Appl Microbiol Biotechnol (2013) 97:50555067

DOI 10.1007/s00253-013-4748-6

APPLIED MICROBIAL AND CELL PHYSIOLOGY

Growth of the yeast Kluyveromyces marxianus CBS 6556

on different sugar combinations as sole carbon and energy
source
Gustavo Graciano Fonseca &
Nuno Miguel Barbosa de Carvalho &
Andreas Karoly Gombert

Received: 9 October 2012 / Revised: 31 January 2013 / Accepted: 31 January 2013 / Published online: 23 February 2013
# Springer-Verlag Berlin Heidelberg 2013

Abstract The yeast Kluyveromyces marxianus has been
pointed out as a promising microorganism for a variety of
industrial bioprocesses. Although genetic tools have been
developed for this yeast and different potential applications
have been investigated, quantitative physiological studies
have rarely been reported. Here, we report and discuss the
growth, substrate consumption, metabolite formation, and
respiratory parameters of K. marxianus CBS 6556 during
aerobic batch bioreactor cultivations, using a defined medi-
um with different sugars as sole carbon and energy source,
at 30 and 37 C. Cultivations were carried out both on single
sugars and on binary sugar mixtures. Carbon balances
closed within 95 to 101 % in all experiments. Biomass and
CO2 were the main products of cell metabolism, whereas
by-products were always present in very low proportion
(<3 % of the carbon consumed), as long as full aerobiosis
was guaranteed. On all sugars tested as sole carbon and
energy source (glucose, fructose, sucrose, lactose, and ga-
lactose), the maximum specific growth rate remained be-
tween 0.39 and 0.49 h1, except for galactose at 37 C,
which only supported growth at 0.31 h1. Different growth
behaviors were observed on the binary sugar mixtures
G. G. Fonseca : N. M. B. de Carvalho : A. K. Gombert (*)
Department of Chemical Engineering, University of So Paulo,
PO Box 61548,
CEP 05424-970, So Paulo, SP, Brazil
e-mail: gombert@usp.br
Present Address:
G. G. Fonseca
Laboratory of Bioengineering, Faculty of Engineering,
Federal University of Grande Dourados, Dourados, MS, Brazil
investigated (glucose and lactose, glucose and galactose,
lactose and galactose, glucose and fructose, galactose and
fructose, fructose and lactose), and the observations were in
agreement with previously published data on the sugar
transport systems in K. marxianus. We conclude that K.
marxianus CBS 6556 does not present any special nutrition-
al requirements; grows well in the range of 30 to 37 C on
different sugars; is capable of growing on sugar mixtures in
a shorter period of time than Saccharomyces cerevisiae,
which is interesting from an industrial point of view; and
deviates tiny amounts of carbon towards metabolite forma-
tion, as long as full aerobiosis is maintained.
Keywords Kluyveromyces marxianus . Yeast physiology .
Nonconventional yeasts . Crabtree effect . Diauxic growth .
Sugar transport
Introduction
The yeast Kluyveromyces marxianus is a GRAS organism
(Caballero et al. 1995) which converts sugar substrates into
biomass with high yields (Bellaver et al. 2004). It has various
potential industrial applications (Fonseca et al. 2008), ranging
from the production of oligosaccharides, oligopeptides, ribo-
nucleotides, enzymes (Hensing et al. 1994; Cruz-Guerrero et
al. 1995), aroma compounds (Fabre et al. 1998; Wittmann et
al. 2002), and heterologous proteins (Berkamp et al. 1993;
Hensing et al. 1995; Cai et al. 2005; Rocha et al. 2010, 2011),
to its use as an anticholesterolemic agent (Yoshida et al. 2004,
2005) and in the removal of heavy metals (Yazgan et al. 1993;
Yazgan and Ozcengiz 1994). A prerequisite for this yeast to be
5056
the object of metabolic engineering strategies, with the aim of
improving different bioprocesses, is to increase the knowledge
on its physiology, which includes the central metabolic func-
tions, such as carbon metabolism.
The consumption of sugars by yeasts has been the aim of
several studies, especially to verify the restraining of some
cellular functions by their presence (Johnston 1999). In
some species, high initial sugar concentrations cause catab-
olite repression phenomena and trigger alcoholic fermenta-
tion even when oxygen is present (Hensing et al. 1995).
However, it is not yet well known how strong is catabolite
repression by sugars in K. marxianus and which cellular
functions are affected by this mechanism. Moreover, many
gaps still need to be filled in the direction of examining the
behavior of K. marxianus in terms of the consumption of
different sugars. What is known in general is that the kinet-
ics of growth and sugar consumption depend qualitatively
and quantitatively on the yeast species, type and concentra-
tion of the sugar, oxygen availability, and other environmen-
tal parameters, which include temperature, pH, medium
composition, and presence of uncoupling metabolites
(Postma and van der Broek 1990; Verduyn 1991; van
Djiken et al. 1993).
Microorganisms are frequently exposed to more than
one carbon source, in both natural and industrial environ-
ments. Nevertheless, the utilization of mixed substrates
by yeasts has received little attention. Some exceptions
are the study of ethanol production on sugar mixtures
(glucose/xylose) by Pachysolen tannophilus (Kruse and
Schugerl 1996), the consumption of xylose in a complex
mixture of sugars by several Saccharomyces cerevisiae
strains (van Zyl et al. 1989), and the utilization and
transport of hexoses and pentoses in Debaryomyces han-
senii (Nobre et al. 1999).
This work aimed at characterizing the physiology of the
yeast K. marxianus during exponential growth, more spe-
cifically in terms of the maximum specific growth rate
(max), the specific rate of substrate consumption (S), and
the biomass yield on substrate (YX/S). A synthetic medium
was employed with different sugars as single carbon sources
or with binary sugar mixtures. Cells were grown in a biore-
actor, which enabled monitoring of dissolved oxygen con-
centration and pH control.
Materials and methods
Strain, maintenance, and pre-cultures
K. marxianus CBS 6556 (= ATCC 26548, NCYC 2597,
NRRLy 7571) was kindly provided by Prof. Marcos Morais
Jr., Departamento de Gentica, Universidade Federal de
Pernambuco (UFPE), Recife, Brazil. After growth at 30 C
Appl Microbiol Biotechnol (2013) 97:50555067
in shake-flask culture on 100 ml YPD medium (yeast extract,
10 gl1; peptone, 20 gl1; glucose, 20 gl1) until late expo-
nential phase, glycerol was added to a final concentration of
15 % (v v1), and 1 ml aliquots of this culture were stocked
frozen at 80 C. Pre-cultures were prepared by transferring a
frozen stock vial to 50 ml of liquid YPD medium held in a
500-ml baffled Erlenmeyer flask. After 8 h of growth in an
orbital shaker (200 rpm) at 30 or 37 C (depending on the
temperature of the subsequent cultivation, which was the
same), cells were centrifuged, washed twice with 5 ml of a
0.9-M NaCl solution, and resuspended in the synthetic medi-
um described below.
Synthetic medium for pre-cultures and bioreactor
cultivations
The mineral medium contained per liter of distilled water:
(NH4)2SO4, 5.0 g; KH2PO4, 3.0 g; MgSO47H2O, 0.5 g; trace
elements (EDTA, 15 mg; ZnSO 4 7H 2 O, 4.5 mg;
MnCl22H2O, 0.84 mg; CoCl26H2O, 0.3 mg; CuSO45H2O,
0.3 mg; Na2MoO42H2O, 0.4 mg; CaCl22H2O, 4.5 mg;
FeSO47H2O, 3.0 mg; H3BO3, 1.0 mg; KI, 0.1 mg); and
silicone antifoam, 0.05 ml (Verduyn et al. 1992). After auto-
claving (121 C, 20 min), the medium was cooled to room
temperature and a filter-sterilized solution of vitamins pre-
pared in demineralized water was added, to a final concentra-
tion per liter of D-biotin, 0.05 mg; calcium pantothenate,
1.0 mg; nicotinic acid, 1.0 mg; myo-inositol, 25 mg; thiamine
HCl, 1.0 mg; pyridoxine HCl, 1.0 mg; and para-aminobenzoic
acid, 0.20 mg (Verduyn et al. 1992). The different sugars
(glucose, fructose, sucrose, lactose, galactose, xylose, or cel-
lobiose) were sterilized separately and added to a final con-
centration of 10 gl1 (as the unique carbon source) or to a final
concentration of 20 gl1 (10 gl1 for each of sugar, in a binary
combination). Glucose was also studied at 20 gl1. The con-
centration of all medium components was also doubled with
the carbon source (except when explicitly indicated).
Bioreactor cultivations
Cultivations were performed in batch mode in a Biostat
B bioreactor (Braun Biotech International, Germany) and
started by adding a certain volume of the washed 8-
h grown pre-culture, so that the initial cell concentration
in the bioreactor was 0.001 absorbance units at 600 nm
(Abs600). Cultivation conditions were 30 or 37 C and
4 l working volume with pH monitored by an electrode
and controlled at 5.0 by automatic addition of 1 M
KOH. The culture was sparged with air at a flow rate
of 4 l min1 and stirred at 700 rpm. The dissolved
oxygen concentration was continuously monitored with
an oxygen probe (Mettler-Toledo) and was never less
than 60 % of saturation with air (except when explicitly
Appl Microbiol Biotechnol (2013) 97:50555067
indicated). Eventually, the stirring speed was increased
via an automatic system up to 1,100 rpm, in order to
keep the dissolved oxygen concentration above 60 % of
saturation with air. With the aim of minimizing loss of
ethanol, a mixture of ethanol/water at 8 C was circu-
lated through the outlet gas condenser.
Gas analysis
The inlet gas flow rate was adjusted using the bioreactors
rotameter. The molar fractions of CO2 and O2 in the outlet gas
were analyzed via an infrared detector (Fuji Electric) and a
paramagnetic detector (Beckman Industrial, Model 755), re-
spectively. Detectors were calibrated 24 h before the cultiva-
tions started using synthetic air containing a defined
concentration of carbon dioxide. The oxygen uptake rate and
the CO2 evolution rate were calculated from the O2 and CO2
contents in the inlet and exhaust gases, respectively. The outlet
gas flow rate was calculated from the inlet gas flow rate,
applying a mass balance for N2 (Heinzle and Dunn 1991).
Determination of extracellular metabolites and biomass
concentration
Sugars, ethanol, acetic acid, and glycerol were separated by
HPLC using a Bio-Rad HPX-87H column (Hercules, CA,
USA). Samples taken from the bioreactor were immediately
filtered with positive pressure and injected into the HPLC,
with the aim of minimizing loss of ethanol. The column was
eluted at 65 C, with 0.0075 M H2SO4 at a flow rate of
0.6 ml min1. These compounds were detected by means of
a Waters 410 differential refractometer coupled to a data
module (Milford, MA, USA). Detection limits were 0.01 g
l1 for the sugars and 0.025 gl1 for ethanol, glycerol, and
acetate. The dry cell weight of 10 ml culture samples was
determined using 0.45 m membrane filters and a micro-
wave oven (180 W, 15 min) (Olsson and Nielsen 1997).
Determination of parameters during the exponential growth
phase (EGP)
The EGP was identified as the linear region on an ln(X)
versus time plot, where X is the cell concentration in terms
of dry cell weight per volume. The maximum specific
growth rate (max) was determined as the slope of this linear
region. The biomass yield on substrate (YX/S) was deter-
mined as the slope of the line on an X versus S plot,
exclusively including points belonging to the EGP, where
S is the substrate (sugar) concentration. The specific rate of
substrate consumption (S) was calculated according to the
following equation:


