BBA - General Subjects 1866 (2022) 130154

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BBA - General Subjects

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Xylose and yeasts: A story beyond xylitol production

Alejandra Karina Estrada-Ávila a, Juan Carlos González-Hernández b, Martha Calahorra a, Norma
Silvia Sánchez a, Antonio Peña a, *
a
Departamento de Genética Molecular, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Circuito Exterior s/n, Ciudad Universitaria, 04510,
México City, México
b
Tecnológico Nacional de México / Instituto Tecnológico de Morelia, Departamento de Ingeniería Química y Bioquímica, Av. Tecnológico # 1500. Colonia Lomas de
Santiaguito, 58120 Morelia, Michoacán, México

A R T I C L E I N F O A B S T R A C T

Keywords: Six different yeasts were used to study their metabolism of glucose and xylose, and mainly their capacity to
Yeast produce ethanol and xylitol. The strains used were Candida guilliermondii, Debaryomyces hansenii, Saccharomyces
Xylose cerevisiae, Kluyveromyces marxianus, Meyerozyma guilliermondii and Clavispora lusitaniae, four isolated from a rural
Xylitol
mezcal fermentation facility. All of them produced ethanol when the substrate was glucose. When incubated in a
Sugar transport
Respiratory pathway
medium containing xylose instead of glucose, only K. marxianus and M. guilliermondii were able to produce
ethanol from xylose. On the other hand, all of them could produce some xylitol from xylose, but the most active
in this regard were K. marxianus, M. guilliermondii, C. lusitaniae, and C. guilliermondii with the highest amount of
xylitol produced. The capacity of all strains to take up glucose and xylose was also studied. Xylose, in different
degrees, produced a redox imbalance in all yeasts. Respiration capacity was also studied with glucose or xylose,
where C. guilliermondii, D. hansenii, K. marxianus and M. guilliermondii showed higher cyanide resistant respiration
when grown in xylose. Neither xylose transport nor xylitol production were enhanced by an acidic environment
(pH 4), which can be interpreted as the absence of a proton/sugar symporter mechanism for xylose transport,
except for C. lusitaniae. The effects produced by xylose and their magnitude depend on the background of the
studied yeast and the conditions in which these are studied.

1. Introduction
Xylose, a pentose sugar, is one of the primary products of lignocel­
lulose degradation. It is, after glucose, the second most abundant car­
bohydrate in nature [1]. The importance of this sugar lies in the ability
of certain bacteria, yeast or fungi that not only ferment glucose but also
xylose into ethanol [2] which can be employed in biofuel production. As
for yeast, strains from Pichia and Candida are known as pentose fer­
menting yeasts [3]. Nevertheless, certain yeasts can also convert xylose
into xylitol and other compounds [4]. Xylitol is widely used as a
sweetener recommended for diabetic patients [5]. Highest yields of
xylitol obtained from xylose have been reported for Candida sp. with up
to 17 g L− 1 [4].
For xylitol to be produced, xylose must be first transported inside the
cell, and this represents the first bottleneck for xylose fermentation. A
common pattern has been reported in Candida shehatae [6], Rhodotorula
glutinis [7], D. hansenii [8], K. marxianus [9] and Candida intermedia
[10,11]. Under repression by glucose, cells present a low affinity facil­
itated diffusion xylose/glucose transporter (Km > 100 mM). Reported
genes for this transporter are SUT1 (sugar transporter 1) in Pichia stipitis,
and GXF1 (glucose/xylose facilitator 1) in C. intermedia. When grown in
xylose with or without glucose they express a high affinity sugar/proton
symporter. Genes such as GXS1 (glucose/xylose symporter 1) in
C. intermedia and XYLH in D. hansenii have been studied [12,13]. In
S. cerevisiae, facilitated diffusion of xylose is carried out by transport
proteins encoded by the HXT (hexose transporter) genes. However,
transport is much less efficient for pentoses than for glucose since pro­
teins encoded by HXT1, HXT6 and HXT7 genes show higher affinity for
glucose [13], but after several mutations in these proteins and expres­
sion of transporters genes from C. intermedia, P. stipitis and Arabidopsis
thaliana in S. cerevisiae; an increase in xylose uptake has been reported
[14–16].
Once xylose is inside the yeast, it is reduced into xylitol by a NADPH-
dependent enzyme: xylose reductase [XR]; subsequently xylitol is

* Corresponding author.
E-mail address: apd@ifc.unam.mx (A. Peña).

