Enzymatic Fatty Acid Hydroxylation in a LiquidâLiquid Slug Flow

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Industrial & Engineering Chemistry Research

Enzymatic fatty acid hydroxylation in a liquid-liquid slug flow microreactor Ion Iliuta, Alain Garnier, Maria C. Iliuta* Department of Chemical Engineering, Laval University, Québec, Canada, G1V 0A6

Abstract. Fatty acids omega hydroxylation biocatalytic process into an intensified liquid-liquid slug flow microreactor with immobilized or aqueous solution-phase enzyme was proposed and analyzed numerically. Hydroxylation of the tetradecanoic acid by the recombinant P450foxy enzyme produced by an Escherichia coli was chosen as a case study. The liquid-liquid reaction system includes an aqueous continuous liquid phase containing buffer, cofactor and enzyme (when biotransformation occurs in aqueous phase) and an organic dispersed liquid phase which behaves as a substrate (tetradecanoic acid) reservoir facilitating a constant mass transfer between the organic dispersed and aqueous continuous liquid phases without deactivating the enzyme. The behaviour of the intensified microreactor was analyzed through simulation via two-scale, isothermal, unsteady-state models accounting for detailed hydrodynamics whereupon were tied thermodynamics and kinetics of fatty acid hydroxylation catalyzed by immobilized or aqueous solution-phase P450foxy enzyme. The effects of key operating parameters as well as the contribution of P450foxy enzyme on the performance of fatty acid hydroxylation process are highlighted. The intensified microreactors with liquid-liquid reaction systems offer a promising option for fatty acids hydroxylation biocatalytic process because of high specific enzymatic activity as a result of the constant mass transfer of the substrate between the dispersed organic and continuous aqueous liquid phases.

Keywords Liquid-liquid slug flow microreactor; fatty acids hydroxylation; P450foxy enzyme; modeling.

*Corresponding author. Tel.: 1 418 656-2204; fax: 1 418 656-5993. E-mail address: [email protected] (M.C. Iliuta)

ACS Paragon Plus Environment

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1. Introduction Hydroxy fatty acids are composed of a series of straight-chain carboxylic acids that contain one or more hydroxyl groups substituted on the hydrocarbon portion of molecule. Hydroxy fatty acids are saturated or may contain one or more unsaturated bonds in the hydrocarbon chain. They are found in nature as components of cerebrosides, triacylglycerols, waxes and other lipids in animals, plants (chain from 12 up to 24 carbon atoms), and microorganisms (chain up to 80 carbon atoms).1-4 Hydroxy fatty acids are widely used in chemical, food and cosmetic industries as starting materials for the synthesis of polymers and as additives for the manufacture of plasticizers, emulsifiers, stabilizers, grease, paint, and coating.4 They have been applied to renewable energy sources for biodegradable oil-based lubricants and biodiesel. Hydroxy fatty acids are used as precursors in the production of flavour and fragrance lactones by bacteria and yeast. They have antibiotic, anti-inflammatory, and anticancer activities and for that reason can be applied for medicinal uses. So, the industrial utilization of hydroxy fatty acids, as environment-friendly and sustainable technology for the future, offer potential ways to solve current energy resource crises by replacing petroleum oil.4 The demand in chemical, agricultural, food, cosmetic, and pharmaceutical industries are steadily increasing, but hydroxy fatty acids mass production and diversification remain limited. Hydroxy fatty acids are produced using wild-type and recombinant whole cell conversion, fermentation, and enzymatic conversion processes.4 The substrates used to produce hydroxy fatty acids include saturated and unsaturated free fatty acids (palmitic, palmitoleic, oleic, linoleic, and α-linolenic acids) and oil hydrolyzates, which are hydrolyzed to fatty acids from olive, soybean, and waste oils by lipase or hydrolysis methods.5,6 The enzymes used as biocatalysts to produce


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hydroxy fatty acids include P450, lipoxygenases, hydratases, 12-hydroxylases, and diol synthases.4 With NAD(P)H as a cofactor, P450s catalyze the insertion of one oxygen atom from molecular oxygen into an organic substrate through electron transfer.7 Hydratases catalyze the irreversible addition of a hydrogen atom and a hydroxy group from water to the carbon–carbon cis-double bond of unsaturated fatty acids at the C9 and C10 positions, respectively, to make 10hydroxy fatty acids.8 12-Hydroxylases convert oleic acid to ricinoleic acid and catalyze the sitespecific hydroxylation of the 12-position of oleic acid in the presence of NADH using O2.4 Lipoxygenases catalyze the insertion of molecular oxygen into polyunsaturated fatty acids containing one or more cis,cis-pentadiene units to produce the corresponding hydroperoxy fatty acids, which are reduced hydroxy fatty acids.9 The bacterial diol synthases catalyze the fatty acid di-hydroxylation process.4 Monophasic aqueous reaction systems or biphasic aqueous/organic reaction systems are used for fatty acids hydroxylation.10,11 In monophasic aqueous reaction systems the high hydrophobicity of the fatty acids hinders their accessibility for water-soluble enzyme. In order to overcome this problem and to enhance substrate accessibility a very large aqueous reaction volume12, addition of water-miscible cosolvents and emulsifiers promoting the solubility of fatty acids11 or biphasic aqueous/organic reaction systems have been applied.10,11 However, the possible inactivation of the enzyme by solvent in a monophasic reaction system can outweigh the increase of solubility of fatty acid (a suitable cosolvent for the biocatalytic process must be nontoxic, highly water soluble and easily hydrolysed at low pH values). On the other side, even if the biphasic aqueous/organic reaction systems, where the organic phase serves as a substrate reservoir facilitating a constant phase transfer between organic and aqueous phase, are appropriate for the fatty acids hydroxylation process, a very poor substrate transfer rate can lead


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to inferior results compared to the monophasic aqueous reaction system with water-miscible cosolvents or emulsifier. In this case a water-miscible cosolvent can be added to the reaction mixture to improve the solubility of fatty acid in the aqueous solution.11 Unfortunately, application of fatty acids hydroxylation with aqueous solution-phase enzyme is not always suitable and optimal because of the large volume of enzyme required. Binding of fatty acids hydroxylation enzyme in a porous washcoat structure is an attractive modification of its application having several advantages, including easier separation of the reaction products without catalyst contamination, ability to recover and reuse the enzyme, increase of the enzyme stability and operational lifetime, continuous operation of enzymatic processes and flexibility of the reactor design.13,14 However, attaching the hydroxylation enzymes in a porous washcoat layer may lead the enzyme to behave differently because: (i) the immobilization may cause the enzyme molecules to adopt a different conformation; (ii) the immobilized enzyme exists in an environment different from that when it is in solution-phase; (iii) there is a partitioning of substrate between the solution and support, with the result that the substrate concentration in the neighborhood of the enzyme may be significantly different from that in the bulk solution; (iv) diffusional effects play a more important role with immobilized enzymes.14,15 The present contribution investigates the biocatalytic fatty acids omega hydroxylation process into an intensified liquid-liquid slug flow capillary microreactor with immobilized or aqueous solution-phase enzyme. Hydroxylation of the tetradecanoic acid by the P450foxy enzyme produced by recombinant Escherichia coli was chosen as a case study. The two-liquid reaction system includes an aqueous continuous liquid phase and an organic dispersed liquid phase. The aqueous continuous liquid phase contains buffer (2-[2-morpholino]ethanesulfonic acid - MES), cofactor (NADPH) and enzyme (when biotransformation occurs in aqueous phase).


