ARTICLE

Simultaneous Clostridial Fermentation,Lipase-Catalyzed Esterification, and EsterExtraction to Enrich Diesel with Butyl Butyrate

Corjan van den Berg, Arjan S. Heeres, Luuk A. M. van der Wielen, Adrie J. J. Straathof

Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BCDelft,

The Netherlands; telephone: 31-15-2782330; fax: 31-15-2782355;

e-mail: a.j.j.straathof@tudelft.nl

ABSTRACT: The recovery of 1-butanol from fermentationbroth is energy-intensive since typical concentrations infermentation broth are below 20 g L�1. To prevent butanolinhibition and high downstream processing costs, we aimedat producing butyl esters instead of 1-butanol. It is shownthat it is possible to perform simultaneously clostridialfermentation, esterification of the formed butanol to butylbutyrate, and extraction of this ester by hexadecane. The veryhigh partition coefficient of butyl butyrate pulls the esterifi-cation towards the product side even at fermentation pHand relatively low butanol concentrations. The hexadecaneextractant is a model diesel compound and is nontoxic to thecells. If butyl butyrate enriched diesel can directly be used ascar fuel, no product recovery is required. A proof-of-prin-ciple experiment for the one-pot bio-ester production fromglucose led to 5 g L�1 butyl butyrate in the hexadecane phase.The principle may be extended to a wide range of esters,especially to longer chain ones.

Biotechnol. Bioeng. 2012;xxx: xxx–xxx.

� 2012 Wiley Periodicals, Inc.

KEYWORDS: biofuels; ester; partitioning; clostridia; fed-batch; butanol

Introduction

The sustainable production of biofuels such as 1-butanol iscurrently one of the main strategies in developing a greenereconomy. 1-Butanol can be produced by fermentation but itseverely inhibits growth and metabolism of microorganisms(Liu and Qureshi, 2009). The achieved product concentra-tions in the fermentation broth are only 10–30 g L�1. In

industrial 1-butanol production processes, separation of 1-butanol from water is performed using distillation (Ni andSun, 2009). This involves heating all fermentation liquid toits boiling point, including �980 g L�1 of water, and part ofthis water will also be evaporated, making this separationenergy intensive. Different alternative 1-butanol recoverymethods have been evaluated for their energy efficiency(Oudshoorn et al., 2009). The energy requirements forsteam stripping and distillation were estimated to be 66% ofthe combustion energy of the recovered 1-butanol whileliquid–liquid extraction and adsorption showed the bestenergy efficiency with losses of 25% and 22%, respectively.Extractive acetone–butanol–ethanol (ABE) fermentationshave been studied numerous times using organic solvents(Barton and Daugulis, 1992; Groot et al., 1990) and ionicliquids (Ha et al., 2010) as extractant. However, all testedextractants with a high 1-butanol partition coefficient weretoxic towards Clostridium acetobutylicum. Groot et al.(1990) found a 1-butanol partition coefficient of 3.9 usingoleic acid, being the highest found for a non-toxic solvent tothis date. These data indicate main limitations of in situ 1-butanol extraction. However, back extraction may beunnecessary if a fuel is used as extractant. Li et al. (2010)used biodiesel as in situ extractant and enriched it by 1-butanol. This improved biodiesel properties such as adecreased cold filter plugging point, acid number, and anincreased cetane number.

In situ extraction should be more effective for fermenta-tion products that are more hydrophobic than 1-butanol.We considered converting 1-butanol enzymatically intobutyl esters during extractive fermentation. Butyl butyrate,which is the main biofuel in this study, should have excellentproperties both as gasoline as well as diesel component, byanalogy with propyl valerate (Lange et al., 2010).

Volatile short-chain esters have been frequently reportedin food microbiology papers (Liu et al., 2004; Sumby et al.,2010). The feasibility of enzymatic production of esterssuch as butyl butyrate by transgenic C. acetobutylicumand Escherichia coli has been demonstrated (Horton andBennett, 2006), but literature on them as fuel constituents is

The authors have no conflict of interest to declare.

