ORIGINAL ARTICLE

The influence of nitrogen and biotin interactions on theperformance of Saccharomyces in alcoholic fermentationsJ.C. Bohlscheid1, J.K. Fellman2, X.D. Wang3, D. Ansen4 and C.G. Edwards1

1 Department of Food Science and Human Nutrition, Washington State University, Pullman, WA, USA

2 Department of Horticulture and Landscape Architecture, Washington State University, Pullman, WA, USA

3 Health Science Center, McMaster University, Hamilton, ON, Canada

4 Chambre d’Agriculture du Bas-Rhin, Obernai, France

Introduction

While the specific nutritional requirements of Saccharomy-

ces for growth and completion of alcoholic fermentations

depend on many factors including species/strain (Monk

1994; Shinohara et al. 1996; Vaughan-Martini and Martini

1998), an interdependency may exist between the essential

nutrients, yeast assimilable nitrogen (YAN) and biotin.

YAN concentrations in Vitis vinifera reportedly range from

40 to 1360 mg l)1 (Spayd and Anderson-Bagge 1996;

Butzke 1998), while biotin concentration ranges from 0Æ6to 60 lg l)1 (Amerine 1980; Hagen, in preparation). In

grape must, both these nutrients can be limiting with

respect to completing an optimal or problem-free alco-

holic fermentation (Agenbach 1977; Davenport 1985;

Winter et al. 1989; Henschke and Jiranek 1993). While the

nitrogen content of a medium is an important global

regulator of yeast gene expression and metabolism (Coo-

per 1982; Jones and Fink 1982), the role of biotin in nitro-

gen and lipid metabolism has been established (Oura and

Suomalainen 1978; Lynen 1979; Keech and Wallace 1985).

Although the individual effects of these nutrients on

alcoholic fermentations have been studied (Ough et al.

1989; Henschke and Jiranek 1993), there has been little

research on potential interactions. Ough and Kunkee

(1968) suggested that the concentration of biotin along

with total nitrogen in a grape must be a better predictor

of alcoholic fermentation rates than nitrogen alone. How-

ever, this study only addressed general fermentation rates

and did not investigate the effect of critical levels of the

nutrients towards growth, fermentation kinetics or vola-

tile production by Saccharomyces.

Keywords

biotin, esters, fermentation, higher alcohols,

hydrogen sulfide, nitrogen, Saccharomyces.

Correspondence

Jeffri C. Bohlscheid, Department of Food

Science and Human Nutrition, Washington

State University, Pullman 99164-6376,

WA, USA.

E-mail: jbohlscheid@wsu.edu

2005/1501: received 19 December 2005,

revised 10 May 2006 and accepted 25 May

2006

doi:10.1111/j.1365-2672.2006.03180.x

Abstract

Aim: To study the impact of assimilable nitrogen, biotin and their interaction

on growth, fermentation rate and volatile formation by Saccharomyces.

Methods and Results: Fermentations of synthetic grape juice media were con-

ducted in a factorial design with yeast assimilable nitrogen (YAN) (60 or

250 mg l)1) and biotin (0, 1 or 10 lg l)1) as variables. All media contained

240 g l)1 glucose + fructose (1 : 1) and were fermented using biotin-depleted

Saccharomyces cerevisiae strains EC1118 or UCD 522. Both strains exhibited

weak growth and sluggish fermentation rates without biotin. Increased nitrogen

concentration resulted in higher maximum fermentation rates, while adjusting

biotin from 1 to 10 lg l)1 had no effect. Nitrogen · biotin interactions influ-

enced fermentation time, production of higher alcohols and hydrogen sulfide

(H2S). Maximum H2S production occurred in the medium containing

60 mg l)1 YAN and 1 lg l)1 biotin.

Conclusions: Nitrogen · biotin interactions affect fermentation time and vola-

tile production by Saccharomyces depending on strain. Biotin concentrations

sufficient to complete fermentation may affect the organoleptic impact of wine.

Significance and Impact of the Study: This study demonstrates the necessity to

consider nutrient interactions when diagnosing problem fermentations.

Journal of Applied Microbiology ISSN 1364-5072

390 Journal compilation ª 2006 The Society for Applied Microbiology, Journal of Applied Microbiology 102 (2007) 390–400

ª 2006 The Authors

A shortage of YAN and biotin during fermentation

may diminish the quality of a wine. Nitrogen deficiencies

in grape must can lead to sluggish alcoholic fermentations

and/or excessive production of hydrogen sulfide (H2S)

(Agenbach 1977; Hallinan et al. 1999; Bisson and Butzke

2000; Spiropoulos et al. 2000). A lack of biotin may also

be of concern (Monk and Costello 1984; Boulton et al.

