/. Insl. Brew., November-December, 1992, Vol. 98, pp.

509-513

THE FORMATION OF ESTERS AND HIGHER ALCOHOLS DURING BREWERY

FERMENTATION; THE EFFECT OF CARBON DIOXIDE PRESSURE

Bv R. S. Renger, S. H. van Hateren and K. Ch. A. M. Luyben

(Department of Biochemical Engineering, Delft University of Technology, Julianalaan 67, 2628 BC

Delft, The Netherlands)

Received 17 March 1992

The influence of the size and geometry of brewery fermentation vessels on beer flavour and aroma
formation is generally attributed to carbon dioxide pressure. In order to study this pressure effect,
brewery batch fermentations were carried out on the laboratory scale with Saccharomyces cereviaiae.
The formation rates and yields of esters and fusel alcohols were studied in relation to the growth of
metabolically active biomass. The results indicate that the observed reduction in the formation of esters
and fusel alcohols with increased carbon dioxide pressure is mainly caused by reduced yeast growth.
The overall formation of fusel alcohols is less affected than the formation of esters.

Key Words: Saccharomyces cerevisiae, brewery fermentation, TABLE I. Concentration and taste thresholds of some esters
flavour, ester, fusel alcohol, carbon dioxide, pressure. and fusel alcohols found in various lager beers
(ll<
Introduction
The importance of carbon dioxide for the growth and metab Compound Concentration Taste threshold
olism of microorganisms has been recognized for several
decades. In many organisms, including yeasts, carbon dioxide Esters
seems to have an inhibiting effect on growth, at least under
ethyl acclalc 8-48 33
high carbon dioxide partial pressure5. In aerobic cultures, npropyl acetate 30
for instance during baker's yeast production, the relatively isobuiyl acetate 0.03-0.25 1.6
low carbon dioxide partial pressure does not significantly isoamyl acetate 0.8-6.6 1.6
affect yeast growth4. However, in beer production, high dis 2-phenyl-elhyl acetate 0.1-1.5 3.8
solved carbon dioxide concentrations occur due to the ethyl hcxanoate 0.1-1.5 0.23
absence of aeration. The inhibition of growth under these elhyl octanoatc 0.1-0.9 0.9
circumstances actually results in a favourably high efficiency
Fusel alcohols
of the conversion of sugars to ethanol. However, the pro
duction of ethanol is not the main objective of brewery n-propanol 4-17 800
fermentation. The flavour and aroma of the beer is a vital isobutanol 4-57 200
aspect of its quality and they are also known to be affected active amyl alcohol 7-34 65
by carbon dioxide pressure. isoamyl alcohol 25-123 70
2-phcnyl cthanol 5-102 125
Flavour and aroma of beer
Many compounds contribute to the flavour and aroma of
beer. Due to the low taste thresholds for some of these
compounds, supposedly insignificant variations in their con to be mediated by co-enzyme AM. The formation of the acyl-
centrations may result in an entirely different flavour for the CoA species proceeds either through fatty activation, which
beer. Thus, for pilsner beer, at least twenty compounds are involves ATP, oxidative decarboxylation of a-keto acids or
recognized as being important". These compounds include through fatty acid synthesis. Since two different types of
several esters, fusel alcohols, vicinal diketones and organic esters are present in beer (the ethyl esters of medium chain
sulphur compounds. The latter two, which are present in length fatty acids and acetate esters of ethanol and higher
'green' beer, are significantly reduced during lagering. The alcohols), only two of these acyl-CoA formation pathways
remaining fusel alcohols and the esters, especially, contribute are important. The acyl-CoA formation for ethyl esters, such
significantly to the quality of the finished beer. In order to as ethyl hcxanoate and ethyl decanoate, depends on fatty
evaluate the significance of the individual esters and fusel acid synthesis. The acetyl-CoA for the acetate esters, such
alcohols, taste thresholds and ranges of concentrations of as isoamyl acetate and ethyl acetate, is mainly formed by
some esters and fusel alcohols found in various lager beers oxidative decarboxylation of pyruvate. The regulation of
are summarized in Table Ifi. The following compounds are ester synthesis has been reported to be linked to lipid metab
considered to be the most important: isoamyl alcohol, ethyl olism172".
acetate, isoamyl acetate, ethyl hexanoate and ethyl octano- Understanding of the effect of carbon dioxide pressure on
ate. However, it would be an over-simplification to character the formation of flavour and aroma compounds is limited.
ise the taste of beer only by the analytical determination of The conclusion that carbon dioxide pressure inhibits the
these five compounds. In practice, the flavour of one com formation of flavour compounds has been indicated by sev
pound may be suppressed by other compounds18. eral investigations2-7-'"-14. However, in many cases it cannot
The biochemical formation pathways for esters and fusel be determined whether this inhibition is caused by the
alcohols are essentially known. The catabolic or Ehrlich path reduction of yeast growth, or by a specific effect on the
way of fusel alcohol formation starts by transformation of an formation rates. In order to study the effect of carbon dioxide
amino acid and a-ketoglutarate3. This results in a correspond pressure on specific formation rates during fermentation,
ing a-keto acid and glutamic acid. The a-keto acid is success data concerning the biomass composition are also needed.
ively decarboxylated and reduced to a fusel alcohol. Thus, This paper describes the investigation of the effect of
starting with leucine as the amino acid, isoamyl alcohol is carbon dioxide pressure on the flavour and aroma of beer.
formed. The formation of esters now is generally recognized The formation of four important esters and fusel alcohols in

