Process Biochemistry 45 (2010) 493–499

Kinetic study of the velum formation by Saccharomyces cerevisiae (beticus ssp.)during the biological aging of wines

Patricio Gutierrez, Ana Roldan *, Ildefonso Caro, Luis Perez

Department of Chemical Engineering and Food Technology, Faculty of Sciences, University of Cadiz, Campus Rıo San Pedro, P.O. Box 40, Puerto Real, 11510 Cadiz, Spain

A R T I C L E I N F O

Article history:

Received 2 June 2009

Received in revised form 4 November 2009

Accepted 8 November 2009

Keywords:

Biofilm growth

Yeast culture

Kinetic model

Wine biological aging

A B S T R A C T

The main objetive of this work was to evaluate and model the biofilm growth of the Saccharomyces

cerevisiae (beticus ssp.) yeast during the biological aging of some types of wines. Thus, we have study how

the biofilm growth, the glycerine is consumed and the acetaldehyde is produced, and how this

phenomena are affected by the media ethanol concentration (0–17%, v/v), under experimental

conditions similar to the industrial ones. In consequence, the growth of the S. cerevisiae (beticus ssp.)

biofilm on the surface of the liquid was studied and kinetically modelled. Growth curves were fitted by

using general kinetic models that include biomass and substrate inhibition factors. The alcohol content

of the medium for the fastest growth rate of biofilm was found to be 4.3%, v/v. The proposed kinetic

models for biomass growth, glycerine consumption and acetaldehyde formation fit well with the

experimental data.

The growth kinetics of S. cerevisiae beticus ssp. in biofilm phase presents a typical discontinuous

microbial growth profile (with lag, exponential and stationary phases). The glycerine consumption is

directly related to the substrate concentrations (ethanol and glycerine). Finally, the rate of acetaldehyde

formation suggests a model associated with the rate of microbial growth, which is modified by a

substrate-dependent factor. The suggested model can be used for optimization and control processes of

biological aging of wines.

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Process Biochemistry

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1. Introduction

The aging of fino-type Sherry wine (Jerez-Xerez-Sherry, Spain)is a microbiological process that occurs under a biofilm ofSaccharomyces strain yeasts, which grow spontaneously on thefree liquid surface in contact with air. These yeasts are called ‘‘flor’’yeasts and the biofilm formed is known as ‘‘velo de flor’’. This agingstage is fundamental to the Sherry wine production processbecause it produces a wine with special characteristics. Accordingto the standard industrial process, after the must-fermentation ofPalomino fino grapes, the young wine is fortified with wine alcoholto 15%, v/v, clarified and transferred into oak casks for storage [1].During this storage period (5 years average), a thick layer of ‘‘flor’’yeast (1–3 cm) develops on the wine surface, which is responsiblefor the aging process [2]. The intense and continuous metabolicactivity of these microorganisms in the film phase (aerobic) leadsto an important biochemical transformation, which finally givesrise to a unique product called Sherry wine.

At the industrial scale, for the film-forming yeasts to emerge tothe surface, it is necessary that the wine parameters have the

* Corresponding author. Tel.: +34956016554; fax: +34 956 016411.

E-mail address: ana.roldan@uca.es (A. Roldan).

1359-5113/$ – see front matter � 2009 Elsevier Ltd. All rights reserved.

doi:10.1016/j.procbio.2009.11.005

following values: no residual sugars, less than 16%, v/v, ethanol anda temperature of 18–20 8C [3]. Furthermore, the yeast biofilmrequires contact with oxygen to aerobically metabolize ethanol[4,5]. In the beginning of the aging process, the suspended yeastcells in the wine enhance the hydrophobicity of their cellularmembranes, due to the synthesis of hydrophobic wall proteins[6,8]. This increase in hydrophobicity favors the formation of cellaggregates, which retain the CO2 bubbles formed by the microbialmetabolism. The aggregates have lower relative density than thewine, and thus they emerge to the surface and float. Finally, thewine surface is colonized by the ‘‘flor’’ yeast, which forms anextensive biofilm [7]. Once the ‘‘velo de flor’’ has fully formed, thismicrobial film prevents oxygen diffusion into the wine whilemaintaining a reducing atmosphere. These conditions protect thewine from other possible oxidative effects for the remainder of theaging stage [9,10].

