Effect of acetic acid and pH on the cofermentation of glucose and xylose to ethanol by a genetically engineered strain of Saccharomyces cerevisiae

R E S E A R C H A R T I C L E

E¡ectofacetic acidand pHon the cofermentationofglucoseandxylose to ethanol byageneticallyengineered strain ofSaccharomyces cerevisiaeElizabeth Casey1,2, Miroslav Sedlak1,2, Nancy W.Y. Ho1,3 & Nathan S. Mosier1,2

1Laboratory of Renewable Resources Engineering, Purdue University, West Lafayette, IN, USA; 2Department of Agricultural and Biological Engineering,

Purdue University, West Lafayette, IN, USA; and 3Department of Chemical Engineering, Purdue University, West Lafayette, IN, USA

Correspondence: Nathan S. Mosier,

Laboratory of Renewable Resources

Engineering, Purdue University, West

Lafayette, IN, USA. Tel.: 11 765 494 7022;

fax: 11 765 494 7023;

e-mail: [email protected]

Received 8 September 2009; revised 13 January

2010; accepted 22 February 2010.

Final version published online 14 April 2010.

DOI:10.1111/j.1567-1364.2010.00623.x

Editor: Jens Nielsen

Keywords

Saccharomyces cerevisiae; xylose; acetic acid;

ethanol; cellulose; inhibition.

Abstract

A current challenge of the cellulosic ethanol industry is the effect of inhibitors

present in biomass hydrolysates. Acetic acid is an example of one such inhibitor

that is released during the pretreatment of hemicellulose. This study examined the

effect of acetic acid on the cofermentation of glucose and xylose under controlled

pH conditions by Saccharomyces cerevisiae 424A(LNH-ST), a genetically engi-

neered industrial yeast strain. Acetic acid concentrations of 7.5 and 15 g L�1,

representing the range of concentrations expected in actual biomass hydrolysates,

were tested under controlled pH conditions of 5, 5.5, and 6. The presence of acetic

acid in the fermentation media led to a significant decrease in the observed

maximum cell biomass concentration. Glucose- and xylose-specific consumption

rates decreased as the acetic acid concentration increased, with the inhibitory effect

being more severe for xylose consumption. The ethanol production rates also

decreased when acetic acid was present, but ethanol metabolic yields increased

under the same conditions. The results also revealed that the inhibitory effect of

acetic acid could be reduced by increasing media pH, thus confirming that the

undissociated form of acetic acid is the inhibitory form of the molecule.

Introduction

Historically, petroleum has been the major source for liquid

transportation fuels. However, declining oil reserves and

environmental concerns have led to interest in alternative,

renewable energy sources. One promising alternative is the

conversion of plant biomass into ethanol. The primary

biomass feedstocks for the current ethanol industry have

been corn grain and sugar cane. However, interest has

recently shifted to replacing these traditional feedstocks with

more abundant, non-food-based cellulosic biomass feed-

stocks such as agricultural wastes (e.g. corn stover) or energy

crops (e.g. switchgrass). The use of cellulosic biomass as

feedstock for the production of ethanol via biochemical

routes presents many technical hurdles not faced with the

use of corn or sugar cane as feedstock. One significant

challenge is the development of efficient and economical

pretreatment and enzymatic hydrolysis steps for the release

of fermentable sugars from the biomass (Mosier et al., 2005;

Stephanopoulos, 2007). Another obstacle is engineering

robust, process-relevant industrial microorganisms that are

capable of mixed sugar fermentation and are tolerant to

inhibitors (Hahn-Hagerdal et al., 2007).

The complexity of cellulosic biomass hydrolysates as

compared with corn starch or sugar cane hydrolysates leads

to the challenge of engineering a suitable microorganism.

Cellulosic biomass hydrolysates contain a variety of sugars

(primarily glucose and xylose) and inhibitory compounds

such as acetic acid and various phenolic compounds. The

primary microorganism used in industrial fermentations,

Saccharomyces cerevisiae, is not able to ferment xylose

(Barnett, 1976), which represents about 40% of the sugars

found in biomass hydrolysates. This problem of limited

sugar utilization has been successfully addressed through

metabolic engineering of S. cerevisiae with genes for the

xylose metabolic pathways of xylose-fermenting bacteria, i.e.

xylose isomerase (Kuyper et al., 2005), or yeasts, i.e. xylose

reductase and xylitol dehydrogenase (Ho & Chen, 1997;

FEMS Yeast Res 10 (2010) 385–393 c� 2010 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

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Ho et al., 1998; Eliasson et al., 2000; Sonderegger et al.,

2004). Nontraditional microorganisms for industrial etha-

nol production (such as Escherichia coli, Zymomonas mobi-

lis, and Thermoanaerobacterium saccharolyticum) have also

been engineered to convert mixed sugar streams to ethanol

(Lindsay et al., 1995; Zhang et al., 1995; Shaw et al., 2008).

