Antonie van Leeuwenhoek (2011) 100:619–630
DOI 10.1007/s10482-011-9617-7
ORIGINAL PAPER
Physiological diversity within the kluyveromyces marxianus
species
Saul Nitsche Rocha • José Abrahão-Neto •
Andreas Karoly Gombert
Received: 7 February 2011 / Accepted: 21 June 2011 / Published online: 6 July 2011
Ó Springer Science+Business Media B.V. 2011
Abstract The Kluyveromyces marxianus strains
CBS 6556, CBS 397 and CBS 712T were cultivated
on a defined medium with either glucose, lactose or
sucrose as the sole carbon source, at 30 and 37°C.
The aim of this work was to evaluate the diversity
within this species, in terms of the macroscopic
physiology. The main properties evaluated were:
intensity of the Crabtree effect, specific growth rate,
biomass yield on substrate, metabolite excretion and
protein secretion capacity, inferred by measuring
extracellular inulinase activity. The strain Kluyver-
omyces lactis CBS 2359 was evaluated in parallel,
since it is the best described Kluyveromyces yeast and
thus can be used as a control for the experimental
setup. K. marxianus CBS 6556 presented the highest
specific growth rate (0.70 h-1) and the highest
specific inulinase activity (1.65 U mg-1 dry cell
weight) among all strains investigated, when grown
at 37°C with sucrose as the sole carbon source. The
lowest metabolite formation and highest biomass
yield on substrate (0.59 g dry cell weight g
sucrose-1) was achieved by K. marxianus CBS
712T at 37°C. Taken together, the results show a
S. N. Rocha (&)  A. K. Gombert
Department of Chemical Engineering, University of São
Paulo, São Paulo, SP, Brazil
e-mail: sn-rocha@uol.com.br
J. Abrahão-Neto
School of Pharmaceutical Sciences, University of São
Paulo, São Paulo, SP, Brazil
systematic comparison of carbon and energy metab-
olism among three of the best known K. marxianus
strains, in parallel to K. lactis CBS 2359.
Keywords Kluyveromyces marxianus
Yeast physiology  Metabolite formation
Inulinase  Crabtree effect
Introduction
Since Van der Walt (1956) proposed the yeast genus
Kluyveromyces, several works reported on taxonomic
aspects of species and varieties within this genus,
employing different approaches and techniques.
A common aspect among the more recent works has
been the observation of a high heterogeneity among
members of the genus. In 1996, Cai and co-workers
used 18S rRNA gene sequence analysis to conclude
that the members within the Kluyveromyces genus
(at that time) did not form a monophyletic group (Cai
et al. 1996). These authors report that K. aestuarii,
K. dobzhanskii, K. lactis, K. wickerhamii, and
K. marxianus formed a ‘‘highly stable monophyletic
group worthy of separate generic status’’, which
distinguished it from other members of the Kluy-
veromyces taxon. This result was confirmed later by
Belloch et al. (1998a), who employed restriction
map analysis of the 5.8S rRNA gene and the two ribo-
somal internal transcribed spacers as a phylogenetic
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indicator. By employing the analysis of electropho-
retic chromosome patterns, Belloch et al. (1998b)
verified that these same five species mentioned above,
together with K. blattae, K. thermotolerans, and
K. waltii, present common chromosomal patterns,
distinguishing them from other Kluyveromyces spe-
cies. These authors also observed a rich chromosome
polymorphism within the K. marxianus species,
although there seemed to be a basic set of 8
chromosomes resolved into 6 bands in most of the
strains.
More recently, Kurtzman and Robnett (2003) used
multigene sequence analyses, instead of single-gene
analyses, to establish the phylogenetic relationship
among different yeasts. They clearly show that the
early approaches for yeast classification, which were
based on the morphology of vegetative and sexual
cells, as well as on simple fermentation and growth
tests, are poor predictors of genetic relationships
among different species. These authors showed that
the Kluyveromyces species described at that time
(Kurtzman and Fell 1998) are distributed into six
different phylogenetic clades. Still, the species
K. aestuarii, K. dobzhanskii, K. lactis, K. wickerh-
amii, and K. marxianus are part of the same clade,
together with K. nonfermentans. Later, the same
authors proposed that these six species should form
the newly defined Kluyveromyces genus (Kurtzman
2003).
Kluyveromyces marxianus is a homothallic
hemiascomycetous yeast species usually encountered
on dairy products (Fleet and Mian 1987), but also in a
variety of different habitats (Fonseca et al. 2008), and
able to grow on inulin (Rouwenhorst et al. 1988). In
addition, this species is able to grow at temperatures
up to 45°C (Rouwenhorst et al. 1988; Steensma et al.