S max YX =S
5057
where max = maximum specific growth rate (per hour);
S = specific rate of substrate consumption during the EGP
(grams per gram DW per hour); YX/S = biomass yield on
substrate during the EGP (grams DW per gram); DW = dry
cell weight.
Results
Physiology of K. marxianus on different individual sugars
and temperatures
Growth rate, substrate consumption, and biomass yield
The physiological parameters obtained during exponential
growth of the yeast K. marxianus CBS 6556 are presented in
Table 1. All results were obtained on the same defined
medium (Verduyn et al. 1992), with different sugars as
single carbon sources and at either 30 or 37 C. Figure 1
shows the kinetics of growth, metabolite formation, and
consumption of sugars. K. marxianus CBS 6556 was not
capable of growing on mineral medium supplemented with
vitamins and either xylose or cellobiose as the sole carbon
source at 30 C. Consumption of these sugars was not tested
further at 37 C.
The cultivation on glucose at 30 C was performed in
duplicate, with the aim of verifying how reproducible the
experiments are and also to compare the results with those
previously carried out by Bellaver et al. (2004). Considering
the maximum specific growth rate (max), specific rate of
substrate consumption (S), and the biomass yield on glu-
cose (YX/S) obtained for this cultivation (Table 1), we ob-
served no significant difference between the two replicates,
which led us to carry out the subsequent cultivations in
single runs. Comparing our data with the results obtained
by our group previously (Bellaver et al. 2004; Fonseca et al.
2007), it is possible to observe that they are not identical to
neither of the two works. As already discussed in the work
of Fonseca et al. (2007), these differences are probably a
consequence of the different forms in which the cells were
received, propagated, and preserved in the three works.
Since there is a high degree of genetic polymorphism within
the K. marxianus species (Belloch et al. 2002), it could be
that this yeast is particularly rapid in evolving during the
propagation steps involved during stock generation.
Nevertheless, the data presented here and those presented
in the previous works mentioned above indicate that, once a
stock is generated and frozen at 80 C, cultivations become
reproducible and thus different experiments carried out from
the same stock can be compared to each other.
Comparing all experiments performed (Table 1), it is
possible to observe that, at 30 C, max was the highest for
growth on glucose (0.49 h1), whereas this parameter
5058 Appl Microbiol Biotechnol (2013) 97:50555067