This document is provided compliments of the Institute of Brewing and Distilling

www.ibd.org.uk Copyright - Journal of the Institute of Brewing
510 BREWERY FERMENTATIONS [J. Inst. Brew.

brewery batch fermentations was studied, as well as the yield

of these compounds relative to the amount of metabolicully
active biomass formed. The effect of carbon dioxide pressure
on the formation of esters and fusel alcohols during fermen
tation will be explained in physiological terms.

Experimental

Cultivation method
Brewery batch fermentations were carried out at 9°C with
a Saccharomyces cerevisiae industrial brewery strain in 12%
by weight of wort extract medium. The composition of the
medium has been described in a previous paper12. The yeast
was maintained on agar slants at 4°C and precultured at 9°C
using Erlenmeycr flasks fitted with cotton plugs. Fermen
tation experiments were carried out in a Braun Biostat E
fermenter with a 15 I stainless steel vessel. The working bara
volume was 10 I and the stirrer speed was 200 rpm. Before
pressure
inoculation, the wort was aerated until air saturation was
reached. Following inoculation, stirring was stopped in order Fig. 1. Concentrations of biomass at the end of fermentation exper
to minimize diffusion of oxygen from the fermenter head- iments carried out at various carbon dioxide pressures. (•:
carbon dioxide sparging, +: nitrogen sparging).
space to the medium. Upon exhaustion of the dissolved
oxygen by the yeast, the liquid and headspace in the fer
menter were flushed with either nitrogen or carbon dioxide
butanol) were determined by headspace gas chromatography
gas in order to remove any traces of oxygen remaining, and
using a Carlo Erba model 2300 fitted with a Carbowax 400
to produce the selected pressure. During the fermentation
column. The absolute error in the determination is about 0.5
experiments, the pressure was sustained by carbon dioxide
mg/1 for the esters, 5 mg/1 for isoamyl alcohol and 1 mg/l for
formation.
active amyl alcohol.