https://doi.org/10.1016/j.bbagen.2022.130154
Received 22 November 2021; Received in revised form 30 March 2022; Accepted 13 April 2022
Available online 22 April 2022
0304-4165/© 2022 Published by Elsevier B.V.
A.K. Estrada-Ávila et al.
oxidized to xylulose by a NAD+-dependent xylitol dehydrogenase [XDH]
[17]. Both enzymes are induced by xylose [18]. Under oxygen limita­
tion, NADH cannot be reoxidized by the electron transport chain, so its
intracellular concentration increases, and xylitol is accumulated [19].
This may be considered as a second bottleneck if xylitol is not the desired
product; nonetheless, in strains of P. stipitis, C. shehatae and Pachysolen
tannophilus there is a NADH/NADPH-dependent XR, which prevents a
redox imbalance and allows the anaerobic formation of ethanol without
xylitol formation [20–23] (Fig. 1). Previous studies on recombinant
S. cerevisiae harboring genes from P. stipitis XR and XDH proved that
besides the cofactor utilization of XR there is an additional mechanism
or response that establishes a redox balance when xylose is being
consumed, and so the presence of a Cyanide Resistant Respiration (CRR)
was reported in xylose-consuming yeast [24].
In this study, metabolism and effects of glucose and xylose were
analyzed in six different yeasts. We used laboratory wild-type strains of
C. guilliermondii and D. hansenii, and strains from S. cerevisiae, K. marx­
ianus, M. guilliermondii and C. lusitaniae isolated from mezcal fermen­
tations to understand how xylose is transported and transformed,
whether it has consequences at a respiratory level, and how these pro­
cesses vary from yeast to yeast.
2. Materials and methods
2.1. Yeast strains
Six different yeast strains were used in this study: Candida guillier­
mondii NRRL Y-2075 United States Department of Agriculture, Debar­
yomyces hansenii Y-7426 (IFC, UNAM) and four strains that were isolated
from mezcal production in Michoacán, México: Saccharomyces cerevisiae
ITM2014, Kluyveromyces marxianus, Meyerozyma guilliermondii and
Clavispora lusitaniae (strains from ITM were from the collection of Dr.
Juan Carlos González Hernández from Instituto Tecnológico de Morelia
(ITM), México).
2.2. Media and culture conditions
Cells were grown at 30◦ C for 24 h in YPD or YPX (2% sugar: D =
Fig. 1. Proposed pathway for xylose and glucose in yeast described by Hahn-Hägerdal, et al. (1994) [22].
2
BBA - General Subjects 1866 (2022) 130154
Dextrose; X = Xylose, 2% gelatin peptone, 1% yeast extract, 2% agar)
plates, and then maintained at 4◦ C. Cultures were renewed once a month
and used to grow the cells for the experiments. Liquid YPD or YPX
(without agar) were also used to grow the cells. When adjusted pH was
desired, 50 mM tartaric acid-NaOH was used for pH 4, and 50 mM MES-
TEA buffer (morpholinoethane sulfonic acid taken to pH 6 with trie­
thanolamine) for pH 6. Media were autoclaved at 121◦ C, 15 min. A
solution of 20% xylose (≥99%, Sigma-Aldrich ®) was sterilized by
filtration using Stericup Millipore® 0.22 μm, and subsequently added to
YPX media. Cells were grown in 500 mL of medium, in 1 L Erlenmeyer
flasks in an orbital shaker at 30◦ C and 250 rpm for 24–28 h. When
required, cells were fasted by collecting them and washing once with
water by centrifugation, suspended in 250 mL of water and placed again
at 30◦ C and 250 rpm for 24–48 h.
2.3. Carbon source assimilation
To test whether the strains were able to assimilate xylose and grow,
drop tests in Petri dishes with YPD or YPX agar were performed. Strains
were previously grown in liquid YPD or YPX medium for 24 h. Cell
concentration was measured as the optical density (OD) at 600 nm in a
Beckman DU®650 spectrophotometer. Starting with 106 cells mL− 1, ten-
fold serial dilutions were made (105,104,103,102,101 cells mL− 1) on a 96
well plate and spotted on Petri dishes using a sterile multi pin aluminum
device. Plates were incubated at 30◦ C for 48 h.
2.4. Growth and alcohol production
For the growth measurements a loopful of cells was placed in 100 mL
of YPD or YPX medium and grown at 30◦ C and 250 rpm for 12–24 h.
Afterwards, optical density was measured at 600 nm in the spectro­
photometer. The equivalent volume for OD600nm = 0.1 was placed in
100 mL of YPD or YPX medium with and without adjusting pH in
nephelometric flasks. Optical density was then measured with a Klett-
Summerson photocolorimeter every 2 h for 36 h. Measurements were
started at an initial value of 50 Klett Units (OD600nm = 0.1). Growth
curves were obtained with GraphPad Prism 6 software and maximum
growth rates were calculated with the data obtained from these curves.
A.K. Estrada-Ávila et al.
Total biomass, sugar consumption and ethanol production were
analyzed only in the adjusted pH media. 5 mL samples were taken out of
each flask at 0 and 36 h (for YPD media) or 48 h (for YPX media). Total
dry weight biomass was measured with 1 mL of each sample. The
amount of glucose, xylose and xylitol was estimated with D-fructose and
D-glucose Assay Kit, D-xylose Assay Kit from Megazyme® and D-sorbi­
tol/xylitol Assay kit from Boehringer Mannheim/R-Biopharm, respec­
tively. Ethanol was measured by reduction of NAD+ (at 340 nm) in the
presence of alcohol dehydrogenase [25].
2.5. Sugar uptake and transport
Cells were grown in YPD (for glucose transport) or YPX (for xylose
transport) for 24 h, then fasted for 12 h and suspended in water to
achieve a concentration of 500 mg mL− 1 (wet weight). Uptake and
transport of glucose and xylose among the different yeasts was measured
using D-[14C(U)]-Glucose (250 mCi mmol− 1) and D-[14C(U)]-Xylose
(200 mCi mmol− 1) from Perkin Elmer. Uptake was estimated in a final
volume of 2 mL (1 mM14C-glucose or 14C-xylose, pH 4 or pH 6 buffer, 10
mg yeast). 200 μL of each sample were taken out at different times (0,
0.5, 1, 2, 4, 10 and 20 min), passed through a cellulose filter (Millipore,
0.45 mm HAWP) and washed with 10 mM of unlabeled glucose or
xylose. To obtain transport kinetic parameters, cells were washed and
prepared as mentioned above and they were estimated in a final volume
of 1 mL (0.1, 0.5, 1.0 and 2.0 mM 14C-glucose/ 14C-xylose, pH 4 or pH 6
buffer, 10 mg yeast). Within 5 min after the addition of the yeast, 200 μL
were taken out of each sample, passed through a cellulose filter and
washed with glucose or xylose at 10 mM. Filters with each sample were
placed in vials with 5 mL of scintillation liquid (1 L: 257 mL triton X-100,
37 mL ethylene glycol, 106 mL ethanol, 600 mL xylene and 3 g 2,5-
diphenyloxazole). Data were obtained with a Liquid Scintillation
Analyzer Tri-Carb 2910 TR Perkin Elmer.
Uptake rates of both sugars were plotted against time with GraphPad
Prism 6 software. Transport rates of both sugars were plotted against the
concentration of used sugar. Plots were adjusted by non-linear regres­
sion of the Michaelis-Menten equation to calculate Vmax and Km values
with GraphPad Prism 6 software.
2.6. NADH-NADPH fluorescence
Changes of NADH-NADPH fluorescence in response to the carbon
source (glucose or xylose) were measured as previously described by
Peña et al. (2015) [26] following the fluorescence increase at 340–450
nm in a spectrofluorometer (SLM-Olis) at 30◦ C with 10 mM MES-TEA
pH 6 and 50 mg of cells, using 200 μM NaCN and 30 μM octylgallate,
or both as respiratory chain inhibitors. Cells were grown in YPD or YPX
for 24 h and fasted for 24 h, or 48 h for C. guilliermondii. Traces were
followed up for 300 s and plotted in GraphPad Prism 6 software.
2.7. Oxygen consumption
Oxygen consumption was measured with a Clark oxygen electrode in
a closed chamber at 30◦ C. Yeasts were grown in YPD, YPX or YP
(without carbon source) for 24 h and fasted for 24 h. 250 mg of cells
were placed in 10 mM MES-TEA pH 6 and 40 mM of glucose or xylose.
Traces were followed up for 350 s. NaCN (200 μM at 100 s) and octyl­
gallate (30 μM at 150 s) were added as respiratory chain inhibitors to
compare their effects with glucose and xylose on respiration of each
yeast. Oxygen values are given as nanoatoms gram (natg) of oxygen,
considering that the oxygen concentration in water in Mexico City
(2250 m above the sea level) is 400 natg O mL− 1. Data were plotted and
statistically analyzed with GraphPad Prism 6 software.
2.8. Medium pH changes
Changes in the medium pH (acidification, or alkalization due to a
3
BBA - General Subjects 1866 (2022) 130154
possible xylose/ H+ symporter), were followed with an electrode con­
nected to a pHmeter and a computer to register data. Based on tech­
niques previously described by Misra and Höfer (1975) [27], Deak
(1978) [28] and Peña et al., (2015) [26]; 9.5 mL of either 2 mM tartaric
acid-NaOH, pH 4, or 2 mM MES-TEA, pH 6 were placed in a water
jacketed glass chamber with constant stirring at 30◦ C. At 10s, 100 mg of
cells, wet weight (grown in YPD or YPX and fasted for 24 h) were added
and at 100 s 1 mM of sugar (glucose/xylose) was added. Traces were
followed up for 250–300 s and data plotted with GraphPad Prism 6
software.
