PRODUCTION OF MICROALGAE BIOMASS (Scenedesmus almeriensis) IN A FARMER GREENHOUSE

Emilio Molina Grima Dpto. Ingeniería Química Universidad de Almería

E-mail: [email protected] Dpt. Chemical Engineering, Univ. Almería, SPAIN

1

1) Microalgas: caracterización y particularidades

Microorganismos unicelulares fotoautotróficos

Microalgas

Más microalgas

LUZ Nutrientes, agua

CO2

O2 Metabolitos

ƒ Microorganismos (diferencia con las macroalgas) ƒ Gran velocidad de duplicación por ser microorganismos ƒ Fotótrofos (aunque flexibles) : su fuente de energía es la luz ƒ Autótrofos: Su fuente de carbono es el CO2

Dpt. Chemical Engineering, Univ. Almería, SPAIN

2

1) Microalgas: caracterización y particularidades

Biomasa de composición compleja: componentes de interés ƒƒ Proteínas Proteínas yy otros otros nutrientes: nutrientes: alimentación alimentación humana humana yy piensos piensos para para ganado ganado ƒƒ Capacidad Capacidad quelante: quelante: biorremediación biorremediación ƒƒ Ácidos Ácidos grasos grasos poliinsaturados poliinsaturados ƒƒ Clorofilas Clorofilas ƒƒ Carotenoides Carotenoides ƒƒ Enzimas Enzimas antioxidantes antioxidantes (SOD) (SOD) ƒƒ Pigmentos Pigmentos fluorescentes fluorescentes ƒƒ Exopolisacáridos Exopolisacáridos ƒƒ Biotoxinas Biotoxinas marinas marinas ƒƒ Compuestos Compuestos bioactivos: bioactivos: antifúngicos, antifúngicos, antivirales, antivirales, citotóxicos citotóxicos ƒƒ ACUICULTURA ACUICULTURA

Componentes Componentes de de gran gran interés interés en en comparación comparación con con otras otras biomasas biomasas de de origen origen vegetal vegetal (plantas (plantas terrestres terrestres o o macroalgas) macroalgas) Dpt. Chemical Engineering, Univ. Almería, SPAIN

3

1) Microalgas: caracterización y particularidades Protoceratium Protoceratium reticulatum reticulatum

Diversidad de especies ƒƒ Muchas Muchas especies especies catalogadas catalogadas yy disponibles disponibles ƒƒ Sólo Sólo unas unas pocas pocas estudiadas estudiadas yy aprovechadas aprovechadas comercialmente comercialmente ƒƒ Gran Gran potencialidad potencialidad de de productos productos yy aplicaciones aplicaciones

Haematococcus Haematococcus pluvialis pluvialis

Dunaliela Dunaliela salina salina

Dpt. Chemical Engineering, Univ. Almería, SPAIN

Phaeodactylum Phaeodactylum tricornutum tricornutum

4

1) Microalgas: caracterización y particularidades

Diversidad de especies Isochrysis Isochrysis galbana galbana

Anabaena. Anabaena.

Phorphyridium Phorphyridium cruentum cruentum

Skeletonema Skeletonema costatum costatum

Dpt. Chemical Engineering, Univ. Almería, SPAIN

Chlorella Chlorella sp. sp.

Tetraselmis Tetraselmis suecica suecica

5

2) Sistemas de cultivo y producción a gran escala

Sistemas abiertos: open ponds y raceways ƒƒ Cultivo Cultivo de de Spirulina Spirulina 9 9 Biomasa Biomasa rica rica en en proteína proteína 9 9 Crece Crece aa pH pH muy muy alto alto 9 9 Resistente Resistente aa condiciones condiciones agresivas agresivas

ƒƒ Cultivo Cultivo de de Dunaliella Dunaliella salina salina 9 9 Producción Producción de de β-caroteno β-caroteno 9 9 Halotolerante Halotolerante 9 9 Luminosidad Luminosidad yy salinidad salinidad favorecen favorecen el el proceso proceso

D. salina en open ponds Dpt. Chemical Engineering, Univ. Almería, SPAIN

D. salina en raceways 6

Dpt. Chemical Engineering, Univ. Almería, SPAIN

7

Objective

To study the business possibilities that may offer the tubular photobioreactor technology under a farmer greenhouse, as those existing in Almería, South Spain, to produce algal biomass.