1988), possesses a high capacity of converting
substrate into biomass (Bellaver et al. 2004; Fonseca
et al. 2007) and the highest specific growth rate
among eukaryotes (Groeneveld et al. 2009). Further-
more, some strains in the species show dimorphism, a
phenomenon that is described in a few yeast species,
including the widely studied Saccharomyces cerevi-
siae (Hill and Robinson 1988), and the less conven-
tional Wickerhamomyces anomalus (Sundhagul and
Hedrick 1966). The morphology of K. marxianus
CBS 397 is mainly affected by the specific growth
rate. While in the middle-range (higher than 0.15 h-1
and lower than 0.4 h-1), the cells show the dominant
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Antonie van Leeuwenhoek (2011) 100:619–630
yeast-like morphology. On the other hand, at lower or
higher growth rates cells show a filamentous/pseud-
ohyphal shape (O’Shea and Walsh 2000). This
characteristic could be explored industrially for
protein secretion, since filamentous cells are known
to have better secreting properties than yeast-like
cells (McIntyre et al. 2002). To sum up, K. marxianus
is a yeast with several potential biotechnological
applications (Fonseca et al. 2008).
In contrast to studies carried out with K. lactis
CBS 2359, which certainly is a model organism
adopted by an organized scientific community, the
yeast K. marxianus has been investigated differently,
without a reference strain and without an organized
scientific community. If, on the one hand, this has
resulted in a vast metabolic diversity and various
potential biotechnological applications for this yeast
species, on the other hand, it causes difficulty in
interpreting and comparing all the experimental data
available for this organism.
In contrast to the genetic polymorphism within the
K.marxianus species, which has been the aim of several
studies, as discussed above, the reflection of this
diversity at the metabolic/physiological level is an
issue that has not been tackled by many authors. Some
examples include the work of Mahmoud and Kosi-
kowski (1982), who studied biomass formation and
ethanol production in five Kluyveromyces strains
grown on cheese whey concentrate; Rech et al.
(1999) studied b-galactosidase formation by K. marxi-
anus CBS 712T and CBS 6556; Rouwenhorst et al.
(1990) studied inulinase localization in various
K. marxianus strains. Thus, with the aim of bridging
this knowledge gap between genetic and physiological
polymorphism, this work investigated some of the
most popular K. marxianus strains: CBS 6556, CBS
397 (=ATCC 46537, NRRL Y-2415), and CBS 712T
(=NRRL Y-8281, PYCC 3886). The former strain
possesses a high capacity of producing inulinase
(Rouwenhorst et al. 1988) and is cited in a large
number of academic studies involving K. marxianus,
including recent works on the successful expression of
different heterologous proteins (Rocha et al. 2010;
Rocha et al. 2011). K. marxianus CBS 397 presents
dimorphism, depending on the culture conditions
(O’Shea and Walsh 2000). K. marxianus CBS 712T is
the species type-strain and was isolated from grapes by
E.C. Hansen in 1888 (Kurtzman and Fell 1998).
K. lactis CBS 2359 was investigated in parallel to the
Antonie van Leeuwenhoek (2011) 100:619–630
three mentioned strains, since it is the best described
strain within the Kluyveromyces taxon. It also served as
a control for our experiments, because of the vast
availability of literature data on this organism. The
physiological parameters evaluated were (always on
defined media): specific growth rate, biomass yield on
substrate, excretion of extracellular metabolites, and
extracellular inulinase activity.
Materials and methods
Strains, preservation and pre-cultures
Kluyveromyces lactis CBS 2359 and K. marxianus
PYCC 3886 (=CBS 712T, NRRL Y-8281) were kindly
provided by Prof. Lucı́lia Domingues, University of
Minho, Braga, Portugal. Professor Marcos Morais Jr.,
from Federal University of Pernambuco, Recife,
Brazil, provided the strain K. marxianus CBS 6556.
The strain K. marxianus NRRL Y-2415 (=CBS 397,
ATCC 46537) was provided by Dr. Cletus Kurtzman
from Agriculture Resources Services (Peoria, IL,
USA). Right after arrival, each strain was submitted to
an overnight growth in YPD medium (yeast extract
10 g l-1; peptone, 20 g l-1; glucose, 20 g l-1) in
baffled Erlenmeyer flasks. Sterile glycerol was added
to a final concentration of 15% and cells were stored
in cryogenic vials at -80°C. Pre-cultures were
prepared by first transferring some cells from the
frozen stock vials to an YPD agar plate, using a
platinum inoculating loop. After 24 h incubation at
30°C, isolated colonies were visible on the plate. Cells
from one single colony were used to inoculate a
500 ml baffled Erlenmeyer flask filled with 100 ml of
mineral medium plus vitamins, containing the same
carbon source that would be used subsequently in the
cultivation. The pre-cultures were always incubated at
the same temperature of the subsequent cultivation, in
a New Brunswick (Edison, NJ, USA) orbital shaker
(300 rpm).
