Analysis and Design of Extractive Fermentation Processes Using a Novel Short-cut Method

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Analysis and Design of Extractive Fermentation Processes Using aNovel Short-Cut MethodLuis F. Gutierrez,

, O scar J. Sanchez,, and Carlos A. Cardona*,

,

Department of Engineering and Institute of Agricultural Biotechnology, Universidad de Caldas, Calle 65 No. 26-10, Manizales,ColombiaInstituto de Biotecnologa y Agroindustria, Departamento de Ingeniera Qumica, Universidad Nacional de Colombia sedeManizales, Cra. 27 No. 64-60, Manizales, Colombia

*S Supporting Information

ABSTRACT: One means of increasing the productivity of microbiological processes is to remove toxic inhibiting products asthey are formed through extractive fermentation. The aim of this work was to develop a short-cut tool to allow the design andanalysis of extractive fermentation processes. For this purpose, a rigorous model considering both the fermentation kinetics andthe application of a thermodynamic phase equilibrium model was developed for batch and continuous regimes. The outcomes

and regularities found during the analysis of extractive fermentation using the rigorous model were considered to develop theshort-cut tool. The short-cut method was based on the principles of topological thermodynamics and was applied to several casestudies in this article. This method enables preliminary information to be acquired and used during the subsequent rigoroussimulation, enabling a decrease in the calculation efforts and in the number of experimental runs.

1. INTRODUCTION

Most industrial microbiological processes are carried out in thebatch regime. However, the features of microbial metabolismare among the main causes for the low concentration of targetmetabolites obtained in this regime. This is explained by theinhibition effect caused by the end products on either the cellgrowth rate or the product biosynthesis rate, as occurs in the

production of acetone and butanol, lactic acid, and ethanol.

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Similarly, different metabolic intermediate or byproducts can beinhibitory, as in the case of acetic acid and lactate duringethanologenic fermentation using Clostridium thermocellumfrom cellulosic substrates.4,5 On the other hand, highconcentrations of the carbon source, particularly glucose, caninhibit both the growth and product formation rates, as in thecase of alcoholic fermentation.6 A rational approach toenhancing the fermentation performance is to remove thetoxic inhibiting products as they are formed in the culture

broth. To reach this aim, a biocompatible extracting agent(solvent) can be added to the medium in such a way that theproduct migrates to the solvent phase. This is the principle ofextractive fermentation combining liquidliquid extraction with

the fermentation process (reaction

separation integration).Continuous processes have several advantages compared tobatch processes, mainly because of their higher productivities,reduced capital costs, lower maintenance and operationrequirements, and better process control. In addition,continuous processes allow the product inhibition effect to bedecreased through a cascade of continuous reactors.2 Never-theless, end-product inhibition cannot be completely neutral-ized by implementing continuous processes. Extractivefermentation is an attractive alternative to overcome thisdifficulty.

Extractive fermentation can be considered as an integratedprocess in which a reaction process (fermentation) is combined

with a separation operation (liquid extraction).5 Thus,extractive fermentation could have the key to reduce thecosts of fuel ethanol production.7,8 In the early 1980s, Minierand Goma9 reported the operation of a continuous extractivefermentation system for the conversion of a glucose-basedmedium into ethanol using immobilized yeast cells and n-dodecanol as the solvent. In that work, the solvent improvedthe fermentation performance by reducing the inhibition effect

of ethanol and enabling the use of concentrated solutions ofglucose (up to 409 g/L). At this point, it is worth noting thatthe relationship between the glucose concentration and the

water activity imposes an additional upper limit because of theavailability of free water needed for biomass growth. In thiscase, the minimum water activity for yeast growth is about0.90,10which corresponds to 485.4 g/L glucose in the broth, sothe increase in the glucose concentration due to productremoval by extractive fermentation does not limit the yeastgrowth rate. To enhance ethanol production, a process withfurther integration through the simultaneous achievement ofsaccharication and extractive fermentation (in the case ofpolymeric carbohydrate substrates such as starch or cellulose)

was proposed by Moritz and Duff.11 This kind of integrated

processes has also been applied for the production of othermetabolites. Wayman and Parekh12 demonstrated an increasein the substrate conversion due to the effect of the extractingagent (dibutylphthalate) on the fermentation of culture mediafor producing a mixture of acetone and butanol.

The task of modeling such integrated processes as extractivefermentation is crucial for the appropriate design of the process

Received: May 17, 2012Revised: April 21, 2013Accepted: August 6, 2013Published: August 6, 2013

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M in eq 1) and amount of the extract is introduced in thebalances.

The conguration of continuous extractive fermentationinvolves the continuous feeding of culture medium and solventinto the reactor. Similarly, both aqueous and solvent phases arecontinuously removed from the system in a separate way (seeFigure 1). Stream Q leaving the bioreactor contains cells,culture medium, and solvent. During the phase splitting in thedecanter, the cells and exhausted culture medium remain in theeffluent aqueous stream (Q A), whereas the effluent solventstream (QE) removes the extracted product. Depending on theproduct properties, the organic phase with the extractedproduct can be sent to a ash vaporization unit or otherseparation device, where it is collected and regenerated solventis obtained. The solvent can be recycled back to the extractivefermentation bioreactor. The aqueous stream leaving thesystem can be sent to wastewater treatment or to additionaldownstream operations if a valuable product is present. Ifneeded, the cells can be recovered from this stream. In general,the mass ow rate (in liters per hour) of inuent aqueousstream (FA) is greater than the ow rate of stream QA(denotedasQA) because of the migration of the extractable product tothe solvent phase. Likewise, the ow rate of the inuent solventstream (FE) is less than the ow rate of the effluent solventstream (QE). The material balance can be applied for the case

when the solvent contains small amounts of the product as aresult of incomplete regeneration of the extracting agent. For adetailed representation of the mass balance equations of thisprocess, see Appendix A (Supporting Information). One of themain problems during chemostat operation is the lowconcentration of the cell biomass. To overcome this drawback,recycling of the cell biomass can be implemented. In this way,this recycling provides some decoupling from hydraulics,leading to the compensation of toxicity effects and improvingthe volumetric productivity. The proposed rigorous model can

be slightly modied to consider such types of recirculation

streams.During batch extractive fermentation, the culture medium

loaded into the bioreactor contains the nutrients required bythe microbial cells. Once the fermentation starts, solvent isadded to extract the end product formed. The bioreactor isstirred to disperse the solvent phase. At the end of cultivation,the medium is discharged, and then the solvent and aqueousphases are separated in a decanter. The solvent can beregenerated to be used for subsequent batches. The volumeratio of the solvent and aqueous phases (RV) was changed toanalyze its effect on the extraction parameters. The features ofrigorous modeling for batch extractive fermentation are shownin Appendix A of the Supporting Information.