Corjan van den Berg’s present address is Present address: TNO Earth Environment &

Life Sciences, Utrechtseweg 48, 3704 HE Zeist, The Netherlands.

Correspondence to: A. J. J. Straathof

Received 27 April 2012; Revision received 9 July 2012; Accepted 16 July 2012

Accepted manuscript online 25 July 2012;

Article first published online in Wiley Online Library

(wileyonlinelibrary.wiley.com).

DOI 10.1002/bit.24618

� 2012 Wiley Periodicals, Inc. Biotechnology and Bioengineering, Vol. xxx, No. xxx, 2012 1

scarce. Significant efforts have been made on the productionof so-called fatty acid ethyl esters (FAEEs). During ethanolfermentation by Saccharomyces cerevisiae, oleic acid andlipase have been added to produce FAEE in situ (Oliveiraet al., 2001). Alternative to this extracellular production,ethanol fermentation by E. coli was used, with intracellularesterification of added oleic acid (Elbahloul and Steinbuchel,2010). Although ethanol inhibition was prevented, theFAEEs had to be extracted from the cells after thefermentation, since most FAEEs remained intracellular(Elbahloul and Steinbuchel, 2010). In an extractivefermentation, the acetone required for this extraction wouldhave killed the cells.

For producing 1-butanol esters, we considered clostridialfermentations. It is well established that ABE-producingClostridia possess two distinct characteristic phases in theircatabolic pathway, being the acidogenic and solventogenicphase (Jones et al., 1981; Husemann and Papoutsakis, 1987).Typically, during acidogenesis, cell growth is exponentialand products are acetic and butyric acid along with ATPformation. Undissociated acids can diffuse passivelythrough the membrane causing the pH gradient to collapse.At high butyric acid concentrations, this results in a lowATP/ADP ratio. However, this does not completely stopsubstrate assimilation and cell metabolism. Once a criticalundissociated butyric acid concentration is reached(�1.4 g L�1), cells shift to solventogenesis. Then, cell growthenters the stationary phase, the organic acids are reutilized,and acetone, 1-butanol, and ethanol are produced (Joneset al., 1981; Husemann and Papoutsakis, 1987). It should benoted that, due to thermodynamic constraints, onlyundissociated butyric acid (pKa¼ 4.82, see Equation 1)will react with 1-butanol in aqueous media, making itfavorable to perform this reaction at low pH from anesterification point of view.

Ka ¼CaqB� � Caq

Hþ

CaqHB

¼ 1:51� 10�5 mol=L (1)

The aqueous amount concentration of undissociatedbutyric acid ðCaq

HBÞ can be calculated from the total aqueousbutyric acid ðCaq

totBÞ amount concentration usingEquations (1) and (2) and the pH value.

CaqtotB ¼ C

aqHB þ C

aqB� (2)

Here, we propose a new process in which we aim at: (1)alleviating 1-butanol inhibition; (2) diminish downstreamprocessing costs; (3) obtain an ester biofuel blended into ahydrocarbon that is typical of a diesel fuel; and (4) performall of this simultaneously in one reactor (Fig. 1). The butyricacid may be produced during fermentation or may beadded. To estimate the potential of the concept ofesterification/extraction for ABE fermentations, we mea-sured the esterification equilibrium constant (Kaq) alongwith the extraction behavior of the solutes (Pi).

Subsequently, we evaluated different media and buffers inshake flasks and proved the concept in fed-batch fermenta-tions. This is the first investigation on direct productremoval during clostridial fermentation involving a ‘‘one-pot’’ combination of esterification and extraction.

Materials and Methods

Ester Reaction Equilibrium Determination

All chemicals were from Sigma-AldrichQ1 and had a purityof>99%. Butyric acid and 1-butanol (0.23mmol; 17mg) orbutyl butyrate (0.41mmol; 59mg) were dosed to a 100-mLvessel by pipetting the required volume into a biphasicreaction medium (30mL of aqueous phase and 30mL ofhexadecane). The amount of added substrate was measuredon an analytical balance. Subsequently, the pH of theaqueous medium was set to 4.7 using 1mol L�1 sodiumphosphate buffer of pH 4.7. After addition of 300mgimmobilized Candida antarctica lipase B (CaLB; Novozym435, Novozyme), the flask was continuously stirred at200 rpm in a water bath at 378C for 48 h, after which thephases were allowed to settle. Samples of the aqueous andorganic phases were analyzed for solute concentrations andwere assumed to be at equilibrium since concentrations werethe same when using longer reaction times.