Defined medium for cultivations
The defined medium (Verduyn et al. 1992) was
prepared as follows. The nutrient solution was
composed by: (NH4)2SO4, 10.0 g l-1; KH2PO4,
6.0 g l-1; MgSO4 7H2O, 1.0 g l-1. Potassium
hydrogen phthalate (used here as buffer) was added
621
to this solution to a final concentration of 0.2 M and
the pH was adjusted to 5.0 with KOH. A carbon
source solution containing 20 g l-1 of the sugar to be
studied (glucose, lactose or sucrose) was prepared in
pure water. This solution was separately sterilized
from the nutrient solution (121°C, 20 min) and both
were cooled to room temperature. Then, 50 ml of the
nutrient solution were mixed with 50 ml of the
carbon source solution. Finally, to the nutrient and
carbon source mixture, 0.1 ml of a pre-sterilized
(121°C, 20 min) trace elements solution and 0.1 ml
of filter-sterilized (0.22 lm) vitamin solution were
added, completing the composition of the medium.
Trace elements solution composition was, in g l-1:
EDTA, 15; ZnSO47H2O, 4.5; MnCl22H2O, 0.84;
CoCl26H2O, 0.3; CuSO45H2O, 0.3; Na2MoO42H2O,
0.4; CaCl22H2O, 4.5; FeSO47H2O, 3.0; H3BO3, 1.0;
KI, 0.1. The vitamins solution composition was, in g
l-1: D-biotin, 0.05; calcium pantothenate, 1.0; nicotinic
acid, 1.0; myo-inositol, 25; thiamine HCl, 1.0; pyri-
doxine HCl, 1.0; and p-aminobenzoic acid, 0.20.
Batch cultivations in baffled Erlenmeyer flasks
Batch cultivations were started by adding a certain
amount of exponentially growing cells from the pre-
culture, in such a way that the initial absorbance (at
600 nm) of the cultivation was 0.1. Cells were
harvested from the pre-culture, centrifuged (90309
g, 1 min), washed with fresh culture medium, cen-
trifuged again (90309g, 1 min), resuspended in fresh
culture medium and then transferred to a baffled
500 ml Erlenmeyer flask containing 100 ml of
medium (the baffles aid in avoiding oxygen limitation
during the cultivations). All steps were performed
under sterile conditions. The four yeast strains were
cultivated in mineral medium plus vitamins, supplied
with glucose (GLC), lactose (LAC) or sucrose (SUC),
at 30 or 37°C. The pH was kept at 5.0 by buffering
the medium with phthalate-KOH (0.1 M final con-
centration), according to Blank and Sauer (2004).
Cultivations were carried out in duplicate and in
random order.
Sampling occurred every hour, without the need of
removing the flasks from the incubator, through a
syringe coupled to a tube inside the flasks. For
K. marxianus CBS 6556, sampling occurred every
30 min during the exponential growth phase. Samples
were taken to determine absorbance at 600 nm, the
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Fig. 1 A typical kinetic experiment performed in this study. Graphs show a duplicate cultivation of K. marxianus CBS 6556 grown
on a defined medium with sucrose as the sole carbon source at 30°C
concentration of extracellular metabolites, besides
inulinase activity (in cultures with sucrose as the
carbon source). Except for biomass determination,
samples were rapidly filtered using a 0.22 lm-pore
Millex filter (Millipore, Billerica, MA, USA) and
immediately frozen at -20°C.
An example of how the measured variables
behaved along a cultivation is shown in Fig. 1. The
graphs show experimental data from a duplicate
cultivation, in order to illustrate the reproducibility
attained in these kinetic studies.
Determination of extracellular metabolites
and biomass concentration
Glucose, lactose, ethanol, acetic acid, succinic acid,
pyruvic acid and glycerol were detected by HPLC
(Waters) analysis using a Biorad (Hercules, CA,
USA) HPX-87H column. Column temperature was
maintained at 60°C while samples were eluted with
5 mM H2SO4 at a flow rate of 0.6 ml. These
compounds were detected by means of a Waters
410 differential refractometer (Milford, MA, USA)
coupled to a data module. Detector temperature was
constant at 35°C and the sensitivity value was 32.