Moreover, a comparative test was run based on the techniques from
Leandro et al. [11]. 5 mL of H2O and 20 mM of sugar (glucose/xylose)
were placed in a water jacketed glass chamber with constant stirring at
30◦ C. Tests were performed at pH 4 and pH 5. At 10s, 50 mg of cells, wet
weight (grown in YNB with amino acids and 2% xylose for 24 h or 48 h)
were added. Traces were followed up for 300–800 s and data plotted
with Excel software.
3. Results
3.1. Carbon source and growth
As a first approach to study xylose utilization by the different yeasts,
they were grown in plates with glucose or xylose after previous incu­
bation in liquid YPD or YPX; in order to define whether growth in solid
medium was influenced by the medium they came from. In Supple­
mentary Fig. 1, yeasts were grown 24 h in YPD and then spotted on
either YPD or YPX plates. Excepting D. hansenii and S. cerevisiae, with
less growth, all other yeasts grew well in YPD. When grown in YPX, very
little growth was observed for S. cerevisiae, D. hansenii and C. lusitaniae.
Supplementary Fig. 2 shows results when yeasts had been grown in YPX
for 24 h; no difference between those previously grown with YPD was
found.
3.2. Growth kinetics
In YPD, as presented in Fig. 2, all yeasts grew rather similarly, except
for D. hansenii, that showed a larger lag phase and slower growth,
reaching however a similar maximum growth after 36 h.
In YPX, growth was slower for all strains, particularly D. hansenii,
C. lusitaniae and more so S. cerevisiae, which showed a much slower, and
Fig. 2. Growth curves in YPD. Yeasts were grown in YPD medium at 30◦ C and
250 rpm. Readings were made every 2 h for 36 h in a Klett-Summerson pho­
tocolorimeter. Initial reading was of 50 Klett Units (KU) = 0.1 OD600 nm. Data
were plotted with GraphPad Prism 6 software. n = 3. ( ) C. guilliermondii, ( )
S. cerevisiae, ( ) D. hansenii, ( ) K. marxianus, ( ) M. guilliermondii, (x)
C. lusitaniae.
A.K. Estrada-Ávila et al.
lower final growth than the other strains (Fig. 3).
Medium pH is known since very long to affect metabolism of
S. cerevisiae [29,30], and D. hansenii [31]. Therefore, it was considered
important to observe the growth behavior of the several yeasts in both
YPD and YPX to which pH was adjusted to 4 and 6 (Suppl. Figs. 3–6). In
YPD at pH 4, D. hansenii clearly showed a slower, but similar final
growth as compared to the other yeasts (Suppl. Fig. 3). Additionally,
M. guilliermondii showed the fastest and highest final growth values. At
pH 6 (Suppl. Fig. 4) again, D. hansenii showed a slower, but also lower
final growth than the other strains.
In YPX, mainly lower growth rates than in YPD were evident at both
pH values. As expected, D. hansenii, and S. cerevisiae, displayed dimin­
ished growth rates and low total yield at pH 4 (Suppl. Fig. 5). At pH 6,
C. lusitaniae also showed lower growth rate and yield (Suppl. Fig. 6).
Growth rates at both pH values with YPD or YPX are shown in Table 1.
In another experiment, biomass production, as well as ethanol yields
per glucose consumed during 36 h were measured at pH 4 and 6
(Table 2). Regarding biomass produced, in g L− 1, values were rather
high and similar for all yeasts, except for K. marxianus and D. hansenii,
with significant lower values, and also a significant higher value for
M. guilliermondii. Besides, in all cases, except for M guilliermondii, g L− 1 of
biomass were higher at pH 6 than at pH 4. As for the biomass yield per g
of glucose consumed at pH 4, the lower values were for D. hansenii and
S. cerevisiae. At pH 6 once more, D. hansenii and K. marxianus gave the
lowest values, and again, M. guilliermondii displayed the highest value in
terms of biomass produced per g of glucose consumed. Regarding the
ethanol yield per g of glucose consumed, all yeasts gave similar values,
except for D. hansenii, which clearly produced the lowest value at both
pH values. Besides, the net fermentation was similar at both pH values.
An additional but similar experiment was performed using YPX as
the growth medium, but incubation was carried out during 48 h, in order
to obtain higher biomass values in g L− 1, (Table 3). In this case the
lowest values were for S. cerevisiae, followed by D. hansenii and
C. lusitaniae. D. hansenii, besides, gave a still lower value at pH 6 than at
pH 4. The highest values were observed for M. guilliermondii and
C. guilliermondii.
Regarding biomass yield, all strains gave similar values, excepting
S. cerevisiae at pH 4, which may be explained because this yeast has an
almost null capacity to use xylose. As to the production of ethanol from
xylose, only K. marxianus and M. guilliermondii produced a small amount.
Finally, xylitol was produced in small amounts, as compared to
Fig. 3. Growth curves in YPX. Yeasts were grown in YPX medium at 30◦ C and
250 rpm. Readings were made every 2 h for 36 h in a Klett-Summerson pho­
tocolorimeter. Initial reading was of 50 Klett Units (KU) = 0.1 OD600 nm. Data
were plotted with GraphPad Prism 6 software. n = 3. ( ) C. guilliermondii, ( )
S. cerevisiae, ( ) D. hansenii, ( ) K. marxianus, ( ) M. guilliermondii, (x)
C. lusitaniae.
4
BBA - General Subjects 1866 (2022) 130154
ethanol with glucose. Nevertheless, it was produced in significant
amounts, in decreasing order, by C. guilliermondii, C. lusitaniae,
K. marxianus, M. guilliermondii, and surprisingly, by D. hansenii, more so
at pH 6.
3.3. Sugar consumption
Consumption of glucose (Table 4) was measured after 12 h in YPD
and found between 1.46 and 2.08 g L− 1. C. lusitaniae had the lowest
consumption rate, but it was also interesting that D. hansenii was that
with the highest consumption capacity. As for xylose (YPX), even though
values were obtained after 48 h, rates were much lower, from 0.10 to
0.64 g L− 1 (Table 4). Consistent with its low growth in YPX, the lowest
xylose consumption was shown by S. cerevisiae, and the highest by
M. guilliermondii. No large differences between pH 4 and pH 6 were
found for any sugar. Besides, glucose was depleted by all yeasts after 12
h, while at 48 h only 50% of xylose was consumed (data not shown).
3.4. Sugar transport
Transport of both sugars was measured at pH 4 and pH 6 with fasted
yeasts grown in both YPD for glucose assays and in YPX for xylose as­
says. A final concentration of 1 mM (glucose or xylose) was used to
analyze transport.
For glucose, (Suppl. Fig. 7), in C. guilliermondii, S. cerevisiae and
C. lusitaniae the curve saturated at 5 min transporting approximately 15
μmol of glucose per gram of yeast. D. hansenii transported the same
amount with saturation at 10 min. However, K. marxianus and
M. guilliermondii transported only around 5 μmol g− 1, saturating shortly
before 5 min. None of them displayed a significantly different transport
of glucose at pH 4 or 6.
Regarding xylose transport, Suppl. Fig. 8 shows that C. guilliermondii
transported more than 15 μmol g− 1 in 20 min. Next was D. hansenii with
4 μmol g− 1, C. lusitaniae transported up to 3 μmol g− 1, while
K. marxianus and M. guilliermondii less than 2 μmol g− 1. None of the
above presented saturation at 20 min. S. cerevisiae transported less than
1 μmol g− 1. No significantly different transport of xylose at any pH value
was observed.
3.5. Sugar transport kinetics
To get more information about the uptake of both sugars, it was
considered relevant to determine the kinetic constants (Table 5).
Transport kinetics were carried out for 5 min, using sugar concen­
trations from 0.01 mM to 2 mM. Vmax and Km were calculated from the
plots (Suppl. Figs. 7 and 8) obtained from these experiments. As shown
in Table 5, Vmax for glucose transport went from 7.25 μmol (g min)− 1 up
to 55.9, being D. hansenii the yeast with the lowest value at pH 4 and the
second lowest at pH 6. For xylose, the lowest Vmax value was for
S. cerevisiae (1*10− 13 and 5*10− 13 μmol (g min)− 1 for pH 4 and pH 6
respectively, these values are only a representation of the transport ki­
netics behavior, indicating an almost null transport of xylose (accurate
values could not be adjusted by non-linear regression of the Michaelis-
Menten equation), and the highest were of 33.79 and 22.36 μmol (g
min)− 1 for C. lusitaniae. C. guilliermondii and D. hansenii had similar Vmax
at pH 4 and 6 for glucose and xylose, and for S. cerevisiae, K. marxianus
and M. guilliermondii, Vmax diminished when xylose was transported,
contrary to C. lusitaniae.
The lowest Km for glucose was displayed by D. hansenii at both pH
values, nevertheless Km values for glucose transport were not higher
than 10 μM. As for xylose, lowest values of Km were those of
C. guilliermondii at pH 4 and 6, but no great difference was seen in those
of K. marxianus pH 4 and M. guilliermondii pH 6. S. cerevisiae had the
highest Km values for xylose transport and the lowest ones for Vmax.
However, all this information, although useful, except for
S. cerevisiae, has no meaning for the growth and fermentation
A.K. Estrada-Ávila et al. BBA - General Subjects 1866 (2022) 130154