2.1. Case Study 1: Modeling of Ethanol Production by

Extractive Cofermentation. In this case study, ethanolproduction by an extractive cofermentation process wasanalyzed using detoxied lignocellulosic hydrolyzate. Thishydrolyzate was obtained through several steps: pretreatmentof the lignocellulosic biomass, detoxication of the pretreated

biomass to remove compounds such as furfural formed duringthe previous step, and hydrolysis of the exposed cellulosecontained in the biomass.

2.1.1. Batch Regime. The kinetic model of batchalcoholicfermentation was taken from Leksawasdi et al.,20 whodeveloped it from experimental data. This model describesthe simultaneous consumption by a recombinant Zymomonasmobilis strain of the two main substrates contained in the

detoxied lignocellulosic hydrolyzates: glucose and xylose. Inaddition, it takes into account the biomass growth and ethanolproduction. The fatty alcoholn-dodecanol was selected as thesolvent. Similar analysis was performed using oleyl alcohol asthe solvent.

Based on the model of Leksawasdi et al.,20 the simulation ofthe batch alcoholic fermentation from lignocellulosic hydro-lyzate was performed with an initial glucose concentration of100 g/L and an initial xylose concentration of 50 g/L. Theculture behavior can be observed in Figure2a, where a decline

in cell concentration because of the relatively high amounts ofethanol in the broth can be seen after 25 h. The model of theseauthors captures the inhibition of the growth rate due to thepresence of ethanol. According to this model, if the ethanolconcentration is greater than the threshold ethanol concen-tration (28.9 g/L), the end-product inhibition effect can beclearly noticed (see the model in Appendix A of the SupportingInformation).

The use of more concentrated culture media leads to theunderutilization of expensive feedstocks that cannot be

Figure 2. Comparison of conventional and extractive batchcofermentations for ethanol production using Z. mobilis. The curveswere calculated by the rigorous model using the kinetic expressionstaken from Leksawasdi et al.20 (a) Batch cofermentation with nosolvent addition. (b) Batch extractive cofermentation with addition ofn-dodecanol as the extraction agent (RV= 2.0); the addition of the

solvent was performed at 5 h of cultivation. S 1, glucose; S2, xylose; X,cells; P, ethanol in the aqueous phase; P*, ethanol in the solvent phase.

Industrial & Engineering Chemistry Research Article

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transformed into ethanol despite their availability in the broth.According to the model of Leksawasdi et al.,20 which alsocaptures the limitation in growth rate at high substrateconcentrations, when a medium containing up to 400 g/Lfermentable sugars is employed, an ethanol concentration ofabout 71.2 g/L is reached only after 80.5 h of cultivation.

The addition of solvent allows the removal of ethanol fromthe aqueous phase, as depicted in Figure2b. Ifn-dodecanol isadded to the medium once at 5 h of cultivation, the ethanolconcentration in the aqueous phase is reduced, leading to alower end-product inhibition effect. In this case, the solvent isnot removed from the culture broth until the total depletion ofthe substrates. At that time, ethanol-enriched solvent isregenerated byash vaporization. The total ethanol productiv-ity for batch extractive fermentation (evaluated as the mass ofethanol recovered from both phases per liter of workingaqueous volume per unit of time) using n-dodecanol dependson the solvent volume/aqueous volume ratio (RV). Therigorous model showed a total ethanol productivity of 1.89g/(L h) when the substrates were exhausted. Considering onlythe solvent phase, the ethanol productivity reached 0.82 g/(Lh). For comparison, the ethanol productivity when no solvent

was used reached 1.49 g/(L h). On the other hand, theconcentration proles of both substrates and cells in theaqueous phase indicated that the extractive effect of the solventenabled the reduction of the cultivation time for the same initialsubstrate concentration until both sugars were exhausted: from44 h without solvent addition to 27 h when n-dodecanol wasadded (see Figure 2). For oleyl alcohol, the total ethanolproductivity and ethanol productivity from the solvent phase

were 1.98 and 0.63 g/(L h), respectively.The behavior of productivity as a function of the solvent

volume/aqueous volume ratio (RV) was also assessed. Higherratios led to lower productivities. For high ratios (RVabove 8.1for n-dodecanol), the algorithm developed by the authors ofthis article (based on the rigorous model) predicts the

formation of homogeneous mixtures that do not allow theextraction of ethanol. Modeling results for volume ratios lowerthan 0.54 predict a homogeneous mixture as well. Theseoutcomes were obtained using the UNIFAC equations. At thispoint, the algorithm enables the activity model used to beshifted to calculate the liquidliquid equilibrium of the systemunder evaluation. Thus, the nonrandom two-liquid (NRTL)equation predicts homogeneous mixtures for the system

waterethanoln-dodecanol where phase splitting existsaccording to the experimental data provided by Kirbaslar etal.,21 as can be seen in theSupporting Information(Figure B1).In the case of the UNIQUAC (universal quasichemical) model,the liquidliquid envelope is located above the experimentalenvelope. The UNIFAC model was demonstrated to be

appropriate for describing the liquidliquid equilibrium ofthe analyzed system by comparison to the data of Kirbaslar etal. Consider the analysis of one raffinate point with thefollowing composition (in mass fraction): 0.024 n-dodecanol,0.423 ethanol, and 0.553 water. UNIFAC predicts a point withthe composition 0.01 n-dodecanol, 0.43 ethanol, and 0.56

water, whereas UNIQUAC predicts the composition 0.00 n-dodecanol, 0.43 ethanol, and 0.57 water. NRTL does notpredict any phase splitting at this point. A similar analysis foroleyl alcohol was also performed, giving a similar behavior.

