However, due to differences existing in microalgae

species, it’s hard to conclude that cultivation of all microalgal strains is only favorable to mixotrophic

conditions.

Optimization of cultivation parameters

The production process (upstream process) of phycobiliproteins from microalgae depends on various

environmental factors (such as medium content i.e. nutrients availability, temperature, light intensity,

pH, CO2, residence time, etc.) that influence the growth of microalgal cells and the composition of

the microalgal biomass produced due to changes in the metabolism [60, 92]. Even though the

production of microalgae biomass has less cost than the downstream process, it has very crucial

implication in the biosynthesis of the microalgae-derived metabolites especially phycobiliproteins.

The maximum accumulation of metabolites here considered as valuable products implies an absolute

optimization of cultivation parameters affecting the growth of cultivated microalgae stains [1].

Generally, there are two main steps involved in the biosynthesis of phycobiliproteins. The first step

consists in the growth of the microalgae cells under optimal conditions for an efficient production of

biomass, followed by a second step where stress factors (such as lower nitrate concentrations in

fed-batch process, a moderate light intensity) are applied in order to induce synthesis of the

phycobiliproteins and other metabolites [57]. Therefore, depending on microalgal strains used in the

cultivation process, it is very important to provide and optimize an adequate culture medium and

culture conditions that favor both microalgal cells growth and effective phycobiliproteins

accumulation. Furthermore, many microalgae have the ability to survive harsh environmental

conditions due to different forms of adaptation, but they also need optimum culture media and

production conditions in order to avoid changes that may occur in the microalgae physiology thus

leading to the production of different metabolites as part of the microalgae adaptation strategies [5]. It

is very important to elucidate how algae react during different forms of stress [5, 93]. Based on the

experimental results and literature, Jelica B. Simeunović et al. [80] reported that changes of the

cultivation conditions (such as intensity and quality of light, temperature, concentration of media

content particularly nitrogen and carbon sources, etc.) could influence the pigment production in all

14
strains and combining appropriate cultivation conditions can lead to the synthesis of high amount of

phycobiliproteins [80].

Some efficient culture media and microalgae species commonly used for phycobiliproteins

production are highlighted in this review (see Table 3). Some culture media such as Artificial Sea

Water (ASW) medium, BG11 medium, Zarrouk synthetic medium etc. have been reported to be very

convenient and effective for microalgae growth, biomass production and phycobiliproteins

accumulation [13, 41, 94]. Therefore, the selection of culture media is very crucial criteria to be

considered depending on microalgae species. Referring to the metabolic pathways involved in the

biosynthesis of phycobiliprotein (described in section 3.1), it is obvious to use the media containing

the substrates such as glutamate, succinyl CoA, aminolevulinic acid, etc., which could participate and

facilitate in the biosynthesis of the phycobiliprotein via its precursors like heme, biliverdin and

phycobilins. In addition, it is very important to provide suitable carbon and nitrogen sources as both

contribute a lot in the metabolism of biomass production and phycobiliprotein accumulation. Carbon

source provide the large amounts of energy required for the growth of the microalgae cells (case of

heterotrophic), however, microalgae growth can also be supported by the energy from the light

(mixotrophic case) [83]. The depletion of nitrogen creates a chain reaction that decompensates energy

metabolism and amino acid biosynthesis, thereby affecting photosynthesis, respiration and growth

[95]. For example, the assimilation of nitrate, as nitrogen source, contributes to the production of two

molecules of glutamate where one of them re-enters to the assimilation pathways as substrate for

glutamate synthesis and the other is exported to the cytoplasm and will participate either in the

biosynthesis of heme or transamination reactions with alpha-ketoacids to produces other amino acids

and proteins [96]. During the optimization of culture media, different variables have to be taken into

consideration in order to control both the microalgal cells growth and the accumulation of high

content of phycobiliprotein. The most important variables to be focused on during culture media

optimization are nitrogen and carbon sources, without forgetting other culture media compositions

such as salinity, vitamins, trace metal etc. and the enrichment of culture media with CO2 flow rates i.e.

air supply, (for example, CO2 carrier can be NaHCO3 provided as a supplement in the culture

medium) [28, 97, 98].

15
Optimum culture media play a vital role in the microalgae growth under specific conditions in order

to promote the high-valued products, as phycobiliprotein, increasing productivity and products

quality [41, 99].