During the aging process, the yeast in the biofilm phase has anoxidative metabolism which decreases the concentrations ofethanol, acetic acid, ethyl acetate, glycerine, amino acids andorganic acids. In contrast, the yeast biofilm increases othercompounds concentration like acetaldehyde, superior alcohols,acetoin and 2,3-butanediol [11,12]. Once the alcohol fermentationprocess was finalized, in the absence of sugars, the yeasts use theethanol as the main carbon source (oxidative pathway) until

P. Gutierrez et al. / Process Biochemistry 45 (2010) 493–499494

acetaldehyde formation occurs. The acetaldehyde accumulates inthe medium and some is oxidized by an aldehyde-dehydrogenase(AlDH) to give acetate and then acetyl-CoA, which is incorporatedin the Krebs cycle. Finally, the yeast also metabolizes ethanol andincorporates its metabolites directly into the cellular structure ascarbohydrates, fatty acids or proteins by means of the pyruvic acidcycle [12,13].

Four subspecies of velum Saccharomyces cerevisiae yeast havebeen isolated from the industrial systems: beticus, cheresiensis,montuliensis and rouxii [3]. The subspecies montuliensis and rouxii

consume the most ethanol, and therefore, produce greaterquantities of acetaldehyde [2,6,7]. On the other hand, Sacchar-

omyces beticus is the most abundant yeast in the wine biologicalaging of the Sherry region. Since acetaldehyde is an intermediatecompound in cellular metabolism, it can give rise to a wide range ofsecondary products by means of collateral reactions; theseproducts include acetate, acetoin, 2,3-butanediol, diacetal or, onceagain, ethanol [14].

In industrial production, acetaldehyde usually accumulates atlevels around 300–400 mg/L, and depending on the cultivationconditions and the type of yeast strain, it can reach levels of 700–800 mg/L [15,16]. The final concentration of acetaldehyde dependson the balance between production and consumption by biofilmand also on the storage conditions affecting other phenomena,such as evaporation and diffusion. Moreover, the metabolism ofethanol is accompanied by a simultaneous consumption ofglycerine. This consumption represents about 80% of the decreaseof the wine dry extract, which occurs during biological aging [17].However, the quantity of metabolized glycerine also depends onthe case-specific aging conditions [18].

So far, biological aging studies have focused on the micro-biological and biochemical characterization of the species involvedin the ‘‘velo de flor’’ formation. However, kinetic studies on thevelum yeast growth have not been performed until now andmodels that reflect the kinetics of the industrial process have notbeen still developed. This work describes the influence of thealcohol concentration on the yeast growth in the biofilm phase.Thus, an experimental methodology for biofilm kinetic studies hasbeen established. As a result, the influence of the ethanolconcentration of wine on the consumption of ethanol and glycerineand on the production of acetaldehyde has been quantified.General theories of microbial growth have been applied in thisstudy to develop a general kinetic model and adjust the predictionsto the experimental data. Thus, the model may allow a betterestimation of the kinetics of the velum yeast formation. Accord-ingly, we can reach a more precise optimization and control of thebiological aging of wines.

2. Materials and methods

2.1. Yeast strains

First, pure cultures of the four velum biofilm yeasts (subspecies beticus,

cheresiensis, montuliensis and rouxii), were isolated and selected. The yeast samples

were taken from an industrial biological aging system for Fino-type wine in the

Sherry area winery (Bodegas Domecq, Jerez de la Frontera, Spain). The strain

isolation was performed according to the serials dilutions technique and several

inoculations were completed in Petri dishes with YPD agar (yeast extract, peptone,

glucose) culture media. The Petri dishes were held in an incubator, at 28 8C for 4–5

days [19]. After pure cultures had been obtained, the appropriate taxonomic tests

were performed to identify species and subspecies following the procedure of

Barnet el al. [20] and Martınez et al. [6]. All experimental studies in this work were

carried out using a strain of S. cerevisiae (beticus ssp.) (B17, Bodegas Domecq, Jerez

de la Frontera, Spain) which was previously isolated from the industrial aging

system above mentioned. This strain was chosen because it is the subspecies most

commonly found at the industrial biological aging systems [14]. This strain was

preserved on slope agar until use.