Sucrose concentration was determined by measur-
ing the glucose concentration after total hydrolysis,
using the Boehringer–Mannheim (Ingelheim, Ger-
many) Sucrose/D-Glucose/D-Fructose test combina-
tion kit (Cat nr. 10 716 260 035).
The biomass, represented as dry cell weight (DW),
was determined at the end of each cultivation, in order
to correlate with the absorbance measurements. Cells
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Antonie van Leeuwenhoek (2011) 100:619–630
were filtered through a previously dried 0.45 lm-pore
membrane, washed twice with distilled water and
dried in a microwave oven (180 W, 15 min). The
sample volume filtered was 5 ml, which always led to
less than 30 mg of dry cell weight on the membrane,
as recommended by Olsson and Nielsen (1997).
Determination of inulinase activity
Inulinase activity was measured by the same method
described by Rouwenhorst et al. (1988), determining
the rate of appearance of fructose and glucose with
the Boehringer–Mannheim (Ingelheim, Germany)
Sucrose/D-Glucose/D-Fructose test combination kit,
in the presence of 2% (w w-1) inulin from chicory
(Sigma) in a 0.1 M sodium acetate buffer, pH 4.5, at
50°C. One unit of inulinase activity is defined as the
amount of enzyme catalyzing the formation of 1 lmol
of fructose min-1 under the conditions mentioned
above. Specific enzyme activities of cultures are
expressed per mg DW (milligram of cell dry weight).
Calculation of physiological parameters
The exponential growth phase (EGP) was defined as
the linear region on a ln (X) versus time plot for batch
cultivation data. The maximum specific growth rate
(lmax) was determined as the slope of this linear
region. The biomass yield on substrate during the EGP
(YEXP
X/S ) was determined as the slope of the line on an X
versus S plot, only including points belonging to the
EGP, as determined above. The global biomass yield
on substrate (YNET
X/S ) was the ratio between the total
Antonie van Leeuwenhoek (2011) 100:619–630
biomass formed and carbon source consumed, until
1 h before carbon-source exhaustion. In addition, the
specific rate of substrate consumption during the
exponential growth phase (lEXP S ) was calculated
according to the following equation:
lmax
lEXP
S ¼ EXP
YX=S
where lmax = maximum specific growth rate (h-1);
X = biomass concentration in the flask (g DW l-1);
lEXP
S = specific rate of substrate consumption during
the EGP [g (g DW h)-1]; S = substrate concentration
in the flask (g l-1); YEXP X/S = biomass yield on
substrate during the EGP.
Results and discussion
Specific growth rate
Recently, Groeneveld et al. (2009) discussed the
importance of the growth rate in organisms consid-
ered as ‘‘living factories’’. As an example, high
growth rates are interesting for single-cell protein
production, and K. marxianus is considered an
efficient platform for this application (Bergkamp
et al. 1993; Hensing et al. 1995). Furthermore,
K. marxianus has been labelled the fastest-growing
eukaryote (Groeneveld et al. 2009). Taking these
aspects into account, a study of the intraspecific
variation of the maximum specific growth rate within
the K. marxianus species is interesting for at least
three purposes: to evaluate how this important
physiological parameter varies within the species, to
pinpoint a promising strain for industrial single-cell
protein production, and to identify the fastest strain of
the fastest-growing eukaryote on Earth.
The maximum specific growth rate is determined
under conditions of unlimited nutrient supply and is
therefore only constrained by the biological proper-
ties of the cell itself. In the case of this work, the
maximum specific growth rates were calculated for
cells growing in mineral medium supplemented with
vitamins and a single carbon source. Under these
conditions, growth rates are usually lower than the
ones observed in complex media, since cells have to
synthesize, from a sole carbon source, all the building
blocks needed to make up the macromolecules to be
assembled in new cells. According to Table 1,
623
K. marxianus CBS 6556 has shown, in general,
higher maximum specific growth rates (lmax) than the
other strains, including K. lactis CBS 2359.
The highest lmax values were observed when cells
grew on sucrose as sole C-source, at 37°C
(0.70 ± 0.05 h-1). The data obtained here under the
other conditions tested are in accordance to values
described in literature. Fonseca et al. (2007) observed
a maximum specific growth rate of 0.56 ±
0.02 h-1for the strain K. marxianus CBS 6556, when
grown on a defined medium, with glucose as the sole
C-source at 30°C. This is very close to the value
obtained here (0.59 ± 0.01 h-1, Table 1). Groene-
veld et al. (2009), for this same strain and conditions,
estimated this value to be 0.60 h-1. Rouwenhorst
et al. (1988) cultivated the same strain in the same
medium at 33°C, and calculated lmax to be 0.69 h-1.