Apart from the optimum culture media, there are other various cultural conditions (such as initial pH,

temperature, residence time, light intensity photoperiod, etc.) [41, 98]. Those environmental

conditions need to be optimized for suitable growth and considerable accumulation of

phycobiliprotein. Irradiance is a determining factor in the high content phycobiliproteins

accumulation [100]. In general, microalgae with phycobilisomes may prefer low light intensities (i.e.,

~10-50 µmol photons·m-2·s-1) while some other algal strains (e.g., most of dinoflagellates) often

need higher light intensities (~60–100 µmol photons·m-2·s-1) [99]. Culture medium pH is an

important factor for the growth of all living microorganisms as it affects the metabolic processes and

biochemical composition of the cells [41, 98]. Various authors have reported the effects of change in

pH on various processes inside and outside of cell, such solubility, transport of substances across

cytoplasm membrane, nutrients bioavailability, activity of both intra and extracellular enzymes,

photosynthetic electron transport and osmotic potential of the cytoplasm [101-103]. The latter

processes are most factors that influence the metabolic reactions of phycobiliproteins biosynthesis

[61, 104]. The pH values for phycobiliproteins biosynthesis range from pH 6-10, but the most

suitable is alkaline depending on the microalgae strain, for example, cyanobacteria have efficient pH

range between 7.1-7.5 [98]. Generally, pH does not affect much the microalgae growth as it affects

significantly phycobiliproteins synthesis.

Temperature is also among the most fundamental factors that influence growth and composition of

cells for all living organisms [105, 106]. The optimal growth temperature and tolerance to the

extreme values usually vary from strain to strain [94]. The lower and higher temperature values

reduce the phycobiliproteins synthesis significantly reason why it is crucial to optimize the

temperature value depending on the microalgal strain [105]. The temperature range for

phycobiliproteins synthesis varies between 20-37 °C [107]. Meanwhile, incubation temperatures

16
higher than 20 °C should be combined with increased light intensities to prevent photo-inhibition

[99]. Furthermore, culture media nutrients level and production conditions such as pH, temperature,

light intensity and residence time were reported to influence the phycobiliprotein concentrations and

ratios [41, 108]. The choice of environmental culture conditions is also influenced by the targeted end

products and their application, as they affect their characteristics [109]. The optimum production

conditions lead to the reduction of production cost by obtaining the high productivity [110].

After cultivation process, microalgal biomass should be recovered from a relatively dilute culture

through the process known as the harvesting process, also known as biomass recovery process.

4. Overview on biomass harvesting, and extraction, purification and characterization of

phycobiliprotein

After the production period in which microalgae biomass increases and phycobiliproteins

accumulation , the harvesting /dewatering step is needed to collect the biomass which for

phycobiliproteins isolation. An efficient and effective harvesting method must be able to process

large volumes of microalgal cultures at minimal possible cost without any environmental concern.

Centrifugal recovery of the microalgae biomass is feasible for high-value products [55, 111, 112].

Centrifuges can process large volumes rapidly and the biomass can remain fully contained during

recovery [111]. The centrifugation method is expensive and energy-intensive in case there is a large

volume of culture medium has to be processed and targeted end product from biomass is taken as

low-value product such as biofuels [112]. The novel methods with low energy consumption should be

evaluated and applied [113] but their applicability would be implemented with analysis of quality of

targeted end products.

Generally, flocculation, also known as coagulation, is a method that is used to separate a small

amount of impurities from the large volume of liquid by using the flocculants to coagulate those

impurities; concerning the microalgae biomass harvesting, flocculation is a harvesting method used

during the first step to concentrate diluted suspended biomass followed by a dewatering method such

as mechanical, drying methods, etc. [112]. Flocculation can be carried out in several ways such as

physical flocculation, autoflocculation, etc [112].

17
Flocculation is one among microalgae harvesting methods, considered as a promising low-cost

harvesting method; ideally, the flocculants should be inexpensive, nontoxic, and effective in low

concentration and they should not hinder downstream processing. Harvesting methods should be

efficient costly, environmental friendly and facility to the further downstream processes. Therefore,

bioflocculants are recommended for flocculation of microalgae biomass to avoid negative effects that

may be caused by traditional chemical flocculants [111, 114-116].

A further consideration in selecting a suitable harvesting and dewatering method is the acceptable

level of moisture in the product because too much moisture in the harvested biomass can

substantially influence the economics of product recovery by imposing further downstream processes

[55]. Recovery and processing of microalgae biomass from a culture media can be carried out by

using individual (single stage) or in combination (interconnected and interdependent multiple stage

techniques) depending on the microalgae species, desired end product concentration and quality

[117]. Concisely, as each conversional method for microalgae biomass harvesting has advantages and

disadvantages reason why the harvesting method should be selected by paying much attention on the

microalgae strain nature, the cost benefit, energy requirements, environmental friendly, biomass

application targeted, etc. It is better to minimize waste -thus contributing to the reduction of waste

treatment cost, reason why this paper recommends that more emphasis should be on the green and

environmental friendly microalgal biomass harvesting methods such as bio-flocculation in further

researches.

