Brazilian Journal of Microbiology 44, 2, 551-558 (2013) Copyright © 2013, Sociedade Brasileira de Microbiologia

ISSN 1678-4405 www.sbmicrobiologia.org.br

Research Paper

Yeast biomass production: a new approach in glucose-limited feeding strategy

Érika Durão Vieira1, Maria da Graça Stupiello Andrietta2, Silvio Roberto Andrietta2
1
Faculdade de Engenharia Química, Universidade Estadual de Campinas, Campinas, SP, Brazil.
2
Centro Pluridisciplinar de Pesquisas Químicas, Biológicas e Agrícolas,
Universidade Estadual de Campinas, Paulínia, SP, Brazil.

Submitted: June 27, 2011; Approved: September 10, 2012.

Abstract

The aim of this work was to implement experimentally a simple glucose-limited feeding strategy for
yeast biomass production in a bubble column reactor based on a spreadsheet simulator suitable for in-
dustrial application. In biomass production process using Saccharomyces cerevisiae strains, one of
the constraints is the strong tendency of these species to metabolize sugars anaerobically due to
catabolite repression, leading to low values of biomass yield on substrate. The usual strategy to con-
trol this metabolic tendency is the use of a fed-batch process in which where the sugar source is fed
incrementally and total sugar concentration in broth is maintained below a determined value. The
simulator presented in this work was developed to control molasses feeding on the basis of a simple
theoretical model in which has taken into account the nutritional growth needs of yeast cell and two
input data: the theoretical specific growth rate and initial cell biomass. In experimental assay, a com-
mercial baker’s yeast strain and molasses as sugar source were used. Experimental results showed an
overall biomass yield on substrate of 0.33, a biomass increase of 6.4 fold and a specific growth rate of
0.165 h-1 in contrast to the predicted value of 0.180 h-1 in the second stage simulation.

Key words: yeast, biomass, fed-batch, process simulation, Saccharomyces cerevisiae.

Introduction ever, besides this simple concept, industrial production

aims at efficient conversion of sugar feedstock into yeast
Microbial biomass is a suitable supplemental protein biomass mainly in the later stages where biomass volume is
source obtained from processes in which bacteria, yeasts, high. An efficient transformation of sugar in yeast protein
other fungi or algae are cultivated in large quantities. Yeast requires that anaerobic metabolites production such as eth-
biomass is extensively used as human or animal protein anol and acetaldehyde (Bauer et al., 1999) is minimized,
supplement in animal feed or in human nutrition (Halasz i.e., that sugar metabolism is deviated to the oxidative path-
and Lasztity, 1991). Common strains used as single cell way to achieving maximum ATP energy yield and biomass
protein source includes Saccharomyces cerevisiae and formation (Van Hoek, Van Dijken and Pronk, 1998). More-
Candida utilis strains. Typical microbial biomass products over, fermentation products such as ethanol and, in particu-
include bakers’ yeast and yeast extracts where lar, acetaldehyde are toxic. This problem becomes espe-
Saccharomyces cerevisiae strains are the host for hetero- cially relevant during cultivation at high biomass densities
logous-protein production (Beudeker et al., 1990; Hensing (Hensing et al., 1995; van Dijken, Weusthuis and Pronk,
et al., 1995). 1993).
Basically, the industrial process concept relies on
propagating cells from pure culture agar slants to large It’s generally known that Saccharomyces cerevisiae
bioreactors increasing volume in each propagation stage till species tends to metabolize glucose glycolytically under
the final bioreactor volume (Rose, 1979; Burrows, 1979; glucose excess even in fully aerobic conditions producing
EPA, 1995; Rendez-Gil, Sanz and Priteo, 1999; Di Serio et ethanol, a phenomenon known as the Crabtree effect
al., 2001; Di Serio, Tesser and Santacesaria, 2001). How- (Käppeli, 1986; Sonnleitner and Käppeli, 1986; Verduyn,

Send correspondence to E.D Vieira. School of Chemical Engineering, University of Campinas, Cidade Universitária “Zeferino Vaz”, C.P. 6066,
13083-970 Campinas, SP, Brazil. E-mail: [email protected].
552 Vieira et al.