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The organic dispersed liquid phase (cyclohexane) behaves as a substrate reservoir facilitating a constant mass transfer between the organic dispersed and aqueous continuous liquid phases without deactivating the enzyme and simultaneously acts as an organic solvent for extraction of the reaction product (cyclohexane was proven not to be a substrate of P450foxy – Kitazume et al.7). The work objective lies on exploring the possibility to use this intensifying mass transfer microreactor system as a tool for further understanding and development of biphasic hydroxy fatty acid synthesis process. The behaviour of the intensified system was analyzed through simulation via two-scale, isothermal, unsteady-state models accounting for relatively detailed hydrodynamics whereupon were tied thermodynamics and kinetics of fatty acid hydroxylation catalyzed by immobilized P450foxy enzyme or free enzyme in aqueous continuous liquid phase. 2. Model for the immobilized enzyme microreactor The microreactor system consists of a circular tube with P450foxy enzyme uniformly attached in a porous washcoat layer. Two immiscible liquids flow continuously upflow in slug flow regime through the micro-channel. The aqueous continuous liquid phase contains the buffer (MES) and cofactor (NADPH) and the organic dispersed liquid phase (cyclohexane) acts as a substrate (tetradecanoic acid) reservoir facilitating a constant mass transfer between the dispersed organic and continuous aqueous liquid phases. Also a substantial amount of reaction products accumulates in dispersed liquid phase which can be continuously removed using extraction, distillation or adsorption to resins. Fatty acid hydroxylation was performed under constant air bubbling at a very low velocity. The physical flow model of the microreactor is sketched in Fig. 1. Under the microreactor operating conditions presented in Table 1 the expected liquid-liquid flow pattern is Taylor flow.16 Because the reaction takes place inside the washcoated wall pores, the liquid and gas


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reactants have to transfer from the bulk to the catalyst surface. So, different mass transport phenomena in liquid-liquid slug flow inside the microreactor were considered: dispersed liquidsolid (dispersed liquid reactant diffusion through the thin film surrounding the droplet), dispersed liquid-continuous liquid and continuous liquid-solid mass transfer (Fig. 1). Additionally, gasliquid and liquid-liquid (between the continuous aqueous and dispersed liquid phases) mass transfer of oxygen was taken into account. In the hydroxylation microreactor model the hydroxylated products are lumped into a pseudocomponent in order to decrease the computational effort even if the recombinant P450foxy enzyme catalyzes the subterminal (ω-1, ω-2, and ω-3) hydroxylation of fatty acids.7 Axial dispersion was considered for the continuous liquid phase and plug flow for the dispersed organic phase. The unsteady-state mass, continuity and momentum balance equations are formulated below (the gas phase was neglected in the formulation of the hydrodynamic model): •

species mass balance equations in dispersed liquid phase (substrate, product, oxygen)

∂C ∂ ∂ ε d CS ,d ) + ( ε d ud C S ,d ) = − DSeff, w S , w ( ∂t ∂z ∂r ∂C ∂ ∂ ε d CP,d ) + ( ε d ud C P, d ) = − DP,effw P, w ( ∂t ∂z ∂r

)

(

Ld − kd ad ( CS , d − C S ,cmS ) Luc

(1)

as , w

Ld + kd ad ( CP,c m P −C P,d ) Luc

(2)

r = R1

r = R1

∂C ∂ ∂ ε d CO2 ,d + ε d ud CO2 ,c = − DOeff2 ,w O2 ,w ∂t ∂z ∂r

(

as , w

)

as , w r = R1

 mO ,d Ld − kd ad  CO2 , d − CO2 ,c 2  Luc mO2 ,c 

  

(3)

 CO , g  + ( kl a )l = d  2 − CO2 , d   mO ,d   2 

•

species mass balance equations in continuous liquid phase (substrate, oxygen, cofactor, product)


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∂C ∂ ∂ ∂2 ε c CS ,c ) + ( ε c uc CS ,c ) = Dl 2 ( ε c CS ,c ) + kd ad ( CS ,d − CS ,c mS ) − DSeff, w S , w ( ∂t ∂z ∂z ∂r ∂C ∂ ∂ ∂2 ε c CO2 ,c + ε c uc CO2 ,c = Dl 2 ε c CO2 ,c − DOeff2 ,w O2 ,w ∂t ∂z ∂z ∂r

(

)

(

)

(

)

 mO , d + kd ad  CO2 , d − CO2 ,c 2  mO2 ,c 

as , w r = R1

r = R1

Lc Luc

Lc Luc

(4)

(5)

  CO , g   + ( kl a )l =c  2 − CO2 ,c    mO2 ,c 

∂CCF , w ∂ ∂ ∂2 eff ε cCCF ,c ) + ( ε cuc CCF ,c ) = Dl 2 ( ε cCCF ,c ) − DCF ( ,w ∂t ∂z ∂z ∂r

(6)

as , w r = R1

∂C ∂ ∂ ∂2 ε C + ε u C = D ε C − kd ad ( CP,c mP − C P,d ) − DPeff, w P , w ( ( c P ,c ) c c P ,c ) l 2 ( c P ,c ) ∂t ∂z ∂z ∂r

•

as , w

as , w r = R1

Lc Luc

(7)

oxygen mass balance equation in gas phase

C  C  ∂ ∂ ε g CO2 , g + ε g u g CO2 , g = − ( kl a )l = c  O2 , g − CO2 ,c  − ( kl a )l = d  O2 , g − CO2 ,d   mO ,c    ∂t ∂z  2   mO2 , d 

(

)

(

)

(8)

The initial and boundary conditions for the transport of species j in the dispersed and continuous liquid and gas phases are as follows: t = 0, z > 0

C j ,α = C inj ,α

t > 0, z=0

C j , d = C inj ,d

t > 0, z = L •

∂C j ,c ∂z

(9)

uc ε c C j , c

z = 0−

= uc ε c C j , c

z = 0+

− Dlε c

∂C j ,c ∂z

z = 0+

CO2 , g = COin2 , g

=0

(10)

(11)

z=L

Continuity for the dispersed and continuous liquid phases

∂ ∂ ( ε d ρ d ) + ( ε d ρ d ud ) = 0 ∂t ∂z

(12)

∂ ∂ ( ε c ρ c ) + ( ε c ρ c uc ) = 0 ∂t ∂z

(13)


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•

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Momentum balance equations for the dispersed and continuous liquid phases

∂ 2u ∂ ∂ ∂P ( ρ d ε d ud ) + ud ( ρ d ε d ud ) = ε d µd 2d − ε d -ε d ρd g − Fll ∂t ∂z ∂z ∂z

(14)

∂ 2 uc ∂ ∂ ∂P ( ρcε cuc ) + uc ( ρcε c uc ) = ε c µc 2 − ε c -ε c ρc g − Fls + Fll ∂t ∂z ∂z ∂z

(15)