2.1.2. Continuous Regime. Some simulation results ofcontinuous ethanologenic fermentation without solventaddition using the model of Leksawasdi et al.20 and considering

a glucose concentration of 100 g/L and a xylose concentrationof 50 g/L in the feed stream are provided in the SupportingInformation (see Figure A1). The dilution rate (D = F/V,

where F is the ow rate of the inuent or effluent stream, inliters per hour, and V is the volume of culture medium, inliters) corresponding to cell wash-out is about 0.32 h1. Theimprovement of the process when the solvent is employed isevident in Figure3, where the distribution of ethanol between

the two immiscible liquid phases occurs. Thus, the addition of

n-dodecanol leads to a 3-fold increase in total ethanolproductivity compared to maximum ethanol productivity forthe process without any solvent [5.17 g/(L h) atD= 0.15 h1].This comparison was done for the extractive fermentationprocess with a dilution rate of the feed aqueous stream (DAi=

FA/VA, whereVAis the volume of the aqueous phase, in liters)equal to 0.25 h1. Considering productivity only from thesolvent phase, the increase reaches 78.2%. These same valuesfor the case when oleyl alcohol was used as solvent are 2.29times and 9.9%, respectively. In addition, the concentrations of

both substrates at maximum total ethanol productivity arelower than in the case of conventional continuous culture,

where substrates are not completely utilized. These results wereobtained for a ratio between the inlet solvent stream and the

inlet culture medium (aqueous) stream (R) of 2.0. The coupledalgorithm was employed for simulation of the process with

varyingRvalues for an inlet dilution rate of 0.25 h1, that is, adilution rate corresponding to the maximum ethanolproductivity in the previous simulations. For n-dodecanol, the

best results were obtained for values above 4, corresponding toan increased amount of consumed substrates. Both the totalproductivity and the productivity for ethanol recovered fromthe solvent phase approach constant values: 18.2 and 15.5 g/(Lh), respectively. Note that the substrate consumption wasfound to increase with increasing feed ow rate ratio, indicatingthe possibility of using more concentrated media in thecontinuous regime.

Figure 3. Continuous extractive cofermentation of a lignocellulosichydrolyzate for ethanol production. Effect of aqueous dilution rate ofinuent stream (DAi) on the effluent concentrations of glucose (S1),

xylose (S2), cells (X), ethanol in the aqueous phase (P), and ethanol inthe solvent phase (P*), usingn-dodecanol as the extractive agent. Theratio of the inlet solvent stream to the inlet culture medium (aqueous)stream (R) was 2.0. Concentrations of sugars in the feed aqueousstreams: glucose, 100 g/L; xylose, 50 g/L.




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Based on the proposed modeling approach, the simulation ofthe extractive cofermentation process was performed modifying

both R and DAi. The results for the case of n-dodecanol are

presented in Figure4, where the positions of the pseudoinitialcompositions of the system and trajectories through which itmoves for each set of inlet conditions are represented in theternary waterethanoln-dodecanol diagram. In this gure,each point on the curves represents one pseudoinitial mixturefor given inlet conditions of R and DAi. Then, the mixture

separates into two liquid phases in equilibrium (extract and

raffi

nate) that are considered as the steady states of the system.The points with identicalDAivalues are represented by a single

line. The zone of manipulated variables where higherproductivities were obtained was found to correspond todilution rates near wash-out conditions (below 0.32 h1) and tohigher values ofR(near the solubility limit of the extract, i.e.,7.8). The solubility (binodal) curve obtained using the rigorousmodel agreed well with the experimental data obtained byKirbasl ar et a l.21 (see Appendix B of the SupportingInformation).

The results presented in this section were obtained for acomposition of the feed aqueous stream (nutritive medium) of100 g/L glucose and 50 g/L xylose. If these concentrations

were changed, the analysis of the system would become more

complex.17

The simulation of the process utilizing moreconcentrated culture broths is illustrated in the SupportingInformation(see Table C1 in Appendix C). The best values forthe total productivity and productivity of ethanol recoveredfrom the solvent phase were found to correspond to an inletconcentration of total sugars of approximately 600 g/L.

3. DEVELOPMENT OF A SHORT-CUT METHOD FORANALYSIS OF EXTRACTIVE FERMENTATION

In the past 20 years, the analysis of statics that is based on theprinciples of a thermodynamic-topological approach has beensuccessfully applied to integrated processes such as reactivedistillation. This methodology denes whether this type of

process can be carried out,as well as the spectrum of feasibleinitial operating conditions.18 However, the analysis of staticshas not yet been applied to extractive fermentation processes.In a previous work,17 the rst steps for developing a short-cuttool were accomplished, but without the validation of the dataobtained using actual experimental data for both the batch andcontinuous regimes.

Starting from the well-known representation of liquidliquidequilibrium in ternary diagrams showing the state of the systemin terms of the fermentation product, water as the main

component of the culture medium, and solvent as the extractiveagent, it is possible to add another key component involved inthe biochemical transformation, namely, the substrate. Thus, aquaternary diagram is obtained (see Figure 5). In this sense,any point in the quaternary diagram can represent thepseudoinitial composition of the system. If this compositionfalls into the space of heterogeneous mixtures (the goal of an

Figure 4.Representation in the ternary diagram of the steady states achieved during the rigorous simulation of alcoholic extractive cofermentationusingn-dodecanol for different operating conditions. Concentration of sugars in feed aqueous stream: glucose, 100 g/L; xylose, 50 g/L. The diagramwas amplied in the lower part. The units of the inlet aqueous dilution rate (DAi) are h

1.

Figure 5. General representation of extractive fermentation in theconcentration simplex. The dashed lines represent the processtrajectory.