1991; Dynesen, 1998). Additionally, not only glucose but simulated experimentally using sugar cane molasses as car-
also fructose has shown to triggers catabolite repression on bon source.
Saccharomyces cerevisiae strains (Dynesen, 1998). This
catabolite repression renders low biomass yield when culti- Materials and Methods
vating Saccharomyces cerevisiae in batch cultures and neg-
atively affects biomass yields due to the low ATP yield Operational system
from alcoholic fermentation. Despite this, biomass forma- The operational system is shown in Figure 1. The re-
tion can be achieved by innumerous metabolic pathways actor design used in this work was a cylindrical acrylic ves-
(Frick and Whitmann, 2005) and biomass yield on substrate sel of 5.5 liter volume with a conic bottom. At the end of the
can reach values up to 50% in pure oxidative growth conic bottom it was connected a 1 cm tube for air entrance
(Akinyemi, Betiku and Solomon, 2005). without any kind of air distributor. Five equally spaced
To overcome these constraints on yeast biomass pro- transversal 4-hole baffles were added to the design to create
duction two important variables are of major importance: turbulence and shear which could break up the air bubbles.
oxygen transfer rate and glucose concentration in the broth. The design also included small outlet tubes from were mo-
In heterogeneous gas-liquid reactions, e.g., in aerobic fer- lasses could be fed and cells could be circulated out through
mentation, the liquid phase controls mass transfer pro- a glass coil heat exchanger connected to a 34 °C thermo-
cesses due to the relative insolubility of gases. This limita- static bath. Aeration was estimated by scale down of typical
tion is minimized by the use of bubble column reactors due industrial conditions: 0.3 vvm for the first stage and
to its good oxygen transfer with low cost operation when 1.3 vvm for the second stage. The scale down was based on
compared to stirred tank reactors (Kantarcia, Borakb and the criterion of geometric similarity of the reactors and
Ulgen, 2005). The minimum sugar concentration in broth identical mass transfer coefficient (kLa1 = kLa2) (Aiba,
can be reached by the use of a fed-batch process. This pro- 1971). It resulted in 2.5 vvm (9 lpm) for first stage and
cess concept is the current one in industrial scale and ren- 1.3 vvm (approximately 30 lpm) for the second stage. The
ders good biomass yields when appropriate process control reactor was washed previously with 5% sodium hydroxide
strategy is used. Traditionally, in industrial production, mo- solution to prevent excessive bacterial contamination.
lasses or another feedstock feeding follows a strategy built
Microorganism
on the basis of factory historic data and so it is peculiar to a
determined strain and other process conditions. Nowadays, The microorganism evaluated in this assay was a
not only for economic reasons but also because of environ- commercial baker’yeast strain of Saccharomyces
mental policy, some industries are investing in new strate- cerevisiae named Y167 from CPQBA yeast collection.
gies of process control to avoid emission of toxic pollutants This strain was chosen due to its known characteristic of
(EPA, 1995). In scientific literature, many articles can be possessing a high cell growth rate. The microorganism was
found about baker’s yeast production dealing with yeast preserved in potato-dextrose agar (PDA) submerged in
growth modeling and aiming at different goals such as pro- mineral oil and stored at 4 °C. The propagation was initi-
ductivity, yield and yeast quality as well as new and robust ated by transferring the preserved microorganism to a fresh
online sensors (Reyman, 1992; Rigbom, Rothberg and PDA slant that was incubated at 32 °C for 24 h.
Saxen, 1996; Rendez-Gil, Sanz and Priteo, 1999; Jones and
Kompala, 1999; Di Serio et al., 2001; DiSerio, Tesser and
Santacesaria, 2001; Soley, 2005). However, there are few
simple theoretical models for yeast biomass production that
could fit to any strain and process and could be applicable
to industrial scale.
The objective of the present work was, therefore, to
develop and implement experimentally a simple theoretical
model for yeast growth based on few parameters aiming at
achieving a glucose-limited feeding strategy applicable to
industrial scale. For this purpose, a spreadsheet was devel-
oped in which the theoretical model could predict at each
time interval the nutritional growth needs of yeast cell
based on two major input data: the theoretical specific
growth rate and initial cell biomass. The model was vali-
dated in a laboratory scale experiment. Biomass production
was performed in a 5-liter bubble column reactor using a Figure 1 - Operational system: (1) acrylic vessel, (2) molasses flask, (3)
commercial baker’s yeast strain. The first two stages of glass coil heat exchange, (4) thermostatic bath, (5) rotameter, (6) Mas-
yeast cell propagation, common in industrial process, were terflex peristaltic pumps and (7) two digits balance.
Yeast biomass production 553