With strains now capable of mixed sugar fermentation, the

primary challenge has become the development of industrial

microorganisms with tolerance to the inhibitors that are

formed and released as biomass is broken down in the

pretreatment and hydrolysis steps (Hahn-Hagerdal et al.,

2007). The inhibitors found in biomass hydrolysates can be

primarily classified as weak acids, furan derivatives, or

phenolic compounds. These inhibitors have been shown to

negatively impact the fermentative performance (cell

growth, ethanol yield and productivity, and/or sugar con-

sumption rates) of non-pentose-fermenting strains of

S. cerevisiae (Palmqvist & Hahn-Hagerdal, 2000; Klinke

et al., 2004; Almeida et al., 2007) and Z. mobilis (Lawford &

Rousseau, 1993), as well as non-ethanol-producing strains

of E. coli (Luli & Strohl, 1990). The previously studied

strains are not suitable for industrial ethanol production

because of their substrate and product limitations. It would

be more valuable to understand how inhibitors impact

microorganisms that have been engineered to ferment

multiple substrates to ethanol. For example, Bellissimi et al.

(2009) recently published a study discussing the effect of

acetic acid on a strain of S. cerevisiae that was engineered for

xylose fermentation with bacterial xylose isomerase. How-

ever, few inhibition studies of engineered organisms have

been published.

Acetic acid is a weak acid generated from the deacetyla-

tion of hemicellulose during pretreatment (Palmqvist &

Hahn-Hagerdal, 2000; Klinke et al., 2004; Almeida et al.,

2007). It is known to inhibit microbial growth and has been

used as an antimicrobial agent in the food and beverage

industries (Luck & Jager, 1997). Acetic acid is present in

varying concentrations in all types of biomass, for example

corn stover and poplar contain 5.6% and 3.6% acetyl by

mass, respectively (Lu et al., 2009). When studying the

inhibitory effect that acetic acid can have on microorgan-

isms, process-relevant conditions for the production of

cellulosic ethanol at an industrial scale must be considered.

Assuming that the minimum concentration of ethanol for

economic distillation is 5% ethanol, the initial unhydrolyzed

biomass concentration at the start of the process must be

approximately 20% by weight. This would result in theore-

tical acetic acid concentrations of 11.2 and 7.2 g L�1 in the

hydrolysates of corn stover and poplar, respectively, assum-

ing no accumulation due to recycling of process streams. An

actual acetic acid concentration of 13 g L�1 has been ob-

served in dilute acid-pretreated corn stover hydrolysate (Lu

et al., 2009). The removal of acetic acid and other inhibitors

would add cost to the overall process. Therefore, a detailed

study of the effect of these inhibitors on ethanol yields and

production rates, especially for xylose fermentation, is

important for ongoing microorganism development efforts

and cellulosic ethanol commercialization.

The goal of the study reported in this paper was to

determine the effect of acetic acid at relevant industrial

process concentrations on the cofermentation of glucose

and xylose under controlled pH conditions by S. cerevisiae

424A(LNH-ST), a polyploid industrial yeast strain capable

of fermenting glucose and xylose. Saccharomyces cerevisiae

424A(LNH-ST) was genetically engineered for xylose meta-

bolism by overexpressing xylose reductase, xylitol dehydro-

genase from Pichia stipitis, and xylulose kinase from

S. cerevisiae (Ho et al., 1998; Ho et al., 2000). We report the

effects of acetic acid and pH on biomass growth, substrate

consumption rates, and ethanol production rates.

Materials and methods

Yeast strain

All fermentations utilized S. cerevisiae 424A(LNH-ST), a

recombinant industrial yeast strain capable of the cofermen-

tation of glucose and xylose (Ho et al., 1998; Ho et al., 2000).