The downstream process (i.e. extraction, purification processes and characterization of the end

product) is one of the most important required processes to obtain high purity phycobiliproteins from

microalgal biomass, which comes after the microalgal biomass production and harvesting process.

The phycobiliproteins may be extracted from wet or dry microalgae biomass [13]. It has been

reported that the extraction of phycobiliproteins from wet biomass is more suitable, as it helps to

avoid loss in pigment by various drying methods [13, 118]. Furthermore, the extraction from wet

18
biomass is not expensive as dry biomass extraction because of other costs associated with drying

processes [55]. For example, extraction from dry biomass can result in approximately 50 % loss of

phycocyanin and finally can cause the variations in spectra compared to phycocyanin from wet

biomass [118]. The recovery of phycobiliproteins from microalgal biomass is carried out through

various steps which can be summarized into four main steps, namely, cells disruption, extraction,

purification and characterization of phycobiliproteins as end products and each step has various

methods/techniques[55].

The cell disruption is a crucial step in the intracellular metabolites reason why that is step can be well

carried out [119]. Generally, microalgal biomass should be treated by one or a combination of

physical (like nitrogen lysis, high pressure exposure, cavitation, sonication, osmotic chocks,

homogenization, repeated freezing thawing at different temperatures, etc), and chemical (like usage

of acid, alkali, detergents, enzymes, etc.) methods for the microalgal cells disruption[13, 119, 120].

After cell disruption step, the supernatant (commonly named: crude extract) has to be collected for

further extraction and purification steps [80] including precipitation, aqueous two phase extraction,

ultrafiltration, gel filtration, chromatography column depending on chosen method. The

phycobiliproteins should be purified from desalted extract by using available column

chromatography methods such as ion exchange chromatography, adsorption chromatography [27].

Finally, the phycobiliprotein yielded have to be characterized using different conventional

characterization methods such as sodium dodecyl sulfate-polyacrylamide gel electrophoresis

(SDS-PAGE), absorption spectra (at wavelengths 620, 652 and 562 nm for phycocyanin,

allophycocyanin, and phycoerythrin respectively) [27].

Phycocyanin has been selected as a representative case study of phycobiliproteins extracted from wet

microalgae biomass due to its various pharmacological and biological properties. Briefly,

phycocyanin is commonly extracted using diluted sodium phosphate or potassium phosphate buffer

(5-50 mM, pH 6.0-7.5) from freshly harvested microalgal biomass, followed by precipitation by

salting-out with ammonium sulfate [121]. The precipitated crude extract is desalted by dialysis or gel

19
filtration, and the phycocyanin is purified by column chromatography, like ion exchange

chromatography on DEAE-cellulose DE-52 and hydroxyapatite, from desalted extract [17]. The

purity of phycocyanin obtained is measured by purity number that describes the ratio between

absorbance from phycocyanobilin at 620 nm and all proteins in the sample at 280 nm using a

UV-spectrometer [121], and the polypeptide components of purified phycocyanin is analyzed by

SDS-PAGE using polyacrylamide slab gel, run at 50V, 12.5 A [17]. Proteins standard of medium

range 97-14 kDa, 180-10 kDa, 260-10 kDa, etc. can be used as molecular weight markers and the

phycobiliproteins (molecular weight range 24-15 kDa for individual subunits (α, β), 260-44 kDa for

complex mix subunits and 215kDa for oligomer subunits) can be visualized by staining with

coomasie brilliant blue G-250 [17, 122, 123].

Devendra K. et al. [37] harvested and isolated the C-phycocyanin from Arthrospira platensis

CCC540 using freezing and thawing methods considered as a shorter and good extraction. Authors

reported that the purity of C-phycocyanin produced reached 4.58, which was in range as the one

reported by Niraj Kumar Singh et al. [43] for C-phycocyanin from Phormidium ceylanicum, where

authors used stirred ultrafiltration cell (UFC) method.

Phycocyanin is considered pure when the absorption ratio of visible maximum to 280 nm is greater

than 4 [11]. Moreover, criterion of purity for phycocyanin is considered in case the allophycocyanin

is not observed at 650 nm [11]. Light blue color is a characteristic color of phycocyanin, absorbing

orange and red light, particularly near 620 nm and emits fluorescence at about 650 nm [26, 28].