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extractive fermentation process), the system, after havingundergone some biochemical transformation, will move towardthe extract and raffinate compositions following the tie lines ofthe liquidliquid equilibrium. This displacement from thestarting point toward the product streams can be considered asthe trial trajectory of the process analogously to the trialtrajectory of a reactive distillation process.19 Thus, the systemmoves from the pseudoinitial point to the corresponding steadystates (extract and raffinate), namely, the product streams. The

basis for the extension of these principles from reactivedistillation to extractive fermentation were comprehensivelydiscussed in a previous work.22

The procedure for locating the steady states in theconcentration simplex using the proposed short-cut methodcan be conceptually divided into the following steps:

Locate the point representing the composition of thefeed aqueous stream (nutritive medium). This point islocated on the edge representing the binary compositions

watersubstrate (see point A in Figure 5). Convert the substrate into the fermentation product

(microbial transformation). This conversion is repre-

sented by the displacement of point A toward point Blocated in the plane representing the ternary composi-tions substrateproductwater.

Add the extracting agent (feed solvent stream). This isrepresented by line BC in Figure 5. The addition of thesolvent is accomplished until point D is reached withinthe zone of heterogeneous compositions.

Separate the system into two liquid phases. Thecompositions of the effluent solvent stream, or extract(E), and effluent aqueous stream, or raffinate (W), areobtained through the tie lines.

The initial information needed to apply the proposed short-cut method includes data on the xed points (pure substances)and the solubility curve of the system (liquidliquid

equilibrium). The nal aim of the method is to determinethe zone of feasible operating conditions with highproductivities. This is illustrated in the following cases studies.

3.1. Case Study 2: Application of the Short-CutMethod for Analysis of Ethanologenic ExtractiveCofermentation.In ref17, the basis of the proposed short-cut method was preliminarily applied to the production ofethanol by extractive fermentation using n-dodecanol as thesolvent. In this work, some complementary issues are disclosedand applied to other systems in a systematic way. The maincomponents (sugarswaterethanoln-dodecanol) are repre-sented in a quaternary diagram to determine the processtrajectory, including the biochemical transformation. For therepresentation of the reaction trajectory, considering that the

overall fermentation process is irreversible, a stoichiometricapproach was employed. In this case, the cultivation processcan be described as

+C H O 2C H OH 2COZ mobilis

6 12 6glucose

.

2 5ethanol

2

+3C H O 5C H OH 5COZ mobilis

5 10 5xylose

.

2 5ethanol

2

The substrate is represented as the sum of both sugars(glucose and xylose) to simplify the graphical representation ofthe process trajectory. The location of the pseudoinitial mixtureis determined considering a substrate concentration of 150 g/L

(point A in Figure 6). The maximum stoichiometric productyield from substrate (0.511 g of ethanol/g of sugar consumed)

was considered as well. This implies the total conversion of thesugars, and therefore, the system behavior can be represented inthe ternary diagram ethanolwatern-dodecanol (see Figure7). Thus, the complete transformation of sugars into ethanol is

shown by line AB (Figure6), where B is the state of the systemwhere the total amount of produced ethanol is represented.The ternary diagram requires the inclusion of the binodal curve.Experimental data or commercial simulators can be used toobtain this curve separating the zone of homogeneous andheterogeneous mixtures. In this work, this curve was obtained

by the algorithm developed.Point B is the starting mixture for the liquidliquid

equilibrium. Line BC represents the addition of the solvent(n-dodecanol) to the aqueous medium containing the product(ethanol), as shown in Figure 7. In this gure, vertical linescorrespond to the points representing the operating conditionsrelated to the feed solvent stream/feed aqueous stream ratio(R). This fact was deduced from the modeling results shown inFigure4. The intersection of these vertical lines with line BC(the pseudoinitial mixture indicated by point D) represents thetheoretical conditions corresponding to the mixture beforephase separation (equivalent to the feed mixture in a liquidextractor). The location of point D determines the value of

Figure 6. Application of the short-cut method to the ethanologenicextractive cofermentation represented in the quaternary diagram. Thecompositions are shown in mass fraction.

Figure 7. Application of the short-cut method to the ethanologenicextractive cofermentation represented in the ternary diagram. Thecompositions are shown in mass fraction.




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ratioR, and vice versa. To graphically represent the conditionslinked to R in the ternary diagram, mass balance equationsshould be employed. The sum of the mass ow rates of the feedaqueous stream (FA) and the feed solvent stream (FE) gives themass of the overall mixture before phase separation

+ =F F MA A E E (1)

whereMis the mass

ow rate of the pseudoinitial mixture (ingrams per hour) and Aand Eare the densities (in grams perliter) of the feed aqueous and solvent streams, respectively. Thecorresponding equation for the outlet streams (extract andraffinate), assuming that there are no signicant changes in thedensities of the two phases, is

+ =Q Q MA A E E (2)

The product balances for the pseudoinitial mixture are given by

+ = *F x F x MxA A A E E E (3)

+ = *Q x Q x MxA A A

RE E E

E(4)

wherexAis the product mass fraction corresponding to point G

in Figure 7, namely, the product composition when thesubstrate contained in the feed aqueous stream is convertedinto the product before the virtual mixing leading to thepseudoinitial mixture, that is, the amount of product appearingin the system due to the fermentation before the liquidliquidextraction;xE is the product mass fraction in the feed solventstream (equal to zero when fresh solvent is used); xA

R is theproduct mass fraction in the raffinate (point W in Figure7),xE

E

is the product mass fraction in the extract (point E); and x*isthe composition of the pseudoinitial mixture (point D).