Inoculum Second stage of cell growth

The inoculum used was prepared in 250 mL Erlen- The cleaned reactor was filled with 2 liters of distilled
mayer flasks with 100 mL of sterilized medium (15 min water. This amount was calculated based on the spread-
121 °C) composed of : 150 g/L sucrose, 6 g/L yeast extract, sheet data to complete the final working volume of 3.5 li-
5 g/L potassium phosphate monobasic, 5 g/L ammonium ters. This operation of water addition is usual in industrial
chloride, 1 g/L magnesium sulphate and 1 g/L potassium yeast biomass production in order to minimize the ethanol
chloride. The yeast grown in PDA slant for 24 h at 32 °C concentration produced in the first stage propagation which
was resuspended in sterilized water and inoculated at a 10% could inhibit cell growth in the next stage. Subsequently,
concentration of total volume of the fermentation medium. the reactor was inoculated with 560 mL (16% of 3.5 liters)
The flasks were incubated in a shaker for 24 h at 32 °C and of the fermentation broth from the first stage which had
150 rpm. its pH previously adjusted to 4.5 with HCl 2N. The second
stage of cell growth was carried out for 12 hours with an
Feed Substratum aeration level of 30 lpm. Diluted molasses (40°Brix) previ-
ously sterilized for 10 min at 121 °C was fed incrementally
The carbon source used in all assays came from one by a peristaltic pump. The feeding rate was controlled by
sole batch of sugar cane molasses from a Brazilian Sugar weighting the molasses flask on a two digits balance. At
Mill. This molasses sample was an 80°Brix with total sugar each hour, a specific aliquot of 10% (wt/wt) potassium
around 56% and was diluted conveniently with distillated phosphate monobasic solution and 10% (wt/wt) ammo-
water to be used in the assays. Potassium phosphate mono- nium chloride solution was added to the fermentation broth.
basic (KH2PO4) was used as phosphorus source and ammo- The molasses mass and nitrogen and phosphorus solutions
nium chloride (NH4Cl) as nitrogen source. Other vitamins aliquots fed in each time interval were calculated based on
and nutrients are already present in sufficient amounts in the biomass production spreadsheet simulator.
the sugar cane molasses so that the fermentation broth was
only enriched with those cited salts to guarantee nitrogen Bioreactor sampling
and phosphorus supply. In the first stage, two 10 mL samples were collected at
the 0 h and at the 16 h and, in the second stage, a 10 mL
First stage of cell growth
sample was collected at each hour to measure biomass
The reactor was filled with 3.5 liters of diluted sugar (standard method for dry cell mass) and fructose, glucose
cane molasses with approximately 15° Brix previously ster- and sucrose analyses (HPLC). The 10 mL sample was cen-
ilized for 10 min at 121 °C. Molasses was enriched with trifuged at 4000 rpm and 4 min in graduated centrifuge
KH2PO4 and NH4Cl and had the pH adjusted to 4.5, the opti- tubes. The supernatant was diluted for HPLC analysis in a
mum pH for most Saccharomyces cerevisiae strains (Rose, Dionex chromatography system with a CarboPac PA1 col-
1987). The necessary quantity of raw molasses and nitrogen umn and pulsed amperometric detection. The wet cell vol-
and phosphorus salts were calculated based on the spread- ume was used to estimate cell concentration in the broth
sheet simulator. An amount of 45 mL of the yeast grown in considering that dry cell mass (g) is approximately equal to
the flask was incubated in the reactor. This first stage of cell 25% of wet cell volume (mL). Centrifuged cells were
growth was carried out for 16 hour with an aeration level of washed with distilled water and centrifuged two times after
9 lpm. This stage was carried out without periodical sam- being transferred to pre-weighed dishes. Cells were dried at
pling and with carbon source in excess once the goal of this 60 ± 5 °C to a constant weight.
stage is to obtain a maximum increase in biomass in detri-
ment to a loss in biomass on substrate yield (estimated in Biomass production spreadsheet simulator
0.22) and a high productivity. After the 16 hours, the reactor A spreadsheet was developed to simulate yeast cell
was emptied and rinsed with water and part of the fermenta- growth and predict its nutritional needs in a fed-batch pro-
tion broth of the first stage was used as inoculum to the sec- cess under limited-glucose concentration. Cell growth pat-
ond stage. In a industrial process, there is a gradual tern was set as exponential following the general equation
increment in bioreactors volume so that the volume of the X=X0 EXP(v0.t), where X denotes microbial biomass at
first stage bioreactor is designed to be around 15-20% of the time t, X0 microbial biomass at time t = 0, t time in hours (h)
second stage bioreactor volume and all fermentation broth of and v0 the specific growth rate (h-1). Biomass increase at
the first stage is transferred to the second stage bioreactor to each hour interval could be calculated once inputted the ini-
continue the yeast biomass propagation. Due to the absence tial cell mass (X0) an±te (m). The nutritional needs (carbon,
of a larger bioreactor in laboratory, the second stage assay nitrogen and phosphorus source) were calculated based on
was carried out in the same acrylic vessel of the first stage (i) cell average composition of the elements (%w/w) car-
and an amount of fermentation broth from the first stage, cor- bon, nitrogen and phosphorus (Atkinson and Mavituna,
respondent to 16% of final working volume (3.5 liter), was 1991; Lange and Heijnem, 2001) and (ii) yeast cell mass in
used as inoculum for the second stage. the reactor at each time (X(t)). It was considered that only
554 Vieira et al.