Fig. 1 Kinetics of growth, metabolite formation, and sugar consumption

presented values around 0.41 h1 for growth on the remain-
ing sugars. The behavior at 37 C is somewhat different.
Again, growth on glucose occurred at the highest specific
rate (0.45 h1), with intermediary rate values for growth on
fructose, lactose, and sucrose (around 0.41 h1). Growth on
galactose at 37 C was the slowest, when compared to the
other sugars investigated. This suggests that galactose up-
take is less efficient than the uptake of other sugars in K.
marxianus CBS 6556. This behavior has been reported for
S. cerevisiae (Ostergaard et al. 2000).
In general, growth on a sugar seems to be more rapid at
30 C than at 37 C. The biggest difference in the max value
was observed for galactose, when the two temperatures are
compared. Bajpai and Margaritis (1987) reported that in K.
marxianus UCD (FST) 5582, the specific growth rate
remained almost constant over the range 30 to 35 C and
dropped sharply at temperatures either higher than 35 C or
lower than 30 C and at pH values higher or lower than 5.0.
The specific rate of substrate consumption (S) presented
similar values for growth on glucose, fructose, and galactose
(only at 30 C), whereas these values were somewhat
smaller for growth on the disaccharides sucrose and lactose.
This is probably a consequence of the fact that cells need to
perform a hydrolysis step before disaccharides can enter the
glycolytic pathway, which might decrease the rate with
which the substrate is metabolized. Galactose presented a
much slower specific consumption rate at 37 C, when
during cultivations with a glucose/30 C, b glucose/37 C, c fructose/
30 C, d fructose/37 C, e sucrose/30 C, f sucrose/37 C, g lactose/30 C,
h lactose/37 C, i galactose/30 C, j galactose/37 C. Filled square, X
(biomass, gram per liter); filled circle, GLC (glucose, gram per liter);
filled diamond, FRU (fructose, gram per liter); empty circle, SAC (su-
crose, gram per liter); filled triangle, LAC (lactose, gram per liter); empty
diamond, GAL (galactose, gram per liter); empty square, ETH (ethanol,
gram per liter); empty triangle, GLY (glycerol, gram per liter); asterisk,
ACE (acetate, gram per liter); times symbol, PYR (pyruvate, gram per
liter); minus sign, SUCC (succinate, gram per liter); plus sign, pO2
(dissolved oxygen concentration, milligram per liter)
compared to 30 C. Together with the low max value
observed for growth under this condition, it seems that
galactose consumption at 37 C is severely hindered in K.
marxianus CBS 6556.
Comparing the substrate consumption rates at 30 and 37 C,
it is interesting to note that there seems to be an increase in the
values of these parameters with increasing temperature, when
cells are grown on the disaccharides sucrose and lactose. For
the monosaccharide fructose, the values do not change much
when the two temperatures are compared, whereas a decrease
in the S value with increasing temperature is observed for
growth on glucose and galactose.
These values of max and S supply indications that the
cell activities of growth and substrate consumption are de-
pendent on both the type of sugar and the temperature. It is
difficult to identify a trend when inspecting the YX/S values,
except for the fact that there seems to be a slight decrease in

Table 1 Physiological parameters during exponential growth of K. marxianus CBS 6556 on different sugars and temperatures

C- S0 T DT max S YX/S Xmax qmetabolites qO2 qCO2 RQ Carbon Reference

source (g l1) (C) (h) (h1) (g g (g DW g1) (g DW l1) (mmol g1 h1) (mmol g1 h1) (mmol g1 h1) recovered
DW1 h1) (%)

GLC 10 30 1.39 0.495 0.908 0.5450.005 5.400.04 0.0250.005 11.80.7 12.70.4 1.08 98.80.1 This work
0.005 0.001 0.03
GLC 10 37 1.54 0.45 0.85 0.53 5.32 0.03 11.5 12.1 1.06 96.8 This work
FRU 10 30 1.65 0.42 0.86 0.49 5.05 0.06 11.8 12.0 1.02 98.4 This work
FRU 10 37 1.69 0.41 0.87 0.47 5.28 0.01 11.8 11.8 1.00 99.4 This work
SUC 10 30 1.61 0.43 0.68 0.63 5.73 0.03 9.9 10.1 1.02 95.7 This work
SUC 10 37 1.65 0.42 0.75 0.56 5.73 0.05 10.6 10.8 1.02 95.4 This work
LAC 10 30 1.73 0.40 0.71 0.55 5.00 0.03 10.1 10.6 1.05 101.3 This work
LAC 10 37 1.77 0.39 0.75 0.52 5.00 0.04 10.7 11.1 1.04 96.1 This work
GAL 10 30 1.69 0.41 0.84 0.49 5.04 0.05 11.6 11.8 1.01 96.3 This work
GAL 10 37 2.24 0.31 0.66 0.47 4.80 0.03 9.7 9.7 1.00 95.3 This work
GLCa 20 30 1.41 0.49 0.92 0.53 9.36 0.11 9.9 10.7 1.09 96.1 This work
GLC 10 30 1.24 0.56 1.095 0.510.02 5.05 111 12.10.6 1.09 96.4 Fonseca et
0.02 0.005 al. (2007)
GLC 10 30 1.58 0.44 0.90 0.49 5.16 13.5 Bellaver et
al. (2004)
LAC 10 30 1.58 0.44 0.98 0.45 5.66 Bellaver et
al. (2004)