Dpt. Chemical Engineering, Univ. Almería, SPAIN

8

Starting-up

• Discovery of new strain, Scenedesmus almeriensis. • Local bloom: adapted to environment • Extraordinary producer of Lutein (and Zeaxantin) • Clean carotenoid profile

Dpt. Chemical Engineering, Univ. Almería, SPAIN

9

1.- S. almeriensis and the interest of lutein: the new strain

Scenedesmus almeriensis CHARACTERIZATION: rDNA analysis Prof. Thomas Friedl Exp. Phycol. Cult. Collect. Algae SAG, Gottingen 37073 Germany • 18S rDNA and ITS rDNA sequencing was employed • Sequences deviated from the most closely related species by 11 sequences position in the 18S rDNA exon region and in the two group I introns • The new strain Scenedesmus almeriensis has been deposited in the Culture Collection of Algae and Protozoa (CCAP) code CCAP 276/24

TITULO: Nueva especie de microalga y su aplicación para consumo animal, humano y en la obtención de carotenoides NÚMERO DE PATENTE: Solicitud Nº P200500374 FECHA: 5 de Febrero de 2005 SOLICITANTE: Cajamar, Universidad de Almería.

Dpt. Chemical Engineering, Univ. Almería, SPAIN

10

1.- S. almeriensis and the interest of lutein: LUTEIN

• An adequate intake of this product might help to prevent or ameliorate the effects of degenerative human diseases, such as age-related macular degeneration (AMD) • Supplements containing lutein enriched extracts are usually prescribed for these patients in order to supply the recommended daily intake of lutein (6 mg/day) • Using microalgal biomass makes possible to formulate lutein complements with only 1 g of dry biomass, that supply the recommended daily dose of lutein •Potential market of lutein is around 90 millions people in the world and increasing

Lutein is the major carotenoid present in the biomass of Scenedesmus almeriensis Dpt. Chemical Engineering, Univ. Almería, SPAIN

11

1.- S. almeriensis and the interest of lutein: OTHER SOURCES Comparison with commercial sources of lutein

Comparison with dietary sources of lutein

Species

Free lutein (mg/100 g)

Mono/diest ers (mg/100g)

Total lutein (mg/100 g)

Source food

Lutein content (mg/100g)

Kale

38.5

Tagetes patula

3.6

128.8

132.4

Spinach

12.2

Tagetes erecta

1.2

67.2

68.4

Cress

12.1

Calyces (mean)

0.38

4.29

4.67

Chard

11.9

0

569

569

Collard

8.9

Champion orange (T patula)

S. almeriensis

600*

Mixed species (T. erecta)

2.8

137.3

140.1

S almeriensis

-

-

600*

*Average content

Digestibility and effective absorption of most dietary sources is unknown and may vary with patient, clinical condition and food elaboration.

*Preliminary data

Piccaglia et al (1998) “Lutein and lutein ester content in different types of Tagetes patula and T. erecta” Industrial Crops and Products, 8, 45-51

Lutein content of Scenedesmus almeriensis greatly overpasses the dietary and commercial sources of this compound Dpt. Chemical Engineering, Univ. Almería, SPAIN

12

1.- S. almeriensis and the interest of lutein: OTHER SOURCES Microalgal sources of lutein Microalgae

Content (mg/100g)

Lutein productivity and conditions

Chlorella zofigiensis

342

Laboratory scale.

Muriellopsis sp

430

Outdoors 50 L external tubular photobioreactor 170 mg/m2 day

Chlorella protothecoides

535

Heterotrophic, laboratory scale, productivity 49 mg/L day

Scenedesmus almeriensis

600*

Outdoors 4000 L external tubular photobioreactor 386.66 mg/m2 day a

*Average content

referred to the land area shaded by the tubes

Del Campo et al., (2000) J. Biotechnol. 76, 51–59 Del Campo et al., (2001) J. Biotechnol. 85, 289-295 Shi et al. (2002) Biotechnol. Prog. 18, 723-727

In addition to Scenedesmus almeriensis, very few other microalgae strains has been proposed as lutein producers, S. almeriensis being the most promising of them Dpt. Chemical Engineering, Univ. Almería, SPAIN