To determineFEfrom a givenFAhaving chosen a point D online BC, the values of xA and x* are directly read from theternary diagram and used in the equation

=

*

*

F

F x x

x x( )( )

E A A

E

A

E (5)

In this way, the ratio R = FE /FA is readily calculated. Thevertical line representing the ratio R links the base of theternary diagram to point D (line R2in Figure7). Alternatively,for given FAandR, the location of point D in the diagram isdetermined from eq5 by solving for the product composition

* =

+

+x

F x F x

F F

A A A E E E

A A E E (6)

To determine the ow rates of the outlet streams (QE andQA), the points corresponding to the extract (E) and raffinate(W) can be located in the diagram by using the tie line passing

through point D. With the compositions of W and E read fromthe diagram, the ow rates can be calculated from eqs24asfollows

=* +

Q

x x F F

x x

( )( )

( )AEE

A A E E

A AR

EE

(7)

= + Q F F Q ( )E E

E

AA A

(8)

Extractive fermentation is aimed at reducing end-productinhibition. To take this effect into consideration, it is necessaryto locate the inhibition boundary in the ternary diagram. If the

composition of the raffinate is above this boundary, themicroorganisms limit their growth because of an excessiveconcentration of the product in the aqueous phase. Therefore,the ethanol concentration in the outlet aqueous stream(raffinate) should not be greater than the ethanol inhibition

boundary whose location is determined by point I in Figure7.The intersection of the tie line passing through point I with the

balance line BC (point J) represents the working conditionsrelated to the ratioR limited by ethanol inhibition (line R2 inFigure7). The pseudoinitial mixtures located to the left of lineR2are not advantageous for the system, and therefore, the zoneof feasible operating conditions can be limited by this line. InFigure8, this zone is represented by the area delimited by the

line R1 and the right branch of the binodal curve (the grayshaded zone). When the concentrations of sugars in the feedaqueous stream increase up to values lower than the maximumsolubility of these sugars in water to avoid crystallization (asshown in Figure6), the total amount of product formed basedon the stoichiometry increases as well, and therefore, point B isdisplaced toward product along the edge of the ternary diagramcorresponding to waterproduct binary mixtures. This newpoint should be below the line corresponding to the substratesolubility boundary determined by point H (Figure 8). This

boundary is shown as a shaded plane in Figure6, although itshould be strictly represented by a surface. In this way, theoperating zone for extractive cofermentation can be constrainedto a more reduced area delimited by line R3 and the right

branch of the binodal curve (the dark gray shaded zone untilthe bottom edge of the diagram in Figure 8). When the initialsubstrate concentrations in the feed stream are changed, the

vertical lines showing the values of R are displaced incomparison to the original for starting point B in Figure7.

We now analyze the extreme case when the feed aqueousstream has the maximum allowable concentration of sugars.From the stoichiometry of ethanologenic fermentation, thiscondition corresponds to an inlet concentration in the feedaqueous stream of approximately 600 g/L of total sugars.

Assuming the maximum theoretical yield, the total amount ofethanol that could be produced from this amount of sugars is0.511 g/g. This yield implies a theoretical starting ethanolconcentration of 306.6 g/L, which corresponds to an ethanolmass fraction of about 0.31 (point L). This value determinesthe position of the substrate solubility boundary for waterethanol mixtures (point H in Figure8). Since the concentrationof ethanol in the aqueous phase (raffinate) should not be above

Figure 8.Determination of the zone of feasible operating conditionsfor the ethanologenic extractive fermentation process in the ternarydiagram. The compositions are shown as mass fractions.




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the ethanol inhibition boundary (approximately 10% w/w), theoperation conditions represented by the ratio R3should be suchthat the ethanol content in the raffinate be equal or less thanthe ethanol content of the point I to avoid product inhibition.Thus, the zone delimited by the dark gray shaded area (untilthe bottom edge of the diagram) is the zone of feasible steadystates for given operating conditions of the extractivecofermentation process with the maximum allowable concen-

trations of sugars in the aqueous feed stream.For instance, let us consider a continuous ethanologenicextractive fermentation process usingZ. mobiliswith a 0.11 L/hfeed aqueous stream containing 295 g/L glucose, an inletsolvent stream ow rate/aqueous stream ow rate ratio of 4.0, a

working aqueous volume of 4.8 L, and a working solventvolume of 1 L (the conditions reported by Bruce et al.23). Thelocation of the pseudoinitial mixture (point D in Figure 8) can

be found by using eq6. Then, the corresponding compositionsof the extract and raffinate can be located using thecorresponding tie line (indicated by two rhombuses on the

binodal curve in Figure8). Thus, the ethanol content of pointD and the compositions of the extract and raffinate read fromthe diagram are 3.3%, 2.5%, and 4.5%, respectively (by weight).

According to these data, the total ethanol productivity of thesystem reaches 15.92 g/(L h). The total ethanol productivitycalculated by the rigorous model using the developed algorithmis 15.13 g/(L h), which is an acceptable difference for a short-cut method. The experimental data reported by Bruce et al.23

are close to the results of the proposed short-cut method (seeTable 1). Hence, the proposed method enabled thedetermination of the feasibility of operating parameters andan estimation of the productivities. From the rigoroussimulation, this zone corresponds to the regimes with highertotal ethanol productivities (see Figure4).

3.2. Case Study 3: Application of the Short-CutMethod for Analysis of Lactic Acid Extractive Fermenta-

tion. Lactic acid is a biotechnological commodity with manyapplications in the food, chemical, and pharmaceuticalindustries. One of the main challenges in lactic acid production

by fermentation is the end-product inhibition effect. Extractivefermentation has the potential to reduce this effect, therebyimproving the separability and recovery of this acid fromculture broths.

The main results obtained from the rigorous modeling of an

extractive fermentation process for lactic acid production areshown in Appendix D of the Supporting Information. Theshort-cut method presented here was applied to the lactic acidextractive fermentation of a glucose-based medium using amixture of tri-n-decylamine (TDA) and oleyl alcohol as theextracting agent. To establish the lactic acid inhibition

boundary, an inhibitory product concentration of 100 g/Lwas assumed. The analysis followed the same procedure asdescribed in the preceding section for ethanol extractivefermentation. Figure8can be used to explain the applicationof this method. From the stoichiometry of lactic acidfermentation, a maximum theoretical yield of 1 g of lacticacid/g of glucose was considered. The equivalent starting lacticacid concentration after microbial conversion was 10% (w/w),

which is represented by point B. In this case, the feed aqueousstream ow rate was set to 0.5 L/h. To delimit the operatingzone, the ratioRdened by the lactic acid inhibition boundary

was calculated using eq5, reading thexAvalues of point G andthex*values of point J from the ternary diagram. The ratio Robtained was 1.40 (line R1). Then, the zone constrained by thesubstrate solubility boundary was also dened using the sameequation and considering the xAvalues of point H and the x*

values of pointK. The corresponding ratioRwas 6.7 (line R3).In this way, the operating zone with high productivities for themaximum allowable glucose concentration in the feed aqueousstream (600 g/L) was located to the right of line R3until theright branch of the binodal curve. This zone ensures that no

