1

Theodore Sotiroudis, Fragiskos Kolisis

Institute of Biological Research and BiotechnologyThe National Hellenic Research Foundation

Athens, Greece

Spirulina: A Green Factory

Spirulina (Arthrospira) :

Is a photosynthetic, filamentous, spiral- shaped,multicellular, blue-green microalga

It belongs to Cyanobacteria, Oscillatoriaceae (family),Arthrospira (genus)

Most commercial production systems are based on large shallow raceways in which spirulina cultures aremixed by a paddle wheel under controlled conditions

It has a long history of use as a safe health food and as an important source of protein.

Arthrospira sp. cultured in our Lab

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ALCALINE SALT

NUTRIENTS

CO2

WATER

Solar Radiation

Spirulina culturePOND

SEPARATION

WASHING

DEHYDRATION

DRYING

PACKAGINGPRODUCTS

Recycle of medium

NUTRIENTS

A Schematic diagram of production system of Spirulina

Filtration/Centrifugation

Algal Mass

tablets powder

HEATING

They represent a cheap and renuableenergy form that is sequestered in underground aquifers

They come from high depths

They have high temperatures

They usually contain dissolved CO2

Geothermal Fluids used for Spirulina Production in Greece

The geothermal energy used in this way in greenhouse cultivation pondscontribute to increasing the daily yield by 20-30%

The use of freely released geothermal CO2 reducesthe production costs of Spirulina biomass by over 25%.

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Location map of the Nigrita area with the sites of the geothermal production well (Th-13b) andthe plant for cultivation of Spirulina

ALGAE A.C. Spirulina production pond

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5% 3%

65%

20%

7%

Composition of Spirulina

Protein and Amino Acids

Carbohydrates

Minerals

FatsMoisture

The highest protein content of any natural food

Spirulina: The Green Factory

Cosmetics Nutraceuticals

Fluorescent Markers/ Restriction Enzymes Hydrogen Production

Food/Feed

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Potential Applications of Spirulina as Nutritional and Therapeutic Supplement in Health Management

Antioxidant

Anticancer effects

Immunomodulation effects

Anti-viral effects

Health benefits from Spirulina are mainly dueto some major bioactive components:

Phycocyanin ( Phycocyanin C/Allophycocyanin proteins)

Spirulan (sulphated polysaccharide : MW~220,000 ; rhamnose (52%), uronic acids (16%))/

Immulina (High MW polysaccharide(~107) ; rhamnose (35.4%)

Gamma Linolenic acid (GLA)(ω-6 PUFA)

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Ribbon representation of spirulina platensisPhycocyanin C subunits

20.5 kDa 23.5 kDa

Phycocyanobilin

(Phycocyanin C: Trimer of α/β heterodimers)

α-84

β-84β-155

Phycocyanobilin (PCB) Chromophore

PCB is covalently attached to cysteines of the apoprotein via a thioether bond to C-31 on ring A

The final step in phycobiliprotein biosynthesis is the covalent chromophore attachment to the apoprotein by heterodimeric lyases

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Old and newBlue smarties (Nestlé)with phycocyanin (2008)(artificial coloring was removed)

Phycocyanin extract

A Spirulina high Mr PolysaccharideNutraceutical

Commercial products containing Spirulina bioactive components

Chocolate confectionary

Commercial GLA preparationscontain plant-based oil GLA

Present Research activities

Studies of Spirulina bioactive components in order to evaluate theirpotential use in biomedicine and cosmetics

Lipid components

Sulphated polysaccharides

Phycocyanin and phycocyanobilin (PCB)

Enzyme activities present in dried microalgal biomass

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Lipid Components

What is the chemical composition and Bioactivityof Volatile Lipid Components in Commercial Spirulina Dry Biomass ?

Major Volatile Lipid Components of Spirulina Dry BiomassIdentified by GC-MS

heptadecane (77.88%) 6,10,14-trimethyl-2-pentadecanone (6.09%)

L-linalool (10.5%) 9(Z)-octadecenoic acid (10.19%) hexadecanoic acid (5.0%) cis-linalool oxide (4.08%)

hexadecenoic acid 1,1΄-bicyclohexyl2(4H)-benzofuranone

Volatiles by Hydrodistillation

Pentane Extract

Fractions of Si gel Chromatography of Dichloromethane extract

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22(0.0001)

21(0.001)20(0.01)NTNTNTNTNTNT5-flucytocine

232220NTNTNTNTNTNTItraconazole

NTNTNT16(0.01018(0.008)17(0.00816(0.088)19(0.004)17(0.004Netilmicin

10981011101013121Bicyclohexane

101081011111013121Fraction 268

101071110111011121Fraction 223-224

1110711910912111Fraction 169-170

10108109101012121Fraction 148-151

99899101012111Fraction 50-56

99891010910121Fraction 70-74

9109101011101415Methanol extract

89NA9NANANA1111Dichloromethane extr.