The solution of Eqs. (1-6) requires knowledge of the species concentration profiles within the porous washcoat layer. These concentration profiles are governed by the diffusional flux within the porous washcoat and the kinetics of consumption of substrate by the enzyme immobilized in washcoat. Formulation of equations describing the simultaneous transport and reaction within the porous washcoat layer is based on the following assumptions: the enzyme is uniformly immobilized over the surface of the porous washcoat (i); the washcoat layer is a homogeneous porous medium of constant thickness wherein the reactants and products diffuse according to Fick’s law (ii); internal wetting of washcoat is complete, and reactants’ diffusion inside the washcoat occurs in the liquid phase (iii). According to the above assumptions, the simultaneous transport and consumption of species j within the washcoat is described as:

εw

εw

εw

εw

∂CO2 , w ∂t

∂CS , w ∂t

∂CP , w ∂t

 ∂ 2CO2 , w 1 ∂CO2 , w  +   - RO2 2 r ∂r   ∂r

(16)

 ∂ 2CS , w 1 ∂CS , w  = DSeff, w  +  − RS 2 ∂ r r ∂ r  

∂CCF , w ∂t

=D

eff O2 , w

=D

eff CF , w

(17)

 ∂ 2CCF , w 1 ∂CCF , w  +   - RCF 2 r ∂r   ∂r

(18)

 ∂ 2CP , w 1 ∂CP , w  = DPeff, w  +  + RP 2 r ∂r   ∂r


(19)

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The boundary conditions at the washcoat-liquid (liquid) interface couples the dispersed and continuous liquid axial mass balances to the internal mass balances, and reflects that the reaction rate catalyzed by the immobilized enzyme equals the flux to the washcoat:

C j , w = C inj ,c

t = 0, R1 0, r = R2

∂r

r = R1

C L Lc + kl' s , S  S ,d − CSs , w  d Luc  mS  Luc

(21)

(22)

r = R1

∂CO2 , w

(

= kls ,O2 CO2 ,c − COs 2 , w

)

(23)

r = R1

∂CP, w ∂r

(20)

s = kls ,CF ( CCF ,c − CCF ,w )

∂r

∂C j , w ∂r

= kls , S ( CS ,c − CSs , w )

∂CCF ,w

∂r

j = O2, S, CF, P

= kls ,P ( CP,c − CP,s w ) r = R1

=0

C L Lc + kl' s ,P  P,d − CP,s w  d Luc  mP  Luc

(24)

j = O2, S, CF, P

(25)

r = R2

2.1. Tetradecanoic acid hydroxylation kinetics. The kinetic model developed by Kitazume et al.7 was used to describe the tetradecanoic acid hydroxylation:

RS =

kcat CE 0CS  C  K m + CS  1 + S   Ks 

(26)

The kinetic model with substrate inhibition was developed for tetradecanoic acid hydroxylation in the presence of free cytochrome P450foxy enzyme in solutions which contains buffer (2-[2morpholino]ethanesulfonic acid) and cofactor (NADPH). The kinetic model is expected to be suitable under immobilization enzyme conditions as in the case of other enzymes.17 The rate constants at 30°C are: kcat = 1300 min −1 , K m = 0.019 mmol/L and K s = 1.1 mmol/L .7


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The stoichiometry between the consumption of NADPH and O2 was 1.3:1, consistent with the theoretical value for 2 electron reduction coupling to monooxygenation of the substrates.7 2.2. Interfacial Drag Forces. The continuous liquid-solid drag force, Fℓs, was determined from the formulation of the liquid flow in the laminar regime in empty pipes: Fls =

 4 4  1 4  16 1  τ w,lε c =  f Rel ,dc ρ l vsc2  ε c =  ρ l vsc2  ε c 2 dc dc  d c  Rel ,dc 2  

(

)

(27)

The expression for the continuous liquid-dispersed liquid interaction force per unit reactor volume, Fℓℓ, was obtained from the drag force exerted on a single liquid droplet in the viscous flow, fdc, multiplied by the number of droplets per unit reactor volume in the Taylor flow regime. The drag force exerted on an isolated (cylindrical) liquid droplet is defined as: 1 f dc = CD ρ c ( ud − uc ) ud − uc Ad 2

24 Re d

CD =

where

Re d =

Ad =

(28)

viscous flow18

(29)

d d ρ c ( u d − uc )

(30)

µc

π d d2 4

+ π d d Ld

(31)

The number of liquid droplets per unit reactor volume is defined as:

nd =

εd π d d2 4

(32)

Ld

and the droplet diameter was evaluated taking into account the film thickness (see Fig. 1): d d ≈ d c − 2δ f

(33)


10

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The correlation of Bretherton19 obtained for no-slip boundary conditions at the interface (which agrees very well with CFD simulations of liquid–liquid Taylor flow in microchannels20) was used to evaluate the film thickness between the liquid droplet and the wall channel:

δf dc / 2

= 1.34Ca 2 / 3

(34)

Finally, by introducing the dispersed liquid drift flux, jd, the resulting Fℓℓ expression is:

Fll =

 L  12 µc jd  4 d + 1   εd   dd  1 −  d d Ld ε  

(35)

 ε  where jd = ε d 1 − d  ( ud − uc ) ε  

(36)

2.3. Auxiliary Relationships. The continuous liquid slug length in Taylor flow was evaluated using the correlation developed by Kreutzer21:

ψc =

Lc εc = d c −0.00141 − 1.556ε c2 ln ( ε c )

(37)

Assuming that the volume of aqueous continuous liquid in the film between the organic droplet and the wall channel is negligible, the average unit cell and dispersed liquid droplet lengths can be estimated from the following relationships22:

Lc = Luc (1 − ε d )

(38)

Ld = Luc − Lc

(39)

Liquid-solid mass transfer from the liquid slug and for the liquid droplet was estimated from the film theory23: kls , j = kl' s , j =

D j ,l

(40)

δf


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Kreutzer et al.23 showed that below Ca =0.01, the film thickness is comparable for the liquid slug and for the droplet and the mass transfer through the liquid film is about as fast for the liquid slug as for the droplet. Dispersed phase mass transfer coefficient and liquid-liquid specific interfacial area were estimated using the following equations24:

 dd  0.371 0.371 4 D j , d  Ld − d d + 3.14  ρ u d  µu  k d 2  Shd = d m = 31.4  c d m   c d   2 D j ,d d m ud  µc   σ c     ad =

     

−0.338

 µd   ρ d D j ,d

  

−0.125

(41)

4d d ( Ld − d d ) + 3.14d d2

(42)

Luc d c2

Gas-liquid mass transfer coefficient was estimated using the following correlation based on the Higbie’s penetration theory25: kl a =

4.5 D j ,l u g dc Luc

(43)

The partition coefficients between the water and a solvent were given by the ratio of solubilities of a solute in the solvent and in water.26 The solubilities of fatty acids in water and cyclohexane were taken from Ralston and Hoerr27 and Hoerr and Ralston.28 The liquid diffusivity coefficients were estimated with Vorlop29 and Wilke-Chang (Reid et al.30) correlations. The effective diffusion coefficient in washcoat was evaluated assuming tortuosity according to granular media theory31): Τ=

εw 2/3 1 − (1 − ε w )

(44)

The extent of back-mixing in the liquid phase was quantified in terms of an axial dispersion coefficient which was evaluated from the experiments of Thulasidas et al.32