Table 1 - Backbone of the spreadsheet simulator.

Column Variable Symbol Unit Formula

1 time t h -
2 Cell Mass at time t X(t) g = X0 * exp (m * t)
3 Carbon Mass in Cells at time t C(t) g = CF * X(t)
4 Nitrogen Mass in Cells at time t N(t) g = NF * X(t)
5 Phosphorus Mass in Cells at time t P(t) g = PF * X(t)
6 Carbon Needs DC(t) g = C(t+1) - C(t)
7 Total Reducing Sugar Needs DTRS(t) g = DC(t) / (H * YX/S)
8 Diluted Molasses Needs DM(t) g = DTRS(t) * d / MP
9 Nitrogen Needs DN(t) g = N(t+1) - N(t)
10 10% NH4CL solution aliquot DNS(t) mL = DN(t) * W / NFS
11 Phosphorus Needs DP(t) g = P(t+1) - P(t)
12 10% KH2PO4 solution aliquot DPS(t) mL = DP(t) *W / PFS

50% of the carbon consumed was transformed in biomass, Table 2 - Definition of the variables.
i.e., biomass yield on substrate (YX/S) equal to 0.5. Table 1
Variable Value Definition Unit
shows the spreadsheet backbone implemented in Microsoft
Excel Software. Table 2 shows the definitions of the vari- X0 To be set Initial cell mass g
ables used in the spreadsheet simulator. m To be set Specific growth rate h-1
FC 0.47 Carbon weight fraction in yeast cell -
Results NF 0.085 Nitrogen weight fraction in yeast -
cell
The calculated spreadsheet used to estimate the nec- PF 0.0113 Phosphorus weight fraction in yeast -
essary amount of nutrients in the first stage assay is shown cell
in Table 3. The needed quantity of raw molasses and nitro- H 0.4 Carbon weight fraction of hexose -
gen and phosphorus salts were estimated considering a 70 g sugars
biomass content in the reactor at the end of the first stage. YX/S 0.5 Biomass yield on substrate -
This consideration was made on the basis of a estimative of d To be set Molasses dilution factor -
biomass increase of 100-fold, from 0.7 g to 70 g of total bio- MP To be set Molasses sugar purity (TRS per Mo- wt/wt
mass, not considering the exponential growth model. The lasses, g/g)
calculated spreadsheet used in the second stage assay is NFS 0.26 Nitrogen weight fraction in NH4Cl -
shown in Table 4. It was adopt a specific growth rate of PFS 0.23 Phosphorus weight fraction in -
0.18 h-1 and an expected initial cell mass of 7.0 g estimated KH2PO4
by centrifuged wet cell volume of the inoculum. The molas- W 10 Salt concentration %wt/wt
ses purity was 56% as determined by sugar HPCL (fruc-