Concerning the strain K. marxianus CBS 397, Varela
et al. (1992) calculated the lmax of K. marxianus
NRRL Y-2415 (same strain) growing on cheese whey
permeate to be 0.28 h-1. In our work, we calculated
this parameter to be 0.46 ± 0.00 h-1 for cells grow-
ing on mineral medium supplemented with vitamins
and lactose as the sole C-source, at 30°C (Table 1).
The difference might be due to some nutrient
limitation in the cheese whey.
Lages et al. (1999) calculated a lmax value of
0.39 h-1 for the strain K. marxianus CBS 712T, when
cells grew in a complex medium, while in this work
the lmax for the same strain was 0.31 ± 0.00 h-1. In
this case, the difference can be explained by the
medium composition: complex in the former and
defined in the latter case. For K. lactis CBS 2359, the
lmax value on defined medium with glucose as the
sole C-source was 0.37 ± 0.00 h-1, while in similar
conditions Zeeman et al. (1999) calculated 0.42 h-1,
and Kiers et al. (1998), 0.40 h-1. When comparing
all these values to those obtained with the most
investigated yeast species, S. cerevisiae, for which
van Hoek et al. (1998) report a lmax of 0.41 h-1for
the CEN.PK113-7D strain grown under similar
conditions, we observe that K. lactis CBS 2359
grows at a similar rate, K. marxianus CBS 6556 and
CBS 397 grow much faster and K. marxianus CBS
712T grows slower.
The data in Table 1 also show that, in general,
K. marxianus grows faster when cultivated at 37°C
than at 30°C, with the exception of the CBS 397
strain grown on glucose, for which the lmax value
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Table 1 Specific growth rate, specific substrate consumption rate and biomass yield on substrate during the exponential growth phase, and net biomass yield on substrate and
inulinase specific titers obtained during shake-flask cultivations of different K. marxianus and K. lactis strains grown on a mineral medium with different single carbon sources

123
Strain CS, Temperature lmax (h-1) lEXP
s (h-1) YNET
X/S YEXP
X/S TEXP (h) PT Inulinase
(g DW/gCS) (g DW/gCS) (U/mg DW)

K. marxianus CBS 6556 Glucose, 30°C 0.59 1.34 0.44 0.44 6 8 –

Glucose, 37°C 0.66 1.43 0.45 0.44 5 7 –
Lactose, 30°C 0.40 0.72 0.54 0.55 8 8 –
Lactose, 37°C 0.63 1.19 0.53 0.53 6 8 –
Sucrose, 30°C 0.58 – 0.39 – 6 9 1.24
Sucrose, 37°C 0.70 – 0.49 – 4 6 1.65
K. marxianus CBS 397 Glucose, 30°C 0.55 1.28 0.42 0.40 7 7 –
Glucose, 37°C 0.52 1.53 0.41 0.34 5 5 –
Lactose, 30°C 0.46 1.02 0.40 0.42 8 8 –
Lactose, 37°C 0.59 1.55 0.40 0.38 6 7 –
Sucrose, 30°C 0.52 – 0.39 – 7 6 0.59
Sucrose, 37°C 0.67 – 0.47 – 6 5 0.83
K. marxianus CBS 712T Glucose, 30°C 0.31 0.75 0.45 0.46 11 10 –
Glucose, 37°C 0.47 0.85 0.58 0.55 8 8 –
Lactose, 30°C No growth detected
Lactose, 37°C No growth detected
Sucrose, 30°C 0.33 – 0.45 – 10 11 0.46
Sucrose, 37°C 0.45 – 0.59 – 0.50
K. lactis CBS 2359 Glucose, 30°C 0.37 0.78 0.49 0.47 10 10 –
Glucose, 37°C No growth detected
Lactose, 30°C 0.37 1.16 0.30 0.32 10 11 –
Lactose, 37°C No growth detected
Sucrose, 30°C 0.40 – 0,52 – 10 11 0.00
Sucrose, 37°C No growth detected
Initial C-source concentration was 10 g l-1. Experiments were carried out in duplicate. The results shown are the mean values. The values for which the relation [(Deviation of the
mean) (Mean Value)-1)] was higher than 15% are highlighted in bold. For all other values, this relation was \15%
-1 EXP
lmax maximum specific growth rate, lS specific substrate consumption rate [lEXP
S = lmax (YEXP
X/S ) ], YNET
X/S net biomass yield on substrate, YX/S biomass yield on substrate during
EXP
the exponential growth phase, T duration of exponential growth phase, PT number of experimental points during the exponential growth phase, DW biomass dry weight,
CS carbon source, Temp.: cultivation temperature, ND not detected (below the detection limit)
Antonie van Leeuwenhoek (2011) 100:619–630
Antonie van Leeuwenhoek (2011) 100:619–630
was basically the same under both conditions.
