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Empleo de catalizadores magnéticos para la obtención de Biodiesel a partir de lípidos microalgales

Juan Luis Serra & María Jesús Llama Grupo de Tecnología Enzimática y Celular Facultad de Ciencia y Tecnología Universidad del País Vasco (UPV/EHU) [email protected]

Jornada Final de Presentación de Resultados Vitoria, 27 de Marzo de 2014

WP 2.4. Transformación enzimática de aceite de microalgas en Biodiesel

Resumen de actividades realizadas por la UPV/EHU desde el 1 de Abril de 2012 hasta Marzo de 2014


WP 2.4. Transformación en Biodiesel (UPV/EHU & CENER) ACTIVIDAD 2.3.1. Transformación por vía enzimática El objetivo de la actividad es la obtención de biodiesel por (trans)esterificación de aceite crudo de microalgas y/o de los ácidos grasos aislados del mismo. El aceite se extraerá por métodos convencionales (en disolventes orgánicos como n-hexano) o por vía húmeda y lo proporcionarán otros socios. Como catalizador se empleará lipasa comercial (CALB) que se inmovilizará en soportes magnéticos desarrollados en el laboratorio de la UPV/EHU.


Objetivos 1. Obtención y funcionalización de MNPs de magnetita y su caracterización estructural y

magnética (M1-M8) 2. Obtención y caracterización de CLEAs magnéticas (mCLEAs) de lipasas (M3-M8) 3. Obtención a microescala de biodiesel catalizada por lipasa inmovilizada en MNPs y por mCLEA y

optimización de las condiciones de esterificación con alcoholes (M3-M12) 4. Obtención de biodiesel en un reactor enzimático catalizada por lipasa inmovilizada en MNPs y

por mCLEAs (M13-M18) 5. Optimización de las condiciones y operación para obtener biodiesel en un reactor enzimático

(M18-M24)


Outline

1. Synthesis and characterization of MNPs

2. Use of lipase CALB

3. Obtainment of magnetic Biocatalysts (mCLEAs and MNP) of lipase

4. Characterization of magnetic Biocatalysts

5. Use of magnetic Biocatalysts to obtain Biodiesel

6. Stability and reutilization of mCLEAS

7. Conclusions


Lab Synthesis of MNPs

Morales et al. (1999) Chem. Mater. 11: 3058-3064


Reunión de consorcio Toulouse 22-23 de Noviembre 2012

Video of Lab Synthesis of MNPs


A drop of freshly prepared MNPs in suspension

(a) (b)

(d)

(c)

Dryed MNPs without a magnetic field

Dryed MNPs subjected to a magnetic field

Detail of (c)

Morphology of MNPs


At different enlargements

TEM Images of MNPs


NANO3.1 Information of: • Magnetite/maghemite composition • Crystallite size ≃10 nm

X-ray Diffraction Patterns of MNPs


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• Superparamagnetic character • No hysteresis (no remanence) • High saturation magnetisation • Size about 10 nm • Surface covered with amine groups • Easily pelleted with a magnet

Main Properties of MNPs


Superparamagnetic Properties of MNPs and mCLEAs

MNP-OH MNP-NH2

MNP-CALB mCLEA-CALB


Outline







7. Conclusions


Candida antarctica LIPASE B (CALB)

Enzyme Used

Lipozyme® CALB L commercially available from Novozymes


Outline







7. Conclusions


(A) Standard immobilization (MNPs) (B) Magnetic Cross-Linked Enzyme Aggregates (mCLEAs)

Enzyme Immobilization to MNPs

(A)


(A) Standard immobilization (MNPs) (B) Magnetic Cross-Linked Enzyme Aggregates (mCLEAs)

Enzyme Immobilization to MNPs

(B)

(A)


Outline







7. Conclusions


mCLEA-CALB

MNP-CALB

TEM Images of MNPs, MNP-CALB and mCLEA-CALB


MNP

Parameter Soluble MNPs mCLEAs

KM (mM) 1.7 4.5 1.9

VM (U/mg prot) 1416.2 18.3 36.1

VM (U/mg MNPs) ----- 0.2 7.2

Catalytic efficiency 833 4.1 19

Using p-nitrophenyl acetate as substrate

Properties of Soluble and Immobilized Lipase

MNPs mCLEAs

(A) Kinetic properties

Property MNPs mCLEAs

Maximum protein amount loaded?