Due to differences existing in microalgae species, it’s hard to name one extraction method as the best

choice for a phycobiliprotein produced by different microalgae species [17]. Therefore, most of the

common factors that affect the phycobiliproteins extraction are here under highlighted, and this

information can serve as a guide for future researchers. Furthermore, some conventional

phycobiliproteins extraction methods are also highlighted (Table 4). The chromatography techniques

such as ion exchange chromatography are used to increase the purity of the phycobiliproteins [17, 24].

20
Other factors that affect the phycobiliproteins extraction include abiotic conditions such as

biomass-solvent ratios, temperature, pH, etc. [17, 23, 52].

Optimization of cell disruption method is also very important for the maximum extraction because it

facilitates the release of phycobiliproteins into extracting medium. The freezing and thawing method

has been reported as the best for cell disruption and produced high amount of phycobiliproteins [17].

Selection of suitable buffer for phycobiliproteins extraction is also a very crucial factor for obtaining

the high yield and quality phycobiliproteins [7]. Among different buffer evaluated for the maximum

phycobiliproteins extraction, sodium phosphate buffer was found to be the best [17]. Once the

suitable buffer for phycobiliproteins extraction is selected, its optimum concentration should be

evaluated [10]. Freezing and thawing cycles is the mostly used method and it is among the most

effective and suitable methods for efficient lysis of some microalgae strains, however the process can

be quite lengthy [13, 118]. Another reason supporting the efficiency of freezing and thawing as

extraction method is that, when the cell is frozen, there is an inevitable intracellular ice formation,

resulting in cell damage, which promotes a better extraction of the intracellular substances [13, 124].

Currently, the novel methods for phycobiliproteins extraction and purification have been applied to

obtain phycobiliproteins of various purity indexes (food, reactive, and analytical grades); where new

steps replace/supplement the existing steps with purpose of reducing the number of steps employed

in purification process. Various processes, steps sequences involving ATPE and ultrafiltration can be

found in the following examples: the ultrafiltration (principle of ultrafiltration is similar to that of the

dialysis for desalting)[125, 126], the aqueous two phase extraction (ATPE) [43, 125, 127-129],

various types of chromatography (such as hydrophobic Chromatography, ion exchange

chromatography) [24]. The ultrafiltration (UF) is grown technology for biomolecules separation of

natural sensitive compounds such as phycobiliproteins and it became more interesting and attractive

technology because it does not require stages of precipitation, centrifugation and dialysis which

dilute samples [126]. Moreover, ultrafiltration can be used at large scale while it is able to filter the

large amount of extracts. The drawback of this technology is the retention all compounds bigger than

21
membrane molecular weight cut-off.

The aqueous two phase systems (ATPS), also known as Aqueous biphasic systems, is fundamentally

a liquid–liquid extraction technique employing two aqueous phases comprising either two immiscible

mixing polymers or a polymer and a salt [130]. ATPS can be considered as incorporated technique (a

single step combining extraction, precipitation and primary purification) and it have been utilized as

an efficient alternative to traditional purification systems in biomolecules separation in

biotechnological and chemical industries [131-134]. The ATPS become more attractive due to their

advantages: low processing time, high product yield in terms of yield and purity, easy in scale-up and

reliable because the working conditions at small scale are not significantly change on large scale, etc.

[132, 133].

The ATPE is especially used in the early stages of operation for the partitioning and recovery of

targeted product and its efficiency is highly based on the best choice of aqueous two phase systems

(such as polymer-polymer, polymer-salts) to be used [132] and it is influenced by many factors

suchlike polymers molecular weight and concentration, degree of polymerization, ionic strength of

salts, temperature, pH of the system [134, 135]. The polymer-salts systems were reported to be more

suitable than other systems for purification of phycobiliproteins especially phycocyanin [127]. Some

drawbacks of ATPS technology are: it may form the wastewater streams from polymer-salts with

high concentration of phosphate/ammonium ions, it requires more knowledge about the

understanding the technique and the mechanism necessitated in the portioning process, high cost of

phase forming polymer, it may denature the quality of end product in case organic solvent are being

used, cost of materials involved may be a major disadvantage [133, 134].

In the most cases, an aqueous two-phase system (ATPS) coupled with ultrafiltration for the extraction

and purification of phycobiliprotein facilitate the remove ATPS components (polymers, salts) [127]

that contribute to the minimization of purification steps thus overcoming some hindrances that affect

the downstream process of phycobiliproteins (such as the loss of the product yield, difficulties and

22
high cost in establishment of the downstream processes at large scale) [43, 127].

Selection of a suitable extraction method is influenced by the microalgal strains used in the

production process and the quality of phycobiliproteins required, moreover the chosen method should

be optimized to suit the product requirements, maximize the yield and minimize the steps required in

the extraction process [13, 17, 24]. In some cases, the succession of extraction steps and methods

may contribute further increase in purity of phycobiliproteins [127, 128].