12

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3. Model for microreactor with free enzyme in aqueous continuous liquid phase The microreactor system consists of a circular tube with an aqueous continuous liquid phase (where biotransformation occurs) containing the enzyme, buffer and cofactor and a dispersed liquid phase containing substrate. The two-liquid reaction scheme is presented in Fig. 2. A liquid-liquid Taylor flow regime14 with small gas bubbles in both phases (very low gas holdups: 0.02-0.04) is assumed and the physical flow model of the microreactor is sketched in Fig. 1 (without washcoat). The unsteady-state mass, continuity and momentum balance equations are (the gas phase was neglected in the formulation of the hydrodynamic model): •

species mass balance equations in dispersed liquid phase - no-reaction (substrate, product, oxygen)

∂ ∂ ε d CS ,d ) + ( ε d ud C S ,d ) = −kd ad ( CS , d − C S ,cmS ) ( ∂t ∂z

(45)

∂ ∂ ε d CP,d ) + ( ε d ud C P,d ) = kd ad ( CP,c mP − C P,d ) ( ∂t ∂z

(46)

C   m ∂ ∂ ε d CO2 ,d + ε d ud CO2 ,c = ( kl a )l = d  O2 , g − CO2 ,d  − k d ad  CO2 ,d − CO2 ,c O2 ,d  mO , d   ∂t ∂z mO2 ,c  2  

(

•

)

(

)

  

(47)

species mass balance equations in continuous liquid phase (oxygen, substrate, cofactor, product)

 m ∂ ∂ ∂2 ε c CO2 ,c + ε c uc CO2 ,c = Dl 2 ε c CO2 ,c − RO2 + kd ad  CO2 ,d − CO2 ,c O2 ,d  ∂t ∂z ∂z mO2 ,c 

(

)

(

)

(

)

  

(48)

 CO , g  + ( kl a )l = c  2 − CO2 ,c   mO ,c   2  ∂ ∂ ∂2 ε c CS ,c ) + ( ε c uc CS ,c ) = Dl 2 ( ε c CS ,c ) + kd ad ( CS , d − CS ,c mS ) − RS ( ∂t ∂z ∂z


(49)

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∂ ∂ ∂2 (ε cCCF ,c ) + ∂z (ε cucCCF ,c ) = Dl ∂z 2 (ε cCCF ,c ) − RCF ∂t

(50)

∂ ∂ ∂2 ε C + ε u C = D ( c P,c ) ∂z ( c c P,c ) l ∂z 2 (ε cCP,c ) − kd ad ( CP,cmP − CP,d ) + RP ∂t

(51)

•

oxygen mass balance equation in gas phase

C  C  ∂ ∂ ε g CO2 , g + ε g u g CO2 , g = − ( kl a )l = c  O2 , g − CO2 ,c  − ( kl a )l = d  O2 , g − CO2 ,d   mO ,c    ∂t ∂z  2   mO2 , d 

(

)

(

)

(52)

The initial and boundary conditions for the transport of species j in the dispersed and continuous liquid and gas phases are: t = 0, z > 0

C j ,α = C inj ,α

t > 0, z=0

C j , d = C inj ,d

(53)

uc ε c C j , c

z =0

−

= uc ε c C j , c

z =0

+

− Dlε c

∂C j ,c ∂z

(54) z =0

+

CO2 , g = COin2 , g t > 0, z = L •

∂C j ,c ∂z

(55)

=0

(56)

z=L

Continuity for the dispersed and continuous liquid phases

∂ ∂ ( ε d ρ d ) + ( ε d ρ d ud ) = 0 ∂t ∂z

(57)

∂ ∂ ( ε c ρ c ) + ( ε c ρ c uc ) = 0 ∂t ∂z

(58)

•

Momentum balance equations for the dispersed and continuous liquid phases

∂ 2 ud ∂ ∂ ∂P ( ρ d ε d ud ) + ud ( ρ d ε d ud ) = ε d µd 2 − ε d -ε d ρd g − Fll ∂t ∂z ∂z ∂z

(59)

∂ 2u ∂ ∂ ∂P ( ρcε cuc ) + uc ( ρcε c uc ) = ε c µc 2c − ε c -ε c ρc g + Fll − Fls ∂t ∂z ∂z ∂z

(60)


14

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The kinetic model, interfacial drag forces and auxiliary relationships are similar with those used in the model for the microreactor with immobilized enzyme.

4. Method of solution In order to solve the system of partial differential equations, we discretized in space and solved the resulting set of ordinary differential equations. The spatial discretization is performed using the standard cell-centered finite difference scheme (at the microreactor level) and the method of orthogonal collocation (at the porous washcoat level). The number of collocation points specified for the porous washcoat layer was restricted to 10. The GEAR integration method for stiff differential equations was employed to integrate the time derivatives. The relative error tolerance for the time integration process in the present simulations is set at 10-5 for each time step.

5. Results and discussion Model validation Examination of Figs. 3 and 4 provides the ability to compare the model predictions to experimental data obtained in an immobilized enzyme microreactor with a mono-liquid aqueous reaction system. The figures present the average outlet product concentration for different inlet substrate concentration and liquid velocity for the enzymatic CO2 hydration in the presence of immobilized human carbonic anhydrase II (hCA II) in an intensified microreactor. Experimental data were obtained in our laboratory by Hanna et al.33 at low enzyme loadings and small residence times. The intensified microreactor coated with hCA II included a helical screw which generates “concentration plug flow” under laminar flow conditions because of the significant convective mass transfer enhancement. The agreement between predictions and measurements is very good: the average relative deviation between calculated and experimental data is 4.4% for all tests involving immobilized enzyme process.


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At high liquid-liquid mass transfer fluxes, the specific enzymatic activity in fatty acids hydroxylation biocatalytic process into intensified microreactors with two-liquid reaction systems and free enzyme in aqueous continuous liquid phase is similar to specific enzymatic activity obtained by Nakayama et al.34 and Kitazume et al.7 using the native enzyme (≈1200 mol NADPH/min/mol enzyme).

Simulations The performance of the microreactor with a two-liquid reaction system is much higher than the performance of the microreactor with a mono-liquid aqueous reaction system (for the operating conditions listed in Table 1, the specific enzymatic activity is 15 times higher for the two-liquid reaction system with free enzyme in aqueous continuous liquid phase) because of the constant substrate mass transfer between the dispersed organic and continuous aqueous liquid phases. The model was initially used to compare the performance of the capillary microreactor with free (in aqueous continuous liquid phase) or immobilized enzyme under the same P450foxy enzyme loading. Figs. 5 and 6 show typical hydroxy fatty acid time-dependent and steady-state concentration profiles in the dispersed liquid phase and NADPH and oxygen axial steady-state concentration profiles in the aqueous continuous liquid phase obtained under the same operating conditions (Table 1). Diffusional effects are more important with immobilized P450foxy enzyme and the result is a lower fatty acid conversion. With the increase of P450foxy enzyme loading (Fig. 7), microreactor performance is improved in accordance with higher hydroxylation reaction rate leading to higher hydroxy fatty acid concentration. The amplification is less important under immobilized enzyme conditions because fatty acid hydroxylation process is mass-transfer limited by the diffusion through the liquid film surrounding the washcoat and inside the washcoat.