Yes (up to25 µg/mg MNPs)

No Up to (200 µg/mg MNPs)

Stability Medium High

Steric problems Less probable More probable

Reusable Yes Yes

(B) Running properties


Outline







7. Conclusions


Palm oil80%

Soybean oil10%

Olive oil10%

Chlorella sp. Scenedesmus sp.

Real Oil Model Oil


Biodiesel Production Palmitic (C16:0)

23,6%

Stearic (C18:0)4,1%

Oleic (C18:1)52,1%

Linoleic (C18:2)12,4%

Linolenic (C18:3)7,8%

Palmitic (C16:0)28,2%

Oleic (C18:1)2,8%



Real FFAs Model FFAs mixture

TEKNALIA + CENER CENER UPV/EHU

UPV/EHU

mCLEAs

Alcohols

Methanol Ethanol

2-Propanol

Biodiesel

Olive Oil (Control)

Scenedesmus sp.

Chlorella sp.

Chlorella sp.

FAMEs FAEEs FAPEs

Biodiesel Production Reactions


From Microalgal Oil

From Microalgal FFAs

Transesterification

H H2O Free fatty acid Alcohol Ester (biodiesel) (FFA)

Esterification


Intelli-Mixer RM-2, Elmi Ltd.

Sonoreactor Hielscher UTR200

Radleys Carousel 12 Plus

Ultrasound stirring

Types of Stirring and Reactors Used in This Work

Rotatory mechanical stirring

Magnetic stirring

http://www.google.es/url?sa=i&rct=j&q=&esrc=s&frm=1&source=images&cd=&cad=rja&uact=8&docid=TmNWSn7-OPpfLM&tbnid=PgSxvQgHMe63UM:&ved=0CAYQjRw&url=http%3A%2F%2Fwww.pocdscientific.com.au%2Felmi_Intellimixer_RM-2_custom-rocker-mixer-shaker-blotter.php&ei=NbguU6qGKeHG0QWB_oBo&bvm=bv.62922401,d.d2k&psig=AFQjCNG59H7keofEjNAfX4sGtqj5Sr8vYA&ust=1395657105036771


TLC (Thin Layer Chromatography) HPLC (High Performance Liquid Chromatography)

Analytical Techniques

Waters™ Corporation, Milford, MA, USA. C18 column (5 µm, 4.6 × 250 mm, Tracer Lichrosorb RP18)

100% pure methanol (phase A) and a mixture of propanol:n-hexane (5:4 v/v) (phase B)

Eluting phase: n-hexane:ethyl acetate:acetic acid (90:10:1 v/v/v). Stained with 0.02% (w/v) Coomassie Blue R-350

Silica gel 60 coated plates (Merck, Darmstadt, Germany)

Palm oil80%

Soybean oil10%

Olive oil10%

Biodiesel


Real Microalgal Oil

Model Microalgal Oil


Biodiesel Production from Microalgal Oils Transesterification

Model Mixture of FFA (FFA_M): Palmitic: 28.19% Oleic: 2.83 % Linoleic: 22.32%

Linolenic: 46.66%

Samples Used for Biodiesel Production

Type Amount approx.