Fig. 4. NADH/NADPH production with YPD-grown yeast. Yeasts were grown in YPD medium for 24 h and fasted for 24 h (C. guilliermondii 48 h). Reduction of
NAD+/NADP+ was observed by fluorescence (340–460 nm) in MES-TEA 10 mM pH 6 buffer without sugar (–), after glucose addition ( ) and after xylose addition
( ). Sugars were used at 20 mM and added after 30 s. Traces were followed up for 300 s. NaCN (200 μM) and octylgallate (30 μM) were used as respiratory in­
hibitors. Yeasts (50 mg, wet weight) were added. Representative data of n = 3. Data were plotted with GraphPad Prism 6 software.

Fig. 5. NADH/NADPH production in YPX-grown yeast. Yeasts were grown in YPX medium for 24 h and fasted for 24 h (C. guilliermondii 48 h). Reduction of NAD+/
NADP+ was observed by fluorescence (340–450 nm) in MES-TEA 10 mM pH 6 buffer without sugar (–), after glucose addition ( ) and after xylose addition ( ).
Sugars were added after 30 s at 20 mM. Traces were followed up for 300 s. NaCN (200 μM) and octylgallate (30 μM) were used as respiratory inhibitors. Yeasts were
used at a final concentration of 50 mg mL− 1. Representative data of n = 3. Data was plotted with GraphPad Prism 6 software.

experiments that were performed in either YPD or YPX, in which sugars 3.6. Ethanol and xylitol production
were performed with concentrations of sugars higher than 0.1 M.
In these experiments, the pH of the medium was also adjusted to 4 or
6.Table 6 shows the ethanol production in YPD and YPX at both pH

5
A.K. Estrada-Ávila et al. BBA - General Subjects 1866 (2022) 130154

Fig. 6. Medium acidification, YPD-grown yeasts. External pH was observed in MES-TEA 2 mM pH 6 buffer with yeasts grown in YPD for 24 h and fasted for 24 h
without sugar ( ), after the addition of glucose ( ) or xylose ( ). Sugars were used at 20 mM and added where the arrow indicates. Representative data of n = 3.
Data was plotted with GraphPad Prism 6 software.

Table 1 Table 2
Maximum growth rate of yeasts in the different media. Biomass produced, and biomass and ethanol yields per glucose consumed in
Yeast μmax (h¡1)
YPD, 36 h.
Yeast Biomass Biomass yield Ethanol yield
YPD YPX YPD YPD YPX YPX
(g L− 1) (g g− 1) of glucose (g g− 1) of glucose
pH 4 pH 6 pH 4 pH 6
pH 4 pH 6 pH 4 pH 6 pH 4 pH 6
0.75 0.71 0.86 1.09 1.06 1.18
C. guilliermondii
± 0.06 ± 0.08 ± 0.05 ± 0.20 ± 0.08 ± 0.04 4.92 5.37 0.163 0.170 0.590 0.580
C. guilliermondii
0.88 0.45 0.84 0.90 1.10 0.49 ± 1.8 ± 2.2 ± 0.1 ± 0.1 ± 0.1 ± 0.1
S. cerevisiae
± 0.07 ± 0.06 ± 0.10 ± 0.08 ± 0.03 ± 0.03 4.33 5.50 0.116 0.186 0.580 0.510
S. cerevisiae
0.44 0.45 0.57 0.60 0.88 0.64 ± 2.4 ± 0.9 ± 0.1 ± 0.0 ± 0.1 ± 0.1
D. hansenii
± 0.16 ± 0.09 ± 0.18 ± 0.15 ± 0.09 ± 0.19 2.70 3.40 0.093 0.116 0.262 0.243
D. hansenii
1.20 0.74 0.97 1.03 1.08 0.95 ± 0.6 ± 0.8 ± 0.0 ± 0.0 ± 0.1* ± 0.0*
K. marxianus
± 0.20 ± 0.09 ± 0.10 ± 0.12 ± 0.04 ± 0.25 3.07 3.37 0.206 0.123 0.500 0.500
K. marxianus
0.97 0.73 1.02 0.86 1.32 0.92 ± 1.7 ± 1.1 ± 0.0 ± 0.1 ± 0.1 ± 0.0
M. guilliermondii
± 0.13 ± 0.02 ± 0.25 ± 0.13 ± 0.23 ± 0.31 6.10 3.77 0.286 0.475 0.510 0.455
M. guilliermondii
1.01 0.48 0.98 0.86 1.01 0.94 ± 2.4 ± 2.8 ± 0.1 ± 0.1* ± 0.1 ± 0.1
C. lusitaniae
± 0.13 ± 0.07 ± 0.07 ± 0.07 ± 0.11 ± 0.07 3.60 4.15 0.295 0.263 0.400 0.380
C. lusitaniae
± 1.1 ± 0.1 ± 0.1 ± 0.2 ± 0.2 ± 0.1
Data from experiments described in Figs. 2 and 3, and Supplementary Figs. 3–6.
n = 3. Total biomass was obtained from the dry weight of 1 mL of each sample after 36
h. Yields were obtained by measuring glucose and ethanol after 36 h. Glucose in
values and xylitol production in YPX. Ethanol levels in YPD reached up the medium was analyzed with an assay kit, and ethanol was measured by the
reduction of NAD+. Biomass is expressed as g L− 1, dry weight. Biomass yield is
to about 16 g L− 1by M. guilliermondii. No difference was observed be­
expressed as g per g of glucose consumed; ethanol yield, as ethanol produced per
tween pH 4 and pH 6. D. hansenii produced the lowest amounts at both
g of consumed glucose. n = 2–4. *Statistical difference.
pH values.
When the sugar was xylose (YPX), ethanol production was zero for all
for growth. Yeasts were also fasted to deplete them as much as possible
strains, except for small values with K. marxianus and M. guilliermondii.
of endogenous substrates.
On the other hand, xylitol production was significantly larger, except for
Changes with YPD grown cells are seen in Fig. 4. As expected, when
S. cerevisiae, which produced a very low amount and there was no dif­
no substrate was added, the fluorescence changes were low, and
ference between pH 4 and pH 6.
remained so in all tracings. Upon the addition of glucose, an immediate
increase was observed in most strains, in the following order:
3.7. NADH changes C. lusitaniae > S. cerevisiae > M. guilliermondii > C. guilliermondii >
K. marxianus > D. hansenii. However, only in S. cerevisiae, the large
Metabolism of both glucose and xylose should reduce NAD+ or initial fluorescence increase was followed by an also large and rapid
NADP+ to NADH and NADPH, which when excited at 340 nm fluoresce decrease, indicating that glycolysis was functioning normally: the initial
at 450 nm. Yeasts were grown both in YPD and YPX to determine if there increase produced by the reduction of glyceraldehyde-3-phosphate
was any influence in NADH production according to the medium used producing NADH, was followed by a decrease by the reduction of

6
A.K. Estrada-Ávila et al. BBA - General Subjects 1866 (2022) 130154

Table 3
Biomass production, biomass and fermentation yields per xylose consumed in YPX, 48 h.
Yeast Biomass Biomass yield Ethanol yield Xylitol yield
(g L− 1) (g g− 1) of xylose (g g− 1) of xylose (g g− 1) of xylose

pH 4 pH 6 pH 4 pH 6 pH 4 pH 6 pH 4 pH 6

C. guilliermondii 7.93 ± 1.70 7.10 ± 1.30 0.35 ± 0.06 0.44 ± 0.06 0 0 0.28 ± 0.04 0.20 ± 0.08
S. cerevisiae 3.50 ± 0.51 3.77 ± 0.68 0.13 ± 0.04 0.22 ± 0.03 0 0 0.04 ± 0.04* 0.02 ± 0.00*
D. hansenii 5.30 ± 0.96 4.10 ± 1.21 0.23 ± 0.07 0.15 ± 0.04 0 0 0.10 ± 0.04 0.17 ± 0.05
K. marxianus 8.50 ± 0.10 7.43 ± 1.93 0.37 ± 0.15 0.28 ± 0.16 0.11 ± 0.06 0.07 ± 0.04 0.18 ± 0.07 0.15 ± 0.04
C. guilliermondii 7.27 ± 1.02 8.60 ± 1.31 0.28 ± 0.07 0.28 ± 0.14 0.04 ± 0.12 0.06 ± 0.02 0.12 ± 0.06 0.13 ± 0.06
C. lusitaniae 5.50 ± 1.27 4.50 ± 0.70 0.43 ± 0.05 0.32 ± 0.12 0 0 0.18 ± 0.09 0.19 ± 0.10

Total biomass was estimated from the dry weight of 1 mL of each sample after 48 h. Yields were obtained by measuring the amount of biomass, xylose or ethanol after
48 h. Xylose and xylitol in the medium were analyzed with an assay kit, and ethanol was measured by the reduction of NAD+. n = 2–4. *Statistical difference.