Analyses
Results and Discussion
'Dry weight: Dry weights of cultures were determined
using 0.45 /x Sartorius cellulose nitrate niters and a Sharp R-
Effects of experimental procedures
7200 microwave oven. A 10 ml sample was filtered on a
Since limited amounts of molecular oxygen are essential
preweighed filter and washed with demineralized water. The
for growth of brewer's yeast under anaerobic conditions'•",
filter was dried in the microwave oven, and after equilibration
considerable attention was paid to secure a reproducible
with local humidity, the filter was weighed on a microbalance.
supply of oxygen, as described in the materials and methods.
The absolute error made in this determination was about
Figure 1 shows the biomass concentration at the end of
0.05 g/1. In addition to yeast biomass, the wort usually con
fermentation as a function of carbon dioxide pressure in
tained other dry matter. The measured dry weight concen
fermentation experiments which involved either carbon diox
tration was therefore corrected for the initial wort dry weight
ide or nitrogen gas sparging at the beginning of fermentation.
concentration. The concentration of metabolically active
The biomass yield was reduced by 30 to 100% by using
biomass was determined by correction for the glycogen con
carbon dioxide instead of nitrogen. Nitrogen sparging pro
tent. This is an adequate method to account for major
vided the conditions favourable for yeast growth, and it
changes in biomass composition during brewery fermen
would also stimulate the formation of ethyl acetate if it
tation12.
merely reduced the carbon dioxide concentration. However,
'Glycogen content: Fresh cell samples were washed with
using nitrogen instead of carbon dioxide significantly reduced
demineralized water and freeze dried using FTS Flexi-Dry
the concentrations of ethyl acetate measured at the end of
freeze drying equipment. The glycogen content of the
fermentation (figure 2). The opposing effect of nitrogen and
biomass was determined using the method described by
Quain1'. Freeze dried cell samples were extracted with hydro
chloric acid and sodium carbonate solutions. The extracts
ethyl acetate concentration
were incubated with amyloglucosidase and the resulting glu
cose was determined using a Skalar SA40 glucose autoana-
30
lyzer. The absolute error of this determination was about
2%. mg/l
'Total sugar concentration: The total sugar concentration
was calculated from the density of the cell-free culture
medium, as measured with a Paar DMA40 digital density
meter. The measurement was corrected for the ethanol con
centration using the Tabarie formula16. The total sugar con
centration was calculated from the corrected culture density
using a polynomial described by Ruppert15. The absolute
error made with this determination was about 1 g/1. The
results were consistent with total organic carbon determi
nations and HPLC measurements of sugar concentrations.
'Ethanol concentration: Ethanol was determined using a
Hewlett-Packard HP437 gas chromatograph fitted with a bara
Chromosorb 101 column. The relative error of this determi
pressure
nation was less than 5%.
'Ester and fusel alcohol concentrations: Esters (ethyl acet Fig. 2. Concentrations of ethyl acetate at the end of fermentation
ate and isoatnyl acetate) and fusel alcohols (isoamyl alcohol experiments carried out at various carbon dioxide pressures.
or 3-methyl-l-butanol and active amyl alcohol or 2-methyl-l- (•: carbon dioxide sparging, +: nitrogen sparging).

This document is provided compliments of the Institute of Brewing and Distilling

www.ibd.org.uk Copyright - Journal of the Institute of Brewing
Vol. 98, 1992] BREWERY FERMENTATIONS 511

carbon dioxide sparging on yeast growth and ethyl acetate fermentable sugars

formation suggests that oxygen was involved. Oxygen has ethanol active biomass

been known to promote yeast growth and reduce the forma 1O0 1 •>
tion of esters7. Using carbon dioxide instead of nitrogen 9/I g/i
may have removed the air from the fermentor much more
effectively because of the formation of a relatively dense eo

carbon dioxide blanket over the liquid surface. In the fermen

tation experiments described hereafter, carbon dioxide sparg
60
ing was used.

Effects of carbon dioxide pressure on final concentrations 40-

Batch fermentation experiments were carried out under
0, 1 and 2 carbon dioxide over-pressure. At the end of
fermentation, ester and fusel alcohol concentrations were 20- t■■
determined (Table II). Both esters and fusel alcohol concen *.••»-
trations descreased as a result of increasing pressure. This »'*/""*
was partially caused by a reduction of biomass growth, which 50 100 150 200 250 300
h
was about 70% at 2 bar carbon dioxide over-pressure. In
addition, the overall yields of esters and fusel alcohols on
total biomass were affected (table 2). Due to the equal
reduction of total and active biomass growth, similar results Fig. 3. Concentrations of active biomass, corrected for the glycogen
were found for the yields on active biomass. In the case of content (•), fermentable sugars (T) and ethanol (A) during
the fusel alcohols, the yields were significantly increased by two duplicate brewery fermentation experiments.
carbon dioxide pressure. In the case of ethyl acetate, the
yield reached a minimum at 1 bar carbon dioxide over
pressure. It is not clear whether this also holds for isoamyl
acetate because of the low accuracy of the calculated yields. fusel alcohol concentration ester concentration