tose, glucose and sucrose) and Brix analysis. The distillated
water volume added in the reactor was calculated by de- because of the loss of water stripped by aeration. The final
ducting the nutrient volume to be added until the end of the volume was approximately 3 liters and all the concentration
12 h and the inoculum volume from the total operational data was corrected based on this effect.
volume of the reactor. Diluted molasses density was con- The compiled data of the first stage and second stage
sidered to be 1.06 g/mL. It is worth to mention that final of cell growth assays are shown in Table 5. In the first stage,
volume in the second stage did not reach the expected 3.5 L biomass yield reached 0.20 as expected in an environment

Table 3 - First stage spreadsheet data built with the following input variables: X0 = 0.7 g/L, YX/S = 0.22, MP = 0.56 and d = 1.

1 2 3 4 5 6 7 8 9 10 11 12
t X(t) C(t) N(t) P(t) DC(t) DTRS(t) DM(t) DN DNS DP DPS
(h) (g) (g) (g) (g) (g) (g) (g) (g) (g) (g) (g)
0 0.70 0.33 0.06 0.01 32.57 370.1 660.9 5.89 22.49 0.78 3.44
16 70.0 32.90 5.95 0.79 - - - - - - -
Yeast biomass production 555

Table 4 - Second stage spreadsheet data built with the following input variables: X0 = 7.0 g/L, m = 0.18 h-1, MP = 0.56 and d = 2.

1 2 3 4 5 6 7 8 9 10 11 12
t X(t) C(t) N(t) P(t) DC(t) DTRS(t) DM(t) DN DNS DP DPS
(h) (g) (g) (g) (g) (g) (g) (g) (g) (mL) (g) (mL)
0 7.0 3.29 0.60 0.08 0.65 3.2 11.6 0.12 4.5 0.02 0.7
1 8.4 3.94 0.71 0.09 0.78 3.9 13.9 0.14 5.4 0.02 0.8
2 10.0 4.72 0.85 0.11 0.93 4.7 16.6 0.17 6.4 0.02 1.0
3 12.0 5.65 1.02 0.14 1.11 5.6 19.9 0.20 7.7 0.03 1.2
4 14.4 6.76 1.22 0.16 1.33 6.7 23.8 0.24 9.2 0.03 1.4
5 17.2 8.09 1.46 0.19 1.60 8.0 28.5 0.29 11.0 0.04 1.7
6 20.6 9.69 1.75 0.23 1.91 9.6 34.1 0.35 13.2 0.05 2.0
7 24.7 11.60 2.10 0.28 2.29 11.4 40.8 0.41 15.8 0.05 2.4
8 29.5 13.89 2.51 0.33 2.74 13.7 48.9 0.50 18.9 0.07 2.9
9 35.4 16.62 3.01 0.40 3.28 16.4 58.5 0.59 22.6 0.08 3.5
10 42.3 19.90 3.60 0.48 3.93 19.6 70.1 0.71 27.1 0.09 4.1
11 50.7 23.83 4.31 0.57 4.70 23.5 83.9 0.85 32.5 0.11 5.0
12 60.7 28.53 5.16 0.69 - - - - - - -
Total - - - - - 126.19 450.68 4.56 174.3 0.61 26.7
Initial Data
Volume of added nutrients (mL) 1186
Inoculum volume (mL) 560
Total Final Volume (mL) 3500
Distilled water volume (mL) 2314