Cells of K. marxianus CBS 6556 were grown as described in Materials and methods
GLC glucose, FRU fructose, SUC sucrose, LAC lactose, GAL galactose, S0 initial substrate concentration, T temperature, DT doubling time, max
maximum specific growth rate, S specific substrate consumption rate, Xmax maximum biomass concentration, YX/S biomass yield on substrate
a
All other nutrients were also doubled. The kinetics of this experiment are shown in Fig. 2b
Appl Microbiol Biotechnol (2013) 97:50555067 5059

a b

c d

e f

g h

i j
5060
this parameter with increasing temperature. In some culti-
vations, e.g., on sucrose at 30 C, high biomass yields on
substrate were obtained (0.63 g DW g1, Table 1). This
parameter was calculated from the slope of an X (biomass
concentration) versus S (substrate concentration) plot, only
including data points belonging to the exponential growth
phase. In all other situations investigated, a clear straight
line was obtained. However, for the particular case of su-
crose (both at 30 and 37 C), the data points did not align as
perfectly (data not shown). This means that YX/S is not
constant during the exponential growth phase in this partic-
ular situation (growth on sucrose). Interestingly, during
growth on lactose, which is also a disaccharide, YX/S was
clearly constant during the exponential growth phase (data
not shown). This difference could be attributed to the fact
that sucrose metabolism starts extracellularly via inulinase
activity (which also hydrolyzes sucrose; Rouwenhorst et al.
1990), whereas lactose is first transported into the cells and
subsequently hydrolyzed in the cytoplasm (Bacci Jnior et
al. 1996).
Rech et al. (1999) suggested that K. marxianus strains
CBS 712 and CBS 6556 are thermostable, contrasting with
Furlan et al. (1995), who reported maximum growth rate at
30 C for the same strains, concluding that an increase in
temperature slows cell growth. Rech et al. (1999) also
demonstrated that the strains showed no significant differ-
ence in terms of growth and biomass formation, when
cultivated at 30 or 37 C. On the other hand, at 30 C, the
consumption of sugars was faster than at 37 C. In our study,
however, we observed that K. marxianus CBS 6556 metab-
olism is temperature dependent, with higher growth rates at
30 C than at 37 C and different specific rates of substrate
consumption at 30 and 37 C, depending on the sugar. Thus,
the influence of temperature on growth in general is sugar
dependent.
It should be noted that it was necessary to activate control
of the dissolved oxygen concentration, in order to keep it
above 60 % of saturation with air, during some cultivations
carried out at 30 C (glucose, fructose, sucrose, lactose,
galactose) (results not shown). This reflects the more active
aerobic metabolism at this temperature, than at 37 C, in
which it was not necessary to increase the stirring speed.
Carbon dioxide evolution, oxygen utilization, and respiratory
quotient
The specific rates of CO2 production (qCO2) and O2 con-
sumption (qO2) oscillated around 1011 mmolg1 h1 for the
different sugars and temperatures studied (Table 1).
Comparison of the specific growth rates, substrate uptake
rates, and oxygen consumption rates was previously demon-
strated to be directly proportional to each other (Schulze and
Lipe 1964). Typically, an increase in the specific growth rate
Appl Microbiol Biotechnol (2013) 97:50555067
should lead to higher carbon dioxide production and oxygen
consumption rates (Fiechter et al. 1981). The respiratory quo-
tient was always between 1.00 and 1.11, indicating that the
metabolism of K. marxianus was purely respiratory.
Metabolite production and carbon balances
The formation of extracellular metabolites was very low in
all cultivations performed and never exceeded the sum of 34
C-mmol of products per C-mol of sugar (Table 2).
Considering that the biomass yield reached values around
0.53 0.05 g g1 (average standard deviation) for all
experiments performed with an initial carbon source con-
centration of 10 gl1, these data confirm the strong tendency
of K. marxianus to generate energy via respiration and to
convert sugar substrates into biomass with high yields. Our
results are in agreement with those published in a detailed
study on nutrient limitation with the K. marxianus DSM
5422 strain (Urit et al. 2012). Although this is not the same
strain as the one utilized in our work, the mentioned study
clearly shows that metabolite formation in this yeast species
is almost absent, as long as the culture medium does not
contain any trace element limitation.
It can also be observed that there is no difference in
terms of ethanol formation at 30 and 37 C for the
different carbon sources studied, once this compound
was not detected at any of the two temperatures, except
for growth on glucose (Fig. 1a, b) or in single samples
for the other sugars, mainly during the exponential
growth phase (Fig. 1c, f, g). In this particular case, the
concentration of ethanol at 37 C was a bit higher than at
30 C. The most abundant metabolite in all cultivations
was glycerol, with a tendency to present higher concen-
trations at 37 C than at 30 C. Known to have a
protective function for the cells, it might be that this
stronger formation of glycerol at 37 C is a consequence
of some stress response. Finally, acetate and pyruvate
were also observed in some cultivations, in agreement
with the data presented by Hensing et al. (1994).
The appearance of ethanol in single samples during some
cultivations might be related to the growth rate, i.e., ethanol
formation would set in for specific growth rates above 0.4
0.45 h1. At values above this interval, ethanol formation
was more evident, e.g., during all growth on glucose; below
this interval, it was absent (growth on galactose).
In Crabtree-positive yeasts, such as S. cerevisiae, under the
same conditions employed in this study, the formation of
ethanol is expected because of a repression of the respiratory
genes by glucose (catabolite repression) and also due to an
overflow of the carbon flux at the pyruvate level (Sonnleitner
and Kappeli 1986; Castrillo and Ugalde 1993). This can easily
be observed by the typical biomass yield on substrate for S.
cerevisiae (YX/S =0.11 g DW g1) during a batch cultivation on
Appl Microbiol Biotechnol (2013) 97:50555067 5061