13

1.- S. almeriensis vs. Marigold

Yield of Marigold vs S. almeriensis Marigold: S. almeriensis:

Biomass

4 harvest/yr, best conditions for commercial cultures (Bosma et al. 2003) 4000 L tubular photobioreactor, occupied land, in greenhouse, current productivity (not optimized)

Marigold:

1200 kg dry petals/yr = 480 g/m2 yr

S. almeriensis:

Lutein

18000 g/m2 yr

Marigold:

22 Kg/Ha yr

S. almeriensis:

1411 Kg/Ha yr

Bosma et al. (2003) “Optimizing marigold (Tagetes erecta) petal and pigment yield” Crop Science, 43

The yield of Scenedesmus almeriensis greatly overpasses the current sources of lutein Dpt. Chemical Engineering, Univ. Almería, SPAIN

14

2.- Characterization of the new strain: Growth model (µ vs Iav) 0.10

n μ max ⋅ I av μ= n n I k + I av

0.09

Growth rate, 1/h

0.08 0.07 0.06 0.05 0.04 0.03

µmax=0.091 1/h

0.02

n=1.8

0.01 0.00 0

50

100

150

200

250

300

350

400

Ik=70 µE/m2s

Iav, µE/m2s

Sanchez et al., (2005) “Characterization of the new strain Scenedesmus almeriensis and potential applications” 6th European Workshop of Microalgal Biotechnology

• The maximum growth rate is high, 0.09 1/h • Ik value is low, 70 µE/m2s, thus indicating a high efficiency of light utilization

The growth rate was a function of average irradiance inside the culture, no influence of external irradiance was observed Dpt. Chemical Engineering, Univ. Almería, SPAIN

15

2.- Characterization of the new strain: Biochemical profile

C = 47.38 %C H = 6.47 %H N = 7.72 %N S = 0.53 %S Proteins = 48.3 %d.wt. Lipids = 10.0 %d.wt. 18:3n3 = 1.78 %d.wt. 18:2n6 = 1.60 %d.wt.

Lutein up to 1.0 %d.wt. • • • • •

Fuente ventajosa Elevado contenido en luteína Pureza elevada Buena digestibilidad (preliminar) Obtención de luteína purificada factible

Lutein is the major carotenoid. Lutein content increased with the dilution rate, irradiance and extreme temperatures Dpt. Chemical Engineering, Univ. Almería, SPAIN

16

3.- Design and setup of the industrial-size photobioreactor

•

Inside a greenhouse

•

Type: tubular, double loop, light captation optimized

•

Hidrodynamic design for light integration regime

•

Enhanced heat and mass transfer

•

Self cleaning, long-term operation

Dpt. Chemical Engineering, Univ. Almería, SPAIN

17

LOCATION: El Ejido, Almería (South of Spain)

SPAIN Almería

El Ejido

Dpt. Chemical Engineering, Univ. Almería, SPAIN

Longitude : 2º 43’ W Latitude : 36º 48’ N Altitude : 155 m 18

3.- Design and setup of the industrial-size photobioreactor Location: El Ejido, Almería (Southern Spain)

• Irradiance inside the greenhouse is 35-40% lower than outdoor • All the solar radiation inside the reactor is disperse radiation due to the composition of the plastic cover used •The mean daily temperature inside the greenhouse is similar to the exterior, although the maximum temperature inside the greenhouse is 3-5 ºC higher than outdoor

Objective: To design, calculate, setup and operate an industrial size photobioreactor for the production of Scenedesmus almeriensis inside a greenhouse Dpt. Chemical Engineering, Univ. Almería, SPAIN

19

3.- Design and setup of the industrial-size photobioreactor Photobioreactor design principles Design and orientation

Geometry

Day of the year

Geographic and climatic localization

Incident solar radiation

Temperature

Light profile, average irradiance

Mass transfer

Light regime Growth rate

Biomass concentration Biomass Productivity

BIOPRODUCT

Fluid-dynamic

Biochemical composition Bioproduct productivity

Harvesting Downstream

Molina et al., (1999) “Photobioreactors: light regime, mass transfer, and scaleup” Journal of Biotechnology, 70, 231-248.