1996) as Saccharomyces cerevisiae strains used in industrial

fermentations are auxotrophic for biotin (Kunkee and

Amerine 1970; Oura and Suomalainen 1978,1982; Monk

1994), and the vitamin is involved in nitrogen metabo-

lism by the yeast (Oura and Suomalainen 1978). Biotin is

required by the enzyme urea carboxylase, which is neces-

sary for the utilization of nitrogen from arginine (Cooper

1982), a key amino acid in grape must (Spayd and

Andersen-Bagge 1996) and storage form of nitrogen for

yeast (Whitney et al. 1973; Jones and Fink 1982). Biotin

also acts as a cofactor for pyruvate carboxylase,

an enzyme that catalysis the transformation of pyruvate

to oxaloacetate (Keech and Wallace 1985). Oxaloacetate is

a precursor for both a-ketoglutarate and aspartic acid, the

key intermediates for nitrogen assimilation and the syn-

theses of other nitrogenous compounds (Cooper 1982).

If nitrogen assimilation and the cellular pool of aspartic

acid are disrupted, cellular functions will be impaired

through diminished synthesis of nucleic acids, proteins

and other important vital compounds (Ahmad et al. 1961;

Moat et al. 1969; Shimada et al. 1978). In addition, the

nutrient concentration and composition of a fermentation

medium affects higher alcohol, ester and H2S production

(Rankine 1967; Rapp and Mandery 1986; Lambrechts and

Pretorius 2000). As biotin is involved in both amino acid

metabolism and lipid synthesis, variations in its level may

impact the production of higher alcohols, esters and med-

ium chain fatty acids (MCFA) (Suomalainen and Keranen

1963; Forch et al. 1975; Lynen 1980). A biotin deficiency

may also influence the production of H2S, as the vitamin

is directly involved in the synthesis of the carbon skeletons

necessary for S2) assimilation from the sulfate reduction

pathway (Jordan and Slaughter 1986; Thomas and Surdin-

Kerjan 1997; Sohn and Kuriyama 2001).

The objective of this research was to determine the

effects of nitrogen, biotin and their interactions on

growth, fermentation rate, H2S and other volatile com-

pound production by wine yeast strains using a synthetic

grape juice medium.

Materials and methods

Yeasts and starter cultures

Saccharomyces cerevisiae strains EC1118 (Prise de Mousse)

and UCD 522 (Montrachet) were supplied by Lallemand

Inc. (Montreal, QC, Canada) in active dry form and

maintained on acidified potato dextrose agar (Difco,

Detroit, MI, USA) at 4�C.

Starter cultures for fermentation were prepared by

streaking the yeasts on wort agar (King and Beelman

1986) and inoculating a single colony into 10 ml 0Æ67%

w/v Yeast Nitrogen Base (Difco) containing 5% w/v glu-

cose and acidified to pH 3Æ5. Cultures were incubated for

24 h at 24�C, harvested by centrifugation (30 min at

2000 g), and then washed and recentrifuged 3· with

10 ml of 0Æ2 mol l)1 phosphate buffer (pH 7). The cells

were resuspended in phosphate buffer and inoculated

(approx. 105 CFU ml)1) into 20 ml of biotin-depletion

medium comprised of 1Æ67% (w/v) vitamin-free yeast

nitrogen base with amino acids and ammonium sulfate

(United States Biological, Swampscott, MA, USA). This

medium was further supplemented with 1 g l)1 peptone,

40 g l)1 glucose, 0Æ2 mg l)1 folic acid, 14 mg l)1 sodium

bicarbonate, 200 mg l)1 myo-inositol, 4 mg l)1 pyridox-

ine, 4 mg l)1 nicotinic acid, 1 mg l)1 thiamin, 0Æ4 mg l)1

riboflavin and 250 lg l)1 pantothenic acid. The media

was then adjusted to pH 3Æ5. Vitamins and glucose were

obtained from Sigma-Aldrich (St Louis, MO, USA), while

peptone was obtained from Difco. Two litres of the med-

ium was dialysed with 1 mg avidin (Inovatech Inc.,

Abbotsford, BC, Canada) placed in a 12 000–14 000

MWC Spectrapor dialysis membrane (Spectrum Medical

Industries Inc., Los Angeles, CA, USA) for 24 h to

remove any residual biotin. The cultures were capped

with BugStopperTM closures (Whatman Inc., Clifton, NJ,

USA) and incubated aerobically for 24 h at 22�C with

agitation provided by a Burrell wrist shaker (Burrell Inc.,

Pittsburgh, PA, USA) to provide a population of approx.