The esterification equilibrium constant (Kaq) value wascalculated, assuming thermodynamically ideal systems,using Equation (3) (Martinek et al., 1981) and C

aqH2O

¼55.49mol L�1.

Kaq ¼CaqBuBC

aqH2O

CaqBuOHC

aqHB

(3)

Figure 1. Evaluated process concept involving esterification reaction between

1-butanol and butyric acid with a simultaneous extraction of butyl butyrate. For

abbreviations see Nomenclature Section. [Color figure can be seen in the online

version of this article, available at wileyonlinelibrary.com]

2 Biotechnology and Bioengineering, Vol. xxx, No. xxx, 2012

Hexadecane/aqueous phase partition coefficients (Pi)were calculated using Equation (4).

Pi ¼ Corgi

Caqi

(4)

For predicting ester concentrations in the organic phaseof a biphasic system, Equation (5) was derived usingEquations (1)–(4):

CorgBuB ¼ PBuB � Caq

BuB ¼ PBuB � Kaq � CaqBuOH � Caq

HB

CaqH2O

¼ PBuB � Kaq � CaqBuOH

CaqH2O

� CaqtotB

1þ ðKa=CHþÞ (5)

Shake Flask Fermentation

The fermentation medium was the same as in Roos et al.(1985) except for a higher glucose concentration (40 g L�1;0.22mol L�1) and additional buffers, being several phos-phate buffers or calcium carbonate. The initial pH was 6.8.Before inoculation, the medium was sparged with (O2

free) N2. Shake flask fermentations were performed in 100-mL vessels. The fermentation broth volume was 25mL andwas kept at 378C. Furthermore, 0.5 g Novozym 435 and25mL (O2 free) hexadecane were added to the flask afterwhich these were inoculated with 1mL C. acetobutylicumATCC824 (obtained from CBS-KNAW Fungal BiodiversityCentre, Utrecht, Netherlands) spore solution, which wasmaintained in a 25% glycerol solution at �808C.Hexadecane was used as solvent since its value of log Po/wexceeds 7, making it non-toxic to C. acetobutylicum (Bartonand Daugulis, 1992).

Fed-Batch Fermentation

The 50mL of overnight-incubated shake flask content wastransferred to the fermentation vessel. The batch volumewas 1.5 L of culture medium and initially no pH control wasapplied. Prior to inoculation, 250mL (O2 free) hexadecanewas added to the fermentation broth. The medium wasthe same as Roos et al. (1985), but contained higher glucoseconcentrations (40 g L�1; 0.22mol L�1) and was keptanaerobic by sparging N2 continuously through it. Themedium also contained 1 g L�1 of CaCO3 for buffering andstimulating 1-butanol production (Kanouni et al., 1998).After the acidogenic phase, where the pH would dropapproximately 2 pH units, the solventogenic phase wouldcommence and initiate a pH increase. After the shift fromthe acidogenic to solventogenic phase 2,500U of lipase(3.31 g CaLB Immobead-150, ChiralVision; Leiden, theNetherlands) was added to the broth. Covalently boundinstead of adsorbed lipase was used in fed-batch experiments

since the vigorous mixing in the fermentation mightresult in lipase dissolving and subsequently quicklyinactivating in the fermentation broth. Also the pH controlwas turned on and set to 5.2, and samples (<5mL) weretaken intermittently. Neutralization and feeding wasperformed using an aqueous solution containing 80 g L�1

(0.44mol L�1) of glucose and 160 g L�1 (1.82mol L�1)butyric acid. The optimal butyric acid/glucose mass ratiofor the feed-fase has been reported to be 1.0 (Tashiro et al.,2004). However, since in our case, half the butyric acid wasused for esterification, we used a butyric acid/glucose massratio of 2.0.

