Fatty Acid Profile of Spirulina platensis Grown Under Different Culture Conditions

Franklin T. Ayran

Mentors: Fabian M. Dayrit, Ph.D.

Rene Angelo S. Macahig, Ph.D.

Why Spirulina?

“The best food for the future.” (UN) fast growing, high nutritional value: fatty acids

(GLA); balanced essential amino acids; about 60% complete protein by weight

Amino Acids(essentials)

Spirulinagrams

Egg Proteinper 100 grams of

IsoleucineLeucineLysine

MethioninePhenylalanine

ThreonineTryptophane

Valine

6.410.4 4.5 2.2 5.4 5.4 1.5 7.5

5.89.0 6.7 3.0 5.3 5.3 1.8 7.2

Easy to collect from the biomass medium (100-200µm): filtration

requires low energy and cost input per kilo of production

The Spirulina Project

Y1 •Growth optimization

Y2 •Technology transfer

Y3 •Isolation of high-end products

Objectives

Culture

• Spirulina platensis in Spirulina medium (Bigelow) with different parameters for 14 days.

Characterize

• fatty acid methyl ester - gas chromatography (FAME-GC) analysis

• GC-MS analysis• total lipids by gravimetric lipid

analysis

Data processing

• effects on the total lipids• fatty acid profile of various

parameters

Parameters

Spirulina medium (Bigelow)

+ 5% v/v young CW

+ 5% v/v mature CW

pH 11 (with HCO3

- or CO32-)

Photoperiod: 12hrs:12hrs

Light color: white

Intensity: 50W/m2 Intensity: 12W/m2

1A 1B 1C 2 3

3 samples 14 days growing

Spirulina: 10mL for UV, temperature and pH measurements, and

cell count

Methodology

0.01g/L seed

(5) Fluorescent light source

12:12 light:dark1L glass bottle

Air tubing

Aluminium foil

Air stone

Air pump

pump

Spirulina Culture Set-up

Centrifuge(4600rpm, 10min) 30 micron

filter

Freeze dry

Spirulina biomass

Spirulina crude extract

Total Lipid Extraction:

Filter

Centrifuge

Dry

0.25g crude extract

5mL distilled water

Cell Lysis Chloroform Extraction

Vortex and Centrifugation

Hexane Extraction

Vortex and Centrifugatio

n

Extraction procedure

5mL chloroform

Centrifuge(4600rpm, 10min)

5mL hexane

40 Hz

CH3OH/NaOH

CH3OH/NaOH

Fatty Acid Methyl Esterification: Standard Method

Firestone, D. Official method Ce 1b-89. In Official Methods and Recommended Practices of the American Oil Chemists’ Society, 4th ed.; Firestone, D., Ed.; AOCS Press: Champaign, IL, 1994.

Alkaline hydrolysis of lipids

Acidic transmethylation:BF3-MeOH catalyst

Phase separation:NaCl

Drying

GC injection: GC-FID

and GC-MS

Lipid Sample0.5 M sodium hydroxide in methanol, at 100 °C for 10 min

5% (v/v) BF3 in methanol (at 100 °C for 2 min)

isooctane (at 100 °C for 30 min).

Na2SO4; rotavap

FAME

lipid

glycerol

FAME

fatty acids

fatty acids

Gas Chromatographic Conditions Instrument: GC-14B (SHIMADZU) GC Column: DB-5(J&W Scientific, 30m×0.25mm

I.D. df=0.25µm) 5% phenyl silicone-methyl silicone --- Slightly

polar

0 5 10 15 20 25 30 35 40 45 500

50

100

150

200

250

300

Temperature program

time, min

tem

pera

ture

, °C

• Duration of run: 50 min• 100°C (2min), 100 – 140°C (5°C/min), 140-200°C

(3°C/min), 200-280°C (8°C/min), 280°C (10min)• Internal standards:

• Undecanoic acid, C11

• Heptadecanoic acid, C17

Parameter

Literature

Control (1A)

young CW (1B)

mature CW (1C)

pH11 (2)

Low int.(3)

Total biomass,

g/L

1.35~7.51* 1.24 2.11 2.15 1.03 0.51

Total Lipid, %

7.72** 9.21 6.28 7.10 7.08 6.60* Soundarapandian & Vasanthi 2008** USDA

Spirulina Parameters compared with Literature

Low intensity

pH 11

Mature CW

Young CW

Control

Literature

0.00 0.50 1.00 1.50 2.00 2.50

Total biomass (g/L)

Total biomass (g/L)

Sam

ple

Soundparandian and Vasanthi (2008)

Low intensity

pH 11

Mature CW

Young CW

Control

Literature

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

Total Lipids (%)

Total Lipid (%)