16

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In the capillary microreactor with immobilized P450foxy enzyme, an important parameter which dictates the mass transfer resistance is the molecular diffusion coefficient. The influence of the diffusion coefficients on the fatty acid hydroxylation process is illustrated in Fig. 8. It is evident that an increase in the molecular diffusion coefficient (a 2-fold increase with respect to estimated values with Vorlop29 and Wilke-Chang (Reid et al.30) correlations) leads to higher mass transfer fluxes transported between the liquid and the catalytic solid surface and higher effective diffusivity inside the washcoat. The result is a higher fatty acid conversion reflected in a higher hydroxy fatty acid concentration in dispersed liquid phase, respectively a lower NADPH and oxygen concentration in aqueous continuous liquid phase (Fig. 8). Fatty acid hydroxylation process is not significantly influenced by the thickness of the porous washcoat layer (Fig. 9). The increase of the washcoat thickness considerably amplifies the mass transfer resistance in the washcoat pores (Fig. 9b) but in the same time the moles of immobilized P450foxy as well (recall that enzyme loading is defined as the number of moles of enzymes immobilized per unit washcoat volume). The reduced mass transfer is compensated by the increased reactor P450foxy loading and as a consequence fatty acid hydroxylation rate does not vary significantly with the raise of the washcoat thickness (the reduction of the fatty acid hydroxylation rate due to increased external and internal diffusion resistance is balanced by the increase of the fatty acid hydroxylation rate due to the increase of the reactor P450foxy loading). Microreactor performance increases with the increase of the superficial gas velocity (Fig. 10a). The influence of the gas velocity is more evident in the capillary microreactor with immobilized P450foxy enzyme because of the oxygen mass-transfer limitation by the diffusion through the liquid film surrounding the washcoat. As expected, at low gas velocity the fatty acid hydroxylation process declines because of the oxygen shortage (Fig. 10b). With the increase of


17


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Page 18 of 46

the dispersed liquid phase velocity, fatty acid hydroxylation process is promoted in accordance with higher mass transfer fluxes between the dispersed and continuous liquid phases and between the gas and dispersed liquid phases (Fig. 11a,b). Hydroxy fatty acid concentration in the dispersed liquid phase decreases with the increase of dispersed liquid phase velocity (Fig. 11c). With the increase of the aqueous continuous liquid phase velocity, fatty acid hydroxylation process is improved because of the higher mass transfer between the dispersed and continuous liquid phases (Fig. 12). Microreactor performance increases significantly with the increase of the fatty acid concentration in the dispersed liquid phase (Fig. 13a) because of the higher substrate mass transfer flux transported between the dispersed and continuous phases (caused by the higher driving force of the liquid-liquid mass transfer). As expected, cofactor concentration in the aqueous continuous liquid phase decreases with the raise of the fatty acid concentration in the dispersed liquid phase (Fig. 13b). The specific enzymatic activity increases with the increase of the fatty acid concentration in the dispersed liquid phase (Fig. 14). This figure suggests that the dispersed liquid phase can be recirculated because the specific enzymatic activity remains elevated within a large range of fatty acid concentration. At high fatty acid concentrations, which assure a high liquid-liquid mass transfer flux, specific enzymatic activity in the capillary microreactor is similar to specific enzymatic activity obtained using the native enzyme.7,34 Fatty acid hydroxylation process is improved by the reduction of the channel diameter (Fig. 15) because of the considerable intensification of the overall mass transfer coefficient due to the reduction of the mass transfer resistance between dispersed and continuous liquid phases24, liquid and the catalytic solid surface (immobilized enzymes - Kreutzer et al.23), gas and aqueous


18

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liquid phase.25 Also, the reduction of the channel diameter reduces the mass transfer resistance in washcoat pores (not shown) in the capillary microreactor with immobilized P450foxy enzyme.

6. Conclusion Fatty acids omega hydroxylation biocatalytic process into an intensified liquid-liquid slug flow microreactor with immobilized or aqueous solution-phase enzyme was proposed and analyzed numerically. Hydroxylation of the tetradecanoic acid by the P450foxy enzyme produced by a recombinant Escherichia coli was chosen as a case study. The liquid-liquid reaction system includes an aqueous continuous liquid phase containing buffer, cofactor and enzyme (when biotransformation occurs in aqueous phase) and an organic dispersed liquid phase which behaves as a substrate reservoir facilitating a constant mass transfer between the organic dispersed and aqueous continuous liquid phases without deactivating the enzyme. This work investigates the behaviour of the intensified microreactor via two-scale, isothermal, unsteady-state models accounting for detailed hydrodynamics whereupon were tied thermodynamics and kinetics of fatty acid hydroxylation, highlighting the effects of key operating parameters as well as the contribution of P450foxy enzyme on the process performance. Microreactor performance increases with the increase of enzyme loading, superficial gas velocity, dispersed and continuous liquid phase velocities and fatty acid concentration in dispersed liquid phase. Fatty acid hydroxylation process is improved significantly by the reduction of the channel diameter because of the considerably intensification of the overall mass transfer coefficient. The intensified microreactors with liquid-liquid reaction systems offer a promising option for fatty acids hydroxylation biocatalytic process because of high specific enzymatic activity as a result of the constant mass transfer of the substrate between the dispersed organic and continuous aqueous liquid phases. The performance of the capillary microreactor declines when P450foxy enzyme is


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Page 20 of 46

immobilized because the hydrodynamic and diffusional constraints do not permit reasonable enzyme utilization.


20

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Notation ad

gas-liquid specific interfacial area, m2/m3reactor

ad

liquid-liquid specific interfacial area, m2/ m3reactor

as,w

liquid-solid specific surface area, m2/m3reactor

Ca

Capillary number, Ca =

CE0

enzyme load, kmol/m3reactor

Cj,α

concentration of species j in α-phase ( α = c, d , g ), kmol/m3

C sj, w

concentration of species j at the surface of washcoat, kmol/m3

dc

channel diameter, m

dd

liquid droplet diameter, m

dm

diameter of a sphere with the same volume as the droplets, m

Dj,α

molecular diffusion coefficient in α-phase, m2/s

D eff j ,w

effective diffusivity of species j inside washcoat, m2/s

Dℓ

axial dispersion coefficient in the aqueous continuous liquid phase, m2/s

µcU TP σ

E1, E2 Ergun constants, – f

friction coefficient

Fℓℓ

liquid-liquid drag force, N/m3

Fℓs

liquid-solid drag force, N/m3 (Iliuta et al.35)

g

gravitational acceleration, m/s2

kl a

volumetric liquid-side gas-liquid mass transfer coefficient, 1/s

kd ad

volumetric liquid-liquid mass transfer coefficient, 1/s

k ls

liquid (continuous phase)-solid mass transfer coefficient, m/s


21


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kl' s

liquid (dispersed phase)-solid mass transfer coefficient, m/s

L

microreactor length, m

Ld

liquid (organic phase) droplet length, m

Lc

liquid (aqueous phase) slug length, m

Luc

unit cell length, m

Page 22 of 46

m j ( j = S , P ) distribution coefficient, m3c /m3d mO2 ,c ( d ) distribution coefficient, m3c(d) /m3g N

mass transfer flux, kmol/m2s

P

reactor pressure, Pa

r

radial position within microreactor, m

R

microreactor radius

Rj

reaction rate, kmol/m3s

R1

channel radius (with washcoat), m

R2

channel radius (without washcoat), m

Rel ,dc Reynolds number based on channel diameter t

time, s

uα

α-phase interstitial velocity ( α = c, d , g ), m/s

UTP

two-phase velocity (total flow rate per unit channel cross-sectional area), m/s

vα

α-phase superficial velocity ( α = c, d ), m/s

z

axial coordinate, m

Greek Letters

δf

continuous liquid film thickness, m


22

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εα

α-phase holdup ( α = c, d , g )

εw

washcoat porosity

µα

α-phase dynamic viscosity ( α = c, d ), kg/m.s

ρα

α-phase density ( α = c, d ), kg/m3

σ

surface tension, N/m

Τ

tortuosity

τ w, l

wall shear stress, N/m2

Subscripts/Superscripts c

aqueous continuous liquid phase

d

dispersed liquid phase

in

microreactor inlet

w

washcoat

Abbreviations CF

cofactor

O2

oxygen

P

product

S

substrate


23


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Page 24 of 46

References

(1)

Kishimoto, Y.; Radin, N.S. Occurrence of 2-hydroxy fatty acids in animal tissues. J. Lipid. Res. 1963, 4, 139–143.