A growth medium was used in order to keep the viable cell population in biofilm

form until required for the experiments. This growth medium consisted of a

standard wine from the industrial system of biological aging (15%, v/v, alcohol),

which was previously filter sterilized (0.22 mm). The medium was refreshed once a

month to keep the yeast in a good state of development (velum phase). This

operation consisted on take out half of the liquid medium and replace it by the same

volumen of young wine. This way, we assured replacing the depleted nutrients of

wine and reducing the inhibition products. During this conservation time, the yeast

does not lose the ability to form velum.

2.2. Culture media

Synthetic medium as Fortachon culture described by Alexandre et al. [8] was

used with some modifications in order to make it more similar to the non-alcohol

fraction of wine. After the addition of these compounds, the composition of the

medium was as follows: yeast extract (1 g), ammonium sulphate (0.5 g), potassium

dihydrogen phosphate (1 g), magnesium sulphate (1 g), calcium chloride (1 g),

glycerine (0.5 g), ferric citrate hydrate (0.03 g), acetic acid (0.25 g), acetaldehyde

(0.06 g), tartaric acid (6 g), sulphur dioxide (0.08 g), all dissolved in 1000 mL of

water. Later, to ensure comparability to industrial media, the pH was adjusted to 3.2

using sodium hydroxide (0.1N). The free amino nitrogen (FAN) levels of this

synthetic medium was determinated using the methodology proposed by Aerny

[21] and resulted in 120 mg/L. These levels were similar to those determinated in P.

fino young wines used for biological aging (123 mg/L).

This alcohol-free medium was used as base to prepare two different media used

in this work: the 0%, v/v, alcohol medium and the 12%, v/v, alcohol medium. The

12%, v/v, alcohol medium was generated by adding wine distilled alcohol (96.3%, v/

v) to obtain the desired grade. The 0%, v/v, alcohol medium was generated by adding

the same quantity of distilled water. The two resulting synthetic media were used

as references to show that the fermentation substrate was ethanol.

Other natural culture media with an alcohol content greater than 12%, v/v, were

prepared, using a young wine from an actual industrial aging system (11.9%, v/v).

The same solution volume with different proportions of water and wine distilled

alcohol was added to this wine, in order to obtain the desired final alcohol content.

This procedure was applied to ensure that the composition of wines (nitrogen

compounds, salts and vitamins principally) was the same in all media used,

regardless of alcohol added, bearing in mind that the composition of wines can

influence the velum evolution [22]. In this way, five different natural media were

prepared containing 12, 13, 14 and 15%, v/v, alcohol.

Finally, other natural media with alcohol contents below 12%, v/v, were

prepared, using the same wine as above. In this case, the wine was first de-

alcoholized by vacuum distillation and later refilled with distilled water, obtaining a

final alcohol content of 3.7%, v/v. Media of different alcohol concentrations were

prepared by adding the same solution volume with different proportions of water

and wine distilled alcohol, as in the previous case. Again five different natural media

were prepared containing 3, 5, 7, 9 and 11%, v/v, alcohol.

The young wines had a mean alcoholic grade of 11.9%, v/v, 0.9930 g/mL density,

6.58 g tartaric acid/L total acidity, 0.3 g acetic acid/L volatile acidity, a pH of 3.1 and

a total sulphur dioxide content of 86 mg/L. This parameters were determined

according to the European Union official methods [23].

2.3. Culture procedures and analysis determinations

The inoculum suspension for each experiment was prepared by taking a portion

of biofilm yeasts from the surface of the conservation medium (approximately

1 mL) and inoculating it into 200 mL of fresh wine (11.9%, v/v, alcohol). The mixture

was then placed in an ultrasonic bath (50 W) for 30 s to obtain a homogeneous

suspension of yeasts. The cell concentration was determined by counting chamber,

resulting in 5–10 Mcell/mL (millions of cells per milliliter). Later, all the

inoculations of media were carried out with such a proportion of these suspensions

that the wanted cell count in the inoculated medium was always obtained. In this

work, the initial cell counts of media was between 10�3 and 1 Mcell/mL in all

experiments, since we had previously verified that this concentration allows follow

the growth of the microbial biofilm with enough accuracy over a reasonable period

of time.

The cultivation experiments were performed in triplicate, using in each case fifty

60 mL cultivation cylindrical glass flasks (diameter 3.5 cm, high 9 cm) containing

20 mL of medium and covered with cotton plug. Ten of the flasks were always

reference samples (without inoculation of starter culture). All the flasks were placed

in a static incubator properly aerated (air with 80% of humidity), temperature

controlled (22 8C) and under total darkness. These conditions were used to

reproduce the industrial biological aging conditions.