107 CFU ml)1. The yeasts were harvested and washed as

described above before inoculation into a fresh biotin-

depletion medium. The growth and cell washing proce-

dures were repeated one more time to provide the

inoculum for the synthetic grape juice media.

Fermentations

A 2 · 3 factorial design based on the synthetic grape juice

of Wang et al. (2003) was used in this study. The syn-

thetic grape juice contained either 60 or 250 mg YAN l)1

(ammonium nitrogen + a-amino nitrogen excluding pro-

line) and either 0, 1 or 10 lg l)1 biotin. The YAN con-

centrations represented the mean extremes of nitrogen

concentration of Cabernet Sauvignon grapes from Wash-

ington State (Spayd and Anderson-Bagge 1996). The

range of biotin concentrations was selected as deficient to

in excess for growth (Castor 1953; Davenport 1985; Win-

ter et al. 1989). The medium also contained 250 lg l)1

pantothenic acid and 240 g l)1 fermentable sugars (1 : 1

J.C. Bohlscheid et al. Performance of Saccharomyces in alcoholic fermentations

ª 2006 The Authors

Journal compilation ª 2006 The Society for Applied Microbiology, Journal of Applied Microbiology 102 (2007) 390–400 391

glucose/fructose) with amino acids, sugars and vitamins

were obtained from Sigma-Aldrich, and the other compo-

nents obtained from Fisher Scientific (Pittsburgh, PA,

USA).

Similar to the culture starter medium, the synthetic

juice components were dialysed with avidin. Media com-

ponents were adjusted to pH 3Æ5 with 10 mol l)1 NaOH

or 50% (v/v) phosphoric acid and sterile filtered through

0Æ22 lm ExpressTM PES bottle top filters (Millipore, Bed-

ford, MA, USA) prior to transfer (3 l) into optimizer

spinner fermentation flasks (Sartorius BBI Systems, Allen-

town, PA, USA). To provide a source of insoluble solids,

SigmaCell� was suspended in phosphate buffer, auto-

claved and then aseptically added to all vessels to produce

a concentration of 0Æ1% (w/v). All media were inoculated

with the starter cultures at approx. 105 CFU ml)1, and

the fermentations were conducted in triplicate at 22�C.

The fermentation vessels were fitted with Cd(OH)2-H2S

traps as described previously (Wang et al. 2003). The ves-

sels were stirred at 75 rev min)1 for 5 min prior to samp-

ling using a syringe bypass sampler (model M1230-6000;

New Brunswick Scientific Co., Edison, NJ, USA). To

equilibrate inside/outside pressures during sampling,

N2 gas was added through a sterile inline 0Æ22 lm

hydrophobic disk filter (Whatman). Fermentations were

considered complete when fermentable sugars reached

£2Æ0 g l)1.

Analytical methods

Cell populations were estimated by spread plate method

using wort agar and an Autoplate� 4000 spiral plater

(Spiral Biotech, Bethesda, MD, USA). Plates were incu-

bated for 48 h at 25�C prior to enumeration. Fermenta-

ble sugars and ethanol concentrations were analysed

using HPLC. Separation was accomplished using a C18

reverse phase column (Hewlett-Packard, Palo Alto, CA,

USA) in series with an ion exchange HPX-87H column

(Bio-Rad Inc., Hercules, CA, USA) and detected by

refractive index (Laurent et al. 1994). Maximum fer-

mentation rates were determined by the method of

Monteiro and Bisson (1992), while H2S evolution was

quantified according to Wang et al. (2003). Esters,

higher alcohols and MCFA were quantified using solid-

phase microextraction-gas chromatography (Bohlscheid

et al. 2006).

Statistical analysis was performed using sas statistical

software version 8.1 (SAS Institute Inc., Cary, NC, USA)

for anova. Least squares analysis was used for the detec-

tion of significant interactions, and Fisher’s protected

LSD was applied towards mean separation. All figures

and tables represent mean values of the fermentation

replicates.

Results

Growth and fermentation rates

Yeast populations in fermentations containing either 1 or

10 lg l)1 biotin exceeded 107 CFU ml)1 within 48 h after

inoculation (Fig. 1). The viability of EC1118 began to

decline rapidly to less than 107 CFU ml)1 when sugar

levels were less than 20 g l)1 (approx. 280 h). In con-

trast, UCD 522 maintained populations greater than

107 CFU ml)1 in the low YAN media throughout fermen-

tation, and the high YAN populations decreased

after 288 h (sugars <20 g l)1), falling to less than

106 CFU ml)1. Without biotin, both yeasts grew poorly.

Growth of EC1118 in the biotin-free media peaked at

populations of 1 log less than those of the fermentations

with either 1 or 10 lg l)1 biotin. However, viable cell

counts of UCD 522 in biotin-free media only increased

0Æ5 log and 1 log for the low and high YAN treatments,

respectively.