Analytical Methods

Determination of 1-butanol, total butyric acid, butylbutyrate, acetone, ethanol, and total acetic acid concentra-tions in aqueous and organic phase was performed usinggas chromatography (Interscience, Breda, Netherlands;HP-INNOWax column 30m and 0.25mm diameter).Total aqueous carboxylic acid concentrations (¼dissociatedþundissociated carboxylic acid) were determined afterdiluting the samples 10 times using a 1mol L�1 HClsolution. The initial oven temperature was 708C and a rampof 108Cmin�1 was initiated up to 1308C, which was held for1min. Cell concentrations were estimated by optical densityusing a cell density meter (Amersham Biosciences, Uppsala,Sweden; Ultrospec 10). Cell dry weight (CDW) wascalculated using a pre-determined correlation betweenoptical density and measured dry weight.

Results and Discussion

Ester Equilibrium Prediction

To achieve esterification equilibrium, CaLB seemed to besuitable considering its activity towards formation ofbutyrate esters and its stability (Lozano et al., 2002). Themeasured esterification equilibrium constant value for thereaction in Figure 1 was Kaq¼ 12.1� 1.8. For the esterifica-tion of decanoic acid with 1-butanol, the reportedequilibrium constant is 35 (Janssen et al., 1993), thus thesame order of magnitude. Furthermore, we determinedpartition coefficients for 1-butanol, butyric acid, and butylbutyrate for the hexadecane/aqueous system (Table I). Sincethe C8-ester is more hydrophobic than its substrates, it isextracted more efficiently. By selective in situ extraction ofbutyl butyrate from the fermentation broth the esterification

Table I. Partition coefficients of solutes in hexadecane/aqueous system

Solute Partition coefficient

1-Butanol 0.44� 0.04

Butyric acid 0.13� 0.06

Butyl butyrate 340� 103

van den Berg et al.: Simultaneous Fermentation, Esterification and Extraction 3

Biotechnology and Bioengineering

equilibrium in Figure 1 can be shifted to the right, therebyremoving 1-butanol, butyric acid, butyrate, and Hþ fromthe fermentation medium.

Using the values of esterification equilibrium constant,partition coefficient of butyl butyrate, and final attainablesubstrate concentrations, a theoretical final concentration ofbutyl butyrate in the hexadecane phase can be estimatedusing Equation (5). For example, at an aqueous butanolconcentration of 15 g L�1 and a total aqueous butyric acidconcentration of 5 g L�1 at pH 4.7, the aqueous undissoci-ated butyric acid concentration would be 2.8 g L�1, and theorganic butyl butyrate concentration would be 70 g L�1.Obviously, achieving at the same time high aqueous phasebutanol and undissociated butyric acid concentrations isrequired for high organic phase butyl butyrate concentra-tions. For C. acetobutylicum, butyric acid production has notbeen optimized but at least 1.5 g L�1 undissociated butyricacid has been achieved (Oh et al., 2009). For Clostridiumtyrobutyricum, butyrate production has been optimized andconcentrations corresponding to undissociated butyric acidconcentrations of 5.4 g L�1 have been achieved (Jiang et al.,2011). However, by removing butyric acid, the pH willincrease, negatively impacting the esterification reactionequilibrium. Therefore, sufficient butyric acid needs to beproduced in situ, or to be fed to the system for an optimizedbutyl butyrate production. For feeding, a stand-alonebutyric acid fermentation might be used (Zigova andSturdik, 2000). Alternatively, bio-based carboxylic acidssuch as valeric acid (Lange et al., 2010) might be fed to thesystem. This might lead to results more favorable than thosedescribed here, because partition coefficients will be higherfor butyl valerate than for butyl butyrate.