The biological aging process in the laboratory was monitored daily once the

flasks were inoculated. Several flasks were removed from the incubator periodically

and analyzed immediately. To determine the biomass growth in the media, the

sample flasks were placed in an ultrasonic bath (50 W) for 30 s, which ensured the

disaggregation of the biofilm and a homogeneous suspension of cells (confirmed by

optical microscopy). Then, the cells were counted under a microscope to determine

total and viable biomass. To perform the remaining analytica determinations the

samples were filtered through a filter of 0.45 mm. A number of reference samples

were also periodically removed from the incubator to determine the possible media

transformations due to other factors like evaporation, oxidation, etc.

The analyses performed in each case were as follows:

Fig. 1. Biofilm cell growth in a natural media with 11.9%, v/v, ethanol, for several

experiments with different inoculum proportions (initial biomass concentration of

10�2, 10�1 and 1 Mcell/mL). Points represent experimental data and lines

theoretical calculations.

P. Gutierrez et al. / Process Biochemistry 45 (2010) 493–499 495

- Total and viable biomass, by staining with methylene blue and subsequent

counting under a light microscope in a haemocytometer (Neubauer counting

chamber) [24] (variability of �1.5%).

- Alcohol concentration by aerometer, according to the OIV method [25].

- Acetaldehyde, quantified by gas chromatograph equipped with a FID detector (HP

5890 Series II), on a Carbowax 20 M column (50 m, 0.25 mm ID, 0.25 mm). The

injector and detector temperatures were 175 8C and 225 8C, respectively. The

carrier gas was hydrogen. The oven temperature was 35 8C for the first 5 min and

then ramped up to 100 8C at 5 8C/min. The internal standard was 4-methyl-2-

pentenol. The sample volume was 20 mL.

- Glycerine, by means of enzymatic test [26].

Ethanol evaporation was observed in all of the cultures during the incubation

period (cultures and reference samples). This was because the liquid surface of each

flask remained open to the atmosphere through the cotton cover in order to allow

the oxygen transfer to the yeast. Therefore, due to the daily variation in total media

volume, it was necessary to apply a correction to calculate the appropriate

concentration variations of each compound present in the sample.

The correction was based on the following equation: Cc = C (V/Vo), where Cc was

the daily corrected concentration, C was the daily experimental concentration, V

was the daily experimental liquid volume, and Vo was the initial liquid volume. The

acetaldehyde, biomass and glycerine concentrations that are discussed later in this

paper were corrected in this way. Thus, the growth, formation or consumption

kinetics were evaluated without the influence of the total evaporation factor.

2.4. Statistical analysis

Means and standard deviations were calculated and significant diferences were

evaluated by the Student test. Statistical processing was carried out using

STATISTICA Release 7 (Statsoft, Inc.-USA) statistical package.

3. Results

3.1. General biomass growth

First of all, several experiments were carried out at differentinitial biomass concentrations, that is to say different inoculumproportion, using the basic culture medium (young wine 11.92%, v/v, ethanol). The object of these tests was the observation of thedifferent phases of the microbial biofilm growth. The obtainedresults are shown in Fig. 1. It is necessary to indicate that, thebiomass is only located on the surface of the wine and notsumerged. Then it is necessary to desegregate the biofilm beforethe biomass measure. The wines are initially saturated withoxygen (8 ppm) and the system was not aerated during aging.

3.2. Alcohol influence in biomass growth

To determine the influence of alcohol concentration on biofilmgrowth, diverse experiments with different ethanol concentrationswere carried out, as it has been mentioned previously. This way,the experiments in the synthetic media (0 and 12%, v/v, ethanol)clearly showed that the yeast formed biofilm on the surface of themedia with alcohol, but did not form biofilm on the surface of themedia without alcohol. However, although biofilm formation wasnot observed on the surface of media without alcohol, a very slightmicrobial growth in suspension was detected.