Fermentation experiments

Batch fermentations were completed in 1-L New Brunswick

BioFlo 110 benchtop fermentors (Edison, NJ) equipped

with pH control. The culture volume was 80% of the

fermentor volume. No sparging of any gas was performed.

Our measurements of dissolved oxygen during fermentation

showed that the oxygen concentration declined to below 1%

of saturation within 40 min of the beginning of the fermen-

tation. The inoculum for the fermentor was prepared by

pregrowing yeast aerobically in a shaker set at 28 1C and

200 r.p.m. in 2-L flasks containing 500 mL YEPD media (1%

yeast extract, 2% peptone, and 2% glucose) (Mallinckrodt

Chemicals, Phillipsburg, NJ).

YEP media (1% yeast extract, 2% peptone) were used as

the fermentation media. YEP was chosen as the fermenta-

tion medium because the extensive prior data for the

performance of this yeast were obtained using this medium.

This allowed for comparisons between this work and prior

work. In addition, YEP is a rich medium. Thus, the most

significant stress on the fermenting yeast was the presence of

acetic acid unencumbered by the metabolic stress due to the

synthesis of minor metabolites. Glucose and xylose concen-

trations in the starting fermentation media were 60 g L�1

each. The acetic acid concentrations examined were 0 (for

control), 7.5, and 15 g L�1. These concentrations were

selected to represent the range of concentrations that can

be expected in hydrolysates from a variety of biomass

FEMS Yeast Res 10 (2010) 385–393c� 2010 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

386 E. Casey et al.

sources (Takahashi et al., 1999). The pH of the media was

adjusted to the desired value (5, 5.5, or 6) with ammonium

hydroxide (28–30% NH3) (Mallinckrodt Chemicals) or

14.8 M phosphoric acid (Fisher Scientific, Pittsburgh, PA).

Once the cell density of the growth culture reached

approximately 6 g dry cells L�1, the culture was centrifuged

for 5 min at 3100 g. The cell pellet was resuspended in YEP

and used to inoculate the fermentor to an initial cell density

of 4.75 g dry cells L�1. The temperature and r.p.m. for the

fermentation were set to 28 1C and 200 r.p.m., respectively.

The media pH was continuously controlled within � 0.1

from the desired value using the pH control system provided

with the BioFlo 110 fermentor using 1 M phosphoric acid

and 1 M ammonium hydroxide. BIOCOMMAND PLUS software

(New Brunswick Scientific Co.) was used to record the real-

time media pH. The fermentation then proceeded for a

maximum of 240 h and all fermentations were carried out in

duplicate.

Analysis of fermentation substrates andproducts

The fermentation metabolites were analyzed by HPLC using

the method outlined by Lu et al. (2009) using a Waters

Alliance 2695 HPLC system with an Aminexs HPX-87H

300� 7.8 mm column (Bio-Rad Laboratories, Hercules,

CA). The HPLC column operating conditions were 60 1C at

a flow rate of 0.6 mL min�1 for the mobile phase, 5 mM

sulfuric acid in water.

Results

We were interested in determining the combined effects of

acetic acid and pH on various fermentation performance

characteristics of S. cerevisiae 424A(LNH-ST). Specifically,

we examined the impact of these factors on biomass growth,

glucose and xylose consumption rates, and ethanol produc-

tivity rates and yields. To accomplish this, a 32 factorial

experimental fermentation design was selected. Two factors,

acetic acid concentration and media pH, at three different

levels were chosen to provide a total of nine different

fermentation conditions, with each fermentation condition

repeated twice. Figure 1 shows the representative fermenta-

tion profiles for each of the nine conditions. A visual

comparison of the profiles revealed the effect of acetic acid

and pH on the cofermentation of glucose and xylose. The

maximum biomass concentrations decreased and the xylose

consumption rates slowed as the acetic acid concentration

increased and pH decreased. The increase in the acetic acid

concentration and the decrease in pH also corresponded to a

decrease in ethanol production rates. Acetic acid had a

minimal inhibitory effect on glucose consumption, with

the exception of the most severe condition tested (pH 5 and

15 g L�1 acetic acid). More detailed results for each of these

fermentation performance characteristics are provided in

the following sections.