followed by an almost similar rapid decrease, and this was also found
Table 4 with K. marxianus. When xylose was added, the main difference with
Sugar consumption.
glucose-grown cells was that with a variable delay, the fluorescence
Yeast Glucose (g L− 1) 12 h Xylose (g L− 1) 48 h increases after the addition of the sugar were clear only with
pH 4 pH 6 pH 4 pH 6 M. guilliermondii, C. guilliermondii, and K. marxianus.
C. guilliermondii 1.57 ± 0.55 1.67 ± 0.51 0.28 ± 0.06 0.34 ± 0.01
S. cerevisiae 1.68 ± 0.30 1.80 ± 0.49 0.10 ± 0.00 0.23 ± 0.00
3.8. Oxygen consumption rates
D. hansenii 2.08 ± 0.32 2.02 ± 0.14 0.41 ± 0.11 0.40 ± 0.09
K. marxianus 1.76 ± 0.46 1.88 ± 0.28 0.49 ± 0.16 0.56 ± 0.17
M. guilliermondii 1.78 ± 0.41 2.02 ± 0.56 0.53 ± 0.20 0.64 ± 0.22 Another pathway for electrons is respiration, which can occur
C. lusitaniae 1.46 ± 0.48 1.56 ± 0.48 0.49 ± 0.24 0.48 ± 0.14 through the main respiratory chain of mitochondria, or through an
Glucose consumption in YPD determined after 12 h. Xylose consumption in YPX alternate oxidase that as pointed before, has been described in several
after 48 h. n = 2–4. yeasts [32,33]. In order to better observe the effects of glucose or xylose
and to understand if the carbon source used for growth influences res­
acetaldehyde to produce ethanol and NAD+. However, when xylose was piratory pathway, after growth in YPD, YPX, or YP (without sugar), the
added, there was an immediate diminution of fluorescence for all yeasts cells were fasted for 24 h. The effect of two respiratory inhibitors on the
except for D. hansenii, and later a long reduction of the nicotinamide main (cyanide) and the alternate electron pathway (octylgallate, OG)
nucleotides; this was not observed for K. marxianus. was measured (See Table 7).
With the cells previously grown in YPX (Fig. 5), still similar, but C. guilliermondii from YPD medium displayed a rate of 165 natg O
lower fluorescence increases were observed upon the addition of min− 1 per 50 mg of cells without added substrate, which increased up to
glucose. Again, in S. cerevisiae, the initial fluorescence increase was 258 if grown in YPX and to 281 when grown in YP. When glucose was
added, rates heightened to 435 in cells from YPD, 543 in YPX and 304 in

Fig. 7. Medium acidification, YPX-grown yeasts. External pH was observed in MES-TEA 2 mM pH 6 buffer with yeasts grown in YPX for 24 h and fasted for 24 h
without sugar ( ), after the addition of glucose ( ) or xylose ( ). Sugars were used at 20 mM and added where the arrow indicates. Yeasts (50 mg, wet weight)
were added at the beginning of the tracings and followed up for 250 s. Representative data of n = 3. Data was plotted with GraphPad Prism 6 software.

7
A.K. Estrada-Ávila et al. BBA - General Subjects 1866 (2022) 130154

Table 6
Ethanol and xylitol production, YPD or YPX.
Yeast YPD YPX

Ethanol Ethanol Xylitol

(g L− 1) (g L− 1) (g L− 1)

pH 4 pH 6 pH 4 pH 6 pH 4 pH 6

10.77 11.30 3.87 4.07

C. guilliermondii 0 0
± 2.2 ± 0.9 ± 0.4 ± 0.1
11.94 12.99 0.33 0.40
S. cerevisiae 0 0
± 3.1 ± 4.4 ± 0.1* ± 0.3*
6.46 ± 6.06 ± 1.70 3.15
D. hansenii 0 0
1.1* 0.7* ± 0.7* ± 0.4
1.72
8.55 ± 14.08 2.20 3.88 3.95
K. marxianus ±
4.0 ± 4.4 ± 0.8 ± 0.1 ± 0.4
0.62
16.39 14.90 1.12 1.60 2.77 2.77
M. guilliermondii
± 1.1 ± 1.5 ± 0.5 ± 0.2 ± 0.9 ± 0.9
9.20 ± 8.37 ± 3.80 1.64
C. lusitaniae 0 0
2.7 0.7 ± 0.2 ± 0.7*

The amount of ethanol and xylitol produced by each yeast in every medium was
Fig. 8. Medium acidification in YNB with amino acids and 2% xylose-grown
estimated at 36 h for glucose media or 48 h for xylose media. Ethanol was
yeasts. External pH was observed in H2O at pH 4 with yeasts grown in YNB
measured by the reduction of NAD+ and xylitol with an assay Kit. n = 2–4.
with amino acids and 2% xylose for 24 h. Sugars ( glucose, xylose) were
*Statistical difference.
used at 20 mM and added where the arrow indicates. Yeasts (50 mg, wet
weight) were added at the beginning of the tracings and followed up for 800 s.
Representative data of n = 3. Data was plotted with Excel software. D. hansenii showed a much lower respiration, which was increased
with glucose, but very little with xylose. Its behavior was similar to that
of C. guilliermondii in response to cyanide and OG; it was increased by
Table 5 cyanide when grown in YPD or YPX, but not in YP.
Sugar transport kinetics. K. marxianus, respiratory rate was lower than that of C. guilliermondii
14
C-glucose when grown in YPD, but much higher when grown in YPX; cyanide did
Yeast Vmax Km not, but OG inhibited respiration.
M. guilliermondii showed low oxygen consumption rate when yeasts
(μmol (g min)− 1) (μM)
were grown in YPD (C, G and X) (126, 190 and 149 natg O min− 1). When
pH 4 pH 6 pH 4 pH 6 YPX was used, rates increased (304, 474 and 354 natg O min− 1),
C. guilliermondii 13.2 ± 1.8 15.1 ± 1.8 0.6 ± 0.2 0.9 ± 0.2 nevertheless after cyanide was added, rates were higher when glucose
S. cerevisiae 48.8 ± 16.8 26.3 ± 3.9 5.2 ± 2.3 2.1 ± 0.5 and xylose where added as substrate, and octylgallate diminished them
D. hansenii 9.0 ± 0.5 8.8 ± 0.5 0.3 ± 0.06 0.3 ± 0.06
when grown in YP, cyanide inhibited oxygen consumption in all
K. marxianus 45.9 ± 37.4 56.0 ± 41.3 6.7 ± 6.7 9.7 ± 8.3
M. guilliermondii 40.4 ± 6.5 18.5 ± 2.5 4.1 ± 0.9 1.3 ± 0.3 conditions.
C. lusitaniae 26.9 ± 4.3 7.3 ± 0.6 2.6 ± 0.6 0.4 ± 0.1 For C. lusitaniae, oxygen consumption rate augmented when glucose
was added in every condition, cyanide diminished it, and more so OG.
14
C-xylose Results show that four strains when grown in YPD presented cyanide-
Vmax Km resistant respiration, except for S. cerevisiae and C. lusitaniae, and
Yeast (μmol (g min) ) (μM) similar results were obtained when grown in YPX. However, all yeasts
¡1

pH 4 pH 6 pH 4 pH 6
showed a cyanide sensitive respiration when grown in YP medium,
C. guilliermondii 11.2 ± 1.1 14.2 ± 1.1 1.1 ± 0.2 1.55 ± 0.2
S. cerevisiae 1*10− 13 5*10− 13 3*1013 1*1014 whether with glucose or xylose as substrate. Cyanide resistant respira­
D. hansenii 7.6 ± 1.9 11.0 ± 3.4 1.4 ± 0.6 2.6 ± 1.2 tion is an indication of an alternative oxidase (AOX), whose develop­
K. marxianus 3.5 ± 0.7 5.2 ± 1.3 1.2 ± 0.5 2.11 ± 0.8 ment depends on the growth conditions of the cells.
M. guilliermondii 4.8 ± 1.9 4.8 ± 2.2 2.1 ± 1.3 1.92 ± 1.5 It has to be mentioned that in any case, respiration, unless the in­
C. lusitaniae 33.7 ± 25.1 22.4 ± 16.4 13.3 ± 11.2 11.5 ± 9.6
cubation media are aerated, more or less rapidly leads to anaerobiosis,
Transport rate plots of both glucose and xylose against time were adjusted by and because of this, is not a factor that may decrease, but on the con­
non-linear regression of the Michaelis-Menten equation to obtain Vmax and Km trary, favor the production of xylitol.
values for each yeast. Plots and adjustments were made with GraphPad Prism 6
software. n = 3–5.
3.9. Changes of pH of the medium