(2)

Bafor, M.; Smith, M.A.; Jonsson, L.; Stobart, K.; Stymne, S. Ricinoleic acid biosynthesis and triacylglycerol assembly in microsomal preparations from developing castorbean(Ricinus communis) endosperm. Biochem. J. 1991, 280, 507–14.

(3)

Jetter, R.; Kunst, L. Plant surface lipid biosynthetic pathways and their utility for metabolic engineering of waxes and hydrocarbon biofuels. Plant J. 2008, 54, 670–83.

(4)

Kim, K-R.; Oh, D-K. Production of hydroxy fatty acids by microbial fatty acidhydroxylation enzymes. Biotechnol. Adv. 2013, 31, 1473–1485

(5)

Heo, S.H.; Hou, C.T.; Kim, B.S. Production of oxygenated fatty acids from vegetable oils by Flavobacterium sp. strain DS5. N Biotechnol. 2009, 26, 105–108.

(6)

Kim, B.N.; Joo, Y.C.; Kim, Y.S.; Kim, K.R.; Oh, D.K. Production of 10-hydroxystearic acid from oleic acid and olive oil hydrolyzate by an oleate hydratase from Lysinibacillus fusiformis. Appl Microbiol Biotechnol. 2012, 95, 929–937.

(7)

Kitazume, T.; Tanaka, A.; Takaya, N.; Nakamura, A.; Matsuyama, S.; Suzuki, T.; Shoun, H. Kinetic analysis of hydroxylation of saturated fatty acids by recombinant P450foxy produced by an Escherichia coli expression system. Eur. J. Biochem. 2002, 269, 2075– 2082.

(8)

Joo, Y.C.; Jeong, K.W.; Yeom, S.J.; Kim, Y.S.; Kim, Y.; Oh, D.K. Biochemical characterization and FAD-binding analysis of oleate hydratase from Macrococcus caseolyticus. Biochimie 2012, 94, 907–15.

(9)

Brash, A.R. Lipoxygenases: occurrence, functions, catalysis, and acquisition of substrate. J. Biol. Chem. 1999, 274, 23679–23682.

(10) Maurer, S.C.; Kuhnel. K.; Kaysser, L.A.; Eiben, S.; Schmid, R.D.; Urlacher, V.B. Catalytic hydroxylation in biphasic systems using CYP102A1 mutants. Adv. Synth. Catal. 2005, 347, 1090-1098. (11) Kuhnel, K.; Maurer, S.C.; Galeyeva, Y.; Frey, W.; Laschat, S.; Urlacher, V.B. Hydroxylation of dodecanoic acid and (2R,4R,6R,8R)-tetramethyldecanol on a preparative


24

Page 25 of 46

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


scale using an NADH-dependent CYP102A1 mutant. Adv. Synth. Catal. 2007, 349, 1451– 1461. (12) Celik, A.; Sperandio, D.; Speight, R.E.; Turner, N.J. Enantioselective epoxidation of linolenic acid catalyzed by cytrchrome P450(BM3) from Bacillus megaterium. Org. Biomol. Chem. 2005, 3, 2688-2690. (13) Kobayashi, T.; Laidler, K.J. Theory of the kinetics of reactions catalyzed by enzymes attached to the interior surfaces of tubes. Biotechnol. Bioeng. 1974, 16, 99-118. (14) Laidler, K.J.; Bunting, P.S. The Kinetics of immobilized enzyme systems. Methods in Enzym. 1980, 64, 227-248. (15) Iliuta, I.; Iliuta, M.C.; Larachi. F. Catalytic CO2 hydration by immobilized and free human carbonic anhydrase II in a laminar flow microreactor - model and simulations. Sep. Purif. Technol. 2013, 107, 61-69. (16) Kashid, M.N.; Agar, D.W. Hydrodynamics of liquid–liquid slug flow capillary microreactor: Flow regimes, slug size and pressure drop. Chem. Eng. J. 2007, 131, 1–13. (17) Bhattacharya, S.; Schiavone, M.; Chakrabarti, S.; Bhattacharya, S.K. CO2 hydration by immobilized carbonic anhydrase. Biotechnol. Appl. Biochem. 2003, 38, 111-117. (18) Drew, D.A.; Passman, S.L. Theory of multicomponent fluids. Springer-Verlag, New York, 1999. (19) Bretherton, F.P. The motion of long bubbles in tubes. J. Fluid Mech. 1961, 10, 166–188. (20) Gupta, R.; Leung, S.S.Y.; Manica, R.; Fletcher, D.F.; Haynes, B.S. Hydrodynamics of liquid–liquid Taylor flow in microchannels. Chem. Eng. Sci. 2013, 92, 180–189. (21) Kreutzer, M.T. Hydrodynamics of Taylor flow in capillaries and monolith reactors, Delft University Press, Delft, The Netherlands, 2003 (22) Liu, H.; Vandu, C.O.; Krishna, R. Hydrodynamics of Taylor flow in vertical capillaries: flow regimes, bubble rise velocity, liquid slug length, and pressure drop. Ind. Eng. Chem. Res. 2005, 44, 4884-4897. (23) Kreutzer, M.T.; Kapteijn, F.; Moulijn, J.A.; Heiszwolf, J.J. Multiphase monolith reactors: Chemical reaction engineering of segmented flow in microchannels. Chem. Eng. Sci. 2005, 60, 5895 – 5916


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(24) Raimondi, N.D.M.; Prat, P.; Gourdon, C.; Cognet, P. Direct numerical simulations of mass transfer in square microchannels for liquid– liquid slug flow. Chem. Eng. Sci. 2008, 63, 5522-5530. (25) Vandu, C.O.; Ellenberger, J.; Krishna, R. Hydrodynamics and mass transfer in an upflow monolith loop reactor. Chem. Eng. Process. 2005, 44, 363-374. (26) Abraham, M.H.; Smith, R.E.; Luchtefeld, R.; Boorem, A.J.; Luo, R.; Acree, W.E. Prediction of solubility of drugs and other compounds in organic solvents. J. Pharm. Sci.