NA8NA10NANANA108Cyclohexane extract

C.glabrata

C. tropicalis

C. albicans

E.coli

K.pneumoniae

E.cloacae

P.aeruginosa

S.epidermidis

S.aureus

Tested samples

Antimicrobial Action of Spirulina extracts and Si-Gel Chromatography Fractions

NA, Not Tested; NA, Not Active; Chromatography 1Fractions of Dichloromethane extract

Antimicrobial activity was assayed against six human pathogenic bacteria and three fungi through both the disc diffusion (inhibition zones (mm)) as well as the dilution methods.

Anionic Polysaccharides

Spirulina anionic polysaccharides expressImmunomodulating and Antiviral activities

Free radical Scavenging Activity ?

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N

N

NO2O2N

NO2

.

H2N

N

CH3

CH3 S S

NN

NN

S S

O

OHO

OH

O

O

H3C

CH3

Scavenging of stable free radicals by Spirulan

EC50= 0.2 mg/ml EC50= 0.3 mg/ml

DMPD ABTS DPPH

No

Polysaccharide Specificity

Chondroitin sulfate, Hyaluronic acid and Heparinexpress very low scavenging activities

(spectrophotometric determination)

3420 3440 3460 3480 3500 3520

+ 0.1 mM oleuropein

DMPD (10mM) +H2O2 (1mM) + POD (80μl) pH 5.5

Magnetic Field (Gauss)

DMPD.+

DMPD.++ Spirulan/Phycocyanin

Scavenging of DMPD.+ by Spirulan or Phycocyaninfollowed by EPR Spectroscopy

Tsapra et al. submitted

.+

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3420 3440 3460 3480 3500 3520

2

1

Magnetic Field, Gauss

Spin-Trapping of Free Radicals in absence (1) and presence (2) of a Spirulina platensis Polysaccharide Fraction

Analysis of the EPR spectrum indicatesthat the polysaccharide scavengeshydroxyl radicals

.CH3

.OH H2O2/NaOH/DMSO system

+DMPO spin trap

Phycocyanobilin (PCB)

Phycocyanin : ~ 14% of Spirulina dry biomass

PCB: ~ 0.66% of Spirulina dry biomass(15 g of Spirulina (a heaping tablespoon) contains about 100 mg PCB)

PCB Preparation

Spirulina Phycocyanin PCB ChromatographyMethanolysis

Phycocyanin exhibits a variety of pharmacological properties

Phycocyanobilin ?

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PCB-mercaptoethanol adductPCB-imidazol adduct

Since a Spontaneous formation of PCB adducts has been observed(Tu et al (2009) JACS, 131, 5399)

Therefore: Our aim was to examine the interaction of PCB with various biomolecules

-200 -150 -100 -50 0 50 100 150 2000

5

10

15

20

25

30

35

40

45 PCB (10 μΜ)

1/V 0

1/[S] (mM)-1

Inhibition of Lipase and Alkaline Phosphataseby Phycocyanobilin (PCB)

-4 0 4 8 12 16 20 240

200

400

600

800

1000 PCB (150μΜ)

PCB (100 μΜ)

1/V

(mM

/s)-1

1/[S] (mM)-1

Lipase + p-nitrophenyl butyrate

Alkaline Phosphatase +p-nitrophenyl phosphate

Ki=18.9 μΜKi=320 μΜ

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PCB irreversibly modified:BSA, Histone H2B, Phosvitin, certain anionic polysaccharides and DNA

PCB interacts with:the anions phosphates and borates as well as with the amine Tris

PCB interacts with surfactants:SDS, Triton X100, CTAB

350 400 450 500 550 600 650 700 750 800 850 9000,0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

0,9

nm

PCB 0 min 15 min 30 min 60 min 90 min 120 min 24 h

A

400 500 600 700 800 9000,0

0,2

0,4

0,6

0,8

1,0 PCB+Histone (0 min) 10 min 60 min 120 min 24 h dialysis (+1% SDS) 24 h dialysis (8Μ urea)

A

nm

PCB+ Histone PCB + Pi (pH 9.0)

All the above interactions may cause autocatalytic PCB covalent modifications

300 350 400 450 500 550 600 650 700 750 800 850 900-0,1

0,0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

0,9

1,0 Φάσμα PCB 0,34 mM SDS 0,68 mM 1,7 mM 3,4 mM 5,1 mM 6,8 mM 10,2 mM 13,6 mM 17 mM 20,4 mM 23,8 mM 27,2 mM 30,6 mM 34 mM