First, decide geometry and design to optimize the irradiance on the reactor surface, then work out adequate fluid-dynamics for heat, mass transfer and light integration Dpt. Chemical Engineering, Univ. Almería, SPAIN

20

3.- Design and setup of the industrial-size photobioreactor Airlift system, heat and mass transfer and liquid velocity

Tube diameter

Pb v = μ ⋅ C b μ max ⋅ I μ= n Ik + I

n av n av

Iav =

Io

φeq

(1−exp(−φ ⋅K ⋅C ))

φeq =

eq

a

b

dt Cos (θ )

π ⋅ dt Pb a = Pb v 4 ⋅ nT

UL

⎛ ⎜ g ⋅ ε r ⋅ hr =⎜ ⎜ ⎛ µL ⎜ ⎜ 0 .3 1 6 ⋅ ⎜ ρ ⎝ ⎝

⋅d ⎞ ⎟ ⎠

1 .2 5 t 0 .2 5

⋅ L eq

⎞ ⎟ ⎟ ⎟ ⎟ ⎟ ⎠

k L k L ⋅ aL (1 − ε r ) = db 6 ⋅εr

β

4/7

εr =

λ+

ub ug + uL

Q = UAΔT Nu = 0.023 Re 0.8 Pr 0.3

External loop, length and liquid velocity

u L ([O 2 ]in − [O 2 ]out ) L= RO 2

Acién et al., (2001) “Airlift-driven external-loop tubular photobioreactors for outdoor production of microalgae: assessment of design and performance” Chemical Engineering Science, 56, 2721-2732.

Equations relating the growth parameters of the microorganism and fluid-dynamic or mass transfer requirements have been previously reported. Dpt. Chemical Engineering, Univ. Almería, SPAIN

21

3.- Design and setup of the industrial-size photobioreactor BASE DESIGN: Two plane, tubular airlift photobioreactor Gas exhaust Cooling water

Medium

Harvest

AIR Probes CO2

Torzillo et al., (1993) “A two plane tubular photobioreactor for outdoor culture of Spirulina” Biotechnology and Bioengineering, 42, 891-898.

The airlift tubular photobioreactor design is selected, with a two-level external loop configuration. Dpt. Chemical Engineering, Univ. Almería, SPAIN

22

3.- Design and setup of the industrial-size photobioreactor External loop: optimizing light capture

18.0 m

0.30 m 0.10 m 0.30 m

4.5 m Acién et al., (2001) “Airlift-driven external-loop tubular photobioreactors for outdoor production of microalgae: assessment of design and performance” Chemical Engineering Science, 56, 2721-2732.

The optimal configuration of the two plane external loop allows to maximize the irradiance on the reactor surface. Dpt. Chemical Engineering, Univ. Almería, SPAIN

23

INDUSTRIAL SCALE PHOTOBIOREACTOR FINAL DESIGN: Frame for the xternal loop

0.10 m

0.10 m

0.10 m

0.10 m

20.0 m

1.25 m

1.25 m

1.25 m

4.0 m Frames and accessories for the setting-up of the external loop of the reactor were designed and installed Dpt. Chemical Engineering, Univ. Almería, SPAIN

24

INDUSTRIAL SCALE PHOTOBIOREACTOR FINAL DESIGN: Airlift system and inoculum bubble columns AIRLIFT SYSTEM

INNOCULUM BUBBLE COLUMN

INTERNAL HEAT EXCHANGERS

0.10 m 0.30 m

4.00 m

3.00 m

0.25 m

0.25 m 3.00 m

0.60 m 0.30 m 0.40 m 0.40 m

0.40 m

0.40 m 0.20 m

Frames and accessories for the setting-up of the inoculum columns and airlift system were designed and installed, as well as the internal heat exchangers for cooling Dpt. Chemical Engineering, Univ. Almería, SPAIN

25

INDUSTRIAL SCALE PHOTOBIOREACTOR SETUP: Build-up

• Ground of the greenhouse was covered with a white plastic sheet to increase the irradiance on the reactor surface

The reactor was set-up inside the greenhouse in two months Dpt. Chemical Engineering, Univ. Almería, SPAIN

26

INDUSTRIAL SCALE PHOTOBIOREACTOR SETUP AND TEST: Hidraulic test

The reactor was setting-up inside the greenhouse in two months Dpt. Chemical Engineering, Univ. Almería, SPAIN

27

INDUSTRIAL SCALE PHOTOBIOREACTOR SETUP AND TEST: Medium and harvest operations

• Medium is prepared on-line by adding a salts stock to the water flow entering the reactor. • Medium is sterilized by 0.2 µm filtration carried out in 5 steps. • Biomass is harvested by centrifugation • Operation of the reactor is AUTOMATICALLY performed.