Table 1Growth kinetic parameters of the yeast biofilm calculated for the different alcohol me

Ethanol concentration

Medium label (%, v/v) Initial phase (g/L) Exponen

3.1 24 23

5.0 39 37

6.8 54 51

8.6 68 64

10.7 84 78

12.0 94 86

12.9 102 93

13.8 109 100

14.8 116 104

In industrial conditions, it is believed that during the lag phase,prior to the formation of the biofilm, the yeast cells are stillsubmerged in the medium and they consume the dissolved oxygento carry out their aerobic metabolism. However, when the mediumlacks ethanol, it is not possible to generate a sufficient submergedpopulation to form the floccules that later colonize the surface.Thus, it is observed that only a certain level of growth can occurbefore the energy reserves of the yeast are exhausted and theyrequire an outside carbon source for survival, ethanol in this case.For high alcohol concentrations, cells first adapt to the extremeconditions by structurally modifying their cellular membrane.Once the high ethanol tolerance has been obtained, the cellaggregates can grow. Then, the floccules can ascend to the surfaceand colonize it [5].

In the experiments carried out in natural media with differentalcohol concentrations, microbial biofilm formation was alwaysclearly observed. The specific growth rate of the exponential phase(mS) obtained in these experiments, determined as it is describedin the next section, is presented in Table 1, for the different ethanolconcentrations studied. Also the maximum biomass concentration(Xm) for each ethanol level is shown.

Another important factor to consider is the biofilm consis-tency, which depends on the metabolic stage of the biomass. Amore consistent biofilm ensures a greater durability andprotects itself against liquid agitation or vessel vibration. From

dia studied.

mS (day�1) Xmax (Mcell/mL)

tial phase (g/L)

0.67�0.012 66.0�4.3

0.74�0.001 70.8�3.9

0.66�0.002 75.9�3.5

0.56�0.015 74.4�2.2

0.44�0.007 69.4�0.8

0.36�0.003 63.4�5.7

0.31�0.001 62.8�2.6

0.24�0.002 61.6�1.6

0.19�0.013 61.5�4.7

P. Gutierrez et al. / Process Biochemistry 45 (2010) 493–499496

the industrial point of view, a biofilm collapse would cause asignificant loss of cell viability, and result in considerable loss ofproductivity. The following observations are based on qualita-tive evaluations of the biofilm consistencies during ourexperimentations. Thus, for ethanol concentrations below 6%,v/v, the biofilm consistency is very low. In contrast, for ethanolconcentrations greater than 12%, v/v, the biofilm consistency ishigh. In general, the stability of the biofilm is greater in higherethanol concentrations and the biomass viability remains highfor longer periods of time. These observations could be relatedto the fact that, in the experimental conditions, when thealcohol concentration is higher, the wall protein synthesis canbe more efficient and the connections among the cells can bestronger [24,41].

3.3. Substrates consumption

To properly determine the decreasing of the ethanolconcentration due to the yeast metabolism, several referenceflasks (10) without inoculum were used in all experiments. Withthese reference samples, loss of ethanol due to evaporationcould be measured. The rate of ethanol loss in the referencesamples can be subtracted from that of the inoculated samplesand the difference corresponds to the biofilm ethanol consump-tion rate. Moreover, if this consumption rate is divided by theviable biomass concentration at each moment (Xv), we obtainthe specific rate of ethanol consumption (�seth). Thus, themathematical definition of this biofilm property is the follow-ing: �seth = �(dS/dt)/Xv, and it is expressed here in this work inmg/Mcell day. The average specific rates of ethanol consumptionare shown in Table 2.

In relation to the other substrate, a specific rate of glycerineconsumption can be defined in a similar way to that for ethanol.Thus, �sgly = �(dG/dt)/Xv, where G is the glycerine concentration(g/L). The experimental data obtained are also shown in Table 2.

3.4. Product formation

The acetaldehyde production was measured in all the experi-ments as it has been previously mentioned. The observed behaviorindicates that acetaldehyde synthesis can be associated with theprimary metabolism of the biofilm yeast [42,43]. Nevertheless, insome cases, slight fluctuations in the concentration were observed,mainly in the final stages of the process. This effect could be due tothe incorporation of acetaldehyde in other competing metabolicroutes, when the acetaldehyde concentration is high, or toevaporation caused by its high volatility. In Fig. 3 are presentedthe evolution of viable biomass concentration, glycerine andacetaldehyde for the experiments with ethanol concentration of11.9%, v/v.

Table 2Biofilm consumption and production rates, for different ethanol concentrations.