Both yeast strains also required biotin to complete the

fermentations (Fig. 2). All fermentations containing biotin

completed 1130 h after inoculation; conversely, those

without biotin still contained excessive levels of sugars

when terminated (1430 h).

The biotin-free fermentations conducted by EC1118

contained concentrations of residual sugars at 40 g l)1

Via

bilit

y (C

FU

ml–1

)

1x105

1x106

1x107

1x108

0 250 500 750 1000 1250 1500 Time (h)

1x105

1x106

1x107

1x108

(a)

(b)

Figure 1 Mean viable populations of (a) UCD 522 or (b) EC1118 dur-

ing fermentation of synthetic grape must with 60 (s, (, n) or 250 (d,

, ) mg l)1 YAN and 0 (s, d), 1 ((, ) or 10 (n, ) lg l)1 biotin.

Performance of Saccharomyces in alcoholic fermentations J.C. Bohlscheid et al.

392 Journal compilation ª 2006 The Society for Applied Microbiology, Journal of Applied Microbiology 102 (2007) 390–400

ª 2006 The Authors

(high YAN) and 10 g l)1 (low YAN), while fermentation

performed by UCD 522 contained 120 g l)1 (high YAN)

and 150 g l)1 (low YAN).

While the biotin-free fermentations were sluggish and

did not complete, only YAN (P < 0Æ001) influenced the

maximum fermentation rates in media containing either

1 or 10 lg l)1 biotin (Table 1). Raising YAN from 60 to

250 mg l)1 increased the maximum fermentation rate by

either yeast, yet increasing biotin at either YAN concen-

tration had no effect. Higher YAN concentrations had a

greater influence on UCD 522 than EC1118; the former

responded with a 26% increase in the maximum fermen-

tation rate, while the latter had only an 8% increase.

In contrast, the time to complete fermentation by the

yeasts in the biotin-containing media demonstrated signi-

ficant YAN · biotin interaction (P < 0Æ001) (Table 1).

Fermentation times decreased at high YAN when biotin

concentrations rose from 1 to 10 lg l)1; yet the increase

had no effect on fermentation times at low YAN. Addi-

tionally, increasing levels of both nutrients had a more

pronounced effect on UCD 522 than EC1118. For exam-

ple, UCD 522 in higher YAN media attenuated fermenta-

tion times by 27% (at 1 lg l)1 biotin) and 44% (at

10 lg l)1 biotin) when compared with the same biotin

concentrations at the lower YAN level. However, fermen-

tation times in media inoculated with EC1118 were only

reduced by 12% and 17% under the same conditions.

Final mean ethanol concentrations in fermentations

containing biotin ranged from 13Æ3% to 13Æ6% (v/v) and

were not significantly different. Mean ethanol concentra-

tions in biotin-free fermentations for UCD 522 and

EC1118 when terminated were 4Æ41 and 5Æ82% (v/v),

respectively.

Hydrogen sulfide production

YAN · biotin interactions influenced H2S production by

the yeasts (Table 2). The highest cumulative H2S produc-

tion (80 lg l)1) occurred for both strains in the low YAN

medium containing 1 lg l)1 biotin. Increases in either

YAN or biotin reduced H2S production by both yeasts

150

100

50

0

250

(a)

(b)

200

Glu

cose

+ fr

ucto

se (

g l-1

)

150

100

50

0

200

250

0 250 500 750 1000 1250 1500Time (h)

Figure 2 Mean changes in total glucose and fructose concentration

during fermentation of synthetic grape must by (a) UCD 522 or (b)

EC1118 with 60 (s, (, n) or 250 (d, , ) mg l)1 YAN and 0 (s,

d), 1 ((, ) or 10 (n, )lg l)1 biotin.

Table 1 Maximum fermentation rates and fermentation times by

yeast strains UCD 522 and EC1118 under varying nitrogen and biotin

concentrations

YAN

(mg l)1)

Biotin

(lg l)1)

Maximum fermentation

rate (g sugar ml)1 h)1) Fermentation time (h)

UCD 522 EC1118 UCD 522 EC1118

60 0 0Æ4g 1Æ9e >1430 >1430

1 6Æ6d 9Æ2c 1130e 700c

10 6Æ8d 8Æ8c 1090e 690c

250 0 0Æ7g 1Æ3f >1430 >1430

1 11Æ7a 10Æ7b 830d 620b

10 12Æ0a 10Æ7b 610b 570a

Mean values within fermentation rate or time categories with

different superscript letters are significantly different at P < 0Æ05 using

Fisher’s LSD.