Impact of acetic acid and pH on biomass growth

Because of the high cell concentration at inoculation,

minimal cell growth was observed. However, the extent of

this growth for each of the nine fermentation conditions was

compared to determine the effect of acetic acid and pH on

the growth of S. cerevisiae 424A(LNH-ST). The maximum

biomass concentrations under each condition are summar-

ized in Table 1. At a given pH, the maximum biomass

concentration was shown to decrease significantly in fer-

mentations with acetic acid as compared with the control

(with the exception of the least severe condition tested, pH 6

and 7.5 g L�1 acetic acid).

Slight reductions in the biomass concentration were also

observed as the pH decreased for fermentations at a given

acetic acid concentration. This effect was not observed in the

control fermentations, suggesting that the reductions in

biomass when acetic acid was present cannot be explained

solely by the decrease in pH. This hypothesis was confirmed

by a previous study that reported that pH had no effect on

biomass growth in the range examined here (Phowchinda

et al., 1995).

The biomass yields observed (0.016–0.025 g g�1 sugar

consumed) were less than the expected yield of 0.055 g g�1

sugar consumed for anaerobic metabolism (Davis et al.,

2006). A carbon balance of all fermentations following the

convention of Wang et al. (1979) closed at or above 90%

(data not shown). Therefore, the lower yield of biomass was

offset by the higher yield of fermentation products such as

ethanol, CO2, xylitol, and glycerol.

Impact of acetic acid and pH on specific glucoseand xylose consumption rates

To explore the effect of acetic acid and pH on substrate

consumption, the initial specific sugar consumption rates

(both glucose and xylose) were calculated for each fermen-

tation condition. This rate was calculated by determining

the slope of the steepest portion of the substrate concentra-

tion curve and dividing that by the average cell concentra-

tion during that period to yield a rate with the units of

g substrate g�1 dry cell h�1. The initial specific glucose con-

sumption rates are summarized in Table 1. The glucose

consumption rates for a single pH condition decreased

significantly with increasing acetic acid concentration, with

the exception of pH 6. Under the most severe condition (pH

5 and 15 g L�1 acetic acid), the glucose consumption rate was

only 12% that of the control. However, the inhibitory effect

of acetic acid on the glucose consumption rates decreased as

pH increased. For example, the glucose consumption rate of

the fermentation with 7.5 g L�1 acetic acid at pH 5 was about


387Effect of acetic acid/pH on xylose fermentation

Fig. 1. Time course profiles for the cofermentation of glucose and xylose by Saccharomyces cerevisiae 424A(LNH-ST) in the presence of varying

concentrations of acetic acid and pH values: (a) 0 g L�1 acetic acid, pH 6; (b) 0 g L�1 acetic acid, pH 5.5; (c) 0 g L�1 acetic acid, pH 5; (d) 7.5 g L�1 acetic

acid, pH 6; (e) 7.5 g L�1 acetic acid, pH 5.5; (f) 7.5 g L�1 acetic acid, pH 5; (g) 15 g L�1 acetic acid, pH 6; (h) 15 g L�1 acetic acid, pH 5.5; and (i) 15 g L�1

acetic acid, pH 5. �, glucose; �, xylose; ., acetic acid; n, ethanol; ~, biomass.


388 E. Casey et al.

half that of the control, compared with almost 90% of the

control when the pH was increased to 6.

The initial specific xylose consumption rates are also

summarized in Table 1. It is evident that xylose consump-

tion was strongly inhibited by acetic acid; a significant

decrease in the rate was observed with both increasing acetic

acid concentrations and decreasing pH values. Under the

harshest condition (pH 5 and 15 g L�1 acetic acid), no

significant xylose consumption was observed. Results from

a similar study (Bellissimi et al., 2009) did not show such a

severe decrease in the xylose consumption rate for the

fermentation conditions of pH 5 with 3 g L�1 acetic acid.

However, the concentrations examined in this study were

2.5–5 times greater than those examined by Bellissimi and

colleagues.

To better understand the source of the inhibition shown

in Table 1, the equilibrium concentration of acetic acid was

calculated for each fermentation. The calculation used the

Henderson–Hasselbach equation to determine the acetic

acid–acetate equilibrium in the medium using a pKa of

4.75 for acetic acid and the pH for each fermentation

condition. The initial specific xylose consumption rate was

plotted vs. the calculated undissociated acetic acid concen-

tration in Fig. 2. This shows a strong correlation between the

data and an exponential decay function in the xylose

consumption rate as the undissociated acid concentration

increases (R2 of 0.95). Similar studies have also observed an

exponential decay in S. cerevisiae-specific growth rate and

fermentation rate for glucose fermentations with increasing

acetic acid concentration (Pampulha & Loureiro, 1989;

Narendranath et al., 2001).