YP. When xylose was added, rate increase was smaller. After cyanide
Changes of the external pH of the medium were measured to obtain
addition, oxygen consumption increased in the cells grown in YPD,
information about two facts: a) whether xylose transport was being
indicating a powerful alternate electron pathway. As expected, the
carried out by a proton/sugar symporter. If such mechanism operates,
further addition of octylgallate reduced considerably the oxygen uptake
after the addition of xylose, should produce an increase of the medium
rate. Results show that: a) it has a very active respiratory chain, when
pH, and b) whether sugars were a good substrate providing energy for
grown in YPD, but more so in YPX; b) its respiration was stimulated by
proton pumping.
cyanide and decreased by OG, and c) its respiration was stimulated by
All cells were fasted for 24 h. With cells grown in YPD, a clear
xylose almost as much as with glucose.
decrease of external pH was observed after the addition of glucose with
With S. cerevisiae, oxygen consumption rate was low in every media
C. lusitaniae, followed by S. cerevisiae, K. marxianus and M. guilliermondii,
and decreased only when cyanide was added. Results indicate that: a) it
(Fig. 6). With cells grown in YPX a small decrease was observed only
has a much less active respiratory chain, whether grown in YPD, or YPX;
with glucose, and practically none with xylose (Fig. 7). Most important
b) its respiration is cyanide sensitive and insensitive to OG, and c) its
is that in none of the conditions, an increase of pH was observed after the
respiration was not stimulated by xylose as it was with glucose.
addition of xylose, as it would be expected if a proton-xylose symporter

8
A.K. Estrada-Ávila et al. BBA - General Subjects 1866 (2022) 130154

Table 7
Oxygen consumption.
C C CN CCN OG G G CN GCN OG X X CN XCN OG

C. guilliermondii
YPD 165 ± 4 178 ± 48 80 ± 13** 435 ± 42 473 ± 36 60 ± 6 269 ± 37 245 ± 57 82 ± 35**
YPX 258 ± 30 229 ± 54 87 ± 12** 543 ± 8 608 ± 97 59 ± 14** 428 ± 24 365 ± 112 76 ± 19**
YP 281 ± 84 142 ± 52* 38 ± 16** 304 ± 55 142 ± 28* 36 ± 16** 287 ± 14 140 ± 60* 38 ± 15**
S. cerevisiae
YPD 51 ± 17 39 ± 41 31 ± 5 185 ± 54 61 ± 33* 38 ± 13 74 ± 32 45 ± 23 39 ± 5
YPX 81 ± 12 61 ± 13 31 ± 16 137 ± 40 61 ± 13* 28 ± 7** 94 ± 21 37 ± 8* 31 ± 11
YP 77 ± 1 54 ± 38 30 ± 5 145 ± 45 50 ± 10* 27 ± 4 88 ± 10 48 ± 15* 29 ± 9
D. hansenii
YPD 91 ± 24 95 ± 27 53 ± 2 235 ± 82 352 ± 35 48 ± 6** 133 ± 15 115 ± 23 57 ± 4**
YPX 65 ± 1 67 ± 21 33 ± 12 132 ± 45 257 ± 23* 32 ± 8** 121 ± 53 135 ± 63 29 ± 5
YP 60 ± 18 41 ± 20 30 ± 13 95 ± 29 45 ± 23* 31 ± 11 68 ± 2 39 ± 30 28 ± 12
K. marxianus
YPD 126 ± 15 69 ± 41 52 ± 42 190 ± 56 222 ± 111 35 ± 9** 149 ± 39 84 ± 59 42 ± 26
YPX 222 ± 76 198 ± 89 110 ± 25** 429 ± 137 305 ± 139 106 ± 27 293 ± 15 273 ± 155 128 ± 36
YP 262 ± 9 130 ± 20* 42 ± 23** 367 ± 9 132 ± 29* 42 ± 21** 388 ± 32 84 ± 28* 46 ± 24**
M. guilliermondii
YPD 126 ± 15 69 ± 41 52 ± 42 190 ± 56 222 ± 111 35 ± 9** 149 ± 39 84 ± 59 42 ± 26
YPX 304 ± 61 231 ± 95 70 ± 47** 474 ± 96 784 ± 135 46 ± 24** 354 ± 93 589 ± 65* 62 ± 30**
YP 213 ± 76 87 ± 20* 26 ± 5** 260 ± 86 84 ± 23* 26 ± 4** 219 ± 93 78 ± 13* 24 ± 6**
C. lusitaniae
YPD 161 ± 65 91 ± 17 60 ± 1** 319 ± 94 176 ± 95 51 ± 5** 241 ± 51 183 ± 75 56 ± 5**
YPX 134 ± 40 62 ± 17* 23 ± 14** 202 ± 91 65 ± 54* 32 ± 15 161 ± 52 76 ± 29* 38 ± 30
YP 180 ± 34 76 ± 20* 476 ± 5 268 ± 49 100 ± 27* 40 ± 9** 227 ± 82 67 ± 40* 35 ± 7

Respiration was measured in 5 mL MES-TEA 10 mM pH 6 buffer at 30◦ C. Glucose or xylose were used at a final concentration of 20 mM. Sodium cyanide (200 μM) and
octylgallate (30 μM) were used as respiratory inhibitors. Rates are reported in natg O min− 1 per 50 mg of cells. Yeasts were grown in YPD, YPX or YP medium for 24 h
and fasted for 24 h, adding 50 mg (wet weight). ANOVA analysis with p < 0.05 was executed in GraphPad Prism 6 software. n = 3–5. *Statistical difference with C, G or
X. **Statistical difference with C CN, G CN or X CN. Abbreviations: C (control, without substrate), G (glucose), X (xylose) CN (sodium cyanide), OG (octylgallate), YP
(medium w/o sugar).