Fatichenti and Berardi (1987) showed that the strain
K. marxianus SS-437 can grow at temperatures
between 3 and 46°C and that the highest lmax values
occur at 41.5°C. Several authors report temperature
values around 40°C to be the ones at which the
duplication time for K. marxianus cells is the lowest.
This is interesting from an industrial point of view,
since it might significantly reduce cooling costs in
bioreactors, which is a real issue in large-scale
fermentations (Rouwenhorst et al. 1988; Hensing
et al. 1995).
Finally, in terms of the preferred C-source or, in
other words, whether cells grow faster on glucose,
lactose or sucrose, it was not possible to observe a
trend in the data presented in Table 1. From an
industrial point of view, it is interesting to note that
K. marxianus grows well on disaccharides, since it
allows flexibility in medium formulation. K. marxi-
anus CBS 712T did not grow on lactose in our
experiments, at least not within 12 h after inoculation,
in spite of the two independent trials we made (results
not shown). This contrasts somewhat with the infor-
mation given on the CBS database (http://www.
cbs.knaw.nl/), where a positive sign (?) is given for
physiological data on lactose. However, incubation
time in CBS tests varies from 3 to 10 days, which is at
least 6 times greater than what was used here. On the
other hand, Rech et al. (1999) showed that this same
strain grows weakly when cells are cultivated on non-
supplemented cheese whey, where the C-source is
also lactose. Thus, it seems that this strain is not
capable of growing on a defined medium with lactose
as the sole C-source, in contrast to its performance on
analogous media containing either glucose or sucrose
as the sole C-source, because it requires some com-
plex nutrient source supply together with lactose.
K. lactis CBS 2359 did not grow at 37°C, as other
K. lactis var. lactis strains, according to the report of
Sidenberg and Lachance (1986).
Conversion of the carbon source into biomass
and extracellular metabolites
The efficiency of cells in transforming substrate into
biomass is represented by the biomass yield on
substrate. Here, two different values of YX/S were
calculated. On the one hand, the net biomass yield
(YNET
X/S ) corresponds to the biomass formed during the
625
whole cultivation (until depletion of the main carbon
source: glucose, lactose or sucrose) and only takes
into account initial and final values of biomass and
substrate concentration. On the other hand, the
biomass yield on substrate during the exponential
growth phase (YEXPX/S ) is calculated using biomass and
substrate concentration data obtained exclusively
during the exponential growth phase. As may be
seen on Table 1, we did not calculate YEXP X/S for the
cultivations carried out using sucrose as the sole
C-source, because this substrate is hydrolysed extra-
cellularly and the resulting glucose and fructose
molecules are not consumed at the same rate at which
sucrose is hydrolysed, probably because in Kluyver-
omyces yeasts hexoses are transported actively, in
contrast to the situation in S. cerevisiae, which is
known to employ a more rapid mechanism for hexose
transport, namely facilitated diffusion (Van Urk et al.
1990). A typical kinetic experiment is shown in
Fig. 1.
Analysing the four strains cultivated on glucose at
30°C (Table 1), K. marxianus CBS 397 presented the
lowest YEXPX/S : 0.40 ± 0.02 g DW g GLC . This
-1
value can be explained by a higher ethanol formation
in this strain, when compared to the others (Fig. 3).
Since K. marxianus is usually considered a Crabtree-
negative yeast, the higher ethanol formation in this
strain might be due to some nutrient limitation which
is specific for this strain (and not for the other ones
investigated here). Although the experiments were
carried out in shake-flasks and dissolved oxygen was
not monitored, an eventual limitation by oxygen,
which could also be the cause of the higher ethanol
formation in the CBS 397 strain, is probably not the
case, since in the cultivations with the CBS 6556
strain, which achieves a higher specific growth rate
than the CBS 397 strain during growth on glucose at
30°C, ethanol formation was much lower than in the
latter case.
Although the YEXP X/S of K. marxianus CBS 397 is
lower than the same parameter obtained for the other
three strains studied here, it still is much higher than
the biomass yield on glucose of S. cerevisiae cells
growing at or close to its lmax. As an example, Nissen
et al. (2000) reported a net biomass yield on substrate
of 0.17 g DW g GLC-1 for S. cerevisiae TN1 cells
growing at a lmax of 0.41 h-1 in batch cultivation,
using the same mineral medium employed in the
present work.