50
Formation of esters and fusel alcohols during fermentation
mg/l
The apparently differing effects of carbon dioxide pressure mg/l

on the formation of esters and fusel alcohols was studied in 40-

more detail. The concentrations of active biomass, ferment
able sugars and ethanol measured during two fermentations
runs atmospheric pressure are shown in figure 3. These exper
iments, which were carried out at constant pH values of 5.2
and 4.1, have already been described in previous papers12-
l3. For this study, these experiments will be regarded as
duplicates. The concentrations of esters and fusel alcohols
gradually increased throughout the fermentation (sec figure
4). However, the formation of the esters, especially ethyl
acetate, was more pronounced towards the end of fermen
tation than was the formation of the fusel alcohols. Differ
50
ences between the esters and fusel alcohols were also
observed in the yields on active biomass as determined during time
fermentation (see figures 5 and 6). The yields of the fusel
Fig. 4. Concentrations of esters and fusel alcohols during the brew-
alcohols on active biomass were essentially constant, whereas
cry fermentation experiments of figure 3 (•: isoamyl alcohol,
the yields of the esters significantly increased during brewery O: active amyl alcohol, ■: ethyl acetate, O: isoamyl acetate).
fermentation. The specific reaction of the esters may be

TABLE II. Final concentrations and yields of esters and fusel

alcohols on total biomass in fermentation fusel alcohol yield on active biomass
experiments at different carbon dioxide pressures
2
(percent reduction as compared to atmospheric
pressure in parentheses) mg/g

Pressure (bara) 1 18-

T T
•

Final concentration (mg/l)

total biomass (MO3) 4.1 3.0 (30) 1.1 (70)

tj-j-f I
■■■

active biomass (*I0') 2.3 1.7 (30) 0.7 (70)

ethyl acetate 28 13 (50) 8 (70) I
isoamyl acetate 4.8 2.2 (50) 1 (80)
1
6-
isoamyl alcohol 73 70 (5) 58 (20) 5 o 0
> o- o- --O---
active amyl alcohol 26 24 (10) 15 (40)

Yield on total biomass (mg/g

DM) 50 100 150 200 250 300

ethyl acetate 6.9 4.2 (40) 7 (0) time

isoamyl acetate 1.2 0.8 (30) 1 (0)
isoamyl alcohol 18 24 (-30) 53 (-200) Fio. 5. Yields of isoamyl alcohol (•) and active amyl alcohol (O)
active amyl alcohol 6.5 8.1 (-20) 14 (-120) on active biomass during the brewery fermentation experiments
of figure 3.

This document is provided compliments of the Institute of Brewing and Distilling

www.ibd.org.uk Copyright - Journal of the Institute of Brewing
512 BREWERY FERMENTATIONS [J. Inst. Brew.
ester yield on active biomass biomass concentration

6
150
mg/g

1OO-

50-

10O 15O 20O 250 300

time pressure

Fig. 6. Yields of ethyl acetate (■) and isoamyl acetate (D) on active Fie. 8. Concentrations of biomass at the end of fermentation exper-
biomass during the brewery fermentation experiments of figure intents carried out at various carbon dioxide pressures (A:
3. Norstedt"1, D: Rice1"1, •: this paper).

explained by their biochemical formation pathway. Since

ethyl acetate and isoamyl acetate, both being acetate esters,
ethyl acetate concentration
are formed by condensation of acetyl-CoA and the corre
sponding alcohol, the different character of their formation
150
during fermentation may be caused by the supply of the
alcohol. Figure 7 shows the effect of dividing the yields
of esters on active biomass by the corresponding alcohol
concentrations. These corrected ester yields were virtually
constant during fermentation. The remaining variations in
corrected ester yields may have been due to changes in the
supply of acetyl-CoA or specific enzymatic activity.

Conclusions
The concentrations of esters and fusel alcohols formed
during brewery fermentations were significantly reduced by
increased carbon dioxide pressure. However, the overall for
mation of fusel alcohols was less affected than that of the
esters. The reduction in ester and fusel alcohol formation bara
was partially caused by the inhibition of growth of mcta-
bolically active biomass. Nevertheless, fermentation runs pressure

operated at different carbon dioxide pressures also produced
significantly different ester and fusel alcohol yields.
Figure 8 compares the results of this study with results
from other investigations. The final concentrations of the
published data were scaled in order to be able to compare
Fig. 9. Concentrations of ethyl acetate at the end of fermentation
experiments carried out at various carbon dioxide pressures (O:
Kumada7, A: Norstedt1", D: Rice14. •: this paper).

total amyl alcohol concentration

150

1O0-

0.0 bara
300

time
Fig. 10. Concentrations of total amyl alcohols at the end of fermen
Fig. 7. Yields of ethyl acetate (■) and isoamyl acetate (D) on active tation experiments carried out at various carbon dioxide press
biomass and corrected for the alcohol concentration during the ures (A: Arcay-Ledezma1. O: Kumada'. D: Rice14, •: this
brewery fermentation experiments of figure 3. paper).