Table 2 Maximum metabolite

formation (C-millimole C-mole C-source S0 (g l1) T (C) Pyruvate Succinate Acetate Glycerol Ethanol Total Kinetic (h)
per substrate) from different aero-
bic batch cultivations on different GLC 10 30 0.00 0.00 13.3 7.79 11.4 32.6 20
sugars and temperatures with K. GLC 10 37 0.00 0.00 8.68 9.03 16.3 34.0 21
marxianus CBS 6556
FRU 10 30 1.90 0.00 0.00 2.37 4.92 9.20 22.5
FRU 10 37 0.00 0.00 2.25 2.00 0.00 4.25 17.5
Cells of K. marxianus CBS 6556 SUC 10 30 0.00 0.00 2.08 1.39 0.00 3.47 20
were grown as described in
SUC 10 37 1.47 0.00 0.00 2.62 4.32 8.40 20.5
Materials and methods
LAC 10 30 0.59 0.00 1.81 1.72 0.00 4.12 21
GLC glucose, FRU fructose,
SUC sucrose, LAC lactose, GAL LAC 10 37 1.51 0.41 0.00 2.76 0.00 4.68 23.5
galactose, T temperature GAL 10 30 0.81 0.00 0.00 2.27 0.00 3.08 18.5
a
All other nutrients were also GAL 10 37 1.89 0.00 0.00 1.05 0.00 2.94 26.5
doubled. The kinetics of this ex- GLCa 20 30 1.63 0.00 11.1 2.34 3.97 19.0 21
periment are shown in Fig. 2b

glucose (van Dijken et al. 2000), which is much lower than the
values around 0.5 g DW g1 obtained for K. marxianus
(Table 1). The maximum specific growth rate (max) of S.
cerevisiae (around 0.4 h-1, van Dijken et al. 2000), grown
under similar cultivation conditions to those applied in the
present work, is also smaller than the values obtained here for
K. marxianus (Table 1), at least considering glucose as the
sole carbon source.
Influence of the initial sugar concentration and of other
nutrients on growth
To investigate a possible growth limitation by nicotinic acid,
as verified by Kiers et al. (1998) for Kluyveromyces lactis
during batch growth on the same synthetic medium used
here, we performed an experiment under the same condi-
tions as those reported above (10 gl1 initial glucose, 30 C,
pH 5.0, pO2 >60 %), except for the nicotinic acid concen-
tration, which was increased fivefold (5 mg l 1 ). We
obtained very similar kinetic profiles for all variables mea-
sured under this condition, when compared to an initial
nicotinic acid concentration of 1 mgl1, which indicates that
nicotinic acid was not limiting the growth of K. marxianus
CBS 6556 under the latter condition (data not shown).
To investigate whether catabolite (glucose) repression
could set in during batch cultivations of K. marxianus CBS
6556, we performed one cultivation with an increased initial
glucose concentration (20 gl1). Accordingly, the concentra-
tions of all other nutrients were also doubled, with the aim of
avoiding an eventual limitation of growth by any of these
nutrients, before the carbon source was exhausted. In a first
experiment, the dissolved oxygen concentration was not con-
trolled, which led to values below 40 % of saturation with air
after 21 h of cultivation, reaching severe oxygen limitation
later on (Fig. 2a). This drop in the pO2 provoked a sudden
increase in ethanol concentration, which had not been ob-
served during the other experiments, in which pO2 was con-
trolled above 60 % of saturation with air. Decreases in the
specific growth rate value (max =0.36 h1) and in the biomass
yield on substrate (YX/S =0.39 g DW g1) were observed, when
compared to cultivations without oxygen limitation (Table 1).
As can be observed in Fig. 2a, ethanol was detected
already when the dissolved oxygen concentration reached
values around 80 % of saturation with air. This was quite
unexpected and it could be that the oxidative metabolism
started suffering a certain degree of catabolite repression at
glucose concentration values between 10 and 20 gl1.
To confirm whether the low dissolved oxygen concentra-
tion (<40 % of saturation with air) was indeed responsible
for the increased ethanol formation after 21 h of cultivation
in the experiment described above, a similar cultivation was
carried out, under exactly the same conditions, except for the
fact that the dissolved oxygen concentration was controlled
above 60 % of saturation with air (Fig. 2b). As can be observed
by comparing the two experimental data sets (Fig. 2a, b), even
under full aerobiosis, ethanol formation occurred, as well as
excretion of organic acids and glycerol (Fig. 2b: ETHmax =
0.33 gl1, GLYmax =0.1 gl1, ACETmax =0.11 gl1, PYRmax =
0.11 gl1). However, we can also conclude that the additional
ethanol production observed in Fig. 2a, starting at 21.5 h of
cultivation (reaching ETHmax =1.09 gl1 at 23 h), was due to a
limitation in oxygen supply. It can also be observed that during
this cultivation carried out with 20 gl1 initial glucose and
under full aerobiosis, the calculated max and YX/S values
during the EGP were very similar to those obtained in experi-
ments with 10 gl1 initial glucose (Table 1).
With the aim of verifying whether nutrient limitation
could play a role in the formation of ethanol during the
experiments carried out in this work, we decided to perform
a cultivation with 20 gl1 initial glucose, without doubling
the concentrations of all other nutrients accordingly
(Fig. 2c). The kinetic profiles observed for this experiment
were very similar to the experiment carried out with all
nutrients doubled (Fig. 2b, c). The maximum concentration
of ethanol was very similar in both experiments (Fig. 2b:
ETHmax =0.33 gl1; Fig. 2c: ETHmax =0.34 gl1). Thus, we
5062 Appl Microbiol Biotechnol (2013) 97:50555067