5. Biomedical, biotechnology and nutraceutical application of phycobiliprotein

Phycobiliproteins have shown crucial importance in various human beings activities many years ago

[136]. Concisely, the phycobiliproteins are high valued photosynthetic accessory pigments in

cyanobacteria and red algae [12, 18], find application in health foods, cosmetics as potential

non-toxic and non-carcinogenic natural colorants (widely used to substitute the synthetic food

colorants) [12, 18, 129], in the biomedical research as fluorescent marker and in oxidative

stress-induced diseases as therapeutic agent [13, 14, 129, 130] because of their potential

pharmaceutical and biological properties. In this regard, various phycobiliproteins applications have

been reported and are still applied.

Those main applications include pharmacological and biological reagents (Table 2), fluorescent

probes, and natural color additives (in food, maquillage, health products, pharmaceuticals, textiles,

detergents and fountain) [10, 11, 137]. Furthermore, other studies showed that phycobiliproteins also

have potential application on memory enhancement and disposal of optics messages, fast

photoelectricity detection and manual nerve networks [25, 136].

Phycocyanin, one of the phycobiliproteins classes, has been reported to be the major phycobiliprotein

in many cyanobacteria and as a secondary phycobiliprotein in some red algae [34]. It could be

extracted from cyanobacteria such as Arthrospira platensis, which has been widely used in

commercial applications such as food, cosmetic industry as a natural blue dye, immunodiagnostics
23
and analytical reagents [13, 30, 137, 138]. Biological and pharmacological properties of phycocyanin

include: water soluble, non-toxic fluorescent protein pigment with potent anti-oxidant,

anti-inflammatory and anti-cancer properties [11, 139]. Furthermore, recent studies demonstrated that

phycocyanin exhibited hepatoprotective and anti-inflammatory properties [11]. Both R-phycoerythrin

and B-phycoerythrin suit classic applications with Kr/Ar and Ar/He (best) lasers, while

allophycocyanin has higher wavelength applications (http://www.interchim.fr/ft/2/28310A.pdf

accessed on 25/12/2014).

Modern medicine has done much so far to eradicate and cure diseases. However, some diseases are

still considered as incurable and many people are suffering and dying because of those diseases, at

least one disease that cannot be cured is suffered by many people in the world every year. Most of top

ten incurable diseases (such as asthma, HIV/AIDS, diabetes, influenza, lupus, polio, Ebola,…) have

been reported (http://listverse.com/2007/10/04/top-10-incurable-diseases accessed on 2015-01-03)

. Apart of acute viral diseases, there are other diseases considered as dangerous, such as hepatitis

(inflammation of the liver caused by a virus or toxin), heart diseases (such as inflammatory heart

disease), etc. Furthermore, many diseases are accompanied or even caused by oxidative stress that is

characterized by excessive formation of reactive oxygen species that cannot be counteracted by the

antioxidant defense systems of the organism, therefore therapeutic use of natural or synthetic

antioxidants appears to be promising [11]. Phycobiliproteins, especially R-Phycoerythrin, are also

used to detect and measure antioxidants as peroxyls radicals. The phycobiliproteins may be a solution

to eradicate such kind of acute viral diseases based on the fact that they have been reported to be very

potential bioactive products depending on their properties/activities (table 2) [10, 50, 140-142]. In

other words, phycobiliproteins, particularly phycocyanin, from various cyanobacterial species have

been reported to exhibit a variety of pharmacological and physiological activities such as antioxidant,

anti-inflammatory [11, 35], hepatoprotective, neuroprotective [11, 36], free radical scavenger [11,

143], anticancer [44], antitumor [144], immunomodulating [10], atheroprotective [142], etc. (table 2).

Extensive studies are highly needed to explore the pharmacological and biological activities

24
(potentialities) of phycobiliproteins and their application to diagnose, prevent and cure those

considered as incurable diseases (table 2). For example, hepatitis is a liver inflammation caused by

virus [145, 146]. It is obvious to consider phycobiliproteins as a prevention and medicine for that

disease because they have antivirus, anti-inflammation and liver-protection activities [11, 147].

Future works on the development of medicines and vaccines from phycobiliproteins for prevention

and treatment of acute viral diseases in general, and hepatitis, Ebola, Influenza, cancers, HIV/AIDS

in particularly, would also be of interest.