Sam

ple

USDA

8 13 18 23 28 33 38 43 48-1000

9000

19000

29000

39000

49000CONTROL

Retention Time, min

Inte

nsit

y

1-C11, undecanoic acid (IS)2-C12:0, lauric acid3-C14:0, myristic acid4-C16:0, palmitic acid5-C17, heptadecanoic acid (IS)

6-C18:3, gamma-linolenic acid7-C18:2, linoleic acid8-C18:1, oleic acid9-cis-13-C18, stearic acid10-C18:0, stearic acid

11-hydrocarbons (~C40-C56)

1

2

3

4

56

7,8,9,10 11

C10 C12 C14 C16 C18 C18:1 C18:2 C18:30.00%

10.00%

20.00%

30.00%

40.00%

50.00%

60.00%

70.00%

%Fatty Acids

Control Young CW Mature CWpH11 Low intensity

Conclusion

High initial pH (at pH 11) and low light intensity (12W/m2) are not good parameters for Spirulina platensis production.

Use of coconut water as additive to the culture: • Increases biomass by >50%• gives high % Lipid• Young CW produced higher amount of GLA

Fatty acid profile of the Spirulina shows a significant amount poly-unsaturated fatty acid GLA, gamma-linolenic fatty acid, C18:3

Recommendations

Utilize the addition of coconut water in the Spirulina medium.

Investigate on other parameters: Illumination conditions

Photoperiod Light frequency

Reference Belay, A. 1997. Mass culture of Spirulina outdoors. –The Earthrise Farms experience. In: Vonshak, A., Ed. The effects of irradiance and photoperiod on the growth

rate of three freshwater green algae isolated from a eutrophic lake Radia Bouterfas, Mouhssine BelkouraAlainDauta. Spirulina platensis (Arthrospira): Physiology, cell-biology and biotechnology. Taylor and Francis. London. pp. 131-158.

Carr, N.G., 1988. Nitrogen reserves and dynamic reservoirs in cyanobacteria, p.13-39. In L.J. Rogers and J.R. Gallon (ed.), Biochemistry of the algae and cyanobacteria. Clarendon Press, Oxford, United Kingdom.

Danesi et. al. Growth and content of Spirulina platensis biomass chlorophyll cultivated at different values of light intensity and temperature using different nitrogen sources

Firestone, D. Official method Ce 1b-89. In Official Methods and Recommended Practices of the American Oil Chemists’ Society, 4th ed.; Firestone, D., Ed.; AOCS Press: Champaign, IL, 1994.

Fereidoon Shahidi and Ying Zhong. Lipid Oxidation: Measurement Methods. Memorial University of Newfoundland, St. John’s, Newfoundland, Canada. 2005

Hyun Yoon et. al. Evaluation of conversion efficiency of light to hydrogen energy by Anabaena variabilis. Seoul University, Seoul, Republic of Korea. 1 August 2005

LECO Corporation. Determination of Fatty Acid Methyl Esters by GCxGC-TOFMS; Saint Joseph, Michigan USA

Luciane Maria Colla, Telma Elita Bertolin, and Jorge Alberto Vieira Costa. Fatty Acids Profile of Spirulina platensis Grown Under Different Temperatures and Nitrogen Concentrations

Othes, S., and PIRE, R. 2001. Fatty acid composition of Chlorella and Spirulina microalgae species. J. AOAC Int. 84: 1708-1714. Morris, I. 1981. Photosynthetic products, physiological state and phytoplankton growth. In: Physiological bases of phytoplankton ecology. T. Platt (ed.). Can. Bull. Fish. Aquat. Sci., 210: 83-102

Peter, P. et al. Studies on the impact of Nitrogen Starvation on the Photosynthetic Pigments Through Spectral Preoperties of the Cyanobacteruim, Spirulina platensis: Identification of Target Phycobiliprotein under Nitrogen Chlorosis. Department of Biochemistry, Sri Venkateswara University, Tirupati-517502, A.P., India.

Ryckebosch, Eline. Extraction of lipids from microalgae:optimization of an analytical method. K.U.Leuven Campus Kortrijk Research Unit Food & Lipids. Belgium. July 2011

Vasuki and Subba Rao. Effect of light intensity, photoperiod, ESP medium and nitrogen sources on growth of marine brown alga Padina boergesenii (Dictyolaes, Phaeophyta). Marine Algal Research Station, Central Salt & Marine Chemicals Research Institute, Mandapam Camp- 623 519, Tamil Nadu, India. 12 September 2001.

Kalpesh K. Sharma, Holger Schuhmann and Peer M. Schenk. “High Lipid Induction in Microalgae for Biodiesel Production” 18 May 2012

http://bioweb.uwlax.edu/bio203/2011/fedor_kara/gallery.htm. Accesed on 3 August 2012.

http://www.sciencephoto.com/media/16234/enlarge. Accesed on 3 August 2012.

http://www.cyberlipid.org/ 7 September 2012 Accesed on 3 August 2012.