Shake Flask Fermentations

In shake flask fermentations containing an organic phase,different buffers were tested to evaluate their effect on butylbutyrate production. Relatively high buffer strengths weretested to prolong the acidogenic phase and reach high totalbutyric concentrations. Table II summarizes the highestachieved concentrations in shake flask fermentation. Other

esters such as ethyl acetate, butyl acetate, and ethyl butyratewere only found in low concentrations, mainly due topoorer extractability along with lower ethanol/acetic acidconcentrations. Therefore, we focused on butyl butyrate inthe latter part of this study.

The use of 0.1mol L�1 phosphate buffer gave relativelygood results in terms of concentration of cells, acid, andsolvent. It was found that high phosphate buffer concentra-tions had a negative effect on clostridial growth and solventproduction.

The highest ester and total acid concentrations werefound in the medium containing CaCO3, which has beenshown to improve C. acetobutylicum fermentations(Kanouni et al., 1998). However, butyl butyrate concentra-tions in the organic phase remained relatively low due to thelow concentration of butyric acid in the aqueous phase. Oneof the problems that had to be overcome to reach higherbutyl butyrate concentrations is the sequential productionof butyric acid and 1-butanol during fermentation.

Fed-Batch Fermentation

The lack of pH control in shake flask fermentations resultedin unpredictable esterification behavior. The esterificationrequires a low pH, whereas for the fermentation the pHshould not be too low. Both processes change the pH bybutyric acid consumption or formation. Therefore, fed-batch fermentations controlled at pH 5.2 were performedusing a feed that contained butyric acid and glucose assubstrates for 1-butanol production and ester formation.Results from a typical fed-batch fermentation coupled withesterification and extraction are shown in Figure 2A and B.

The pH increase in Figure 2A indicates that thesolventogenic phase started after 9 h. The 1-butanolproduction rate was at its highest level at 23 h, being1.03 g L�1 h�1. Furthermore, 1-butanol yields on glucosewere 0.56, 0.41, and 0.45 g g�1 between 19 and 23 h. Thesevalues were close to, or even higher than the theoreticalmaximum of 0.41 g BuOHg�1 glucose (1molmol�1) if onlyglucose would be used as substrate, indicating that 1-butanolwas also produced from added butyric acid. During the time

Table II. Highest concentrations found in shake flask fermentations

Buffer CDW 1-Butanol Total butyric acid Acetone Ethanol Total acetic acid Butyl butyrate

Aqueous phase concentrations (g L�1)

0.1mol L�1 phosphate 7.5 9.1 1 0.52 4.11 1 n.d.

0.2mol L�1 phosphate 1.1 0.48 0.15 0.017 1.08 0.54 n.d.

0.3mol L�1 phosphate 0.25 0.06 0.037 0.007 0.05 0.04 n.d.

0.4mol L�1 CaCO3 3.31 14.23 4.45 0.067 5.67 1.58 n.d.

Organic phase concentrations (g L�1)

0.1mol L�1 phosphate 0 2.24 0.04 0.13 0.53 0.36 0.96

0.2mol L�1 phosphate 0 0.008 n.d. 0.028 0.105 0.47 0.004

0.3mol L�1 phosphate 0 0.007 n.d. 0.016 0.058 0.24 n.d.

0.4mol L�1 CaCO3 0 1.29 0.093 0.026 0.141 0.41 0.51

n.d., not detectable.

4 Biotechnology and Bioengineering, Vol. xxx, No. xxx, 2012

interval of 17–25 h the average butyl butyrate productionrate was 0.43 g L�1 h�1. The esterification stopped atrelatively low butyl butyrate concentrations (4.9 g L�1;0.034mol L�1) in the organic phase. According to equilib-rium calculations 14.8 g L�1 (0.10mol L�1) should havebeen the final butyl butyrate concentration in thehexadecane phase, using in the model Kaq¼ 12.1, partitioncoefficients of Table I, C

aqBuOH ¼ 5.2 g L�1 (0.07mol L�1),

CaqtotB ¼ 3.4 g L�1 (0.039mol L�1) and a pH of 5.08, such as

observed in Figure 2A. At 50 h, fresh lipase (1 g) was addedto the fermentation broth because previously added enzymemight have lost activity. However, since no additionalbutyl butyrate was formed after 50 h it can be assumedthat esterification equilibrium had been reached. UsingEquation (3), and measured pH, aqueous substrates andproduct concentrations, the calculated Kaq values variedbetween 3.2 and 3.9, being much lower than the Kaq valuemeasured previously (12.1). However, a change in Ka valueof butyric acid could have an impact on the calculated Kaq