a formation was increased, max was 0.42 h1, and YX/S was
0.39 g DW g1 (calculated from Fig. 2a). Under both nutri-
ent and oxygen limitations, the decrease in biomass yield
was probably due to the increased relative contribution of
the maintenance energy requirement to the overall energy
budget (van Hoek et al. 1998) and carbon deviation for
metabolite formation (Fonseca et al. 2007), respectively.
Even though we observed some ethanol, glycerol, and
organic acid formation during fully aerobic batch cultiva-
tions of K. marxianus CBS 6556, which could eventually be
b caused by some degree of catabolite repression, as discussed
above, K. marxianus still deviates less carbon towards for-
mation of these excretion products, when compared not only
to a Crabtree-positive yeast, such as S. cerevisiae, but also to
known Crabtree-negative species, such as Saccharomyces
kluyveri. Under similar conditions to those employed here,
and starting with 20 gl1 initial glucose in fully aerobic
batch cultivations, Mller et al. (2002a) measured a maxi-
mum ethanol concentration of 1.4 gl1 and a biomass yield
on substrate of 0.29 g DW g1 for S. kluyveri, whereas in
this work the corresponding values were 0.33 gl1 and
c 0.52 g DW g1 (Fig. 2b, Table 1).

Physiology of K. marxianus on binary sugar mixtures

Glucose plus lactose

In the mixture of sugars GLC + LAC, the presence of

organic acids, glycerol, and ethanol was detected during
the cultivation (Fig. 3). We could also observe the existence
of two independent growth phases, in accordance with two
Fig. 2 Kinetics of growth, metabolite formation, and sugar consumption substrate consumption phases, corresponding to the two
during cultivations of K. marxianus CBS 6556 on the following media (at utilized sugars. Initially, a first growth phase on glucose
30 C): a glucose (at initial 20 gl1) and all other nutrients doubled, pO2 occurred, until 20 h of cultivation, followed by a growth
not always >60 % (4.5 mgl1); b glucose (at initial 20 gl1) and all other
nutrients doubled, pO2 always >60 % (4.5 mgl1); c glucose (at initial phase on lactose (from 20 until 23.5 h). The transition was
20 gl1) and all other nutrients at the same level used in the previous accompanied by a deceleration in respiration, corresponding
cultivations (Fig. 1), pO2 always >60 % (4.5 mgl1). Symbols are the to the increase of pO2 (from 19.5 until 20 h). When the
same as in Fig. 1 microorganism started the new growth phase on lactose,
these values decreased again, indicating that yeast returned
cannot associate ethanol formation in the cultivations car-
ried out at 10 gl1 initial sugar with nutrient limitation.
However, the max and YX/S values in these two experiments
were different (Fig. 2b, c). In the cultivation with 20 gl1
initial glucose and all other nutrients doubled, max was
0.49 h1 (Table 1), whereas in the cultivation with the same
initial glucose concentration, but without doubling all other
nutrients accordingly, max was 0.39 h1. Also, in the former
case, YX/S was 0.53 g DW g1 (Table 1), whereas in the
latter, YX/S was 0.41 g DW g1 (calculated from Fig. 2c).
Thus, although nutrient limitation did not cause increased
Fig. 3 Kinetics of growth, metabolite formation, and sugar consumption
ethanol formation, it limited growth, which is both reflected
during cultivations with K. marxianus CBS 6556 on a binary mixture of
in the growth rate and in the biomass yield on substrate. On glucose and lactose (at initial 10 gl1 each sugar and 30 C). Symbols are
the other hand, when oxygen supply was limited, ethanol the same as in Fig. 1
Appl Microbiol Biotechnol (2013) 97:50555067 5063

recovered (%)
to respiratory activity. In spite of this lag phase of about
30 min, diauxic growth in a stringent sense (Monod 1942) is

Carbon

GLC glucose, FRU fructose, LAC lactose, GAL galactose, DT doubling time, max maximum specific growth rate, S specific substrate consumption rate, YX/S biomass yield on substrate
100.0
not the case here, since lactose consumption started before

1.01 1.00 94.7

1.00 1.00 96.8
99.8

97.6

0.99 1.01 94.9

96.1
glucose was exhausted from the medium, indicating that the
genes required for lactose metabolism were already
expressed before glucose was depleted. There was no lac-

1.01

1.00

1.00

1.09
qCO2 (mmol g-1 h1) RQ
tose consumption while glucose concentration remained
above 6 gl1.
Considering that two distinct growth phases were ob-

10.7
11.1
served, kinetic parameters such as the maximum specific

7.3
growth rate (max), the specific rate of substrate consump-
tion (S), and the biomass yield on substrate (YX/S) were

15.6
10.8
14.9

10.1

10.5

10.3
10.7
calculated for each phase. It could be verified that the yeast
grows with a higher growth rate on glucose than on lactose,

(g g DW h ) (g DW g ) (mmol g h )
as expected (max GLC = 0.43 h1, max LAC = 0.30 h1)

1 1

10.7

7.21
11.1
(Table 3).

Table 3 Kinetic and respiratory parameters from K. marxianus CBS 6556 during aerobic batch cultivations on different sugar mixtures

15.5
10.8
14.7

10.1

10.5

10.4
9.88
qO2
Glucose plus galactose

0.45 0.99
0.48 0.49

0.48 0.49
0.54 (0.63/

0.48 (0.37/

0.47 (0.47/
1
The mixture of sugars GLC + GAL provokes a very similar

0.45)

0.56)

0.47)
growth profile, when compared to GLC + LAC. As on the

0.53
YX/S
latter sugar combination, glucose was the preferred sub-
strate. However, specific growth rate values on glucose 1 1

0.30
0.76

0.76
and galactose were identical (max GLC =0.37 h1, max
GAL
=0.37 h1). The shift in the growth curve is not as clear 0.43 0.30 1.61 2.31 0.96
0.37 0.37 1.87 1.87 0.77
0.76

0.88

0.89

0.37 0.37 1.87 1.87 0.77

0.92
as the one on the mixture GLC + LAC; however, a short lag
S

phase can also be observed (between 18 and 18.5 h) (Fig. 4).