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Fig. 2 Relation between biomass and metabolite formation
during batch cultivations of different K. marxianus and
K. lactis strains grown in shake-flasks on a defined medium
with different single carbon sources (at an initial concentration
of 10 g l-1) and temperatures. The yield of total metabolites on
According to Table 1, K. marxianus CBS 6556
yielded 0.54 g DW g lactose-1 at 30°C. This value is
close to the theoretical one (0.60 g DW g lactose-1),
as modelled by Longhi et al. (2004). For this
particular strain, higher yields were obtained during
growth on lactose than on sucrose. All strains
presented higher biomass yields on sucrose when
grown at 37°C than at 30°C, but only the CBS 712T
strain presented a higher biomass yield on glucose at
37°C than at 30°C.
On Fig. 2, it is possible to verify the relationship
between biomass formation and metabolite produc-
tion. Generally, conditions that led to higher metab-
olite formation also resulted in decreased biomass
yields on substrate, which is expected. Furthermore,
it can also be seen that when K. marxianus strains
were cultivated at 37°C, in general, metabolite
production increased, with respect to 30°C. This
probably occurred due to the higher specific growth
rate that cells achieved at the former temperature,
when compared to the latter. According to Blank and
Sauer (2004), glucose repression of the TCA cycle is
regulated by rates of growth or glucose uptake.
Indeed, the data on K. marxianus CBS 6556
presented by Fonseca et al. (2007) reinforce the
compromise between specific growth rate and ethanol
formation without oxygen limitation (which was
123
Antonie van Leeuwenhoek (2011) 100:619–630
the C-source was calculated as the sum of the carbon content in
all metabolites detected (considering the maximum concentra-
tion of extracellular metabolites formed) divided by the carbon
content in the carbon source
maintained above 50% in their study), as they
observed no ethanol formation at a dilution rate of
0.1 h-1, while at D = 0.25 h-1 by-products were
formed, although to a low extent.
Ethanol formation by yeasts is usually accompa-
nied by the production of other metabolites, including
weak organic acids (Verduyn et al. 1992). Here,
excretion of acetate, and also succinate and pyruvate
to a lower extent, were observed (Fig. 3). This is in
accordance with the results of Fonseca et al. (2007),
who observed acetate and pyruvate excretion to a
larger extent than ethanol when K. marxianus CBS
6556 was grown in bioreactors under full aerobiosis.
Glycerol is also a by-product of ethanolic fermenta-
tion in S. cerevisiae (Nissen et al. 1997) and its
formation plays a role both in the reoxidation of
excess NADH formed in biosynthetic reactions
(Overkamp et al. 2002) and in osmoprotection. As
may be seen from Fig. 3, glycerol was also formed by
all Kluyveromyces strains studied here, albeit to a low
extent (\5 mM).
Although it is not possible to assure that all
cultivations reported here were carried out without
any degree of oxygen limitation, in the cases in which
glucose was the sole carbon source, it was possible to
confirm the Crabtree-negative status of K. marxianus
and K. lactis, due to the small or non-detected ethanol
Antonie van Leeuwenhoek (2011) 100:619–630
Fig. 3 Metabolites produced during batch cultivations of
a K. marxianus CBS 6556, b K. marxianus CBS 397,
c K. marxianus CBS 712T, d K. lactis CBS 2359. Yeast cells
formation, in accordance to previously reported data
(Bellaver et al. 2004; Kiers et al. 1998; Fonseca et al.
2007). The exception was K. marxianus CBS 397,
which produced higher amounts of ethanol than the
other strains, at both tested temperatures: 30°C and
37°C. Since all cultivations were carried out under
exactly the same conditions of agitation, flask
geometry and volume of liquid to volume of flask
ratio, this observation indicates that this particular
strain suffers from a stronger Crabtree effect than the
other ones, as already discussed by Mahmoud and
Kosikowski (1982), who revealed that K. marxianus
CBS 397 was the best ethanol producer among five
Kluyveromyces strains investigated by those authors
(results not shown).
The strain that presented the lowest by-product
formation, among all strains investigated here, was
K. marxianus CBS 712T. Accordingly, this strain also
627
were grown on a defined medium with different single carbon
sources (at an initial concentration of 10 g l-1) and
temperatures
presented high YX/S values and the lowest lmaxval-
ues, among the strains studied (Table 1), which
points to a relation between rate of growth or sugar
consumption and repression of TCA cycle and
respiratory activities, as proposed by Blank and
Sauer (2004). In the cultivations with lactose as the
sole C-source at 30°C, both K. marxianus strains CBS
6556 and CBS 397 excreted fewer metabolites than
the same strains grown on glucose as the sole
C-source at the same temperature. On the other hand,
K. lactis CBS 2359 presented a high ethanol forma-
tion under this condition, around 21 mmol l-1. Some
authors reported ethanol formation by this strain
under aerobiosis using Erlenmeyer flasks (Mulder
et al. 1995) or when complex medium was utilized
(González-Siso et al.1996). Kiers et al. (1998)
described insufficient oxygen transfer in K. lactis
CBS 2359 cultivations carried out in shake flasks.