value, although this effect was not quantified since only thetotal butyric acid amount was determined. The addition ofCaCO3 to the fed-batch experiment might increase the Ka

value of butyric acid by approximately 15% (Partanen andCovington, 2004), resulting in a calculated equilibrium C

orgBuB

of 10.6 g L�1 instead of the expected 14.8 g L�1. Butylbutyrate partitioning between hexadecane and fermentationbroth did not change after 48 h, and PBuB remained between474 and 492. This indicates that extraction equilibrium forbutyl butyrate had been reached. 1-Butanol productionstopped at relatively low concentrations (7 g L�1) in thefermenter even though inhibiting concentrations was notattained yet. A possible explanation for this was thatC. acetobutylicum started forming spores after producing 1-butanol for several hours, which is natural behavior of wild-type C. acetobutylicum (Woods, 1995). Non-sporulatingC. acetobutylicum might be able to produce 1-butanol forlonger periods of time and thereby circumventing thenarrow time window in which Clostridia produce 1-butanol(Sillers et al., 2008).

Obviously, many other improvements and tests arerequired to achieve a viable process. Lignocellulosic feedsmight be used instead of glucose. Immobilized lipase costs(order of magnitude of 500s kg�1) can become acceptableif efficient reuse is obtained. Instead of using immobilizedlipases, it would be advantageous if the fermentingmicroorganism would be capable of producing in situsurface-displayed lipase (Tanino et al., 2007).

Butyl butyrate does not be to be recovered from the organicphase, but the organic phase needs to be separated from theaqueous phase. Upon stopping the stirring, a clear organicphase appeared instantaneously, so this phase can be recoveredwithout de-emulsification efforts. In addition to the 5 g L�1

butyl butyrate shown in Figure 2, the organic phase contained�0.7 g L�1 butanol. Other esters, other alcohols, andcarboxylic acids were below the detection limit (�0.1 g L�1).

Conclusion

A butanol fermentation can easily be transformed into abutyl butyrate producing system. Simultaneous one-pot 1-butanol fermentation, 1-butanol esterification, and esterextraction for the production of butyl butyrate wasdemonstrated on lab scale. The highest ester concentrationachieved in these experiments was 4.9 g L�1 extractantphase. This was lower than expected on the basis of amathematical model that included the reaction andpartitioning equilibria. Nevertheless, this method will bemore generally applicable to overcome product inhibition ofacids/alcohols by esterifying them in situ. If the concomitantin situ extraction occurs by diesel, the esters might be used asbiofuel without having to isolate them.

Nomenclature

C concentration

Ka acid dissociation constant

Kaq aqueous equilibrium constant of esterification reaction

P hexadecane/aqueous partition coefficient

Figure 2. A and B: Results of C. acetobutylicum fed-batch fermentation coupled

with extraction and esterification as described in Materials and Methods Section.

Control of pH at 5.2 was started at 16.5 h using an aqueous solution of 160 and 80 g L�1

glucose. For abbreviations see Nomenclature Section. [Color figure can be seen in the

online version of this article, available at wileyonlinelibrary.com]

van den Berg et al.: Simultaneous Fermentation, Esterification and Extraction 5

Biotechnology and Bioengineering

Subscripts and superscripts

aq in aqueous phase

BuOH butanol

BuB butyl butyrate

B� butyrate

H2O water

Hþ proton

HB undissociated butyric acid

i component i

org in organic phase

totB total butyric acid/butyrate concentration

This research has been supported by the Dutch Technology Founda-

tion STW, Applied Science Division of NWO and the Technology

Program of the Ministry of Economic Affairs. The authors would like

to acknowledge Nick Wierckx for helpful discussions and ChiralVi-

sion for providing us with immobilized enzyme.

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