Accessories facilities were set-up and operated Dpt. Chemical Engineering, Univ. Almería, SPAIN

28

3.- Design and setup of the industrial-size photobioreactor

c,d 2 a,b

3

1

4

1. 2. 3. 4.

Instrumentation: DO2, pH, Temp Innoculum bubble columns Airlift system External loop

a. b. c. d.

Air flowmeter 0-800 L/min CN CO2 flowmeter 0-5 L/min CN Air flowmeter 0-30 L/min CN CO2 flowmeter 0-1 L/min CN

Colour lines: Red, carbon dioxide Blue, water-medium Black, compressed air Green, harvest

The inoculum bubble columns and reactor were distributed in the plant. Dpt. Chemical Engineering, Univ. Almería, SPAIN

29

3.- Design and setup of the industrial-size photobioreactor PLANT LAYOUT: Medium preparation and harvesting waste

5

4

f e

d a

a

9

1 11

b

10

c b

d

7 b

2

8

d 3 h 6

g

a. b. c. d. e. f. g. h.

PVC 50 mm PVC 25 mm PVC 25 mm PVC 25 mm Poliamida 8 mm red Poliamida 8 mm black Electric 3*1.5 mm Electronic, 4-20 mA

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

Filtration: 0.2 µm, manometers Medium tank: 1.5 m3 Harvest tank: 1.5 m3 CO2 Bottles: 8 on two matrix Compressor: 700 L/min CN Electricity supply: 220 v, 16 A Continuous centrifugation Waste tank Prefiltration Mass flowmeter Flowmeter

Colour lines: Red, carbon dioxide Blue, water-medium Black, compressed air Green, harvest Brown, waste

Medium preparation, accessories and harvesting facilities were distributed in the control room Dpt. Chemical Engineering, Univ. Almería, SPAIN

30

2) Sistemas de cultivo y producción a gran escala

Sistemas tubulares horizontales

Dpt. Chemical Engineering, Univ. Almería, SPAIN

31

3.- Design and setup of the industrial-size photobioreactor

Reactor: Reactor:

Volume=4.0 Volume=4.0 m m33 Self-cleaning Self-cleaning system system pH cotrol by CO pH cotrol by CO22 injection injection Temp. Control: recirculating Temp. Control: recirculating water water from from pool pool Liquid Liquid velocity=0.3 velocity=0.3 m/s m/s

External External loop: loop:

Length=400 Length=400 m m Diameter=0.1 Diameter=0.1 m m Polymetilmetacrilate Polymetilmetacrilate Single Single loop, loop, two two levels levels Distance Distance between between tubes= tubes= 0.3 0.3 m m 2 2 Area Area occupied=81 occupied=81 m m (18x4.5) (18x4.5)

Airlift: Airlift:

Diameter=0.30 Diameter=0.30 m m Height=3.5 m Height=3.5 m Tubular Tubular heat heat exchanger exchanger Air Air flow=0-500 flow=0-500 L/min L/min

four months after the commencement of the project, the reactor was inoculated and the first culture was developed Dpt. Chemical Engineering, Univ. Almería, SPAIN

32

4.- Evaluation of the fotobioreactor and the production process AIRLIFT MODE: April-July 2004

a

Date

Dilution, Dilution, 1/h

Io, Io, µEm-2s-1

Iav, Iav, µEm-2s-1

DO2, %Sat. %Sat.