123
628
When cells were cultivated in well-aerated laboratory
fermentors, ethanol concentration was below the
detection limit. Another hypothesis that can explain
ethanol formation by this strain is that growth was
limited by nicotinic acid (vitamin B3). Kiers et al.
(1998) verified B3 vitamin limitation when K. lactis
CBS 2359 grew at high growth rates, in the same
mineral medium employed here. We suppose that one
of the two alternatives above occurred when K. lactis
cells grew on sucrose.
Although we wished to correlate the physiological
data obtained here with the genotypes of the inves-
tigated strains, there is limited information available
in the literature on the genotypes of the CBS 397 and
the CBS 6556 strains (for the CBS 712T strain, there
are data covering roughly 20% of its genome
sequence and for the K. lactis CBS 2359 strain, a
full genome sequence is available; Llorente et al.
2000; Dujon et al. 2004). Only a few genes were
sequenced in all four strains investigated here.
Belloch et al. (2002) sequenced the 5.8S-rRNA gene
and the Internal Transcribed Spacers 1 and 2. The
results show that the CBS 397 strain presents one
nucleotide difference when compared to the CBS
712T and CBS 6556 strains. Besides this, in a
previous work, the same group (Belloch et al.
1998b) had observed a different karyotype for the
CBS 712T strain, when compared to the CBS 397
strain. This limited information does not allow to
correlate physiology with genotype. Thus, future
work will require sequencing of more genes, mainly
those involved in central metabolism and its regula-
tion, in order to elucidate the genotype-phenotype
relationship in the highly polymorphic Kluyveromy-
ces marxianus taxon.
Production of extracellular inulinase
The capacity of growing on inulin as the sole carbon
source is a quite exclusive feature of the yeast
Kluyveromyces marxianus (Lachance 1998). Expres-
sion of the inulinase gene KmINU1 is mainly induced
by inulin, although sucrose and fructose are also
known as inducers (Kushi et al. 2000). Here, the
activity of extracellular inulinase was measured in
each sample taken during the cultivations in which
sucrose was the single carbon source. We decided to
use sucrose as an inducer of inulinase formation,
since this is the main sugar found in cheap raw
123
Antonie van Leeuwenhoek (2011) 100:619–630
materials, such as sugar-cane juice, which may be
commonly used for K. marxianus growth and inulin-
ase production, particularly in places where sugar-
cane or sugar-beet can be cultivated.
According to Table 1, K. marxianus CBS 6556
presented a higher specific extracellular inulinase
activity, when compared to the other two K. marxi-
anus strains studied. At 30°C, the inulinase activity
was 1.24 ± 0.06 U mg DW-1 and at 37°C, the
activity increased to 1.65 ± 0.20 U mg DW-1. The
strains K. marxianus CBS 397 and K. marxianus CBS
712T presented, respectively, maximum activities of
0.59 ± 0.09 and 0.46 ± 0.01 U mg DW-1 at 30°C.
At the higher temperature investigated (37°C), the
activities measured in both strains were 0.83 ± 0.17
and 0.50 ± 0.01 U mg DW-1, respectively. Rou-
wenhorst et al. (1988) determined similar extracellu-
lar inulinase activities for K. marxianus CBS 6556.
Growth temperature influences extracellular inu-
linase production. Rouwenhorst et al. (1988) grew
K. marxianus CBS 6556 at various temperatures and
observed that inulinase activity was higher when the
yeast was cultivated at temperatures close to its
growth optimum, i.e., between 37 and 42°C. Mazutti
et al. (2006) report the highest inulinase production
when K. marxianus NRRL Y-7571 grew at 36°C.
These results agree with our observations, since
inulinase specific activities were always higher at
37°C than at 30°C (Table 1).
The strain K. lactis CBS 2359 was also tested for
inulinase production. As expected, no extracellular
inulinase activity was detected (Rouwenhorst et al.
1990).
Acknowledgments The first author acknowledges a doctoral
grant received from Fundação de Amparo à Pesquisa do Estado
de São Paulo (FAPESP, São Paulo, Brazil). This work was
financially supported by FAPESP and by Conselho Nacional de
Desenvolvimento Cientı́fico e Tecnológico (CNPq, Brası́lia,
Brazil).
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