(g) Partner Extraction Date

Biodiesel conversion (%)

Temperature (˚C)

Time (h)

Oil_1 3 CENER Chlor:MetOH Jun 2012 nd 40 20

Oil_2 50-60 CENER Organic solvents Sep 2012 nd 40 42

Oil_3.1 0.27 TEKNALIA Subcritical Apr 2013 0? 30 24 Oil_3.2 0.12 TEKNALIA Subcritical Apr 2013 0? 30 24 Oil_3.2 0.17 TEKNALIA Subcritical Apr 2013 0? 30 24 FFA_1 1 CENER Organic solvents Feb 2013 61.2 30 24

FFA_2 1 CENER Organic solvents Mar 2013 70.4 30 24

FFA_M --- UPV/EHU --- Jan 2013 74.2 69.5

73.1

30 30 30

24 3 1

Analyzed by CENER

nd: not determined Sonoreactor


C: Orange-yellowish pigments (carotenoids) FAAE: Fatty acid alkyl esters (Biodiesel) TG: Triglycerides DG: Diglycerides MG: Monoglycerides FFA: Free fatty acids G: Greenish pigments (chlorophylls)

Reaction conditions: Crude microalgal oil (30 mg) dissolved in 100 µl n-Hexane and 400 µl 2-propanol incubated at 40ºC mCLEAs: 2 mg Reaction time: 20 and 42 h

0 (Control) 20 42

FAPEs (Biodiesel)

C

TG

FFA DG

MG

Biodiesel Production from Chlorella Oil

G

Reaction time (h)

Oil_1 obtained from CENER


Reaction conditions: 500 µmol (136 mg) FFAs Ethanol:FFAs = 10:1 2 mg mCLEAs 40ºC 20 and 42 h Rotatory stirring

C: Orange-yellowish pigments (carotenoids)

FAAE: Fatty acid alkyl esters (Biodiesel) TG: Triglycerides DG: Diglycerides MG: Monoglycerides FFA: Free fatty acids G: Greenish pigments (chlorophylls)

Reaction conditions: Crude microalgal oil (30 mg) or olive oil (15 mg) dissolved in 100 µl n-Hexane and 400 µl 2-propanol incubated at 40 and 50 ºC mCLEAs: 2 and 4 mg Reaction time: 42 h

FAPEs (Biodiesel)

C

TG

FFA DG

MG G

Oil_2 obtained from CENER

240° 250° 440° 450° 240°250° 440° 450° Cµ Cµ CO CO

Cµ: Control microalgae oil (0 h) CO: Control olive oil (0 h)



Calles: C1: Control de la R1 sin CLEAs 1: Muestra de la R1 C2: Control de la R2 sin CLEAs 2: Muestra de la R2 C3: Control de la R3 sin CLEAs 3: Muestra de la R3

C1 1 C2 2 C3 3

Condiciones de reacción: 24 h a 30˚C empleando 2 mg de CLEAs (lipasa magnética), con etanol 96% (v/v) (relación alcohol:aceite 30:1 mol:mol)

Reacción 1 (R1): Con 75 mg de Oil-3.1 (lote N2)



FAEEs: Fatty Acid Ethyl Esters FFAs: Free Fatty Acids

Oil_3 obtained from TEKNALIA

R1 R2 R3

FAEEs

FFAs




Stearic (C18:0)4,1%

Oleic (C18:1)52,1%




Oleic (C18:1)2,8%



Biodiesel


Model Mixture of FFAs

Scenedesmus sp.

Real Microalgal FFAs

Chlorella sp.


Biodiesel Production from Microalgal FFAs

H H2O Free fatty acid Alcohol Ester (biodiesel) (FFA)

Esterification

FAPEs: Fatty Acid Propyl Esters FAEEs: Fatty Acid Ethyl Esters FFAs: Free Fatty Acids

Reacción 1 (R1): Mezcla de FFA_M*

Calles: C1: Control de la R1 sin CLEAs 1: Muestra con 2-propanol de la R1 2: Muestra con etanol de la R1 C2: Control de la R2 sin CLEAs 3: Muestra con 2-propanol de la R2 4: Muestra con etanol de la R2 C3: Control de la R3 sin CLEAs 5: Muestra con 2-propanol de la R3 6: Muestra con etanol de la R3

Condiciones de reacción: 24 h a 30˚C empleando 2 mg de CLEAs (lipasa magnética), 136 mg de FFA con 2-propanol ó etanol (relación alcohol:FFA 10:1 mol:mol)