Impact of acetic acid and pH on ethanolproduction

To investigate the effect of acetic acid and pH on ethanol

production, ethanol metabolic yields and ethanol

Fig. 2. Relationship between the initial specific xylose consumption rate

and the undissociated acetic acid concentration.Tab

le1.

Sum

mar

yof

bio

mas

san

dsu

bst

rate

consu

mption

resu

lts

under

each

ferm

enta

tion

conditio

nfo

rth

eco

ferm

enta

tion

of

glu

cose

and

xylo

seby

S.ce

revi

siae

424A

(LN

H-S

T)

Tota

lace

tate

1ac

etic

acid

conce

ntr

atio

n(g

L�1)

pH

6pH

5.5

pH

5

07.5

15

07.5

15

07.5

15

Max

imum

bio

mas

s

conce

ntr

atio

n(g

L�1)

6.9

62

AB�

0.0

60

6.5

45

ABC�

0.3

57

6.1

29

CD�

0.1

78

7.1

4A�

0.0

06.3

07

BC

D�

0.0

06.1

58

CD�

0.0

89

7.0

51

A�

0.0

30

6.2

18

CD�

0.0

30

5.7

72

D�

0.0

60

Initia

lspec

ific

glu

cose

consu

mption

rate

(gglu

cose

g�1

dry

cell

h�1)

1.8

67

A�

0.0

09

1.6

51

ABC�

0.1

11

1.6

10

BC�

0.0

34

1.7

29

AB�

0.0

199

1.5

71

C�

0.0

62

1.1

98

D�

0.0

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1.8

20

AB�

0.0

19

0.9

75

D�

0.0

20

0.2

17

E�

0.0

01

Initia

lspec

ific

xylo

se

consu

mption

rate

(gxy

lose

g�1

dry

cell

h�1)

0.3

54

A�

0.0

09

0.2

01

C�

0.0

01

0.1

23

D�

0.0

02

0.2

85

B�

0.0

02

0.1

38

D�

0.0

01

0.0

45

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02

0.2

72

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01

0.0

32

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0.0

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Val

ues

liste

dar

eth

em

ean

and

SEof

two

duplic

ate

ferm

enta

tions

for

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conditio

n.M

eans

with

the

sam

ele

tter

are

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rent

ata

confiden

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velo

f90%

.



volumetric production rates were calculated. The ethanol

metabolic yields, calculated by dividing the observed etha-

nol concentrations by the theoretical ethanol concentrations

(0.51 times the total concentration of consumed glucose and

xylose), are provided in Table 2. If all of the consumed

glucose and xylose was converted to ethanol, the ethanol

metabolic yields would be equal to 1. However, a portion of

the consumed sugars resulted in the production of glycerol,

xylitol, and/or additional cell mass. Thus, the metabolic

yields are all o 1. The presence of acetic acid enhanced

rather than inhibited the yield of consumed sugars to

ethanol, as seen by an increase in the ethanol metabolic

yields as the concentration of acetic acid increased. At pH 5,

ethanol metabolic yield improvements of 20% and 30%

were observed in the presence of 7.5 and 15 g L�1 acetic acid,

respectively, as compared with the control.

In addition, ethanol volumetric production rates

(Table 2) were calculated by dividing the maximum ethanol

concentration by the fermentation time required to reach

that concentration. Significant decreases in productivity

were seen in the presence of acetic acid. Comparing the least

and the most severe conditions (0 g L�1 acetic acid at pH 6

and 15 g L�1 at pH 5, respectively), a 67% decrease in

volumetric productivity was observed. The results also show

a linear increase in the production rates with increasing pH

(R2 of 0.89 and 0.99 for 7.5 and 15 g L�1 acetic acid

concentrations, respectively, plots not shown).

Discussion

The effect of acetic acid on non-pentose-fermenting

S. cerevisiae strains has been studied widely (Maiorella

et al., 1983; Pampulha & Loureirodias, 1989; Phowchinda

et al., 1995; Taherzadeh et al., 1997; Thomas et al., 2002).

