World Journal of Microbiology and Biotechnology (2021) 37:98

https://doi.org/10.1007/s11274-021-03056-9

ORIGINAL PAPER

Comparisons of urea or ammonium on growth and fermentative

metabolism of Saccharomyces cerevisiae in ethanol fermentation
Xinchao Yang1 · Yuling Yang2 · Jiadong Huang1 · Deen Man2 · Maihai Guo2

Received: 20 October 2020 / Accepted: 13 April 2021 / Published online: 10 May 2021
© The Author(s), under exclusive licence to Springer Nature B.V. 2021

Abstract
This work was mainly about the understanding of how urea and ammonium affect growth, glucose consumption and etha-
nol production of S. cerevisiae, in particular regarding the basic physiology of cell. The basic physiology of cell included
intracellular pH, ATP, NADH and enzyme activity. Results showed that fermentation time was reduced by 19% when using
urea compared with ammonium. The maximal ethanol production rate using urea was 1.14 g/L/h, increasing 30% compar-
ing with the medium prepared with ammonium. Moreover, urea could decrease the synthesis of glycerol from glucose by
26% comparing with ammonium. The by-product of acetic acid yields decreased from 40 mmol/mol of glucose (with urea)
to 24 mmol/mol of glucose (with ammonium). At the end of ethanol fermentation, cell number and pH were greater with
urea than ammonium. Comparing with urea, ammonium decreased the intracellular pH by 14% (from 7.1 to 6.1). Urease
converting urea into ammonia resulted in a more than 50% lower of ATP when comparing with ammonium. The values of
NADH/DCW were 0.21 mg/g and 0.14 mg/g respectively with urea and ammonium, suggesting a 33% lower NADH. The
enzyme activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was 0.0225 and 0.0275 U/mg protein respectively
with urea and ammonium, which was consistent with the yields of glycerol.

Keywords Saccharomyces cerevisiae · Ethanol · Urea · Ammonium

Introduction
Nitrogen is an essential nutrient for all life forms. Yeast
can use almost 30 distinct nitrogen-containing compounds,
including amino acids, urea, ammonium, nitrogen bases, and
purine derivatives (Godard et al. 2007). Among the avail-
able nitrogen sources, ammonium and urea are two cheap
nitrogen sources commonly used in large scale fermenta-
tion processes. S. cerevisiae can utilize urea as sole nitrogen
source by degrading it in two steps to ammonia and ­CO2 in
ethanol production (Cooper and Sumrada 1975). Their meta-
bolic pathways are shown in Fig. 1, which shows that ATP
is consumed when urea is converted into ammonia. In addi-
tion, many studies have shown that ammonium is a preferred
source of nitrogen (Marini et al. 1997; Zhao et al. 2016). In
* Xinchao Yang
cse_yangxc@ujn.edu.cn
1 gated the comparison of urea and ammonium used as nitro-
School of Biological Science and Technology, University
of Jinan, Jinan 250022, China
2 S. cerevisiae. The present study examined the effects of
Linghua Group Limited, Jining 272073, China
the medium with urea as the nitrogen source, urea absorption
is not proton-coupled (Cooper and Sumrada, 1975). In con-
trast to ammonium, the assimilation of urea does not cause
acidification of the medium(Hensing et al. 1995).
Then a scientific problem worthy to be studied is the most
suitable nitrogen source being either ammonium or urea for
industrial ethanol production.
Most of microorganisms can sense changes in the
amounts and quality of nutrients, allowing their optimal
utilization in highly competitive environments (Wong et al.
2008). S. cerevisiae can utilize various kinds of nitrogenous
compounds, but with distinct preferences (Zhao et al. 2013).
In the presence of preferred nitrogen sources, transcription
of the genes associated with the transport and utilization of
non-preferred nitrogen are repressed. Within the last decade,
influence of the nitrogen source on S. cerevisiae anaerobic
growth and product formation has gained more and more
attention (Eva Albers, 1996; Kemsawasd et al. 2015; Wang
et al. 2012). However, relatively few studies have investi-
gen source, in particular regarding the basic physiology of