Specific consumption rate of ethanol (�seth), specific consumption rate of glycerine

(�sgly) and coefficient of acetaldehyde yield (aace).

Ethanol

(%, v/v)

�seth (mg/Mcell day) �sgly (mg/Mcell day) aace (mg/Mcell)

3.1 0.07� 0.001 9.9� 0.7 1.57�0.27

5.0 1.03� 0.013 12.1� 0.3 1.76�0.23

6.8 0.98� 0.017 12.8�2.1 2.32�0.30

8.6 1.25� 0.012 13.0�2.7 3.04�0.23

10.7 1.48� 0.026 14.0� 0.5 3.68�0.21

12.0 0.98� 0.028 14.2� 0.5 5.10�0.34

12.9 1.91� 0.047 17.3� 0.5 4.43�0.27

13.8 1.89� 0.055 17.3� 0.2 5.25�0.34

14.8 0.59� 0.015 20.5�2.3 5.09�0.15

4. Discussion

4.1. General biomass growth

As it can be observed in curves of Fig. 1, some of the classicphases of the microbial growth can be clearly detected. In short,the lag phase, the exponential phase and the stationary phase. Thisgrowth profile suggests the use of a kinetic model of general typethat includes the three mentioned phases. Moreover, Fig. 1 showsthat at the end of the exponential phase, the total biomass cellcount is generally the same for all the inocula studied, with anaverage value of 63.4 � 1.9 Mcell/mL (95% confidence interval). Thisindicates that, under the experimental conditions employed, this isthe maximum amount of total biomass that can be reached (Xm),which represents the critical total biomass concentration for biofilmgrowth inhibition. This result agrees with the fact that, along theexponential phase, the cellular viability was greater than 90% and insome cases was even above 95%. However, during the stationaryphase, a progressive decrease in the viability was detected in all cases.

The liquid surface to media volume ratio (g) used in this studywas 0.48 cm�1. This value is similar to that of the typical industrialvessels for the biological aging (wooden casks). For this specificsurface ratio, the Xm value above mentioned leads to a surfacemicrobial population of 132.1 Mcell/cm2, before biofilm growth isinhibited. So, we can consider this value the maximum amount ofbiomass that can be reached per surface unit at these experimentalconditions. Furthermore, if we consider yeast cells spherical, withan average diameter of 10 mm and random packing of the cells inthe biofilm (porosity 0.36), then it can be demonstrated bygeometrical considerations that the above mentioned value of Xm

leads to a maximum biofilm thickness of 1.1 mm, under ourexperimental conditions. This thickness is so small that influenceof diffusional phenomena in the kinetic of the global process doesnot presumably take place under these conditions.

On the other hand, at industrial level we can find biofilmthickness up to 30 times superior, because in the industrial processthe composition of the liquid is periodically renovate and mediumconditions change cyclically. Under those conditions, once biofilmreaches the mentioned maximum thickness, it folds to continuegrowing. Thus, its surface becomes rough with higher number ofpleats and thicker with time, until diffusional limitations stop anyhigher growth. Then, the biofilm thickness stabilizes, balancingbetween cell growth and cell death.

The application of a simple theoretical model for the entire setof phenomena in action described before presents difficulties. Thisis also partly because the yeast growth shows types of inhibitionother than that mediated by biomass, as substrate or product [27–36]. As a consequence, all of these factors should be included in theequations to establish the biofilm stabilization time. With thosefacts in mind, it is possible to establish first a general biomassinhibition model in agreement with the experimental laboratorydata. The model can be expressed first in a mathematical formsimilar to the one used for different inhibition phenomena, as inthe following equation:

dX

dt¼ mX m ¼ mS 1� X

Xm

� �(1)

Here X is the total biomass concentration (Mcell/mL) at the t

time (days) and m is the specific growth rate of biomass (day�1),which is considered approximately constant during the exponen-tial growth phase. Also, Xm is the critical biomass concentrationabove mentioned and mS is the specific growth rate of reference,which here is a factor depending only on the substrateconcentration (ethanol in this case).

Fig. 2. Specific growth rate (mS) as a function of ethanol concentration (S). The

continuous line corresponds to the fitting of Eq. (4) to experimental data.

Fig. 3. Evolution of viable biomass, glycerine and acetaldehyde concentrations for

an experiment with ethanol concentration of 11.9%, v/v.