Prior studies examined a range of acetic acid concentrations

(up to 12 g L�1) under a variety of uncontrolled or con-

trolled pH values (pH 2.8–5.5). A common observation was

that acetic acid resulted in a decrease in biomass yield

coupled to an increase in ethanol yield (Maiorella et al.,

1983; Taherzadeh et al., 1997; Thomas et al., 2002). How-

ever, the increased ethanol yields came at the cost of a

decreased ethanol production rate (Phowchinda et al.,

1995). The decrease in the fermentation rate was explained

by a decrease in intracellular pH, leading to the conclusion

that it is the concentration of the undissociated, uncharged

form of acetic acid that governs the inhibitory effect

(Pampulha & Loureirodias, 1989). Undissociated acetic acid

freely diffuses across the cell membrane and rapidly dis-

sociates because of the higher intracellular pH, resulting in

the release of protons into the cytoplasm. Plasma ATPase

pumps these protons out of the cell at the cost of ATP to

avoid intracellular acidification until the influx of protons

exceeds the cell’s proton-pumping capability and Tab

le2.

Sum

mar

yof

ethan

oly

ield

and

pro

duct

ivity

resu

lts

under

each

ferm

enta

tion

conditio

nfo

rth

eco

ferm

enta

tion

of

glu

cose

and

xylo

seby

Sacc

har

om

yces

cere

visi

ae424A

(LN

H-S

T)

Tota

lace

tate

1ac

etic

acid

conce

ntr

atio

n(g

L�1)

pH

6pH

5.5

pH

5

07.5

15

07.5

15

07.5

gL�

115

gL�

1

Ethan

olm

etab

olic

yiel

d0.7

85

BC�

0.0

12

0.8

57

ABC�

0.0

02

0.8

75

AB�

0.0

08

0.7

93

BC�

0.0

06

0.8

26

BC�

0.0

12

0.8

80

AB�

0.0

08

0.7

55

C�

0.0

02

0.8

92

AB�

0.0

62

0.9

56

A�

0.0

18

Ethan

olv

olu

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ric

pro

duct

ion

rate

(get

han

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1h�

1)

1.2

34

A�

0.0

27

0.7

84

C�

0.0

37

0.5

88

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0.0

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1.1

77

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Val

ues

liste

dar

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em

ean

and

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two

duplic

ate

ferm

enta

tions

for

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conditio

n.M

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with

the

sam

ele

tter

are

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signifi

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rent

ata

confiden

cele

velo

f90%

.


390 E. Casey et al.

acidification of the cytoplasm cannot be avoided (Russell,

1992). ATP is also needed for the removal of excess acetate

from the cytoplasm through the energy-dependent weak

acid efflux pump, Pdr12 (Piper et al., 1998). The need for

ATP to maintain intracellular pH homeostasis and reduce

the internal acetate concentration explains the decreased

biomass and increased ethanol yields; glucose was diverted

from biomass generation to ethanol production to generate

the ATP needed for cell maintenance. Similar to the studies

discussed above, a decreased biomass concentration and

increased metabolic ethanol yields were seen in the glucose/

xylose cofermentations with acetic acid for the given study.

These results can also be explained by the diversion of

carbon from biomass growth to ethanol production for

ATP generation. The ethanol production rates also de-

creased in the presence of acetic acid in this study. Upon

comparison of the results, the rate Phowchinda et al. (1995)

achieved with 6 g L�1 acetic acid and uncontrolled pH

conditions was approximately the rate we achieved under

the harshest condition tested in the present work (15 g L�1 at

pH 5). This suggests that S. cerevisiae 424A(LNH-ST) was

more tolerant to acetic acid than the strain they used and/or

controlling the media pH can significantly mitigate inhibi-

tion by acetic acid.