pH

Temp., °C

Cb, Cb, gL-1

Pb, Pb, gL 1day-1

Efficiencya, % global radiation

1414-April

0.03

581

114

272.0

8.25

24.3

0.99

0.26

1.3

2525-April

0.03

836

105

244.4

8.03

24.0

1.44

0.38

1.7

2-May

0.03

582

117

251.9

8.24

20.8

0.88

0.18

1.7

1111-May

0.04

650

203

194.0

7.84

22.6

0.63

0.23

1.0

2626-Jun

0.02

540

59

369.9

8.02

29.5

1.64

0.39

1.2

1212-Jul

0.03

578

75

262.8

8.27

30.6

1.39

0.50

1.7

2020-Jul

0.05

550

98

191.3

8.30

30.1

0.93

0.56

2.5

Related to the total land area occupied by the photobioreactor (81 m2)

• Liquid velocity 0.32 ms-1 • The culture conditions were not correctly controlled • Excessive dissolved oxygen accumulation • Irradiance inside the greenhouse was low • Biomass productivities of 0.56 gL-1day-1 were measured, with global solar efficiencies of 2.5 % with respect to solar irradiance inside the greenhouse (1.5% with respect to solar irradiance outdoor)

When the reactor was operated as airlift, both LOW biomass productivities and global solar efficiencies were measured Dpt. Chemical Engineering, Univ. Almería, SPAIN

33

5.- Overcoming difficulties

a)- Fixing liquid velocity

Indoor experiments

Centrifugal pump: Liquid flow rate = 9.0 L/min Repump = 65000 Shear rate = 1000 1/s Experiments carried out in continuous mode in two parallel photobioreactors

Outdoor experiments

Centrifugal pump: Liquid flow rate = 420 L/min Repump = 85000 Shear rate = 1350 1/s Experiments carried out in continuous mode in two consecutive steady-state

Centrifugal pump does not damage the cells at both indoor or outdoor conditions Dpt. Chemical Engineering, Univ. Almería, SPAIN

34

5.- Overcoming difficulties: system re-evaluation Centrifugal pump impulsion: October, 2004-May, 2005 Date 1111-octoct-04

Dilution, Dilution, 1/h 0.003

Io, Io, µEm-2s-1 468

Iav, Iav, µEm-2s-1 19

DO2, %Sat. %Sat. 209

7.99

2727-octoct-04

0.029

332

28

182

7.83

23.3

2.15

0.74

6.7%

13.4%

4.3%

1111-novnov-04

0.043

296

42

254

8.19

21.4

1.27

0.66

7.2%

14.4%

4.6%

2222-novnov-04

0.050

322

62

223

8.16

18.9

0.96

0.58

5.3%

10.6%

3.4%

2828-novnov-04

0.043

230

41

259

8.09

19.3

0.89

0.44

8.0%

16.0%

5.1%

8-janjan-05

0.042

295

36

224

8.36

18.0

1.40

0.68

7.3%

14.6%

4.7%

2323-janjan-05

0.042

314

36

210

8.12

19.5

1.44

0.69

7.5%

15.1%

4.8%

7-febfeb-05

0.042

337

43

206

7.81

21.1

1.39

0.67

6.3%

12.5%

4.0%

2020-febfeb-05

0.042

359

41

207

8.02

22.7

1.58

0.76

9.0%

17.9%

5.7%

7-MarMar-05

0.042

408

43

211

8.04

23.3

1.72

0.83

7.2%

14.4%

4.6%

2323-MarMar-05

0.042

478

42

226

8.32

27.3

1.97

0.95

6.3%

12.5%

4.0%

7-aprapr-05

0.042

523

40

236

8.13

26.8

2.18

1.05

5.9%

11.7%

3.8%

1616-aprapr-05

0.042

631

44

258

8.05

28.4

2.41

1.16

5.2%

10.3%

3.3%

1212-maymay-05

0.048

570

52

285

8.02

23.1

2.30

1.10

4.1%

8.2%

2.7%

pH

a

Temp., Cb, Pb, Eficiencya, Eficiencyb, Eficiencyc, Cb, Pb, 1 1 1 °C g L gL day % global % global % global 28.1 4.16 0.32 2.0% 4.0% 1,3%

Related to the total land area occupied by the photobioreactor (80 m2) b Related to the transversal area occupied by the tubes (40 m2) c Related to the total area of the tubes (125 m2)

When the reactor was operated with the centrifugal pump HIGH biomass productivities and global solar efficiencies were measured Dpt. Chemical Engineering, Univ. Almería, SPAIN