existed. As already mentioned, it is well known that pH of the medium pro­

After reviewing these results, a direct comparison to results from
Leandro et al. [11] was suggested given that these authors reported the
increase of pH by addition of xylose in S. cerevisiae strains MJY1 and
MJY2 (carrying Candida intermedia xylose transporters). By following
this methodology, in water, at pH 5 no changes were found. Neverthe­
less, at pH 4 a noticeable difference between xylose and no sugar or even
glucose was found solely for C. lusitaniae (Fig. 8). Two other
C. intermedia strains were also tested with this methodology, and no
xylose/ H+ symport was observed (not shown).
4. Discussion
4.1. Xylose as carbon source for growth in yeasts
Serial drop tests already provided some indications about which of
our yeasts were able to transport and metabolize xylose. S. cerevisiae and
D. hansenii previously grown in YPD showed a lower growth when
spotted on YPX (Suppl. Fig. 1). With YPX as growth medium, a lower
growth was observed with all strains except C. guilliermondii and
M. guilliermondii, and it was practically zero for S. cerevisiae, D. hansenii
and C. lusitaniae.
When cells were previously grown in YPX and spotted on YPD, there
was no difference from those previously grown in YPD, a similar situa­
tion was observed for cell growth in YPX/cells spotted on YPX (Suppl.
Fig. 2). These results are contrary to previous reports [34,35] for
C. lusitaniae and D. hansenii as the best yeasts for a rapid transformation
of xylose into ethanol and/or xylitol. However, this may be due to the
strains used.
In liquid YPD (Fig. 2), growth was similar for all yeasts, except for
D. hansenii. With YPX (Fig. 3), on the other hand, growth rates were
lower, and again, S. cerevisiae, C. lusitaniae and D. hansenii grew much
less after 48 h. Rates obtained in this study for YPX are higher than those
reported for P. stipitis (0.22 h− 1) and C. shehatae (0.14 h− 1) in 20 g L− 1 of
xylose [36,65] and of 0.033–0.201 h− 1 for D. hansenii with 50 g L− 1 of
xylose [35], (analyses of the curves are summarized in Table 1).
duces important changes in metabolism of yeast, both in S. cerevisiae
[30,38] and D. hansenii [39,40], and it has been suggested that xylose is
transported in yeast by a H+-sugar symporter; so, growth which was
followed at both pH 4 and 6 should have been faster and larger at the low
pH, but it was not the case. This again indicates the low ability of the
strains used to use xylose as a substrate, starting from the fact that with
this sugar, to reach maximal growth, it had to be followed for 48 h, and
still so, somewhat lower maximal growth was attained.
Maximum biomass produced in xylose media reached up to 8.5 g L− 1
with a biomass yield of up to 0.4 g g− 1. Furlan and collaborators, in 1997
reported for C. parapsilopsis biomass production of 6.1 g L− 1 in 126 h and
a final yield of 0.28 g g− 1 [41]. Preziosi-Belloy et al., in 2000 reported
for C. guilliermondii biomass yields of 0.1 and 0.3 g g− 1 [42]. Diz et al. in
2002 obtained, in strains of D. hansenii, maximum biomass values of 6 g
L− 1 in more than 90 h of fermentation [43]. With these data we showed
that even though our strains grew in non-optimal conditions they are
good biomass producers from 20 g L− 1 of xylose after 48 h.
Besides, when pH was followed for all the strains, a difference upon
the addition of xylose was found only in C. lusitaniae after using water at
pH 4 instead of buffer. In this experiment, under the addition of xylose, it
still produced an acidification, which however was much lower than
that observed with glucose. Our explanation to this result is that this
strain was among those more effectively transporting xylose, and that if
it is by means of a H+-xylose transporter, it leads to an equilibrium
between H+ pumping by the plasma membrane H+-ATPase, and a pro­
ton influx resulting from the action of this symporter. These results along
with the transport kinetics data might guide to believe that out of all the
yeasts studied; only C. lusitaniae was able to display few evidence of a
xylose symporter yet it did not showed high affinity for this sugar.
4.2. Comparison between glucose and xylose in fermentation yields
As expected, ethanol production and yield were higher when glucose
was used as carbon source. When xylose was the main carbon source,
incubations had to be performed for 48 h. Only K. marxianus and