C1 1 2 C2 3 4 C3 5 6

Reacción 2 (R2): FFA de microalgas de FFA_1**

Reacción 3 (R3): FFA de microalgas del FFA_2**

*FFA_M: 28.19% palmitic, 2.83% oleic, 22.32% linoleic and 46.66% linolenic

R1 R2 R3

**FFA_1: Muestra de Febrero de 2013

FFA_2: Muestra de Marzo de 2013

Biodiesel Production from Chlorella FFAs


FAPEs

FAEEs

FFAs

FAPEs FAEEs

FAMEs

FFAs

FAPEs: Fatty Acids Propyl Esters FAEEs: Fatty Acids Ethyl Esters FAMEs: Fatty Acids Methyl Esters FFAs: Free Fatty Acids

Reacción 1 (R1): Con etanol 99.8% (v/v)

Calles: C1: Control de la R1 sin CLEAs 1: Muestra de la R1 C2: Control de la R2 sin CLEAs 2: Muestra de la R2 C3: Control de la R3 sin CLEAs 3: Muestra con de la R3

Condiciones de reacción: 24 h a 30˚C empleando 2 mg de CLEAs (lipasa magnética), 136 mg de FFA_M* con 2-propanol, etanol ó metanol (relación alcohol:FFA 10:1 mol:mol)

C1 1 C2 2 C3 3

Reacción 2 (R2): Con metanol 99.9% (v/v)

Reacción 3 (R3): Con 2-propanol 99.5% (v/v)

*Mezcla de ácidos grasos comerciales: 28.19% de palmítico, 2.83% de oleico, 22.32% de linoleico y 46.66% de linolénico

R1 R2 R3



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Etanol 99%

Propanol

Metanol

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Biodiesel Conversion from Chlorella FFAs Effect of alcohol used at 30ºC

Ethanol

2-Propanol

Methanol


FAPEs: Fatty Acids Propyl Esters FAEEs: Fatty Acids Ethyl Esters FAMEs: Fatty Acids Methyl Esters

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Biodiesel Conversion from Chlorella FFAs Mechanical stirring vs sonoreactor at 30ºC


FAPEs: Fatty Acids Propyl Esters FAEEs: Fatty Acids Ethyl Esters FAMEs: Fatty Acids Methyl Esters

Biodiesel Production from Oils and FFAs

Sample Partner Date Biodiesel Conv. (%)

Model oils mixture UPV/EHU April 2013 73.9

Model FFAs of Chlorella UPV/EHU April 2013 84.0

Real FFAs of Chlorella batch 3 CENER May 2013 74.0

Real FFAs of Chlorella batch 4 CENER May 2013 73.1

Model FFAs of Scenedesmus UPV/EHU April 2013 98.5

Real FFAs Scenedesmus batch 1 CENER May 2013 96.1

Real FFAs Scenedesmus batch 2 CENER September 2013 90.9

Reaction conditions: 200 mg oil or 500 µmol (136-155 mg) FFAs Ethanol:oil = 30:1 or Ethanol:FFAs = 10:1 2 mg mCLEAs, 30ºC, 24 h


0 0.5 1 24

FAEEs

TG

FFA

MG + DG


Biodiesel Production from Model Oils

Biodiesel

Reaction Time (h)

Reaction conditions: 200 mg model oil (80% Palm + 10% Soya + 10% Olive) Ethanol:oil = 30:1 or Ethanol:FFAs = 10:1 2 mg mCLEAs 30ºC 24 h Rotatory stirring

0 24 0 24 0 24

Reaction Time (h)

Model Chlorella Batch 3 Batch 4



FAEEs Biodiesel

FFAs

Reaction conditions: 500 µmol (136 mg) FFAs Ethanol:FFAs = 10:1 2 mg mCLEAs 30ºC 24 h Rotatory stirring

Biodiesel Production from Scenedesmus FFAs

0 24 0 24 0 24

Reaction Time (h)