It seems that glucose exerts a stronger regulation on
Lag phase Adaptation max (h1) DT (h)

galactose metabolism than on lactose metabolism, since

1.69

1.65

1.65

1.41
galactose consumption only started when glucose concen-
tration reached 2 gl1. In the case of growth on GLC + LAC,

Concentration of sugar below which the consumption of the second sugar started
this value was 6 gl1, as discussed above.
0.41

0.42

0.42

0.49
Lactose plus galactose
time (min)

Considering that lactose and galactose were only con-

30
30

30

sumed after glucose in the previous experiments, we

thought it would be interesting to investigate the growth
behavior of K. marxianus CBS 6556 on a mixture of
Yes
Yes

Total repression Yes

No

No

No

LAC + GAL (Fig. 5). In contrast to what was verified in

the previous cultivations, in which glucose was one of

GAL + FRU Simultaneous No repression
levela (g l1)

the carbon sources, no ethanol was detected throughout

Repression

the whole cultivation. Another observation related to the

excreted metabolites is that only acetate and pyruvate
were detected. In terms of growth curve, a unique growth
6
2
4

phase was observed, in contrast to the two previous

First sugar
consumed

situations. Therefore, the kinetic parameters max, S,

and YX/S were calculated considering the mixture of sug-
GLC + LAC GLC
GLC + GAL GLC
LAC + GAL LAC