Table 2 The effects of YAN (60 or 250 mg l)1), biotin (0, 1 or

10 lg l)1) and nutrient interaction on synthesis of volatile compounds

by UCD 522 and EC1118

Variable

UCD 522 EC1118

YAN B YAN · B YAN B YAN · B

H2S * ** *** *** *** **

Isobutyl alcohol *** *** ** ** *** *

Amyl alcohol *** *** *** NS ** **

Phenylethyl alcohol *** * *** *** *** ***

Hexanoic acid NS NS NS NS *** NS

Octanoic acid ** ** NS * *** **

Decanoic acid ** *** NS NS ** NS

Ethyl hexanoate NS *** NS NS *** NS

Ethyl octanoate NS *** NS NS ** NS

YAN, yeast assimilable nitrogen; B, biotin; YAN · B, YAN and biotin

interaction; NS, not significant.

*P < 0Æ05, **P < 0Æ01 and ***P < 0Æ001 using least squares.

J.C. Bohlscheid et al. Performance of Saccharomyces in alcoholic fermentations

ª 2006 The Authors

Journal compilation ª 2006 The Society for Applied Microbiology, Journal of Applied Microbiology 102 (2007) 390–400 393

(Fig. 3). For EC1118, biotin concentrations of 1 or

10 lg l)1 at high YAN decreased output to the lowest lev-

els of H2S; however, there was no difference between in

production at either YAN level at 0 lg l)1 biotin. With

UCD 522, peak H2S production also fell at 250 mg l)1

YAN and 1 lg l)1 biotin, but increased slightly when bio-

tin was increased to 10 lg l)1. Increasing biotin from 1

to 10 lg l)1 at low YAN resulted in decreased production

by both yeasts; however, the effect did not occur at higher

YAN.

Other volatiles

Higher alcohols production was dependent upon

YAN · biotin interactions, and yeast strain (Table 2), but

there were no consistent patterns of production between

the alcohols. Isobutyl alcohol production peaked for both

yeasts in media containing 0 lg l)1 biotin and high YAN,

followed by 0 lg l)1 biotin and low YAN (Fig. 4). The

additions of 1 and 10 lg l)1 biotin to the media resulted

in progressive decreases of isobutyl alcohol production

for UCD 522, but a similar reduction was not seen with

EC1118. Regarding amyl alcohol formation, EC1118 was

not affected by biotin concentration at low YAN, but

there was a progressive reduction at high YAN with

increasing biotin. In contrast, biotin concentration at high

YAN did not affect production by UCD 522; although it

did result in increased production at low YAN, with the

highest concentration occurring at 1 lg l)1 biotin. Both

yeasts produced similar patterns of phenylethyl alcohol

with respect to nutrient amounts; however, UCD 522 did

yield higher concentrations of the volatile. Fermentations

containing 1 or 10 lg l)1 biotin at low YAN produced

higher concentration of this alcohol than those at higher

YAN. The increase of YAN at 0 lg l)1 biotin resulted in

elevated phenylethyl alcohol production by both yeasts.

Nutrient interactions did not influence MCFA, where

biotin, and to some degree YAN, directed production

(Table 2; Fig. 5). With EC1118, hexanoic acid production

rose nearly tenfold when biotin was present in the

fermentation medium regardless of YAN. Changes in

nitrogen and biotin did not influence hexanoic acid pro-

duction by UCD 522. Nutrient interactions only affected

octanoic acid production by EC1118, with the greatest

01234567

01234567

x

w

xy yy

zz

w xy

ab bbc zz cdd

0

0·6

1·2

1·8

0 1 1 10 0 10

e

zcd z

bcz

dez

aba

y

z

250 mg l–1YAN 60 mg l–1 YAN H

exan

oic

acid

(m

g l–1

)O

ctan

oic

acid

(m

g l–1

)D

ecan

oic

acid

(m

g l–1

)

Biotin (µg l–1)

Figure 3 Concentrations of hexanoic, octanoic and decanoic acids in

synthetic wines produced by UCD 522 (() and EC1118 ( ), with ini-

tial nitrogen concentrations of 60 or 250 mg l)1 YAN and 0, 1 or

10 lg l)1 biotin. Within a given yeast, mean values with different let-

ters are significantly different at P < 0Æ05 for UCD 522 (a–e) and

EC1118 (w–z) using Fisher’s LSD.