Phycobiliproteins are the high quality fluorophores suitable for the conjugation with antibodies or

other probes. Phycobiliproteins function as predominant light-harvesting complexes to absorb the

sunlight in region of 470 to 660 nm, and efficiently transfer the energy to chlorophyll a [19, 32]. In

many cases, the phycobiliproteins are arranged in subcellular structures called phycobilisomes, which

optimize the capture and transfer of light energy [32]. Efficient excitation energy between the

chromophores in the phycobiliprotein trimer/hexamer and between the phycobilisomes in

phycobiliproteins gives them some special spectroscopic properties superior to organic fluorescent

dyes, and these properties render the phycobiliproteins good fluorescent probes applied in various

biotechnological research fields [19].

Phycobiliproteins are considered as efficient fluorescent probes than other organic fluorescent

because they elicit exceptional fluorescent properties for labeling techniques compared to organic

fluorophores, especially when high sensitivity or multicolor detection is required [19]. This is due to

their properties such as broad and high absorption of light suitable with many light sources, they are

10-20 brighter than organic fluorophores, they allow multicolor detections because the relative large

stokes shift gives low background, excitation and emission spectra (Table 1), do not overlap

compared to organic dyes, they allow simultaneous use with conventional chromophores. Moreover,

they are photostable where their fluorescence emission is not easy to quench, because fluorescence

retention period is longer; furthermore, they have very high water solubility, which makes them

stable even after multiple sites conjugation [74]. Three phycobiliproteins including R-phycoerythrin,

25
B-phycoerythrin, and allophycocyanin serve as valuable fluorescent tags with numerous applications

in flow cytometry, fluorescence activated cell sorting, histochemistry and, to a limited degree, in

immunoassay and detection of reactive oxygen species [15].

Phycoerythrin is appreciated and considered as the world’s brightest fluorophore because of its

intense fluorescence, suitability to be excited as fluoresceins and high quantum yield . Moreover,

phycoerythrin is compatible with commonly available lasers and gives exceptional results in flow

cymetry, immunofluorenscent staining and gives less background interference as result of being

sticky than common synthetic fluorephores [148].

The types of phycoerythrin (B-phycoerythrin and R-phycoerythrin) have different applicability

capacity as fluorescent probes, where B-phycoerythrin has a stronger emission than R-phycoerythrin

and R-phycoerythrin has a “spike” in excitation spectrum at around 488 nm chich makes it better for

use with the laser of most Flow Cytometry (FCM) and microarray readers [148, 149].

Phycobiliproteins are promising potential fluorescent probes because they complement each other i.e.

one type may intervene in case of application limitation of another type due to their difference in

properties [148].

Recent researches have proved that natural colorants are more efficient than synthetic dyes because

the natural colorants have no toxicity and carcinogenic properties [12-14]. However, the use of any

natural colorant in foods requires a detailed knowledge of its color stability, particularly towards heat,

light and pH [80]. The phycobiliproteins have been demonstrated to be efficient natural colorants,

thus they can be used as natural colorants in food, drug and cosmetic industries replacing the

synthetic dyes [12, 150]. The phycocyanin has more potential to be used as food additive compared

to other types of phycobiliproteins even though it is unstable in some extents [51, 151]. Phycocyanin

extracted from Arthrospira platensis is used widely as a natural colorant in food such as fermented

milk, ice-creams, deserts, milk shakes and sweet cake decoration [14]. The principles of

commercial and industrial applications of phycobiliprotein are associated to their main functions

26
including natural colorants, fluorescent markers, and therapeutic agents.

6. Future perspectives

The purified phycobiliproteins are required for potential activities along with industrial, biomedical

and pharmaceutical applications. A sequence of steps including precipitation, dialysis,

chromatography columns have been commonly applied but they have some drawbacks including

high cost, long time processing, low yield and hard to scale up but in recent years some new

purification techniques/methods such as the ultrafiltration, the two-phase extraction method,

ion-exchange chromatography have been used [127, 128]. Even though, those new methods have

more advantages compared to those used so far but they still have some hindrances that need to be

improved [134]. Thus, the development of well-adjusted methods (efficient, environmental friendly

and economical large-scale bio-separation methods that preserve the properties of phycobiliprotein)

is very important to achieve the desired phycobiliproteins in high yield, quality and purity.

Phycobiliprotein properties should be determined before its application in food, industrial and

biotechnological process. The literature has shown the potential pharmaceutical and biological

activities of phycobiliproteins (Table 2). Therefore, the novel studies in development of new

medicine and pharmacokinetic and of metabolism as previous stages to clinical trials must be

performed to the phycobiliprotein.