Table 1. Comparison of the Data Calculated by the Rigorous Model and Obtained Using the Short-Cut Method to ExperimentalData Reported for Some Extractive Fermentation Processes

process parameterrigorousmodel

short-cut

methodexperimental

data ref

batch extractive fermentation for lactic acid production using TDAa/oleylalcohol, initial glucose concentration = 40 g/L,S. cerevisiae,RV= 2.0

ethanol in the aqueous phase at theend of fermentation (g/L)

19.10 20.0 Gao et al.24

ethanol in the organic phase at the

end of fermentation (g/L)

40.90 35.0

continuous ethanol production using oleyl alcohol as the solvent, 295 g/Lglucose in the feed stream, Z. mobilis

total ethanol concentration (g/L) 46.93 50.10 55.0 Gutierrez,22

this workres idua l glucose concentrat ion (g/L) 7.87 14.75 3.0

biomass c oncentration (g/L) 8.24 7.0

tota l e thanol productivity [g/(L h)] 15.13 15.92 15.6

continuous ethanol production using oleyl alcohol, 300 g/L glucose in thefeed stream,S. cerevisiae, hollow-ber-based reactor,R= 3.0

increase of ethanol productivityrelative to fermentation withoutsolvent (%)

211.0 171.0 Kang et al.25

continuous lactic acid production using TDAa/oleyl alcohol, 40 g/Lglucose in the feed stream, L. casei,R= 2.0

total lactic acid productivity[g/(L h)]

19.38 20.24 this work

lactic acid in the aqueous phase(g/L)

6.25 1.1

lactic acid in the organic phase (g/L) 16.25 20.24

continuous ethanol production usingn-dodecanol as the solvent, 250 g/Lglucose,R= 2.0

increase of ethanol productivityrelative to fermentation withoutsolvent (%)

300 340b Kollerup andDaugulis15

total ethanol productivity [g/(L h)] 12.11 13.6b

ethanol distribution coefficient 0.600.67 0.0, 0.1,0.5,1.0, b

aTDA = tri-n-decylamine. bData obtained from a mathematical model for ethanologenic extractive fermentation developed by Kollerup andDaugulis.15




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end-product inhibition occurs during the processing of theconcentrated medium.

Consider the case of a feed aqueous stream containing 40 g/L glucose being employed for an extractive fermentationprocess with a ratio Rof 2.0. By applying eq6, the location ofthe pseudoinitial mixture represented by point D in Figure8isdened. From point D and the corresponding tie line, thecompositions of the extract and raffinate can be read directlyfrom the ternary diagram. With this information, theproductivity of this process was estimated by the short-cutmethod to be 20.24 g/(L h). The productivity calculated by therigorous model was 19.38, indicating a very good approx-imation (see Table1).

4. DISCUSSION

This article presents a novel short-cut method for analyzingextractive fermentation processes. Before discussing theadvantages of such a method, it is necessary to evaluate theproposed rigorous model, which provides an appropriatedescription of the phenomena involved during extractivefermentation. This information was crucial for developing the

short-cut tool.In this work, a rigorous model was used to obtain asystematic framework to assess extractive fermentationprocesses under two time regimes. This framework includedthe two main phenomena involved in the process: fermentationkinetics and liquidliquid equilibria. The solution procedureimplied the application of iterative methods to describe thesystem behavior, simultaneously considering the complexnonlinear relationships of the two phenomena. From themathematical point of view, the problem represents severaldifficulties related to the nonsmooth character of somefunctions employed, as in the case of the activity modelsneeded to calculate the phase equilibrium and some kineticexpressions that exhibit a highly nonlinear nature. Theproperties of these functions hinder the convergence ofnumerical methods implemented within the range of validphysical solutions, especially in the case of continuousextractive fermentation where the user should introduce thestarting guess values.

In the case of the batch regime, the proposed modelingframework allows for the generation of the concentrationproles of each of the components involved and thedetermination of the effect of the solvent on the processperformance in terms of productivity. In addition, the modelenables the exploration of the inuence of the amount ofsolvent added to the bioreactor, for example, through theevaluation of the effect of the solvent volume/aqueous medium

volume ratio (RV). The results obtained from the modeling ofbatch ethanologenic extractive fermentation showed a 27%

increase in the total ethanol productivity when n-dodecanol wasused (RV= 2.0) compared to the conventional process withoutsolvent. For oleyl alcohol, the increment reached 32%. The best

values of total productivity correspond to lower values ofRV.Moreover, the productivity for ethanol recovered from theaqueous phase was higher than the ethanol productivity of theprocess without any solvent. In this way, the advantage of thisintegrated reactionseparation process lies in the length of thefermentation process, which can be signicantly reducedcompared to that of the process without solvent. The reductionof the ethanol concentration in the aqueous phase allowed the

biomass to reach a higher nal concentration during thestationary phase in comparison to the corresponding

concentration when no solvent was added (1.8 g/L withoutsolvent, 4.1 g/L for n-dodecanol, and 3.8 g/L for oleyl alcohol;see Figure 2). However, considering the total amount ofethanol produced relative to that from the conventional batchprocess, the results are not conclusive for the integratedprocess. In the latter case, the ethanol should be recovered fromthe two phases by employing a decanter, unlike in theconventional process, where the ethanol is contained in onlyone phase (the culture broth) and a decanter is not needed.