P. Gutierrez et al. / Process Biochemistry 45 (2010) 493–499 497

The integration of Eq. (1), extended across the whole sequenceof phases, gives a typical growth expression, very well known,which is called the logistic curve [37]. This curve has a gentel ‘‘S’’form and its mathematical expression is the following:

X ¼ X0expðmStÞððXm � X0Þ=XmÞ þ ðX0=XmÞexpðmStÞ (2)

where X0 is the biomass concentration at the initial time (t0). Sincewe supposedly know the X0 and Xm values, Eq. (2) can be used tocalculate the mS values at different experimental alcohol con-centrations. To achieve these results a linearization of this equationcan be applied, with the following expression:

ln1

X� 1

Xm

� �¼ ln

1

X0� 1

Xm

� ��mSt (3)

Thus, representing ln ((1/X) � (1/Xm)) versus t, we can directlyobtain the mS value from the slope. The experimental data of Fig. 1lead to an average value of mS equal to 0.371 � 0.046 day�1 (95%confidence interval), under those conditions. As it can be observed,the growth theoretical curves fit well the biomass data.

4.2. Alcohol influence in biomass growth

As it can be observed in Table 1, the Xm data for experimentswith different ethanol concentration oscillate around the samevalue found before, demostrating non dependence of the para-meter with this variable (ethanol concentration). In short, thegeneral average value obtained for the critical biomas concentra-tion (Xm) is 67.3 � 5.5 Mcell/mL (95% confidence interval).

Otherwise, multiple references [28,29,37,38] and the experi-mental results obtained in this work indicate that the kineticmodel for the biomas growth should include a substrate inhibitionterm (ethanol inhibition). Several models for the substrateinhibition term have been published for a wide range ofapplications [29,30,36]. In this case, the data best fit the Luongmodel [38], which is described as follows:

mS ¼ mmax

S

KS þ S1� S

Sm

� �(4)

where mS is the specific growth rate of reference (day�1); S is thesubstrate concentration in the medium (ethanol, %, v/v); Sm is thecritical concentration of substrate (ethanol, %, v/v) at which thebiomass does not grow at all; finally, mmax and KS are the typicalMonod parameters for the biomass growth (maximum specificgrowth rate and biomass-substrate affinity, respectively). Sm is alsoa parameter with physical meaning, so it represents the criticalethanol tolerance of the yeast strain in the experimentalcondiditons. Bibliographical references describe that an alcoholconcentration above 16–18%, v/v, makes it impossible to achievebiofilm growth [40]. For this reason Sm should be within this range.This way, multiple test carried out in our laboratory leads to a valueof Sm = 136 g/L (r2 = 0.9334), which is in agreement with theaforementioned references (136 g/L = 17.2%, v/v).

Therefore, the complete set of data of mS vesus S is registered inFig. 2. The described Monod parameters can be easily calculated bya double-reciprocal representation of the experimental data. Thus,the results obtained are as follows: mmax = 1.44 day�1 andKS = 2.1%, v/v (r2 = 0.9093). Once the kinetic parameters have beenobtained, Eq. (4) determines the ethanol concentration that offersthe fastest biofilm growth rate (Sf), i.e.: the ethanol concentracionfor the highest mS. Thus, by setting the derivative of this equationequal to zero, we obtain a value of Sf = 4.3%, v/v.

4.3. Substrates consumption

The results of the specific rate of ethanol consumption (�seth)obtained in Table 2 are dispersed and a clear trend is not evident. Itis important to note that the ethanol evaporation constituted formore than 90% of the total ethanol reduction. Therefore, theaccuracy achieved for the ethanol consumption rate is conse-quently low. The global average value obtained for �seth is1.1 � 0.5 mg/Mcell day (95% confidence interval).

In relation to the specific rate of glycerine consumption (�sgly),the data of Table 2 show that this variable depends directly on theethanol concentration. Thus, lower ethanol concentrations lead toslower glycerine consumption rates and higher ethanol concen-trations lead to faster glycerine consumption rates. Additionally,the analysis of Fig. 3 suggests that this variable also depends on theglycerine concentration itself, since this consumption ratedecreases as the glycerine concentration decreases. Therefore,the concentrations of both substrates (glycerine and ethanol) seemto influence the glycerine consumption rate.