Image sources http://www.instrument.com.cn/netshow/SH101835/Q1233681.htm

http://www.vitacost.com/natures-way-spirulina-micro-algae Accesed on 3 August 2012.

wellsphere.com Accesed on 3 August 2012.

http://www.fishandfins.co.uk/algae-wafers.htm Accesed on 3 August 2012

http://www.herbwisdom.com/herb-spirulina.html Accesed on 3 August 2012

http://algaeforbiofuels.com/algae-based-biodiesel-current-procedures-and-innovations/ Accesed on 3 August 2012

http://technicolorboogie.wordpress.com/2009/05/08/viral-sephcation-whhaaaa/ Accesed on 3 August 2012

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http://www.igv-biotech.de/mikroalgen-vielfalt.html

http://www.auroville.org/health/food/spirulina.htm

http://www.auroville.org/health/images/spirulina_9.jpg

Acknowledgements

Mr. Elvis T. Chua, and Ms. Maria Krishna D. de Guzman

Philippine Department of Agriculture – Bureau of Agricultural Research (DA-BAR) for funding the research

Ateneo de Manila University for the research and faculty grants

Philippine Institure of Pure and Applied Chemistry for the GC-MS analysis

Alsons’ Aquaculture c/o Mr. Ramon Macaraig for the Arthrospira platensis culture stock.

Fatty Acid Profile of Spirulina platensis Grown Under Different Culture Conditions

Franklin T. Ayran

Mentors: Fabian M. Dayrit, Ph.D.

Rene Angelo S. Macahig, Ph.D.

Fatty Acid Profile of Spirulina platensis Grown Under Different Culture Conditions

Franklin T. Ayran

Mentors: Fabian M. Dayrit, Ph.D.

Rene Angelo S. Macahig, Ph.D.

Spirulina MediumComponent Stock

Solution QuantityMolar

Concentration in Final Medium

Solution I 500 mL --- ---NaHCO3 --- 13.61 g 1.62 x 10-4 MNa2CO3 --- 4.03 g 3.80 x 10-5 MK2HPO4 --- 0.50 g 2.87 x 10-6 MSolution II 500 mL --- ---NaNO3 --- 2.5 g 2.94 x 10-5 MK2SO4 --- 1.0 g 5.74 x 10-6 MNaCl --- 1.0 g 1.71 x 10-5 MMgSO4 7H2O --- 0.2 g 8.11 x 10-7 MCaCl2 2H2O --- 0.04 g 2.72 x 10-7 MFeSO47H2O --- 0.01 g 3.60 x 10-8 MNa2EDTA 2H2O --- 0.08 g 2.15 x 10-7 Mtrace metals solution See next table 1 mL ---

Aiba and Ogawa 1977, Schlösser 1994. Spirulina Medium (modified)

Trace Metal Solution

Component Primary Stock Solution Quantity

Molar Concentration

in Final Medium

Na2EDTA 2H2O --- 0.8 g 2.15 x 10-6 M

FeSO4 7H2O --- 0.7 g 2.52 x 10-6 M

ZnSO4.7H2O 1.0 g L-1 dH2O 1 mL 3.48 x 10-9 M

MnSO4 7H2O 2.0 g L-1 dH2O 1 mL 8.97 x 10-9 M

H3BO3 10.0 g L-1 dH2O 1 mL 1.62 x 10-7 MCo(NO3) 2 6H2O 1.0 g L-1 dH2O 1 mL 3.44 x 10-9 M

Na2MoO4 2H2O 1.0 g L-1 dH2O 1 mL 4.13 x 10-9 M

CuSO4 5H2O 0.005 g L-1 dH2O 1 mL 2.00 x 10-11 M

Figure 1. Lipid induction in microalgae under stress condition.

Kalpesh K. Sharma, Holger Schuhmann and Peer M. Schenk. “High Lipid Induction in Microalgae for Biodiesel Production” 18 May 2012

High initial pH resulted in slightly lower growth.

Initial results: Effect of initial pH

pH9

pH11

Final pH values converge at ~pH10.5.

C10 C12 C14 C16 C18 C18:1 C18:2 C18:30%

5%

10%

15%

20%

25%

30%

35%

40%

45%

50%

%Fatty Acids

Literature Control Young CWMature CW pH11 Low intensity

Questions to ask

Ask for GC-MS graph and the peak identification

How do spirulina make fatty acids and lipids

pH of the controlled spirulina medium

How do the media (chemical components), and light intensity affects

Typically what is the pH level of the medium

How to determine if GLA and not ALA?

WHY can’t we quantify with the GC and GC-MS?