Am

yl a

lcoh

ols

(mg

l–1)

0

40

80

120

160

d

wxa w

wx b xy

c c c

yz z

0

50

100

150

200

250

Isob

utyl

alc

ohol

(m

g l–1

)

y

ab c b z zz d z cd

x

Phe

nyle

thyl

alc

ohol

(m

g l–1

)0

10

20

30

0 1 10 0 1 10

aa

b

cd dwx xy

z z

250 mg l–1 YAN 60 mg l–1 YAN

Biotin (µg l–1)

Figure 4 Concentrations of isobutyl, amyl and phenylethyl alcohols

in synthetic wines produced by UCD 522 (() and EC1118 ( ), with

initial nitrogen concentrations of 60 or 250 mg l)1 YAN and 0, 1 or

10 lg l)1 biotin. Within a given yeast, mean values with different let-

ters are significantly different at P < 0Æ05 for UCD 522 (a–d) and

EC1118 (w–z) using Fisher’s LSD.

Performance of Saccharomyces in alcoholic fermentations J.C. Bohlscheid et al.

394 Journal compilation ª 2006 The Society for Applied Microbiology, Journal of Applied Microbiology 102 (2007) 390–400

ª 2006 The Authors

changes occurring between 0 and 1 or 10 lg l)1. At

higher YAN, octanoic acid output decreased at both 1

and 10 lg l)1 biotin. UCD 522 responded with small

increases of octanoic acid as a result of higher biotin and

YAN. There was no difference in decanoic acid produc-

tion by EC1118 except at 250 mg l)1 YAN and 10 lg l)1

biotin. With UCD 522, decanoic acid concentrations

increased at higher levels of YAN or biotin.

Only biotin influenced ethyl ester production (Table 2).

Overall, EC1118 produced higher concentrations of ethyl

hexanoate, while UCD 522 produced higher concentra-

tions of ethyl octanoate (Fig. 6). Ethyl ester production

by UCD 522 was entirely dependent upon the presence of

biotin, as was ethyl octanoate by EC1118. In this study,

the only changes occurred because of the addition of

biotin to the fermentation medium. Ethyl hexanoate pro-

duction by EC1118 varied because of the changes in

nutrient concentrations, the only differences because of

YAN occurred at 0 lg l)1 biotin. At low YAN, ethyl hex-

anoate production followed the increase of biotin.

Discussion

Growth and fermentation rate

Nitrogen is considered the most important nutrient for

wine yeast to ensure complete and defect-free fermenta-

tions (Salmon 1996); however, biotin is also crucial for

the growth of these auxotrophic strains (Koser 1968;

Kunkee and Amerine 1970). In the current study, both

strains demonstrated faster maximum fermentation rates

and shorter fermentation times at higher YAN, an obser-

vation that is well documented (Agenbach 1977; Ingledew

and Kunkee 1985; Jiranek et al. 1995b; Wang et al. 2003).

The YAN concentrations used, however, did not affect

growth or maximum populations achieved.

Conversely, biotin did affect growth. In fact, maximum

populations and completion of fermentations were

dependent upon the presence of at least 1 lg l)1 biotin;

although concentrations as low as 0Æ25 lg l)1 biotin may

be adequate for growth in a minimal medium (Rogers

and Lichstein 1969). Davenport (1985) noted that con-

centrations of 0Æ7 to 1Æ3 lg l)1 were sufficient for com-

mercial active dry wine yeast strains to complete

fermentations of synthetic grape juice media. However,

these fermentations also contained very high concentra-

tions of YAN (400 mg l)1) that may have altered the

minimum concentrations of biotin required by the yeasts

(Moat and Emmons 1954; Ahmad and Rose 1962b; Oura

and Suomalainen 1978). Maximum growth and fermenta-

tion rates under varying YAN and biotin concentrations

have been reported (Ough et al. 1989; Winter et al. 1989;

Henschke and Jiranek 1993; Shinohara et al. 1996).

Fermentation times by the yeasts exhibited an interde-

pendence of YAN and biotin concentrations. Reduced fer-

mentation times were noted by raising biotin from 1 to

10 lg l)1 in the presence of high YAN, demonstrating the

interaction between YAN and biotin suggested by Ough

and Kunkee (1968). A biotin concentration of 1 lg l)1

0

30

60

90

0 1 10 0 1 10

Hyd

roge

n su

lfide

(µg

l–1)

250 mg l–1 YAN 60 mg l–1 YAN wa

d

y

b

x

y bc

d z z

Biotin (µg l–1)

Figure 5 Cumulative production of hydrogen sulfide in synthetic

wines produced by UCD 522 (() and EC1118 ( ), with initial nitro-

gen concentrations of 60 or 250 mg l)1 YAN and 0, 1 or 10 lg l)1

biotin. Within a given yeast, mean values with different letters are sig-

nificantly different at P < 0Æ05 for UCD 522 (a–d) and EC1118 (w–z)

using Fisher’s LSD.