with excess of glucose where anaerobic respiration is pre- The cell growth pattern of the second stage is shown
ferred by Saccharomyces cerevisiae strains. Biomass in- in Figure 2. Exponential regression curve fitted well within
crease reached 112.8 fold so that the goal of having a high the experimental data. The specific growth rate was
biomass increase on this first stage was fulfilled. In con- 0.165 h-1 what confirms the Y167 strain good growth pa-
trast, in the second stage, the overall biomass yield was rameters and a good initial estimative of 0.18 h-1.
higher reaching 0.33 and biomass increase was 6.4 fold, Figure 3 shows the biomass concentration profile.
both acceptable values in industrial biomass production. Calculated biomass concentration refers to the values cal-
Biomass yield, however, was expected to be closer to 0.5 as culated based on the spreadsheet and experimental biomass
it occurs in industrial bioreactors. concentration refers to the values calculated based on the

Figure 2 - Cell growth profile. Figure 3 - Biomass concentration profile.

556 Vieira et al.

dry cell mass. These data shows that, after the 6th hour, the till the overall value of 0.33. In contrast with sugar profile,
real biomass concentration in the reactor was below the ex- the cumulative biomass yield drop is closely associated to
pected value and nutrients and sugar may have been fed in the TRS increase confirming the strong tendency of yeasts
excess. to shift metabolism from aerobic to anaerobic respiration
The sugar profile of the second stage is shown in Fig- when there is excess of sugar in the broth. Instantaneous
ure 4. TRS concentration was maintained below 1 g/L until biomass yield showed scattered values but it followed the
the 5th hour. After the 6th hour, TRS increased from ap- same tendency of the cumulative values.
proximately 1 g/L to 3 g/L probably due to the biomass con-
centration below the predicted value. Discussion
Other interesting variables that should help the data
In typical biomass production plant, yeast propaga-
analysis are presented in Figure 5. The biomass yield was
tion starts just like the assay presented in this work: from an
calculated as instantaneous and cumulative, i.e., the yield in
agar slant to a seed reactor until a 150.000 liters bioreactors
one hour interval (YX/S Instantaneous in the 5th hour is related to
(bubble column reactors). The yeast growth in the flask rep-
the interval from the 4th hour till the 5th hour) and the yield
resents the yeast growth in the seed reactor where a sterile
from the zero time till the current time respectively. The cu-
growth is necessary to guarantee a pure culture inoculum.
mulative biomass yield starts at a low value of 0.24 reach-
The first stage and second stage in the bubble column reac-
ing a maximum value of 0.45 at the 6th hour. After the 6th
tor represents the first and second stages of yeast propaga-
hour, it can be perceived a slightly tendency of lower values
tion on large bioreactors. In the first stage, the objective is
to obtain a high increase in biomass (productivity) so that
biomass yield on substrate is not an important variable and
there is no need to have a feeding control. In the second
stage, the objective is to maximize biomass yield on sub-
strate by feeding molasses incrementally in order to avoid
catabolite repression. This feeding strategy is used from the
second propagation stage until the final bioreactors where
biomass yield is of major importance on production costs.
The high cell density and high operation volume reached in
these stages of the process turn molasses consumption a
significant cost variable so that a good feeding strategy and
process control is necessary.
Another important variable in biomass production is
the aeration and oxygen transfer once biomass yield de-
pends on achieving a fully aerobic condition. This is not an
easy condition to be simulated in bubble reactors in labo-
ratorial scale due to the small reactor height that leads to a
Figure 4 - Sugar profile. short residence time of the air bubbles. In the case of the as-
say presented in this work, the bioreactor operational height
was too small compared to industrial bioreactors so that air
bubbles residence time was insufficient to achieve maxi-
mum dissolved oxygen in broth. The biomass yield values
showed on Figure 5 evidences that the experimental condi-
tions did not reach fully aerobic conditions. Therefore, the
relatively low biomass yield value obtained in this work is
attributed to a limited oxygen availability caused by a poor
oxygen transfer and a low residence time of the air bubbles
provided by the reactor design and size respectively (Fie-
cher, Käppeli and Meussdoerffer, 1987). Another fact that
supports evidences of the poor oxygenation of the broth
was the intense acetaldehyde odor in the laboratory during
the assay and the presence and continuously growing con-
centration of byproducts as detected by the HPLC analysis
(results not quantitatively computed).
Cell concentration in the broth after the 6th hour was
lower than the calculated value. This suggests that, from the
Figure 5 - Instantaneous and cumulative biomass yield profile. spreadsheet theory, the TRS quantity that was fed from this
Yeast biomass production 557