Model Scenedesmus Batch 1 Batch 2


FAEEs Biodiesel

FFAs

Reaction conditions: 500 µmol (155 mg) FFAs Ethanol:FFAs = 10:1 2 mg mCLEAs 30ºC 24 h Rotatory stirring

Biodiesel Conversion from Scenedesmus FFAs

Oleic acid (C18:1)

Linoleic acid (C18:2)

Linolenic acid (C18:3)

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Reaction conditions: 500 µmol (155 mg) Scenedesmus FFAs Ethanol:FFAs = 10:1 2 mg mCLEAs 30ºC 24 h Rotatory and Ultrasound stirring

Ultrasound Stirring

Rotatory Stirring

Biodiesel Production in Enzyme Reactors

Sonoreactor Hielscher UTR200 Radleys Carousel 12 Plus Reactor

Ultrasound stirring Volume: 5 ml

Magnetic stirring Volume: 5 to 15 ml


Model Scenedesmus FFA mixture FFAs Scenedesmus batch 2

0 0.25 0.5 0.75 1 2 3 5 24 0 0.25 0.5 0.75 1 2 3 5 24


Biodiesel Conversion from Scenedesmus FFAs Reaction conditions: 5 mmol (155 mg) Scenedesmus FFAs 20 mg mCLEAs Ethanol:FFAs = 10:1 30ºC Magnetic stirring

FAEEs Biodiesel

FFAs

Reaction Time (h) Reaction Time (h)

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Oleic acid (C18:1)




Reaction conditions: 5 mmol (155 mg) Scenedesmus FFAs Ethanol:FFAs = 10:1 20 mg mCLEAs 30ºC Rotative and magnetic stirring

Ultrasound Stirring

Rotatory Stirring

Biodiesel Conversion from Scenedesmus FFAs

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FFAs Conversion: Effect of Stirring Type

Oleic acid (C18:1)



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Ultrasound Stirring

Rotatory Stirring

Magnetic Stirring

Outline







7. Conclusions



Reutilization of mCLEAs for Biodiesel Production

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Methanol

Ethanol

Methanol

Ethanol

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Elemental Analysis Composition

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mCLEAs (Control)

mCLEAs (Rotatory)

mCLEAs (Ultrasound)

Ultrasound Stirring (after 36 cycles)

Rotatory Stirring (after 50 cycles)

Outline







7. Conclusions


Conclusions/1

Two types of robust magnetic biocatalysts of CALB (i.e., MNPs and mCLEAs), which are very stable and active at 60ºC or more, have been prepared

Both biocatalysts can catalyze reactions of transesterification of triglycerides and/or esterification of free fatty acids from microalgae (Chlorella sp. and Scenedesmus sp.)

After 24 h reaction, biodiesel conversions of 70, 80 and 96% can be routinely obtained from oils and fatty acids from Chlorella, and from fatty acids from Scenedesmus, respectively

The magnetic biocatalyst (mCLEAs) of CALB can be used for at least 50 consecutive cycles of reaction (at 30ºC, 24 h) without significant loss of activity


The use of ultrasounds in a sonoreactor accelerates the esterification reaction rate of fatty acids and 90% biodiesel conversion can be achieved after 15 min at 30ºC

In a magnetic stirring reactor (5 ml), 90% biodiesel conversion from fatty acids can be achieved after 60 min at 30ºC

mCLEAs of CALB is a robust biocatalyst that can be used efficiently for the production of biodiesel as well as other highly valuable compounds

In this work, the feasibility to obtain biodiesel with more than 90% conversion from microalgal oils and/or fatty acids using magnetic biocatalysts has been demostrated


Conclusions/2

Grupo de Tecnología Enzimática y Celular Facultad de Ciencia y Tecnología Universidad del País Vasco (UPV/EHU)


Juan Luis Serra María Jesús Llama Álvaro Cruz-Izquierdo Carmen López Enrique A. Picó Noelia Villarroel Teresa García-Bárcena Iraide Bejarano

ANY QUESTION....?

Thank you, for your kind attention…!


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