This document is provided compliments of the Institute of Brewing and Distilling

www.ibd.org.uk Copyright - Journal of the Institute of Brewing
Vol. 98, 1992] BREWERY FERMENTATIONS
the pressure dependence with that found in this study. Thus,
effects of other fermentation conditions, such as yeast var
iety, composition of raw materials, temperature, agitation,
etc., are suppressed. The relationships between final biomass
concentrations and carbon dioxide pressure clearly show a
similar trend. Final concentrations of ethyl acetate and total
amyl alcohols have also been reviewed (figures 9 and 10),
and the conclusion drawn in this paper that the final concen
trations of fusel alcohols are less affected by carbon dioxide
pressure than those of the esters is apparently supported by
other investigations. These results indicate that the yield of
fusel alcohols on biomass increases with higher carbon diox
ide pressure, whereas the yield of the esters is not much
affected. The minimum in ester yield at 1 bar carbon dioxide
over-pressure found in this study, does not conflict with other
observations.
References
1. Andrcascn, A. A., Stier, T. J. B. Journal of Cellular and Com
parative Biology, 1954, 42, 271-281.
2. Arcay-Ledezma, G. J., Slaughter, J. C. Journal of the Institute
of Brewing, 1984, 90, 81-84.
3. Chen, E. C. H. Journal of the American Society of Brewing
Chemists, 1978, 36, 1, 39-43.
4. Chen, S. L., Gutmanis, F. Biotechnology and Bioengineering,
1976, 18, 1455-1462.
5. Dixon, N. M., Kell, D. B. Journal of Applied Bacteriology,
1989, 67, 109-136.
This document is provided compliments of the Institute of Brewing and Distilling
www.ibd.org.uk Copyright - Journal of the Institute of Brewing
513
6. Engan, S. Brewing Science, 1981, 93-165.
7. Kumada, J., Nakajima, S., Takahashi, T., Narziss, L. European
Brewery Convention. Proceedings of the 15th Congress Nice,
1975, 615-623.
8. Mcilgaard, M. C. Journal of Agricultural and Food Chemistry,
1982, 30, 1009-1017.
9. Nordstrom, K. Svensk Kemisk Tidskrift, 1964, 74, 9, 1-34.
10. Norstedt, C, Bengtsson, A., Bennet, P., Lindstrom, I., Ayra-
paa, T. European Brewery Convention. Proceedings of the 15th
Congress Nice, 1975, 581-600.
11. Quain, D. E. Journal of the Institute of Brewing 1981, 87,
289-291.
12. Renger, R. S., J. P. van Dijken, K. Ch. A. M. Luyben, Biotech
nology and Bioengineering, in press.
13. Rcnger, R. S., L. Tijhuis, J. S. Vrouwcnvelder, K. Ch. A. M.
Luyben Biotechnology and Bioengineering, in press.
14. Rice, J. F., Chicoye, E., Helbert, J. R., Garver, J. Journal of
the American Society of Brewing Chemists 1977, 35, 1, 35-50.
15. Ruppert, J. R. Journal of the Association ofAnalytical Chemistry
1986, 69, 4, 709-711.
16. Tabarie\ Zeitschrift fur Analytische Chemie 1912, 24.
17. Thurston, P. A., Quain, D. E., Tubb, R. S. Journal of the
Institute of Brewing, 1982, 88, 90-94.
18. Torline, P. A. Technical Quarterly of the Master Brewers Associ
ation of the Americas 1985, 22, 13-18.
19. Visser, W., Scheffers, W. A., Batenburg-Van der Vegte, W.
H., Van Dijken, J. P. Applied and Environmental Microbiology,
1990, 56, 12, 3785-3792.
20. Yoshioka, K., Hashimoto, N. Agricultural and Biological Chem
istry, 1984,48, 1,207-209.