1·6 w

a a a ab b

z

wxxy

wxy

x1·2

0·8

0·4

0

0·8

b zz b

yyyy

aaaa

0·6

0·4

0·2

00 101 0 101

Eth

yl h

exan

oate

(m

g l–1

)E

thyl

oct

anoa

te (

mg

l–1)

250 mg l–1 YAN 60 mg l–1 YAN

Biotin (µg l–1)

Figure 6 Concentrations of ethyl hexanoate and ethyl octanoate in

synthetic wines produced by UCD 522 (() and EC1118 ( ), with ini-

tial nitrogen concentrations of 60 or 250 mg l)1 YAN and 0, 1 or

10 lg l)1 biotin. Within a given yeast, mean values with different let-

ters are significantly different at P < 0Æ05 for UCD 522 (a–c) and

EC1118 (w–z) using Fisher’s LSD.

J.C. Bohlscheid et al. Performance of Saccharomyces in alcoholic fermentations

ª 2006 The Authors

Journal compilation ª 2006 The Society for Applied Microbiology, Journal of Applied Microbiology 102 (2007) 390–400 395

appeared to be limiting with increased YAN. Previous

studies found that the genes involved in biotin uptake

and biosynthesis were expressed at higher rates in fermen-

tations containing high YAN and ‘ample’ concentrations

of 2–3 lg l)1 biotin (Backhus et al. 2001; Rossignol et al.

2003). At high concentrations of YAN, yeast are more

metabolically active (Cantarelli 1957; O’Connor-Cox et al.

1991; Fleet and Heard 1993; Henschke and Jiranek 1993;

Bely et al. 1994; Backhus et al. 2001; Rossignol et al.

2003) and would need to produce greater quantities of

pyruvate carboxylase, urea carboxylase and acetyl-CoA

carboxylase, thus increasing the demand for biotin. The

reduction in total fermentation time, rather than in

maximum fermentation rate, implies that the effect of

increased vitamin availability may be more important

later in the fermentation process.

Nutrient effects on volatile production

Although yeast require specific concentrations of YAN

and biotin for growth and to complete alcoholic fermen-

tation, these concentrations may or may not be optimal

towards the synthesis of desirable aroma compounds.

To date, no previous studies demonstrated the effect of

biotin or YAN · biotin interaction on H2S formation by

yeast. Yeast strain, deficient YAN and pantothenic acid,

along with their interactions, influence H2S evolution

during alcoholic fermentations (Wainwright 1970; Acree

et al. 1972; Rauhut 1993; Jiranek et al. 1995a; Wang et al.

2003). In the current study, yeast supplied with 1 lg l)1

biotin and 60 mg l)1 YAN were able to complete the fer-

mentation; however, they also produced the highest levels

of H2S. This suggests that at low YAN, 1 lg l)1 biotin

was insufficient to control H2S production for the strains.

For UCD 522, the low YAN, 1 lg l)1 biotin medium con-

tained insufficient concentrations of either YAN or biotin

to minimize H2S evolution, as demonstrated by similar

attenuation of H2S production with an increase in either

nutrient. On the contrary, EC1118 was more influenced

by increased YAN than by increased biotin.

H2S production as a response to YAN · biotin interac-

tions may be due to the effect on the concentrations of

aspartic acid or other amino acids involved in sulfur

metabolism. Biotin and nitrogen are required for the pro-

duction of aspartic acid, which in turn is necessary for

the formation of o-acetylhomoserine, the compound that

accepts S2) from the sulfate reduction pathway to form

methionine (Henschke and Jiranek 1993; Thomas and

Surdin-Kerjan 1997; Wang et al. 2003). Without sufficient

amounts of o-acetylhomoserine, H2S is excreted by the

yeast (Hallinan et al. 1999). The synthetic media used in

this study resembled a typical grape must, as it contained

low concentrations of methionine and cysteine (Amerine

1980; Spayd and Andersen-Bagge 1996), thus creating a

demand for the yeast to synthesize amino acids and

organic compounds containing sulfur (Rauhut 1993).

Low YAN and low biotin levels separately may restrict the

production of amino acid precursors for sulfur synthesis,

but the combination of deficient nutrients would have a

synergistic effect as seen with H2S evolution by EC1118.

All of the higher alcohols were all influenced by

YAN · biotin interactions. The production of higher

alcohols is very complex and a result of amino acid and

sugar metabolisms (Chen 1978), but the production of

the metabolites is ultimately a response to the YAN con-

centration of a medium (Ayrapaa 1971; Ough and Bell

1980; Henschke and Jiranek 1993; Webster et al. 1993).