P. Gutierrez et al. / Process Biochemistry 45 (2010) 493–499498

In consequence, for the specific rate of glycerine consumptionthe following equation can be proposed:

�sgly ¼ kglyGS (5)

where kgly is the proportionality constant. Bearing in mind theprevious definition of �sgly, to fit the experimental data to thisequation, we can use the plot of [�dG/dt] versus the mixed variable[G � S� Xv] over time. Thus, this type of plot can lead to a unique kgly

value for each biofilm growth experiment. To reach this result, the[�dG/dt] variable can be numerically evaluated for each timeinterval, from the experimental laboratory data of glycerineconcentration and time. This way, the general average value obtainedfor kgly in all experiments is 3.9 � 10�5� 0.4 � 10�5 (Mcell/mL)�1

(g/L)�1 (day)�1 (95% confidence).

4.4. Product formation

In general, the acetaldehyde formation kinetics (Fig. 3) suggestsa model where product formation is associated with the microbialgrowth and modified by a substrate-dependent factor. The data fitwell with this kind of equations. Moreover, as we mentionedabove, the biofilm growth seems to be inhibited by high productconcentrations.

In consequence, a growth-associated kinetic model can be usedto describe the acetaldehyde production by the yeast, according tothe following expression:

pace ¼ aacem (6)

where pace is the specific production rate of acetaldehyde (mg/Mcell day). This variable is defined in a similar way to the otherspecific rates, as follow: pace = (dP/dt)/Xv, where P is theacetaldehyde concentration (mg/L). Additionally, aace is theacetaldehyde yield coefficient and can be calculated from theintegrated form of Eq. (6), wich is the following: (P � P0) = aace

(X � X0).Fluctuations in the acetaldehyde production can lead to an

oscillating aace value across the biofilm formation phases. There-fore, only the overall data of product formation for eachexperiment has been used to perform the calculations (i.e.: theconcentration increments of P and X from the inoculation to theend of the experiment). The results are shown in Table 2.

As it can be observed, the acetaldehyde yield coefficient isstrongly dependent on the alcohol concentration. Thus, a simplelinear fit can be used to gather this effect, according to thefollowing expression: aace = kace S, where kace is the adjustmentcoefficient. Regression analysis gives a value of kace = 46.3 � 10�6

(Mcell/mL)�1 (r2 = 0.9374).In consequence, Eq. (6) could be expressed in a more general

form, according to the following expression:

pace ¼ kaceSm (7)

The available literature indicates that, under certain condi-tions, high acetaldehyde productivity can be achieved up toacetaldehyde levels of 1.0–1.2 g/L [44,45]. In such cases, thebiofilm growth could be inhibited by these high productconcentrations, which in fact is a toxic factor for microbialgrowth [38,39]. Under these conditions perhaps it would benecessary to include a product inhibition term in the growthkinetic or even any other influence factors like yeast assimilablenitrogen [46]. However, at the moment, we have no enoughexperimental data to include this sort of factors in the proposedmodel.

5. Conclusion

The results indicate that it is possible to apply generalfermentative kinetic theories to the biological aging process ofwine by the S. cerevisiae beticus yeast strain in the biofilm phase.Thus, the end of the lag phase agrees with the time in which theinoculated cells reach the wine surface and then the exponentialphase begins. Once the surface has been colonized, and as a resultof multiple inhibitory phenomena, the cultivation transitions intothe stationary phase.

Under the conditions studied, the yeast formed a biofilm atethanol concentrations ranging from 3 to 15%, v/v. During thisprocess, the yeast consumed ethanol and glycerine in differentproportions to produce acetaldehyde. Analysis of the resultingequation indicates that the fastest biofilm growth occurs at around4.3%, v/v, alcohol.

With regard to consumption rates, the glycerine consumptionkinetics can be represented by a model that is not associated withthe growth, but is directly related to the substrate concentrations(ethanol and glycerine). The corresponding equation shows goodagreement with the experimental data under the different sets ofconditions studied. Finally, with regard to production rates, theacetaldehyde formation occurred in parallel with the microbialgrowth.

Accordingly, the proposed model can be used at the industrialplants in order to estimate the growth time of the biofilm yeastunder certain conditions, and the glycerine consumption or theacetaldehyde formation. Thus, we can schedule the appropriateoperations of process control and we can develop better processoptimization programs.

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