2010, 99, 1500-1515. (27) Ralston, A.W.; Hoerr, C.W. The solubilities of the normal saturated fatty acids. J. Org. Chem. 1942, 7, 546-555. (28) Hoerr, C.W.; Ralston, A.W. The solubilities of the normal saturated fatty acids II. J. Org. Chem. 1944, 9, 329-337. (29) Vorlop, K.D. PhD Thesis, Technische Universit it Carola-Wilhelmina zu Braunschweig, Germany, 1986 (30) Reid, R.C.; Prausnitz, J.M.; Poling B.E. The properties of gases and liquids, fourth ed., McGraw Hill, New York, 1987. (31) Du Plessis, J.P.; Gray, W.G. Pore-scale modeling of interstitial transport phenomena. In: Fluid transport in porous media. Computational Mechanics Publication, Southampton, 1997, pp. 61-104. (32) Thulasidas, T.C.; Abraham, M.A.; Cerro, R.L. Dispesion during bubble-train flow in capillaries. Chem. Eng. Sci. 1999, 54, 61-70. (33) Hanna, J.; Iliuta, I.; Larachi, F.; Iliuta, Maria C. Enzymatic CO2 capture by immobilized hCA II in an intensified microreactor – kinetic study of the catalytic hydration. Int. J. Greenhouse Gas Control 2013, 15, 78-85. (34) Nakayama, N.; Takemae, A.; Shoun, H. Cytochrome P450foxy, a catalytically selfsufficient fatty acid hydroxylase of the fungus Fusarium oxysporum. J. Biochem. 1996, 119, 435-440. (35) Iliuta, I.; Larachi, F. New scrubber concept for catalytic CO2 hydration by immobilized carbonic anhydrase II & in-situ inhibitor removal in three-phase monolith slurry reactor. Sep. Purif. Technol. 2012, 86, 199-214.


26

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Table 1. Microreactor operating conditions (base case) Operating conditions

Data

Channel diameter

0.75 mm

Microreactor length

1.0 m

Washcoat porosity

0.5

Washcoat thickness

50 microns

Active P450foxy loading

2.85 × 10−6 kmol/m3reactor

Microreactor temperature

303 K

Superficial aqueous phase velocity

0.03 m/s

Superficial organic phase velocity

0.015 m/s

Inlet superficial gas velocity

0.0065 m/s

Inlet NADPH concentration in aqueous phase

0.002 mol/L

Inlet fatty acid concentration in organic phase

2 mol/L


27


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Page 28 of 46

Figure captions

Figure 1. Physical model with liquid-liquid slug flow through microreactor; different mass transfer fluxes used in the microreactor model.

Figure 2. Scheme for cytochrome P450foxy-mediated hydroxylation of tetradecanoic acid in a biphasic system. The organic phase serves as a substrate reservoir and extracts the products from the aqueous phase.

Figure 3. Predicted versus experimental data (enzymatic CO2 hydration in the presence of immobilized hCA II in an intensified microreactor; microreactor diameter=2.44 mm; active enzyme loading 0.019 µg hCA II/mm2; experimental data obtained by Hanna et al.33).

Figure 4. Predicted versus experimental data (enzymatic CO2 hydration in the presence of immobilized hCA II in an intensified microreactor; microreactor diameter=2.44 mm; active enzyme loading 0.019 µg hCA II/mm2; experimental data obtained by Hanna et al.33).

Figure 5. Hydroxy fatty acid time-dependent (at z/H=1) (a) and axial steady-state (b) concentration profiles in dispersed liquid phase (free enzyme in aqueous continuous liquid phase, immobilized enzyme, same P450foxy loading – base case operating conditions).

Figure 6. NADPH (a) and oxygen (b) axial steady-state concentration profiles in aqueous continuous liquid phase (free enzyme in aqueous continuous liquid phase, immobilized enzyme, same P450foxy loading – base case operating conditions).

Figure 7. Hydroxy fatty acid axial steady-state concentration profiles in dispersed liquid phase as a function of P450foxy enzyme loading: (a) free enzyme in aqueous continuous liquid phase; b.) immobilized enzyme (base case operating conditions).


28

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Figure 8. Axial steady-state concentration profiles in the capillary microreactor with immobilized P450foxy enzyme – influence of diffusion coefficients (base case operating conditions, P450foxy loading= 1.6 ×10 −4 kmol/m3washcoat = 2.85 × 10−6 kmol/m3reactor ).

Figure 9. Influence of washcoat thickness on hydroxy fatty acid and NADPH exit steady-state concentrations (a) and effectiveness factor (b) (base case operating conditions, P450foxy loading= 1.6 ×10 −4 kmol/m3washcoat = 2.85 × 10−6 kmol/m3reactor ).

Figure 10. Axial steady-state concentration profiles in the capillary microreactor with immobilized P450foxy enzyme – influence of gas velocity (base case operating conditions, P450foxy loading= 1.6 ×10 −4 kmol/m3washcoat = 2.85 × 10−6 kmol/m3reactor ).

Figure 11. Axial steady-state concentration profiles in the capillary microreactor with free enzyme in aqueous continuous liquid phase – influence of dispersed liquid phase velocity (base case operating conditions, P450foxy loading= = 2.85 × 10−6 kmol/m3reactor ).

Figure 12. Axial steady-state concentration profiles in the capillary microreactor with free enzyme in aqueous continuous liquid phase – influence of aqueous continuous liquid phase velocity (base case operating conditions, P450foxy loading= = 2.85 × 10−6 kmol/m3reactor ).

Figure 13. Axial steady-state concentration profiles in the capillary microreactor with free enzyme in aqueous continuous liquid phase – influence of substrate concentration in the dispersed

liquid

phase

(base

case

operating

conditions,

P450foxy

loading= = 2.85 × 10−6 kmol/m3reactor ).

Figure 14. Specific enzymatic activity as a function of substrate concentration in the dispersed liquid phase (capillary microreactor with free enzyme in aqueous continuous liquid phase - base case operating conditions, P450foxy loading= = 2.85 × 10−6 kmol/m3reactor ).


29


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Page 30 of 46

Figure 15. Axial steady-state concentration profiles in the capillary microreactor with immobilized P450foxy enzyme – influence of channel diameter (base case operating conditions, P450foxy loading= 1.6 ×10 −4 kmol/m3washcoat = 2.85 × 10−6 kmol/m3reactor ).


30

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Figure 1.

Washcoat (immobilized enzyme) Enzymatic reaction

Liquid slug Liquid slug Ng-c

LL NNGL Nd-c N NNLS cLS c-s

R2

Liquid droplet Gas bubble Liquid droplet Liquid droplet Ng-d NN dLS d-s

NGS

R1

dcc d

δf LLs c

LbLd Ld


31


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Page 32 of 46

Figure 2.

air

RH (C14H28O2)

ROH (C14H28O3)

Organic dispersed phase (cyclohexane)

aqueous continuum phase RH + O2 + H+ 50 mM MES (pH=6.5) NADP+ P450 foxy

ROH + H2O P450 foxy

NADPH


NADP+

32

Page 33 of 46

Figure 3.

10 experimental

Product (HCO3- ) concentration, mmol/L

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


model

8

6

4

2

0 4

6

8

10

12

14

16

18

Inlet substrate (CO2) concentration, mmol/L


33


Figure 4.

12

Product (HCO3- ) concentration, mmol/L

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 34 of 46

9

6

3 [Buffer] = 20 mmol/L; [ BufferH+] =10 mmol/L [Buffer] = 10 mmol/L; [BufferH+] = 20 mmol/L

0 0.005

0.01

0.015

0.02

0.025

0.03

0.035

0.04

0.045

Liquid velocity, m/s


34

Page 35 of 46

Figure 5.