35

5.- Overcoming difficulties: system re-evaluation Comparison: centrifugal pump vs. airlift impulsion AIRLIFT MODE: Date

Dilution, Dilution, 1/h 140.03 14-April 250.03 25-April 2-May 0.03 110.04 11-May

Io, Io, µEm-2s-1 581 836 582 650

Iav, Iav, µEm-2s-1 114 105 117 203

DO2, %Sat. %Sat. 272.0 244.4 251.9 194.0

pH 8.25 8.03 8.24 7.84

Temp., °C 24.3 24.0 20.8 22.6

Cb, Cb, gL-1 0.99 1.44 0.88 0.63

Pb, Pb, gL-1day-1 0.26 0.38 0.18 0.23

Efficiency, Efficiency, % global radiation 1.3 1.7 1.7 1.0

CENTRIFUGAL PUMP MODE: Date

Dilution, Dilution, 1/h

Io, Io, µEm-2s-1

Iav, Iav, µEm-2s-1

DO2, %Sat. %Sat.

pH

Temp., °C

Cb, Cb, g L-1

Pb, Pb, gL-1day-1

Eficiency, Eficiency, % global

7-April

0.042

523

40

236

8.13

26.8

2.18

1.05

5.9%

1616-April 1212-May

0.042 0.048

631 570

44 52

258 285

8.05 8.02

28.4 23.1

2.41 2.30

1.16 1.10

5.2% 4.1%

When the reactor was operated with the centrifugal pump HIGH biomass productivities and global solar efficiencies were measured Dpt. Chemical Engineering, Univ. Almería, SPAIN

36

5.- Overcoming difficulties: system re-evaluation

600

1.2

500

1.0 400 0.8 300 0.6 200

0.4

100

0.2 0.0 0

100

200

300

400

500

0 600

Air flowrate, L/min

Removing PLASTIC COVER? 1.40 Biomass productivity, g/Lday

1.4

Solar irradiance, µE/m2s

Biomass productivity, g/Lday

Improving PBR MASS TRANSFER

•Mass transfer determines the yield of the system •Minimmum air flowrate of 300 L/min (0.1 v/v/min) is neccesary •Maxium biomass producitvity of 1.2 g/Lday was measured

1.20 1.00 0.80 0.60 0.40 0.20 0.00 0%

20%

40%

60%

80%

100%

120%

Transparency, %

•Solar irradiance determines the yield of the system •No enhancing of the productivity was observed under direct solar irradiance •Covers with minimum light transparency of 65% are required. Dpt. Chemical Engineering, Univ. Almería, SPAIN

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5.- Overcoming difficulties: system re-evaluation CENTRIFUGAL PUMP MODE: October 2004------------

• Lutein content varied from 0.44 to 0.98 %d.wt. •lutein productivities of 8 mg/L day or 386.66 mg/m2 day were measured

Dpt. Chemical Engineering, Univ. Almería, SPAIN

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5.- Overcoming difficulties: system re-evaluation Predicted values for D=0.045 h-1 3.5

Biomass concentration, g/L

3.0

• The predicted biomass concentration varied from 1.3 gL-1 to 3.2 gL-1, during winter and summer respectively.

2.5

2.0

1.5

• The predicted biomass productivity ranged from 0.6 gL-1day-1 to 1.4 gL-1day-1, during winter and summer respectively.

1.0

0.5

0.0 1-Jan

15-Feb 31-Mar 15-May 29-Jun Date

13-Aug 27-Sep

11-Nov 26-Dec

• The expected mean annual biomass productivity is 1.1 gL-1day-1 (60 gm-2day-1)

1.6

Biomass productivity, g/Lday

1.4

• The estimated mean lutein content of the biomass is 0.65 %d.wt., providing a mean annual lutein productivity of 8 mg L-1 day-1 (388 mg m-2 day-1)

1.2 1.0 0.8 0.6

• Experimental values obtained using the centrifugal fit the predicted values

0.4 0.2 0.0 1-Jan

15-Feb

31-Mar

15-May

29-Jun Date

13-Aug

27-Sep

11-Nov

26-Dec

The expected mean annual biomass productivity is 1.1 gL-1day-1 (60 gm-2day-1) Dpt. Chemical Engineering, Univ. Almería, SPAIN