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98 Page 2 of 7
Fig. 1  The metabolic pathways
of urea and ammonium in S.
cerevisiae (GS Glutaminsyn-
thetase, GDH Glutamate dehy-
drogenase, GOGAT​Glutamate
Synthase)
ammonium or urea on ethanol fermentation to further opti-
mize the ethanol fermentation process.
Materials and methods
Microorganism and seed medium
A commercial strain of S. cerevisiae TG1348 for ethanol
production was obtained from Henan Tian Guan Co., Ltd.,
(Henan, Nanyang, China). One loopful of S. cerevisiae was
inoculated into a 500 mL Erlenmeyer flask holding 200 mL
of a solution containing (g/L): glucose 20, yeast extract 8.5,
­(NH4)2SO4 1.3, M ­ gSO4•7H2O 0.1 and C ­ aCl2·2H2O 0.06.
The flask was incubated on a shaker (200 rpm) at 30 °C for
18 h to produce seed medium.
Fermentation medium and ethanol fermentation
Fermentation medium was composed of (g/L): glucose 100,
­MgSO4·7H2O 0.1, C ­ aCl2·2H2O 0.06, dissolved in the appro-
priate process water. The nitrogen content (N) of urea (or
ammonium sulfate) is 0.5 g/L. Urea was sterilized separately
to avoid Maillard reaction. The pH was adjusted to 5.0 using
30% (w/w) ­H2SO4 or 10% (w/v) NaOH. Triplicate fermen-
tations were carried out in 250 mL flasks containing 135
mL of medium. Urea was added before inoculating. A 10%
(v/v) inoculum of seed medium was added to each flask. All
fermentations were carried out at 30 °C without shaking.
Quantification of intracellular ATP and NADH
Duplicate samples for the determination of ATP were col-
lected in the mid-exponential phase. Culture broth (10
mL) was rapidly mixed with pre-chilled methanol (30 mL
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World Journal of Microbiology and Biotechnology (2021) 37:98
at -40 °C) in pre-weighed tubes, as described (Hou et al.
2009). The cells were separated by centrifuging at 8000 ×g
for 20 min at − 4 °C, suspended in 0.5 M H ­ ClO4 at 0 °C,
and then processed by ultrasonic decomposition in an ice
bath for 10 min using cycles of 2 s sonication at 2 s intervals
(SM-650D; Shunma Tech., Nanjing, China). After centrifu-
gation at 12,000×g for 20 min at -4 °C, the supernatant was
filtrated through a 0.22 μm membrane before analysis by
HPLC (Zeng et al. 2014).
Determination of glyceraldehyde‑3‑phosphate
dehydrogenase (GAPDH)
Cells collected were suspended in 0.5 ml of homogenization
buffer (10 mM triethanolamine, 1mM dithioerythritol, 1 mM
EDTA [pH 7.5]) (Blomberg and Adler, 1989) and were then
immediately processed by ultrasonic decomposition in an
ice bath for 10 min with 2 s running and 2 s intervals with
1 g of glass beads (0.5 mm in diameter). Unbroken cells
and debris were removed by centrifugation at 12,000×g for
10 min. The supernatant was used as the cell extraction for
the immediate determination of enzymes. Enzymes assay
was carried out at 30 °C. The protein content of all extract
was measured with BCA (bicinchoninic acid) protein assay
kit (Sangon Biotech, Co. Shanghai, China), using bovine
serum albumin as the standard.
GAPDH (EC1.1.1.8) was assayed in a buffer containing
57 µL of 20 mM imidazole-HCl (pH 7.0), 30 µL of 1 mM
dithioerythritol (DTT), 3 µL of 1 mM ­MgCl2, 30 µL of 0.67
mM dihydroxyacetone phosphate (DHAP), 30 µL of 0.09
mM NADH (Blomberg and Adler, 1989) and 50 µL of cell
extract. The reaction was started by the addition of DHAP
and was linear for at least 5 min. No background NADH
oxidase activity was detected. Specific activities of GAPDH
World Journal of Microbiology and Biotechnology (2021) 37:98 Page 3 of 7 98

were dependent on protein concentration in the cuvette (Edg- Results

ley and Brown, 1983).
The assays were performed in Shimadzu UV-240 dou-
ble-beam spectrophotometer (Shimadzu Co., Kyoto, Japan)
at 25 °C in 1-ml cuvettes. NADH depletion or produc-
tion was monitored spectrophotometrically at 340 nm. To
calculate the enzyme activities, the reaction rate between
30 and 90 s and an absorption coefficient for NADH of
6.2 × ­103 ­mol− 1 ­cm− 1 were used (1 U = 1 umol of NADH
consumed per min).
Intracellular pH (Orij et al. 2009)
Intracellular pH values were assessed by fluorescence
microscopy using the pH-sensing fluorescent probe 5-(and
6-) carboxyl fluorescein diacetate, succinimidylester [5(6)-
CFDA, SE]. Briefly, cells were grown to an ­O D 600 of
approximately 1.0 and centrifuged for 5 min at 4000 rpm.
Then cells were washed three times with PBS and resus-
pended in the PBS buffer. The probe 5(6)-CFDA, SE was
added to the cell suspension (final concentration of 40 mM).
The mixture was mixed and incubated for 30 min at 30 °C.
After fluorescent labelled, cells were centrifuged at 5,500 g
for 5 min (at 4 °C), washed twice with buffer and finally
resuspended in 1 ml of the buffer of pH values ranging from
5.0 to 9.0. Cell suspension was transferred to black 96-well
microtiter plates. The pHluorin fluorescence emission was
measured at 512 nm using Enspire2300, providing excita-
tion bands of 9 nm centred around 390 and 470 nm. Back-
ground fluorescence for control not expressing pHluorin was
subtracted from the measurements. The ratio of emission
intensity resulting from excitation at 390 and 470 nm was
calculated (R390/470) and plotted against the corresponding
buffer pH. In all experiments the control was grown simulta-
neously for background fluorescence at both separate excita-
tion wavelength.
Effect of urea or ammonium on weight loss
of ethanol fermentation
Ethanol fermentation was monitored by measuring the mass
of the flasks, because the mass loss by C ­ O2 evolution is
proportional to the amount of ethanol produced (Wu 2006).
Figure 2 shows the weight loss of C ­ O2 when using either
urea or ammonium as nitrogen source. From Fig. 2 we can
see that the fermentation rate for urea was faster than for
ammonium. Moreover, the weight loss of C ­ O2 for urea was
slightly greater than for ammonium. The results suggested
that urea could accelerate the depletion of glucose. Growth
of yeast is faster and the biomass produced is greater in
media containing urea when compared to media containing
ammonium (Fig. 3).
Effect of urea or ammonium on ethanol
fermentation
The results shown in Fig. 3d suggested that glucose was
depleted after 42 and 52 h respectively with urea and ammo-
nium (Table 1). Therefore, a shorter fermentation time
was achieved when urea was used as the nitrogen source.
Fermentation time was reduced by 19% when using urea
compared with ammonium. Comparing with ammonium,
urea increased the ethanol production at the same time of
fermentation (Fig. 3c). Therefore, ethanol yield per glu-
cose was greater in fermentation medium prepared with