Hydroxy fatty acid concentration-dispersed phase, mol/L

0.0035

(a) 0.003

0.0025

0.002

0.0015

0.001

0.0005

Immobilized enzyme solution-phase enzyme

0 0

20

40

60

80

100

120

Time, s

0.003 Hydroxy fatty acid concentration-dispersed phase, mol/L

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(b) 0.0025

0.002

0.0015

0.001

0.0005

immobilized enzyme solution-phase enzyme

0 0

0.2

0.4

0.6

0.8

1

1.2

z, m


35


Figure 6.

0.003

NADPH concentration-continuum phase, mol/L

immobilized enzyme solution-phase enzyme

0.0025

0.002

0.0015

0.001

0.0005

(a) 0 0

0.2

0.4

0.6

0.8

1

1.2

z, m

0.0003 immobilized enzyme Oxygen concentration-continuum phase, mol/L

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 36 of 46

solution-phase enzyme

0.0002

0.0001

(b) 0 0

0.2

0.4

0.6

0.8

1

1.2

z, m


36

Page 37 of 46

Figure 7.


0.004 Enzyme loading (reactor volume)=1.78e-6 mol/L Enzyme loading=2.85e-6 mol/L Enzyme loading=3.56e-6 mol/L

0.0035

0.003

0.0025

0.002

0.0015

enzyme loading 0.001

0.0005

(a) 0 0

0.2

0.4

0.6

0.8

1

1.2

z, m


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Enzyme loading (washcoat volume)=1.6e-4 mol/L Enzyme loading=2.4e-4 mol/L

0.0035

Enzyme loading=3.2e-4 mol/L 0.003

0.0025

0.002

enzyme loading

0.0015

0.001

0.0005

(b) 0 0

0.2

0.4

0.6

0.8

1

1.2

z, m


37


Figure 8. Hydroxy fatty acid concentration-dispersed phase, mol/L

0.003

(a) 0.0025

0.002

0.0015

0.001

0.0005 Model diffusion coefficients Model diffusion coefficients*2 0 0

0.2

0.4

0.6

0.8

1

1.2

z, m

0.003


(b)

Model diffusion coefficients Model diffusion coefficients*2

0.0025

0.002

0.0015

0.001

0.0005

0 0

0.2

0.4

0.6

0.8

1

1.2

z, m

0.0003 Model diffusion coefficients

(c) Oxygen concentration-continuum phase, mol/L

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 38 of 46

Model diffusion coefficients*2

0.0002

0.0001

0 0

0.2

0.4

0.6

0.8

1

1.2

z, m


38

Page 39 of 46

Figure 9.

0.004

(a)

Hydroxy fatty acid concentration in dispersed phase

Concentration at z/H=1.0, mol/L

0.0035

NADPH concentration in aqueous phase

0.003

0.0025

0.002 0.0015

0.001

0.0005

0 0

20

40

60

80

100

120

Washcoat thickness, microns

0.5

(b) 0.4 Effectiveness factor at z/H=1.0

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0.3

0.2

0.1

0 0

20

40

60

80

100

120

Washcoat thickness, microns


39


Figure 10.


0.0025

(a)

0.002

0.0015

0.001

0.0005

Superficial gas velocity=0.0065 m/s Superficial gas velocity=0.0035 m/s Superficial gas velocity=0.0015 m/s

0 0

0.2

0.4

0.6

0.8

1

1.2

z, m

0.0003

(b) Oxygen concentration-continuum phase, mol/L

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 40 of 46

Superficial gas velocity=0.0065 m/s Superficial gas velocity=0.0035 m/s Superficial gas velocity=0.0015 m/s

0.0002

0.0001

0 0

0.2

0.4

0.6

0.8

1

1.2

z, m


40

Page 41 of 46

Figure 11. 0.003


Superficial organic phase velocity=0.025 m/s Superficial organic phase velocity=0.015 m/s

(a)

Superficial organic phase velocity=0.01 m/s

0.0025

0.002

0.0015

0.001

0.0005

Organic phase velocity 0 0

0.2

0.4

0.6

0.8

1

1.2

z, m

0.0003 Superficial organic phase velocity=0.025 m/s

(b)

Oxygen concentration-continuum phase, mol/L

Superficial organic phase velocity=0.015 m/s Superficial organic phase velocity=0.01 m/s

0.0002

Organic phase velocity

0.0001

0 0

0.2

0.4

0.6

0.8

1

1.2

z, m


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(c)

Superficial organic phase velocity=0.025 m/s Superficial organic phase velocity=0.015 m/s Superficial organic phase velocity=0.01 m/s

0.0035

0.003

0.0025

0.002

0.0015

Organic phase velocity 0.001

0.0005

0 0

0.2

0.4

0.6

0.8

1

1.2

z, m


41


Figure 12.


0.004 Superficial aqueous phase velocity=0.035 m/s

(a)

Superficial aqueous phase velocity=0.03 m/s Superficial aqueous phase velocity=0.025 m/s

0.0035

0.003

0.0025

0.002

Aqueous phase velocity 0.0015

0.001

0.0005

0 0

0.2

0.4

0.6

0.8

1

1.2

z, m

0.0003 Superficial aqueous phase velocity=0.035 m/s Oxygen concentration-continuum phase, mol/L

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 42 of 46

(b)

Superficial aqueous phase velocity=0.03 m/s Superficial aqueous phase velocity=0.025 m/s

0.0002

0.0001

Aqueous phase velocity

0 0

0.2

0.4

0.6

0.8

1

1.2

z, m


42

Page 43 of 46

Figure 13.


0.004 Fatty acid concentration in dispersed phase=2 mol/L Fatty acid concentration in dispersed phase=0.5 mol/L Fatty acid concentration in dispersed phase=0.2 mol/L 0.003

0.002

0.001

(a) 0 0

0.2

0.4

0.6

0.8

1

1.2

z, m

0.003 Fatty acid concentration in dispersed phase=2.0 mol/L NADPH concentration-continuum phase, mol/L

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Fatty acid concentration in dispersed phase=0.5 mol/L Fatty acid concentration in dispersed phase=0.2 mol/L

0.0025

0.002

0.0015

0.001

0.0005

(b) 0 0

0.2

0.4

0.6

0.8

1

1.2

z, m


43


Figure 14.

25

Specific catalytic activity, mol NADPH/mol enzyme/se

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 44 of 46

20

15

10

5

0 0

0.5

1

1.5

2

2.5

Fatty acid concentration in dispersed phase, mol/L


44

Page 45 of 46

Figure 15.


0.003

(a) 0.0025

0.002

0.0015

0.001

0.0005 Microreactor diameter=0.75 mm Microreactor diameter=1 mm 0 0

0.2

0.4

0.6

0.8

1

1.2

z, m

0.003

(b) NADPH concentration-continuum phase, mol/L

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Microreactor diameter=0.75 mm Microreactor diameter=1 mm

0.0025

0.002

0.0015

0.001

0.0005

0 0

0.2

0.4

0.6

0.8

1

1.2

z, m


45


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 46 of 46

For Table of Contents Only


46

Enzymatic Fatty Acid Hydroxylation in a LiquidâLiquid Slug Flow

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