The overall amount of ethanol that can be extracted by batchextractive fermentation is practically the same as that obtainedfrom conventional batch fermentation. This indicates thatliquidliquid extraction is not good enough to neutralize theinhibition effects of ethanol and substrates on the batchcultivation process for the case ofn-dodecanol. The situation isquite similar for oleyl alcohol. Furthermore, the concentrationof ethanol in the solvent phase (16.3 g/L) is too low for a cost-effective separation by ash vaporization or distillation. It isimportant to obtain high relative concentrations of the productin the solvent phase to reduce energy expenditures during thesubsequent separation steps. Thus, the results obtained for

batch ethanologenic extractive cofermentation using n-dodec-anol do not justify the complexity of this integrated process.These values cannot be considered promising when taking intoaccount the fact that ethanol productivities in conventional

batch and fed-batch processes are in the ranges of 13 g/(L h)for batch regime and and 931 g/(L h) for fed-batch alcoholicfermentations.13,26 Therefore, the search for an appropriatesolvent is of paramount importance. Undoubtedly, theproposed model can signicantly contribute to the selectionof a suitable solvent (or mixture of solvents) by employingcomputer-aided techniques such as those described by Wangand Achenie.27

To enhance the effectiveness of batch extractive fermenta-tion, a mixture of solvents can be used. As mentionedpreviously for lactic acid production, one solvent with high

extractive properties (TDA) can be mixed with a solvent withgood biocompatibility (oleyl alcohol). In this case, the overallprocess performance was improved, considering that the lacticacid concentration in the resulting organic phase wassignicantly higher than that in the aqueous phase. This

behavior predicted by the rigorous model (see Appendix D ofthe Supporting Information) can be corroborated in some

works reporting experimental data for analogous processes. Forexample, Gao et al.24 implemented an extractive fermentationprocess employing this same solvent system but using S.cerevisiaeas the lactic acid-producing microorganism because itcan grow well and produce lactic acid efficiently at lower pHthan lactic acid bacteria (see Table1).

Continuous extractive fermentation offers the advantage of

reaching signicantly higher productivities relative to those ofbatch processes. The results obtained in this work show thepossibility of reaching total ethanol productivities as high as30.3 g/(L h) compared to the productivities of conventionalcontinuous ethanologenic fermentations, which are in the rangeof 520 g/(L h).13,26 The simulations performed indicate thatmicroorganisms can grow at higher rates when the inhibitingproduct is continuously removed from cultivation broth.Ethanol would become inhibitory to growth from glucoseand xylose above a threshold levelof approximately 2729 g/Laccording to Leksawasdi et al.20 The ethanol levels in theaqueous phase depend on the changes in the biomass andsubstrate concentrations according to Monod-type kinetics, but




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they are strongly inuenced by the migration of ethanol to thesolvent phase, as can be observed in Figure 3.

For process economy, it is very important to employ theminimum possible amount of the extracting agent to reduce thesolvent costs, pumping expenditures, and energy consumptionduring solvent recovery. Although high values of the ratio Rallow the highest ethanol productivities to be reached,operation of the system at elevatedRvalues is not economically

viable (large amounts of solvent employed). Therefore, for thecase of n-dodecanol, R values near 4.0 are suggestedconsidering the production costs. For oleyl alcohol, therecommended value is 5.5.

The representation of the steady states of the extractivefermentation process in the ternary diagram using the resultsgenerated by the algorithm based on the rigorous modelenables interesting information about the system behavior to beobtained. This information was the basis for the structure andconguration of the short-cut method. Thus, the operatingconditions related to Rcan be represented as vertical lines inthat diagram, where each vertical line represents the geometriclocation of pseudoinitial mixtures with identical inlet conditionsof the feed ow rates (FE and FA). The points representingthese pseudoinitial mixtures can lead to the steady states of thesystem represented by the compositions of the extract andraffinate associated with these points. From Figure 4, it can beobserved that, at low Rvalues, the pseudoinitial points withdifferent inlet aqueous dilution rates lie on the same verticalline in a separate way. Thus, the variation ofDAifor the same

value ofRleads to different ethanol productivities. Again, fromthis diagram, it is evident that higher productivities can beachieved for greater values ofRand DAi, which are limited by

wash-out conditions and the zone of homogeneous mixtures,specically, the curve representing the raffinate compositions.

At high values of the ratioR(above 4.0), pseudoinitial pointswith differentDAivalues tends to be clustered in a very narrow

zone corresponding to higher productivities. This indicates thatvalues of R higher than 4.0 do not provide signicantproductivity increases, whereas they do elevate the productioncosts. In this way, the zone of more favorable operatingconditions can be preliminarily located. The informationderived from Figure4, which, in turn, was obtained using theproposed rigorous model, allowed a new short-cut method forevaluation of extractive fermentation processes to be deducedand developed. This article has illustrated this method anddemonstrated its application to actual experimental systems, asdiscussed later.

Taking into account that one of the main features of mostfermentations (including those used for the production ofethanol and lactic acid) is the impossibility of working with

concentrated media. Continuous extractive fermentation offersthe possibility of employing elevated substrate concentrationsto achieve higher amounts of product. This fact was reected bythe rigorous model and the short-cut method. For the case ofethanologenic extractive cofermentation, high values of theratio R favor the consumption of both substrates, althoughmarginal increases of productivity are reached for values above4.0. The results predicted by the proposed model forconcentrated media are shown in Table C1 of theSupportingInformation. These results can be compared with thoseobtained by Kollerup and Daugulis,15 who used a model in

which the ethanol distribution coefficient (KEtOH) was xed andthe substrate limitation and substrate inhibition effects on both

the growth and ethanol production rates caused by highsubstrate concentrations were not taken into account. Theyconsidered an extremely high glucose concentration of 750 g/Las a means for enhancing the total ethanol productivity, whichreached 82.6 g/(L h) at a very high value of the inuent solventdilution rate of 5 h1. However, calculations using the algorithmdeveloped in this work indicate that substrate inhibition doesnot allow such high productivities to be attained. In addition,the model proposed in the present work takes into account arigorous calculation of liquidliquid equilibrium throughactivity models such as NRTL, UNIQUAC, and UNIFAC,such that the values of KEtOH change for different operatingconditions according to the proposed rigorous algorithm. Acomparison of some key indicators obtained from the model ofKollerup and Daugulis to the data resulting from the rigorousmodeling approach described in this article is presented inTable1.