A

Μήκος κύματος (nm)

0 5 10 15 20 25 30 350

10

20

30

40

50

60

70

0.8 mM

8.6 mM

ΔΑ 6

28 (%

)

SDS (mM)

350 400 450 500 550 600 650 700 750 800 850 9000,0

0,2

0,4

0,6

0,8

1,0

1,2

Φάσμα PCB 0,1 mM CTAB 0,2 mM 0,5 mM 1 mM 1,5 mM 2 mM 3 mM 4 mM 5 mM 6 mM 7 mM 8 mM 9 mM 10 mM

A

Μήκος κύματος (nm)

0 1 2 3 4 5 6 7 8 9 10 110

20

40

60

80

100

0,75 mM CTAB

ΔΑ

629(%

)

CTAB (mM)

nm nm

Titration of PCB by SDS and CTAB

PCB

SDS micelle

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Peroxidase of Spirulina dry Biomass

ROS-scavenging enzymes are increasingly used in skin health care products

Is Peroxidase Activity present in Dry Commercial Spirulina Biomass ?

Peroxidase from Spirulina Dried Biomass

Partial purification by ammonium sulfate fractionation and gel filtration

Molecular mass: 43.500 Da

Two pH optima: pH 4.0 and pH 8.0

Km for ABTS: 46 μΜ (best substrate)

Km for H2O2 : 21.2 mM

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0 5 10 15 20 250

10

20

30

40

50

60

pH 9

pH 8

A61

8(% D

ecre

ase)

min

Spirulina Peroxidase (PO)-induced Spectral Change of Phycocyanin (PC) in presence of H2O2 and ABTS

Bleaching of PC was observed only in presence of ABTS

PO + H2O2 + ABTS ABTS* PCmod

PC

(mediator)

Since Peroxidase activity survives the manufacturing conditions for Spirulina powder production, it may play significant role in cosmetic products in blocking hydrogen peroxide toxicity

Future Research activities

Metabolic Engineering, Metabolic Flux Analysis and determination of

key metabolites using HPLC-MS/NMR techniques will be used for

designing and modifying Spirulina pathways for the overproduction of

important constituents of this microorganism

Synthesis of hydrophobic derivatives of PCB as membrane- targetted

photosensitizers for photodynamic therapy

New cosmetic products containing Spirulina bioactive components

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University of AthensAss.Prof. I. Chinou

University of Malaga Prof. C. JimenezProf. R. Abdala

TEI, AthensProf. E. Nerantzis

NHRFDr A. XenakisDr V. PapadimitriouDr. E. Tzika

G. T. Sotiroudis, MscV. Tsapra, MscP. Koukouraki, MscI.Pontikos, Dipl. Th.

M.Anastasiadis, Dipl. Th.

Bilateral Greece-Spain GSRT 006-γ Programme

01 PRAXE 11 GSRT Programme

ALGAE AC, Nigrita,Serres

Thank you for your attention

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BIOSYNTHESIS OF BILINS

Other genomes for species:

Molecule: DNA

Organism GroupCDDContigs: 1820Topology: other

Completed: 2009/07/20TraceAssemblygMapOthers: None % Coding: 0%

Sequencing center: University of Applied Sciences of Western SwitzerlandBLASTGenePlotPseudo genes:

None GC Content: 44%

Seq.Status: DraftGenBank FTPTaxPlotStructural

RNAs: NoneLength: 4,997,563 nt

Refseq Status: WGSRefseq FTPTaxMapProtein coding: None

GenBank: ACSK00000000

Publications: NoneGenome ProjectCOGGenes: 0Refseq:

NZ_ACSK00000000

Review Info:Links:BLAST homologs:Features:Genome Info:

Arthrospira platensis str. Paraca, whole genome shotgun sequencing project

http://www.ncbi.nlm.nih.gov/sites

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Other genomes for species: Molecule: DNA

Organism GroupCDDContigs: 129Topology: other

Completed: 2008/10/15TraceAssemblygMapOthers: None % Coding: 82%

Sequencing center: US DOE Joint Genome Institute (JGI-PGF)BLASTGenePlotPseudo genes:

None GC Content: 44%

Seq.Status: DraftGenBank FTPTaxPlotStructural RNAs: 38Length: 6,003,314 nt

Refseq Status: WGSRefseq FTPTaxMapProtein coding: 5690GenBank: ABYK00000000

Publications: NoneGenome ProjectCOGGenes: 5728Refseq: NZ_ABYK00000000

Review Info:Links:BLAST homologs:

Features:Genome Info:

http://www.ncbi.nlm.nih.gov/sites/

Arthrospira maxima CS-328, whole genome shotgun sequencing project