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5.- Overcoming difficulties: system re-evaluation

Fotobiorreactores bajo plástico. • Tecnología probada • Ensayos preliminares en campo satisfactorios • Detalles técnicos resueltos (termostatación, mezcla, inoculación, medios, suministro CO2, etc) • Elevada productividad y eficiencia fotosintética • Gran estabilidad en la operación • Biomasa de calidad y composición conocida

Dpt. Chemical Engineering, Univ. Almería, SPAIN

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6.- Economic evaluation of the process. COST ANALYSIS: Considerations Lutein production capacity, mt/annun Lutein content of the biomass, mg lutein/100 g biomass Biomass production capacity, mt biomass/annun

1.00 650 154 0.050 1.076 477 285

Dilution rate, 1/h Pb g/Lday Culture volume, m3 Medium flow rate, m3/day Major Equipment List and Costs (€) Item 1. Photobioreactors (Plexiglas (Plexiglas)) 2. Centrifuge (24" bowl solids discharge, discharge, s.s.) 3. Medium filter unit 4. Medium feed pumps 5. Medium prep tank (SS) 6. Harvest broth storage (SS) 7. Centrifuge feed pumps 8. Air compressors 9. Harvest biomass conveyer belts 10. SpraySpray-dryer 11. Carbon dioxide supply station 12. Weight station 13. Biomass storage Total (2005 €)

Unit capacity 6.54 m3 22.7m3/h

Delivered cost No. of units total cost 4552 95 431345 120000 1 122369 18000 8 138600 6500 2 13000 100 m3 30000 4 120000 3 100 m 30000 2 60000 4550 2 9100 3 600 m /h 26266 4 105065 6000 1 6000 765 kg H2O/h 84000 3 254176 18000 1 18000 5000 1 5000 3 1.3 m 12000 3 36000 1318654

32,7% 9,3% 10,5% 1,0% 9,1% 4,6% 0,7% 8,0% 0,5% 19,3% 1,4% 0,4% 2,7% 100,0%

The major equipments are the photobioreactors, centrifuge and spray-dryers Dpt. Chemical Engineering, Univ. Almería, SPAIN

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6.- Economic evaluation of the process. ECONOMIC ANALYSIS: 80% 70%

Contribution of cost type to the total production cost

Percentage

60% 50% 40% 30% 20% 10% 0% Depreciation

Direct costs

Utilities

Labor/Supervision

Cost

Production cost of supply the daily recommended dosage of lutein

Biomass, Biomass, €/kg

15

Recommeded daily uptake, uptake, g

Cost of daily dosis, €/dosis

1.02

0.015

Extract of carotenoids, €/kg

2496

0.0088

0.018

Lutein purifyed, purifyed, €/kg

5125

0.0060

0.026

Average selling price at pharmacies

0.510

Labor and supervision represents the 70% of the total production cost Dpt. Chemical Engineering, Univ. Almería, SPAIN

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7.- Current situation

Proyecto de desarrollo tecnológico Datos más relevantes de la planta de demostración

•Volumen de cada reactor=3.2 m3 •Configuración tipo valla, alto 2.0 m, largo 40.0 m •Separación entre reactores 1.0 m •Superficie ocupada por reactor=40 m2 •Relación volumen/superficie=80 L/m2 •Volumen total=32.0 m3 •Superficie total de reactores=500 m2 •Superficie total necesaria=1000 m2 •Capacidad de producción=30 kg biomasa/día (10 Tm/año) •Producción de luteína=50 kg/año

Dpt. Chemical Engineering, Univ. Almería, SPAIN

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3) Biomoléculas de interés de origen microalgal

Proyecto Industrial: Instalación y puesta en marcha:

Dpt. Chemical Engineering, Univ. Almería, SPAIN

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3) Biomoléculas de interés de origen microalgal

ACUICULTURA Astaxantina EPA

Polisacáridos Alimentación

Biomasa DHA Biocombustibles (H2, bioetanol, etc.)

Luteína

Ficoeritrinas

Ficocianinas β-caroteno

Proteínas Biotoxinas

Dpt. Chemical Engineering, Univ. Almería, SPAIN

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