It is necessary to assess the suitability of the rigorous modeldeveloped relative to real reported data for continuousprocesses. The rst reported work on extractive fermentationfor ethanol production using microorganisms other than yeasts

was published by Bruce et al.23 They employed a reactor inwhich Z. mobilis cells were cultivated continuously for morethan 225 h using a commercial solvent mostly composed ofoleyl alcohol to remove the ethanol produced by the bacteria.They assessed the process performance at high glucoseconcentrations. Under these conditions, the culture exhibitedan oscillatory behavior that implied difficulties during systemoperation. When solvent was continuously added to the culture

broth, these oscillations ceased, and almost all of the glucosewas consumed, thus increasing the ethanol productivity andyield. The rigorous model was used to analyze this processunder the reported conditions, giving outcomes thatwere nearthe experimental data presented by Bruce et al.23 For thispurpose, the kinetic model of Leksawasdi et al.20 was applied.This model is relevant considering that it allows for the

description of growth inhibition by both glucose and ethanol.In addition, the UNIFAC model was utilized. A comparison ofthe data calculated by the rigorous model and the data obtainedin the experimental work is provided in Table1. The deviationsof the data can be explained by the fact that the kinetic model

was not specically developed for this system. Note especiallythe agreement between the productivity calculated by themodel and the obtained experimental value: 15.1 and 15.6 g/(Lh), respectively.

The proposed model can be used for predicting someperformance indicators of different congurations of extractivefermentation processes. Although the modeling approach wasdeveloped for systems with suspended cells, the liquidliquidextraction phenomenon is the same for several types of

extractive fermenters, and therefore, the software can beemployed for their preliminary performance analysis. Thus, themodel predicted a 2.11-fold increase in ethanol productivity fora process employing a hollow-ber-based fermenter usingimmobilized yeasts under thesame conditions as reported forsuch a process by Kang et al.,25 as shown in Table1.

On the other hand, the selectivity of the extractive agentplays a crucial role in the extractive fermentation process.Comparing the two solvents studied, the selectivity of n-dodecanol (about 28.5) is slightly higher than that of oleylalcohol (about 25.9). This can be evidenced from the modelingdata indicating a 78.2% increase of ethanol productivity fromthe solvent phase (PrE) for n-dodecanol and only a 9.9%




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increase for oleyl alcohol. In the most favorable situation, theaddition of a proper solvent should exhibit higher ethanolconcentrations in the solvent phase than in the aqueous phase.In this way, the ethanol should be mainly extracted from thesolvent phase and not from the aqueous phase, as in the case ofthe two solvents analyzed. This was the case for TDA/oleylalcohol extraction of lactic acid as shown in Appendix D of theSupporting Information. In addition, the use of oleyl alcoholallows the biocompatibility of the solvent system to beimproved, as indicated elsewhere.28,29

The short-cut method presented in this work was tested fortwo continuous systems: ethanologenic extractive cofermenta-tion and lactic acid extractive fermentation. This methodfeatures a high simplicity, requires a minimum of informationabout the process, and allows the feasible zone of operatingconditions to be delimited and some performance indicatorssuch as volumetric productivity to be estimated. The dataobtained by this short-cut method can be used for reducing thespace of inlet operating conditions to be analyzed throughrigorous simulation. In addition, the short-cut method canprovide insight into the impossibility of accomplishing anextractive fermentation process for specic conditions.

The information on the operating conditions correspondingto these delimited zones can be employed as the starting pointfor developing a preliminary optimization strategy. Because theregion of feasible steady states can be determined in the ternarydiagram (see Figure 8), the ranges of such manipulated

variables as the inlet dilution rate, the ratio R, and theconcentration of the sugars in the inlet aqueous stream areknown and can be bounded for solving an optimizationalgorithm. In a previous work,17 GAMS software was employedto nd the optimal values of the mentioned variables thatmaximized the total ethanol productivity. Once a globalpanorama of the space of operating conditions and theiroptimal values for the studied process have been obtained,experimental runs should be performedto conrm the validity

of the given theoretical approach.17

In this manner, theacquired insight into the process will enable the reduction ofexpensive experimental work in the search for optimaloperations, which is the main goal of both short-cut andrigorous simulation methods. As shown in Table 1,experimental data available in the open literature for extractivefermentation allowed for a comparison with the resultsobtained in this work using the rigorous model and theshort-cut method, despite the fact that some reported operatingconditions (immobilized cells, different process microorgan-isms, and coupled systems using a separate extractordecantingunit) were different from those proposed in this work. Finally,the main issues analyzed in this work should be considered as atheoretical development prior to the needed experimental runs

that will be undertaken in the future for specic extractivefermentation systems.

5. CONCLUSIONS

Continuous extractive fermentation has been demonstrated tobe an efficient technology compared to conventional batch andcontinuous fermentations. The coupling of the fermentationkinetics to thermodynamic equilibrium models enables different

ways of improving the productivity and performance ofextractive fermentation to be analyzed. The procedures studiedin this work exemplify this advantage. The determination of the

best values of operating parameters is required to improve theperformance indexes of the process. With this aim, rigorous

modeling is a powerful tool allowing for analysis of the processbefore costly experimental runs at the pilot-plant and industriallevels are executed. The modeling approach for extractivefermentation presented in this article differs from most modelsalready published in the rigor with which it describes thethermodynamic and kinetic phenomena.

The proposed short-cut method for extractive fermentationbased on the principles of thermodynamic-topological analysisallows preliminary information to be obtained and used duringsubsequent rigorous simulation. This approach enables thecalculation time and number of experimental runs to bedecreased. Moreover, it helps to determine which data arerequired and the space of initial conditions where experimentalefforts should be focused.

ASSOCIATED CONTENT

*S Supporting Information

Rigorous model considerations and results with no solventaddition (Appendix A), predictions of several activity modelsfor system ethanolwatern-dodecanol (Appendix B), simu-lation of continuous extractive cofermentation (Appendix C),

modeling of lactic acid extractive fermentation (Appendix D).This material is available free of charge via the Internet athttp://pubs.acs.org/.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected].

Author ContributionsThe manuscript was written through the contributions of allauthors. All authors have given approval to the nal version ofthe manuscript. All three authors contributed equally.

Notes

The authors declare no competing nancial interest.

ACKNOWLEDGMENTS

This work was supported by the Colombian AdministrativeDepartment of Science, Technology and Innovation (Colcien-cias), the National University of Colombia at Manizales, andthe University of Caldas (Colombia).

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Analysis and Design of Extractive Fermentation Processes Using a Novel Short-cut Method

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