GLC + FRU GLC

FRU + LAC FRU

ars as the carbon source (Table 3). Interestingly, even

though a diauxic behavior was not observed for growth,

both sugars were not consumed completely in parallel.
Medium

Lactose was preferred by K. marxianus CBS 6556 over

GLC

galactose. The latter sugar started being consumed when

a
5064
Fig. 4 Kinetics of growth, metabolite formation, and sugar consump-
tion during cultivations with K. marxianus CBS 6556 on a binary
mixture of glucose and galactose (at initial 10 gl1 each sugar and
30 C). Symbols are the same as in Fig. 1
the lactose concentration reached 4 gl1 (Fig. 5). This
result is in agreement with our previous observation that
glucose is more repressive towards galactose than lactose
metabolism.
Glucose plus fructose
During growth on GLC + FRU, the presence of organic
acids, glycerol, and ethanol was detected in similar
concentrations to the ones observed during the other
cultivations employing glucose (GLC + LAC, GLC +
GAL) (Fig. 6). However, in contrast to the latter experi-
ments, no diauxic shift was observed on growth behav-
ior. Furthermore, glucose was only a slightly preferred
sugar over fructose. There was almost no fructose con-
sumption while the glucose concentration was above
8 gl1. From this point onwards, the concentration of
both sugars decreased in parallel, and fructose consump-
tion was accelerated, since there was only a small time
interval between the complete exhaustion of glucose and
fructose (around 45 min, between 20.75 and 21.5 h,
Fig. 6). The kinetic parameters max, S, and YX/S were
calculated considering both sugars as the carbon source,
similar to what was performed with the data from the
cultivation on LAC + GAL (Table 3).
Fig. 5 Kinetics of growth, metabolite formation, and sugar consump-
tion during cultivations with K. marxianus CBS 6556 on a binary
mixture of galactose and lactose (at initial 10 gl1 each sugar and
30 C). Symbols are the same as in Fig. 1
Appl Microbiol Biotechnol (2013) 97:50555067
Fig. 6 Kinetics of growth, metabolite formation, and sugar consump-
tion during cultivations with K. marxianus CBS 6556 on a binary
mixture of glucose and fructose (at initial 10 gl1 each sugar and
30 C). Symbols are the same as in Fig. 1
Galactose plus fructose
During growth of K. marxianus CBS 6556 on the mix-
ture GAL + FRU (Fig. 7), organic acids, glycerol, and
ethanol were detected, although in lower concentrations,
when compared to the other cultivations (except for the
one on the mixture LAC + GAL, Fig. 5). Once again, a
single growth phase was observed. Different from what
was observed for the other cultivations on two sugars,
simultaneous consumption of galactose and fructose oc-
curred, indicating that no regulatory mechanisms are
triggered by galactose or fructose on the consumption
of the other sugar. The kinetic parameters max, S, and
YX/S were calculated considering the mixture of GAL +
FRU as a sole carbon source, with a unique growth
phase (Table 3).
Fructose plus lactose
During growth of K. marxianus CBS 6556 on the mixture
of sugars FRU + LAC (Fig. 8), organic acids, glycerol,
and ethanol were detected in the culture supernatant, in
concentrations comparable to what was observed during
growth on the mixture GAL + FRU (Fig. 7). Fructose
exerted total repression on the consumption of lactose,
Fig. 7 Kinetics of growth, metabolite formation, and sugar consump-
tion during cultivations with K. marxianus CBS 6556 on a binary
mixture of galactose and fructose (at initial 10 gl1 each sugar and
30 C). Symbols are the same as in Fig. 1
Appl Microbiol Biotechnol (2013) 97:50555067
Fig. 8 Kinetics of growth, metabolite formation, and sugar consumption
during cultivations with K. marxianus CBS 6556 on a binary mixture of
lactose and fructose (at initial 10 gl1 each sugar and 30 C). Symbols are
the same as in Fig. 1
since the consumption of the latter sugar only started after
fructose had been exhausted from the medium. This was
somewhat surprising, since neither glucose exerted such a
strong repression on the consumption of lactose, nor fructose
affected the consumption of galactose in such a dramatic
manner. Two independent growth phases were observed, with
a diauxic shift between them (from 20 to 20.5 h of cultivation).
The pO2 increase in the time interval from 20 to 20.75 h
indicates a deceleration of the respiration due to the transition
in sugar metabolism. The kinetic parameters max, S, and
YX/S were calculated separately for each one of the two growth
phases. The max values calculated for each growth phase
were identical (max FRU =0.37 h1, max LAC =0.37 h1)
(Table 3).
Discussion
Sugar transport is the first step in the metabolism of carbohy-
drates (except when there is sugar hydrolysis outside the cell).
In yeasts, this can proceed via a number of different mecha-
nisms. Some mechanisms involved in sugar transport were
described for K. marxianus (Gasnier 1987; de Bruijne et al.
1988; Postma and van der Broek 1990). Gasnier (1987) con-
cluded that glucose can enter K. marxianus cells through two
distinct transporters: a high affinity glucose transporter which
is a protonsugar symporter and a low affinity transporter with
activity not associated with proton movement. de Bruijne et al.
(1988) described four possible transport systems in K. marx-
ianus. One system is specific for glucose and fructose, appar-
ently showing phosphotransferase activity (Postma and van
der Broek 1990). The other mechanisms utilize a proton
symport system specific for fructose, lactose, and glucose/ga-
lactose (de Bruijne et al. 1988).
The data shown here on the growth of K. marxianus on
GLC + LAC indicate that glucose transport occurs using the
low affinity system, whereas the glucose concentration in
the medium is high (e.g., above 6 gl1), provoking repres-
sion of lactose transport. Once glucose concentration
5065
reaches ca. 6 gl1, the high affinity system probably sets
in, relieving repression over lactose transport, allowing for
the co-consumption of both sugars. From the growth data on
the mixture GLC + GAL, it could well be that the higher
repression exerted by glucose on the consumption of galac-
tose, when compared to lactose, is due to the fact that there
is no specific transport system for galactose, as opposed to
lactose (de Bruijne et al. 1988). Thus, galactose competes
with glucose for the same transporter, which usually
presents a lower affinity for the former, when compared to
the latter. This is also corroborated by the slower growth
rates of K. marxianus on galactose as the sole carbon source,
when compared to growth on glucose (Table 1).
Interestingly, although galactose seems to share one of the
transport systems used by glucose (the high affinity system),
when growing on a mixture of lactose and galactose, the
latter was not the preferred carbon source of K. marxianus,
indicating that the repression effect caused by glucose over
lactose (and other sugars) consumption is indeed related to
glucose consumption via the low affinity system or via
signals originating specifically from glucose.
The fact that growth of K. marxianus on the mixtures
GLC + FRU and FRU + GAL occurred with the (almost)
parallel utilization of the two sugars in each mixture also
fits with the results presented by de Bruijne et al. (1988),
i.e., although glucose and fructose share a common low
affinity system, there is a high affinity proton symporter
specific for fructose, which allows for the consumption of
this monosaccharide, in parallel to glucose consumption.
In this case, the repression exerted by glucose on fructose
consumption is certainly weaker than the repression
exerted by glucose over lactose consumption. Also, since
fructose and galactose are transported by different systems
(galactose being transported by the same high affinity
system used for glucose transport), they exert no repres-
sion over the consumption of the other sugar, resulting in
simultaneous consumption. Finally, the results obtained
for growth of K. marxianus on the mixture FRU + LAC
also corroborate the findings of de Bruijne et al. (1988),
i.e., fructose being transported by the low affinity system
(also employed by glucose, when present in the medium)
represses the transport of lactose, which seems to be the
less preferred sugar, among the ones studied here. This
could be due to the fact that lactose is a disaccharide,
which requires an additional metabolic step for its utiliza-
tion, in this case intracellular hydrolysis. Thus, cells might
have developed regulatory mechanisms that favor mono-
saccharide over disaccharide utilization.
Finally, it should be mentioned that galactose is known to
be a slow fermenting sugar in the yeast S. cerevisiae. In
contrast to glucose and fructose, which are directly incor-
porated into glycolysis, galactose needs some additional
metabolic steps, before it can be channeled into glycolysis
5066
(also known as the Leloir pathway). Nevertheless, the rea-
son for slow galactose consumption in S. cerevisiae is
probably related to the transport step (carried out by the
Gal2 protein), since the same enzymatic steps are present in
Kluyveromyces yeasts, which, as shown here, consume ga-
lactose faster than S. cerevisiae.
In K. lactis, which is a closer relative of K. marxianus,
the metabolic response to glucose is different from the one
verified in S. cerevisiae. Respiration is not repressed by
glucose in K. lactis (nor in K. marxianus, Bellaver et al.
2004). However, glucose repression mechanisms also exist,
mainly over the consumption of alternative carbon sources
(Breunig 1989; Goffrini et al. 1995). Nevertheless, few are
the components of the glucose repression pathway identified
in K. lactis, which gives us an incomplete overview of how
the pathway represses the expression of genes necessary for
lactose and galactose utilization, the lactosegalactose reg-
ulon (Zacchariae and Breuning 1993).
From an industrial point of view, simultaneous sugar
consumption by the producing microorganism is highly
desired, since it decreases process time and, consequent-
ly, increases productivity. The results presented in this
work show that K. marxianus presents advantages over
other industrial yeasts, as for example S. cerevisiae. As
an example, S. cerevisiae DGI 342 showed a lag phase
of 6 h between glucose depletion and the start of galac-
tose consumption (Ostergaard et al. 2000; Ronnow et al.
1999). Interestingly, the time interval between glucose
depletion and the start of galactose consumption seems
to be independent from the initial sugar concentration, as
long as both sugars are at the same level when the
cultivation starts (Ostergaard et al. 2000).
Metabolic engineering has enabled yeasts to utilize sugar
mixtures (e.g., glucose/galactose) in a partially simultaneous
manner, i.e., with a decreased lag phase between the ex-
haustion of the first sugar and the start of consumption of the
second sugar. In S. cerevisiae TH1, deletion of the MIG1
and GAL80 genes, which encode proteins directly involved
in the molecular events responsible for glucose repression,
the time interval necessary for the beginning of galactose
consumption, after glucose depletion, decreased by 45 %,
the time interval necessary for the beginning of galactose
consumption, after glucose depletion (Ostergaard et al.
2000). However, this decreased time interval is still much
higher than the 30 min verified for K. marxianus CBS 6556
in our work.
To conclude, we observed that K. marxianus CBS 6556
presents physiological characteristics that make it an excel-
lent candidate organism for employment in biomass-
directed applications. Besides being able to consume a va-
riety of sugars usually found in industrial media at high rates
and with less regulation from one sugar over the consump-
tion of others, when compared to the industrial yeast S.
Appl Microbiol Biotechnol (2013) 97:50555067
cerevisiae, it has a very low tendency to direct the carbon
flux towards the formation of by-products. Besides this, it
does not require a nutritionally more complex medium,
when compared to S. cerevisiae.
Acknowledgments This work was financed by Fundao de Amparo
Pesquisa do Estado de So Paulo (FAPESP, So Paulo, Brazil). We would
also like to thank Prof. Marcos Morais Jr for providing us the strain used
throughout this work.
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