Both the amino acid and sugar metabolic pathways also

involve reactions requiring biotin (Vollbrecht and Radler

1973; Albers et al. 1998; Boulton and Quain 2001). Exces-

sive levels of higher alcohols in wines are typically associ-

ated with YAN-deficient grape must, as seen in amyl

alcohols production by UCD 522 (Fig. 4). Increased con-

centrations of higher alcohols can result from nitrogen

catabolism of valine, leucine, isoleucine and phenylalan-

ine, or by overproduction of a-ketoacids because of a loss

of feedback inhibition (Rankine 1967; Inoue 1975;

Schulthess and Ettlinger 1978; Rauhut et al. 2000; Her-

nandez-Orte et al. 2002). More recent studies, however,

suggest that the anabolic pathway of higher alcohol for-

mation appears to predominate over the catabolism of

amino acids in low YAN media (Beltran et al. 2005), but

the present results indicate a dependence on yeast strain

and higher alcohol. Overproduction of higher alcohols

can be a result of very low or very high YAN, but has

been rarely reported as a result of biotin deficiency (Nor-

dstrom and Carlsson 1965).

Deficiencies of biotin can lead to insufficient synthesis

of a-ketoglutarate as a precursor for amino acid synthesis

and nitrogen assimilation (Ahmad and Rose 1962a,b;

Cooper 1982) and may exacerbate problems for yeast in

an already low YAN medium (Nordstrom 1965). On the

one hand, the decreased production of amyl and phenyl-

ethyl alcohols by UCD 522 (Fig. 4) in the low YAN-bio-

tin-free fermentations suggests that a lack of biotin

affects the salvaging of amine nitrogen from isoleucine,

leucine and phenylalanine which may have contributed

to the decreased growth and fermentation rate. On the

other hand, the high levels of isobutyl alcohol produced

by EC1118 under the biotin-free fermentations indicates

increased catabolism of valine (Dickinson et al. 1998).

The low and high YAN fermentations provided 61 and

440 mg l)1 of valine, respectively (Wang et al. 2003);

while isobutyl alcohol production on a molar basis was

2Æ7 and 0Æ52 times the original valine concentrations in

the media, respectively. Degradation of exogenous valine

Performance of Saccharomyces in alcoholic fermentations J.C. Bohlscheid et al.

396 Journal compilation ª 2006 The Society for Applied Microbiology, Journal of Applied Microbiology 102 (2007) 390–400

ª 2006 The Authors

can account for isobutyl alcohol formation in the high

YAN fermentation, but increased production in the low

YAN fermentations suggests that yeast also produced this

higher alcohol from the anabolic pathway via a-ketois-

ovalerate (Bateman et al. 2002; Hernandez-Orte et al.

2005). The greater production of isobutyl alcohol by

EC1118 over that of UCD 522 in the biotin-free media

is possibly a result of increased growth and metab-

olic activity of the former strain or differences in valine

utilization.

Contrary to nitrogen metabolism, MCFA and their

esters were generally unaffected by nutrient interactions.

Synthesis of these compounds is related to the growth

and general metabolism of yeast (Rattray et al. 1975;

Bardi et al. 1998,1999). As biotin is required for activa-

tion of acetyl-CoA carboxylase, a key enzyme system

de novo fatty acid synthesis, deficient levels of the vita-

min would alter MCFA synthesis and depress growth

(Burkholder 1943; Dixon and Rose 1964; Kuraishi et al.

1971, Hasslacher et al. 1993). Forch et al. (1975) demon-

strated that only very low (<10)2 lg l)1) concentrations

of biotin significantly reduced MCFA along with growth,

which is consistent with the present results. Ethyl ester

synthesis is dependent upon the production of MCFA

and the actions of alcohol acetyltransferase enzymes

(Mason and Dufour 2000); suppression of ester produc-

tion would result from retardation of growth or a short-

age of substrates for the esters inhibited synthesis

(Nordstrom 1965). The limited influence by YAN con-

centration or nutrient interactions in ester formation

indicates that the effect of deficient levels of biotin on

suppressing overall growth has the most pronounced

effect on ethyl ester production.

In conclusion, nitrogen · biotin interactions primarily

affected overall fermentation rates, as well as H2S and

higher alcohol production, which are associated with

nitrogen metabolism. As a result, yeast strains used in

winemaking may also be affected differently owing to dif-

ferent sensitivities to nutrient deficiencies. With biotin

being shown to influence nitrogen metabolism, variations

in vitamin concentrations may have a greater affect on

the strains with higher YAN demands. Biotin levels suffi-

cient for maximum growth and fermentation rate, how-

ever, may not be sufficient to prevent organoleptic defects

in wine. Consequently, because of this nutrient interde-

pendence, there may be an increased need to monitor

grape must components other than nitrogen.

Acknowledgements

This work was supported by research grants from the

Northwest Center for Small Fruits Research and the

Washington State Wine Advisory Committee.

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