Although there were many similarities in the experimen-

tal procedures and results from this study and the previous

studies, the difference in microorganisms (pentose vs. non-

pentose-fermenting strains) makes a direct comparison

difficult. A limited number of studies have been published

that investigate the impact of acetic acid on genetically

engineered S. cerevisiae strains capable of both glucose and

xylose fermentation (Helle et al., 2003; Bellissimi et al.,

2009). Bellissimi et al. (2009) looked at the effect of acetic

acid on an S. cerevisiae strain engineered with the xylose

isomerase pathway as opposed to the xylose reductase and

xylitol dehydrogenase pathway in the strain used in this

study. The conditions they examined were an acetic acid

concentration of 3 g L�1 under controlled pH values of 3.5 or

5. Helle et al. (2003) studied the effect of acetic acid on an

S. cerevisiae strain that was engineered with the xylose

reductase and xylitol dehydrogenase genes for xylose meta-

bolism, similar to the strain used in this study. Their

experimental conditions were 3 g L�1 acetic acid and initial

pH values of 4, 4.7, and 5.5. Similar to the results with the

non-pentose-fermenting S. cerevisiae strains, biomass yields

decreased in the presence of acetic acid for both studies.

Bellissimi et al. (2009) also showed that xylose consumption

rates were more affected by acetic acid than glucose con-

sumption rates, while Helle et al. (2003) noted a decrease in

ethanol production rates from the presence of acetic acid in

the fermentation media. Similar observations were made in

the present study, confirming the impact of acetic acid on

the cofermentation of glucose and xylose. The primary

difference from these results, when compared with the

results using non-pentose-fermenting S. cerevisiae strains,

was the increased inhibitory effect on xylose consumption

vs. glucose consumption. The rate of xylose consumption in

S. cerevisiae 424A(LNH-ST) is approximately 20% that of

glucose and the ATP yield is less (1.67 mol ATP mol�1 xylose

compared with 2.0 mol ATP mol�1 glucose). Thus, the esti-

mated ATP generation rate when xylose is the sole carbon

source is approximately 17% of the ATP generation rate

when glucose is fermented. This is likely a major reason why

the inhibitory effect of acetic acid on xylose consumption is

more severe than on glucose consumption. Bellissimi and

colleagues showed that a continuous feed of glucose at low

concentrations releases some of the inhibition of xylose

consumption, further supporting this conclusion. Bellissimi

et al. (2009) also observed increasing glucose consumption

rates with 3 g L�1 acetic acid present, a trend not seen in this

study. Low amounts of acetic acid have been shown to

stimulate fermentation (Taherzadeh et al., 1997; Thomas

et al., 2002). However, the acetic acid concentrations tested

in this study exceeded those amounts by a factor of 2 or

more.

Conclusions

The presence of inhibitory compounds in biomass hydro-

lysates is a major obstacle facing the cellulosic ethanol

industry because they impact the fermentative performance

of microorganisms negatively. The effect of acetic acid, one

of these inhibitors, on the cofermentation of glucose and

xylose by S. cerevisiae 424A(LNH-ST) under controlled pH

conditions was examined. Acetic acid inhibited biomass

growth, substrate consumption, and ethanol volumetric

productivity. However, acetic acid enhanced ethanol meta-

bolic yield. Significant decreases in the maximum biomass

concentration were observed in the presence of acetic acid,

with this effect becoming less severe as the pH was increased.

Similar trends were noted with glucose and xylose initial

specific consumption rates, with the effect being more

significant on xylose consumption. An exponential relation-

ship was found between the initial specific xylose consump-

tion rates and the concentration of undissociated acetic acid,

confirming that the inhibitory effect of acetic acid is linked

to the undissociated form of acetic acid. Ethanol production

rates decreased considerably in the presence of acetic acid.

However, no inhibitory effect was seen with the ethanol

metabolic yields; rather, acetic acid was shown to improve

these yields. The impact of media pH was also investigated

in this study. The results suggest that increasing media pH

can alleviate some of the inhibitory effect of the acetic acid

as this causes a decrease in the concentration of undisso-

ciated acetic acid, the inhibitory form of acetic acid for

S. cerevisiae fermentations.



Acknowledgements

This work was supported by the US Department of Energy

Biomass Program, Contract GO17059-16649, and Purdue

Agricultural Research Programs. This material is based on

work supported under a National Science Foundation

Graduate Research Fellowship. Any opinions, findings, con-

clusions, or recommendations expressed in this publication

are those of the authors and do not necessarily reflect the

views of the National Science Foundation. The authors

thank Haroon Mohammad for the preliminary work he

completed on this project. They also thank Eduardo Xi-

menes and Chialing Wu for their internal review of this

manuscript.

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Effect of acetic acid and pH on the cofermentation of glucose and xylose to ethanol by a genetically engineered strain of Saccharomyces cerevisiae