9
A.K. Estrada-Ávila et al.
M. guilliermondii produced significant amounts of ethanol. Strains of
K. marxianus producing ethanol from xylose of 3 g L− 1 in 48 h have been
reported, whereas for strains of C. guilliermondii no ethanol was detect­
able at 48 h but at 24 h was approximately 0.75 g L− 1 [44]. This is the
first result where C. guilliermondii and M. guilliermondii displayed
different behavior.
Although ethanol production from xylose is low compared to that
from glucose, we expected that xylitol production would be comparable
at least in those genres reported as good xylitol producers
(C. guilliermondii and D. hansenii). Ojamo et al. (1994) [45] and Roberto
et al. (1994) [46] reported xylitol/xylose yields of 0.78 g g− 1 for
C. guilliermondii with initial concentrations of xylose of 250 and 62 g L− 1.
However, Hallborn et al. (1991) [47] reported yields of 0.95 and 0.81 g
L− 1 for a recombinant strain of S. cerevisiae. Zhang et al. (2014) obtained
K. marxianus transformants with xylitol yields of up to 0.83 g g− 1 [48].
Prakash et al. in 2011, reported yields of 0.78–0.82 g g− 1 for D. hansenii
after 108 h of fermentation with 50 and 100 g L− 1 of xylose [47].
Moreover, yields of 0.26 g g-1 of xylitol and 0.56 g g-1 of ethanol have
been reported for C. tropicalis [37], as an evidence of how a mixture of
xylose/glucose can enhance production of both alcohols.
Maximum production of xylitol reported in this work was of 3.8–4 g
L− 1 by C. guilliermondii, K. marxianus and C. lusitaniae. Several authors
have reported up to 16 g L− 1 for C. guilliermondii, 5 g L− 1 for K. marxianus
from 20 g L− 1 of xylose [44], and up to 71.2 g L− 1 in D. hansenii.
However, these values were after 156 h of fermentation at 40◦ C [49].
Our yeasts produced low xylitol yields but very similar among each
other. If fermentation time were outstretched, temperature adjusted to
an optimal value for each yeast, xylose concentration increased; and,
oxygenation regulated, yields might be improved. However, our results
show that the strain of S. cerevisiae, isolated from mezcal fermentations
is the least indicated for future tests, and not only for the production of
xylitol or ethanol.
4.3. Xylose and the redox imbalance evidence
Bruinenberg et al., in 1983, concluded that anaerobic utilization of
xylose leads to a change in the redox balance which avoids a fast alco­
holic fermentation [21]. Being this “imbalance” the main reason for
xylitol production and not ethanol in certain strains, it is necessary to
know the redox state of yeast with xylose. Yeasts with high alcoholic
fermentation rates, as S. cerevisiae, rapidly reduce and then reoxidize
NAD+ when glucose is added. High xylitol yields are expected when
NADH accumulates, and xylitol NAD+-dependent dehydrogenase is
inhibited. This phenomenon is known as the Custer Effect, result of the
inefficacy of yeast to compensate the excess NADH and without a
transhydrogenase activity [50]. Increased NADH/NADPH levels were
observed after the addition of glucose in all yeasts from YPD and YPX,
although in S. cerevisiae rapidly returned to previous levels. This dif­
ference, although not immediate, was observed only in three cases after
the addition of xylose, with C. lusitaniae from YPD, K. marxianus and
M. guilliermondii from YPX (Figs. 4 and 5). This delayed effect is caused
by the production of ethanol from xylose only detectable in K. marxianus
and M. guilliermondii. Nevertheless, in C. lusitaniae we did not detect
ethanol production from xylose because it was not grown in YPD before.
Here again, we obtained results in which C. guilliermondii and
M. guilliermondii displayed a different behavior.
What is probably more interesting is that the NADH-NADPH levels in
S. cerevisiae, whether previously grown in glucose or xylose, showed an
increase in the tracings, followed by a decrease to previous levels when
glucose was the substrate, as expected from the reduction in the
glyceraldehyde-3-phosphate dehydrogenation step, and then from the
subsequent reduction of acetaldehyde by the ethanol dehydrogenase.
This was also observed with K. marxianus, but only in that grown in YPX.
Apparently, the second step was not observable in the others because it
is too slow. Most of them showed an increase that was not followed by a
decrease, probably due to a slower rate of the second part of glycolysis.
10
BBA - General Subjects 1866 (2022) 130154
Besides, as expected, in the cells grown in YPD, contrary to
S. cerevisiae, most strains showed a fast increase when glucose was added
but was not followed by a fast decrease. Also, the addition of xylose did
not produce any immediate fluorescence increase, but only with
C. lusitaniae, a late increase was seen (Fig. 4). In cells grown in YPX,
probably the most important finding was that occurring upon the
addition of xylose, only in C. guilliermondii and M. guilliermondii (Fig. 5).
However, when measuring the final products, it was found that the
somewhat lower ethanol production was for S. cerevisiae, and the highest
was for C. guilliermondii, K. marxianus and M. guilliermondii. As to xylitol
production, the highest production was with C. guilliermondii, the lowest
was for S. cerevisiae, with intermediate values for the other strains.
4.4. Xylose uptake and transport
There are several reports on the value of Km for glucose and xylose
transport in yeast. Some mention it is 8 times lower for glucose than for
xylose in D. hansenii [51]. For a recombinant S. cerevisiae, xylose
transport Km values are of 143 mM with a 10 mmol g h− 1 Vmax and for
C. intermedia Km = 0.2 mM and Vmax = 1 mmol g h− 1 [52]. These values
change depending on growth conditions and substrate used. Under
glucose repression yeasts display a low affinity xylose transporter
operated via facilitated diffusion, whereas in certain strains of Debar­
yomyces fabryi and D. hansenii grown in xylose and fasted, a proton/
sugar symporter mechanism has been reported [53]. In this study we
used yeast grown in the same sugar as the one measured. In glucose,
D. hansenii was that with the highest affinity for this sugar, while in
xylose C. guilliermondii showed the smallest Km. Nevertheless, we did not
find a statistical difference between transport of glucose and xylose at
pH 4 and pH 6, therefore no symporter mechanism was detected. As to
the importance of these kinetic parameters in the other experiments, it is
clear that Km did not have any problem, because the concentrations of
both sugars in the incubation media were very much above the value of
this constant for all yeasts.
4.5. Xylose transport does not alkalinize the medium in the yeasts studied
If xylose were transported by a proton/sugar symporter into the cell
under fasted conditions, it would be expected that the pH of the external
medium rose as xylose was transported. This mechanism has been re­
ported in Rhodotorula [7] and Candida utilis [54]. Most of the conditions
tested in this study gave out negative results (Figs. 6 and 7), except for
C. lusitaniae which displayed a lower acidification of the media when
xylose was added to the yeasts in water and pH 4, as compared to
glucose (Fig. 8).
Nonetheless, other authors assert that the absence of an observable
alkalization cannot be taken as evidence of the absence of a symporter
[55,56]. The latter may be due to the low transport rate of xylose, which
will imply an imperceptible shift in external pH. A sequence for Xylhp in
D. hansenii CBS 767, a proton/xylose symporter, is found in data bases
but has not been confirmed. When a BLAST sequence alignment was
carried out, no similar sequence was found in any of our yeasts (data not
shown).
4.6. Xylose consumption enhances the expression of an alternative
oxidase
Quantification of oxygen consumption by the different yeasts under
different circumstances gave diverse results characteristic from each
yeast. Model yeast, S. cerevisiae, being fermentative, displayed low levels
of respiration in every condition as well as C. lusitaniae, which came out
as a great ethanol producer from glucose. C. guilliermondii showed a high
and fast respiration in every condition. As for K. marxianus and
M. guilliermondii, changing from YPD to YPX increased oxygen con­
sumption rate with glucose or xylose as substrate. D. hansenii, despite
having a respiratory metabolism [57], displayed low rates. We proved
A.K. Estrada-Ávila et al.
that the carbon source used for growth does have an effect in oxygen
consumption, since the fermentative or respiratory pathways are
enhanced or inhibited according to the used carbon source. When yeasts
were grown in YP, only with K. marxianus an effect was noticeable. The
differences between rates are due to the use of glucose or xylose as
carbon source.
As for the effects of cyanide and octylgallate on the respiratory ca­
pacity, cyanide-resistant respiration (CRR), works as a regulatory
function on the electron over flow when the cytochrome pathway is
limited and is carried out by an alternative oxidase (AOX). Because of
this, respiration is enhanced when cyanide is added but AOXs are
inhibited by hydroxamic acids and n-alkyl-gallates [33]. This respiration
is induced by different conditions as a mechanism that allows survival.
Yeasts from Candida, Debaryomyces, Pichia and Yarrowia show this kind
of respiration under different conditions [58–61]. Our C. guilliermondii
and D. hansenii strains did show CRR both when grown in YPD and YPX
and after glucose and xylose were added, and so did K. marxianus and
M. guilliermondii. The most evident presence of a CRR was in
M. guilliermondii grown in YPX, which again stands out as a difference
with C. guilliermondii in this study. CRR was reported as absent in
Crabtree-positive yeasts [62], such as S. cerevisiae, comparable to our
results. The expression of an alternative oxidase in yeasts grown in YPX
might be explained by an ATP shortage because of the high affinity
transport of xylose, accepting that it consumes 1 mol of ATP per co-
transported proton [63]. Nevertheless, such stress can be caused by
the redox imbalance provoked by the first steps in the conversion of
xylose to xylitol, since our results suggest that such proton/xylose
cotransport does not exist, at least in the strains used for this study.
4.7. Summary of the general properties of the yeasts used
S. cerevisiae showed the expected behavior in YPD, with a high ca­
pacity to consume glucose, starting with the glucose uptake kinetics,
sugar consumption and ethanol production, and also its growth char­
acteristics were as expected. However, it is clear that xylose is far from
being an even slightly adequate substrate, starting with its uptake ca­
pacity and kinetics, growth and ethanol production. Definitely, xylose is
not a suitable substrate for this yeast.
As for the properties, perhaps the next one is D. hansenii, that even
with glucose showed a low growth rate and yield, and even ethanol
production when YPD was the medium. However, it produced a small
but significant amount of xylitol.
K. marxianus may be considered the next in the list, taking into ac­
count its capacity to take up glucose, using it for growth, but also pro­
ducing a small amount of both ethanol (at pH 6), as well as a significant
amount of xylitol when incubated in YPX.
The strain used of C. lusitaniae is a robust yeast, capable of growing at
a similar rate in YPD and YPX, and producing a significant amount of
xylitol, but no ethanol when grown in YPX. It might be an intermediate
candidate for the production of xylitol.
The strain used of M. guilliermondii is also interesting, with the best
values for growth and mass production, as well as glucose uptake, but a
lower uptake of xylose uptake, and a high production of ethanol in YPD,
and a moderate production of xylitol in YPX.
Particularly interesting is C. guilliermondii, with a high growth rate,
as well as fermentation to ethanol in YPD. However, its most important
characteristic is that it can produce the highest values of xylitol in YPX,
but no ethanol. It might be the most adequate to be used for the pro­
duction of xylitol. This result is coincident with that of Winkelhausen
and Kuzmanova [64], who found that high yields of xylitol obtained
from xylose were for Candida sp. with up to 17 g L− 1.
Yeasts from natural fermentations were used in this study with the
sole objective to test if by being previously exposed to a greater extent of
carbon sources than a regular laboratory strain, they could be better
candidates to produce xylitol from xylose. However, with S. cerevisiae,
we confirmed that the intrinsic properties of each strain are the ones that
11
BBA - General Subjects 1866 (2022) 130154
define if a yeast will be capable of using xylose and convert it to xylitol.
We also proved that non-conventional yeasts have a greater ability to
produce this biotechnologically important alcohol. Nevertheless, oppo­
site to what we believed, there was no evidence in our results that
supports the hypothesis that xylose enters these yeasts via a proton
symporter, with the exception perhaps of C. lusitaniae. Further testing
will be required to confirm or deny this.
The studies performed offer the possibility of further experiments by
varying mainly the aeration conditions of the cells, for the production of
both xylitol and ethanol from xylose as substrate.
Funding
This work was supported by CONACYT grants 176199and 238497
for Basic Scientific Research. AKEA was a CONACYT-fellowship holder
(No. 384170) while student from the Biochemical Sciences graduate
program at UNAM.
CRediT authorship contribution statement
Alejandra Karina Estrada-Ávila: Investigation, Methodology, Data
curation, Writing – original draft. Juan Carlos González-Hernández:
Conceptualization, Funding acquisition, Writing – review & editing.
Martha Calahorra: Methodology, Validation, Resources, Writing – re­
view & editing. Norma Silvia Sánchez: Methodology, Validation, Re­
sources, Writing – review & editing. Antonio Peña: Conceptualization,
Funding acquisition, Supervision, Writing – review & editing.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
Authors are grateful with Dr. Alicia González-Manjarrez from IFC,
UNAM for fruitful discussions along the project, to Dr. Cristina Uribe-
Alvarez for helping in the typification of some of the yeasts of this
study and to B. Sc. Alejandro Camacho, from the Microorganisms
Collection of the Chemistry School of UNAM for the donation of two
strains of Candida intermedia, to search for a positive control of the
xylose/H+ symporter.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bbagen.2022.130154.
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