7. Conclusion

Phycobiliproteins applications in different fields required the various purity ratios. There is no other

ways of obtaining the higher purity phycobiliprotein from various microalgal species apart of

optimizing the production process and improving the downstream processes. In other words, the

optimization of cultivation process will lead to the high content of phycobiliproteins in the crude

biomass followed by suitable and adequate biomass and phycobiliprotein recovery process. The

phycobiliproteins from microalgae possess various pharmacological and biological properties that

should be more exploited in diagnosis, treatment and prevention of many diseases. Therefore, this
27
review suggests that future researches on the applications of phycobiliproteins especially in

pharmaceutical and medicine domains should focus on diagnosis, prevention and treatment of

diseases that considered as incurable while the literature shown that the phycobiliproteins have

potentialities to diagnose, prevent and treat them. The harmful microorganisms such as viruses,

bacteria, fungi, etc. are total different with different resistance reason why the phycobiliproteins

should be analyzed for slight variation in efficacy, safety and side effect properties in order to be

applied in diagnosis, prevention and treatment of those diseases considered as incurable. For example,

some future studies should evaluate the efficiency of phycobiliproteins as anti-retroviral to treat or to

prevent Ebola as disease caused by virus. It is obvious that the Arthrospira is more exploited

especially in health food, pharmaceutical and medicine etc. as potential microalgae because it is well

known for its important Source of Nutritional and Medicinal Compounds; in addition to that, the

physiological activities of phycobiliproteins have roots from the microalgae species used in their

production. Therefore, this review suggest that the other microalgae species should be evaluated and

analyzed for their slight variation in efficacy, safety and side effect properties in order to be exploited

in different domains especially health food, pharmaceutical and medicine domains.

Acknowledgement

The authors acknowledge the University of Rwanda-College of Education for giving a PhD study

leave to Mr. MANIRAFASHA Emmanuel and the Government of People’s Republic of China for

financially supporting his PhD studies. This work was also supported by the National High

Technology Research and Development Program 863, China (No. 2014AA021701) and the National

Marine Commonwealth Research Program, China (No. 201205020-2).

28
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36
Figure Captions

Figure 1: Schematic diagram of different steps involved in microalgae-derived metabolites

production. Case study: phycobiliproteins biosynthesis

Microalgae cultivation and biomass production

Downstream processing for microalgae-derived

metabolite recovery
Selecttion and culture
of microalgae strain

Selection and Microalgae biomass Extraction, isolation

production and Harvesting/dewatering
optimization of microalgae biomass of phycobiliproteins
culture media phycobiliproteins
accumulation

Optimization of Cells debris and Purification of

culture conditions residue phycobiliprotein

Characterization of
purified phycobiliprotein

Pure phycobiliprotein

Life cycle assessment

37
Figure 2: Process diagram of Phycocyanin production

Selection of efficient
microalgae strain

Appropriate culture
medium Cultivation

Optimum culture
conditions

Microalgal biomass production

and phycocyanin accumulation

Harvesting/ dewatering

Drying methods such as

Drying
freeze drying

Cell disruption, extraction Extraction solvents

Cell disruption
such as ammonium sulfate
methods and isolation of phycocyanin
to facilitate precipitation

cell debris

Purification methods Purification and characterization Characterization

such as column of phycocyanin methods such as
chromatography SDS-PAGE,

High purity ratio

phycocyanin

38
Figure 3: Phycobilins biosynthetic pathways in cyanobacteria, algae and plant (based on [61, 67, 72])

L-glutamate
glutamate -tRNA ligase pyruvate
5-aminolevulinase
glutamyl-tRNA (GLU)
glutamyl-tRNA
reductase L-alanine
4,5-Dioxopentanoate
(S)-4-amino-5-oxopentanoate
5-aminolevulinic
5-aminolevulinate acid transaminase
synthase
glycine CO2 2H2O
ALA
+ synthase PGB synthase
-aminolevulinic porphobilinogen (PGB)
Succinyl CoA 2x
acid (ALA) 4x hydroxymethylbilane
+ syntase
H CoA-SH
hydroxymethylbilane
uroporphyrinogen III
syntase

uroporphyrinogen III
(-) uroporphyrinogen
4CO2 decarboxylase
coproporphyrinogen III
oxidase
protoporphyrinogen coproporphyrinogen III
protoporphyrinogen
oxidase 2CO2
Protoporphyrin IX
Fe++
ferrochelatase
ferrodoxin
reduction 2H+ oxidoreductase
heme oxygenase (H2Y)
heme biliverdin IX phytochromobilin
ferrodoxin
heme cleavage

oxidoreductase
cy in
(P bil
)

(PebA)
A
s e no
ha a
n t cy

15,16-dihydrobiliverdin
sy yco
ph

ferrodoxin
oxidoreductase
(PebB)
phycocyanobilin
further reduction phycoerythrobilin