time till the end of the experiment was in excess. This can Di Serio M, Tesser R, Santacesaria E (2001) A kinetic and mass
explain the TRS increase in the broth (Figure 4) and the de- transfer model to simulate the growth of baker’s yeast in in-
crease in biomass yield values (Figure 5) together with poor dustrial bioreactors. Chem Eng J 82:347-354.
oxygenation. In higher cell densities conditions, oxygen Dynesen J, Smits HP, Olsson L, Nielsen J (1998) Carbon cata-
uptake can be theoretically calculated and added to the bolite repression of invertase during batch cultivations of
spreadsheet so that aeration is increased as biomass in- Saccharomyces cerevisiae: the role of glucose, fructose and
creases. The oxygenation bottleneck in small scale opera- mannose. Appl Microbiol Biotechnol 50:579-582.
tions turns the simulation of the preceding stages difficult. EPA (1995). Yeast Production In: Compilation of Air Pollutant
It was simulated de 3rd stage (results not shown herein) but Emission Factors: AP-42, Fifth Edition, volume I, chapter
the results showed clearly that oxygen transfer was defi- 9.13.4. Available at: http://www.epa.gov/ttn/chief/ap42.
Acessed June 20, 2011.
cient even when aeration was above 45 lpm. Besides it was
observed that cells were seriously damaged and cell viabil- Fiecher A, Käppeli O, Meussdoerffer F (1987) Bath and Continu-
ous Culture In: Rose, A. H. and Harrison, J.S. (Eds), The
ity drops to 50%. This was attributed to the strong shear
Yeast, Volume 2, 2nd edition, Academic Press, New York,
stress caused by high flow rate of the air bubbles.
pp 99-129.
Frick O, Witmann C (2005) Characterization of the metabolic
Conclusions shift between oxidative and fermentative growth in Saccha-
Industrial yeast biomass production is a well settled romyces cerevisiae by comparative 13C flux analysis. Micro-
and simple process that has been improved along the years bial Cell Factories 4:30.
with the implementation of better process conception, de- Halasz A, Lasztity R (1991) Use of yeast biomass in food produc-
sign and control strategies. The present work focused on the tion. CRC Press, Boca Raton, FL, 312 pp.
process control improvement through a simple glucose- Hensing MCM, Rouwenhorst RJ, Heijnen JJ, van Dijken JP,
limited feeding strategy. The results shown herein are in Pronk JT (1995) Physiological and technological aspects of
good agreement with industrial reality and the spreadsheet large-scale heterologous-protein production with yeasts.
simulator seams to be a useful tool in biomass production Anton van Lee 67:261-279.
process operation and control. van Dijken JP, Weusthuis RA, Pronk JT (1993) Kinetics of
growth and sugar consumption by yeasts. Anton van Lee
63:343-352.
Acknowledgments
Jones KD, Kompala DS (1999) Cybernetic model of the growth
To CAPES, Brazilian Coordination for the Improve- dynamics of Saccharomyces cerevisiae in batch and contin-
ment of Higher Education Personnel, for the fellowship uous cultures, J Biotechnol 71:105-131.
awarded to the first author. Kantarcia N, Borakb F, Ulgen KO (2005) Bubble column reac-
tors. Process Biochem 40:2263-2283.
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