39
Tables
Table 1: Some characteristics of phycobiliproteins
Pigment Absorbance Absorption λexc\λem Fluorescence Molecular weight Absorptivity Molar absorptivity Color Quantu
-1
Maxima range max. emission (L/g-cm) (M.cm) m Yield
-1
(nm) (nm) (nm) (nm) (kda) (g.mol )

R-Phycoerythrin 565 (495) 540–570 498.546.566nm\ 575 240 240 000 8.2 1.53 106 Red 0.84
576nm 1mg/ml:
A566/A280 : >5.5
A566/A498 : ≤1.5
A620/A566:≤0.005
B-Phycoerythrin 545 546.566nm\576n 575 240 240 000 10.0 (545nm) 2.4 106 Red 0.98
m (563nm) 2.33 106
1mg/ml:
A545/A280 : >5.5
A565/A545 : ≤1.0
A620/A545:≤0.005
C-Phycocyanin 615 610–620 620nm\642nm 647 70-110 232 000 7.0 1.54 106 Blue 0.81
R-phycocyanin 617 (555) 637 100 232 000 7.0 0.70 Blue
Allophycocyanin 652 650–655 651nm/662nm 660 100 105 000 7.3 7.3 105 bluish 0.68
1mg/ml: green
A651/A280 ≥ 5.0
A651/A620 >1.5
Phycoerythrocyanin 575 625 100 8.5 0.85 orange
Reference [149, 152] [7] [149] [152] [152] [149] [152] [149] [80] [149]

40
Table 2: Pharmacological and biological properties of phycocyanins: beneficial to human health
action
Microalgae specie Phycocyanin Physiological Reference
property (activity)
Spirulina platensis, C-phycocyanin, novel Antioxidant [10, 11,
Aphanizomenon phycocyanin, 140, 153]
flos-aquae holo-alpha-phycocyanin
Spirulina platensis C-phycocyanin Anti-inflammatory [10, 11]
Arthronema C-phycocyanin Anti-tumor [144]
africanum
(Cyanophyceae)
Phycocyanin Anti-cancer [44]
Spirulina platensis C-phycocyanin Immunomodulating [10]
Spirulina platensis C-phycocyanin, Atheroprotective [142]
phycocyanobilin
Spirulina C-phycocyanin Hepato-protective [11]
Spirulina C-phycocyanin Neuro-protective [11, 36]
Spirulina C-phycocyanin Radical scavenger [11]
Spirulina platensis C-phycocyanin Antihyperalgesic [154]
Spirulina platensis C-phycocyanin Antiviral [147, 155]
Spirulina platensis C-phycocyanin Antifungal [147]

41
Table 3: Common culture media used to produce phycobiliproteins
Culture media Microalgae species Culture Light Reference
period intensity
Modified Spirulina platensis 9 days 4 K.lux [118]
Zarrouks medium (CFTRI)
Artificial sea Porphyridium 12-16 120 and 240 [41]
Water (ASW) purpureum days μE/m2s
BG 11 medium Cyanobacterial strains: 21 days 50 [80]
genera, Nostoc, μmol m-2s-1
Anabaena
SOT medium Spirulina strains 21 days 50 [80]
μmol m-2s-1
BG 11 medium cyanobacterium - 50 mol [17]
without sodium Anabaena sp. photons.m-2.s-
1
nitrate

42
Table 4: Conventional phycobiliproteins extraction methods
Extraction Biomass advantages Limitations Reference
method

Freezing Fresh and dry -Simple, quick (10-12 h), Technology [13, 17,
and thawing reproducible, robust since unproven at 118]
is independent of biomass industrial scale
quantity, free of
corrosible material and
without presenting
significant losses of the
biological capacity of the
protein.
-it is mild and
non-denaturing technique
Sonication Fresh and dry High productivity at Technology [13]
small scale unproven at
industrial scale
fractional Fresh and dry Cheap, best and reliable Uses high frequency [10, 24]
precipitation method as it precipitates (20–50 kHz) of
with ammonium readily and also prevents ultrasound, difficulty
sulfate denaturation of protein in scale up.
Chromatography Fresh and dry are used for high [17, 24]
techniques such as purification of
ion exchange phycobiliproteins
chromatography
Homogenisation Fresh Takes short time i.e. Contamination [118]
(mortar and pestle) quick (10 – 12 h)
Water extraction Fresh Non toxicity Slowest method [118]

Acid extraction Fresh Acid extraction would be It may cause the [118]
useful for direct denaturation of
extraction of pigment phycobiliproteins at
phycocyanobilin from a certain extends.
phycocyanin

43