Analysis

Glucose, ethanol, glycerol and acetic acid concentrations in

fermentation media were determined by high-performance
liquid chromatography (HPLC). Samples were centrifuged
(10,000×g for 10 min) and the supernatant passed through
a 0.20 μm filter prior to analysis. Standards and prepared
samples (20 µL) were injected into a Bio-Rad HPX-87 H
Aminex ion exclusion column. The column was operated at
65 °C and sulfuric acid (0.005 mol/L) was used as mobile
phase at a flow rate of 0.6 mL/min. A refractive index detec-
Fig. 2  Comparisons of the weight loss when using urea or ammo-
tor (Shodex RI-101, Shodex, Tokyo, Japan) was employed.
nium as nitrogen source in ethanol fermentation. Data presented are
Data were processed using Chromeleon software (Dionex, the average of three independent cultivations and error bars represent
CA, USA). Cells were counted using a hemocytometer. standard deviations. (filled square-Urea, filled circle-Ammonium)

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Fig. 3  Effect of urea or ammonium on ethanol fermentation Data presented are the average of three independent cultivations and error bars rep-
resent standard deviations. (filled square-Urea, filled circle-Ammonium)
Table 1  Growth rates and product yields for anaerobic growth of S.
cerevisiae on glucose with different nitrogen source
Parameter and unit Urea Ammonium
Ethanol yield (mol/mol of glucose) 1.86 ± 0.04 1.77 ± 0.02
Glycerol yield (mmol/mol of glucose) 70.00 ± 0.17 94.00 ± 0.22
Acetic acid yield (mmol/mol of glucose) 24.00 ± 0.12 40.00 ± 0.19
Fermentation time (h) 42.00 ± 0.17 52.00 ± 0.22
Maximal ethanol production rate (g/L/h) 1.14 ± 0.03 0.88 ± 0.02
Effect of urea or ammonium on cell physiology of S. cerevisiae
urea than ammonium (Table 1). In addition, Table 1 also
showed the maximal ethanol production rate increased 30%
(from 0.88 g/L/h with ammonium to 1.14 g/L/h with urea).
Glycerol was the main by-product in ethanol fermentation.
Figure 3a showed that ammonium leaded to the increase
of glycerol comparing with urea. From Table 1 we can see
the glycerol yield in medium prepared by ammonium was
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World Journal of Microbiology and Biotechnology (2021) 37:98
94 mmol/mol glucose, where the glycerol yield in medium
using urea as nitrogen source was 70 mmol/mol glucose.
Therefore, urea could decrease the synthesis of glycerol from
glucose by 26%. Acetic acid was another by-product in etha-
nol fermentation. Compared with ammonium, urea could
also decrease the production of acetic acid (Fig. 3b). Ace-
tic acid yields decreased from 40 (with urea) to 24 mmol/
mol (with ammonium) glucose (Table 1). In another words,
urea resulted in a 40% decrease comparing with ammonium.
Our results showed that cell grown was faster in fermenta-
tion medium prepared with urea than ammonium (Fig. 3f).
Moreover, cell number is greater with urea than ammonium.
The presence of urea increased the yeast growth, reflecting
faster glucose uptake rate and subsequent metabolism. The
pH of media decreased rapidly in the first 8 h of fermenta-
tion. The final pH of cultures with ammonium sulfate was
lower than those with urea (Fig. 3e).
Figure 4a showed that intracellular pH (pHi) was 7.1
and 6.1 respectively with urea and ammonium. Thus
World Journal of Microbiology and Biotechnology (2021) 37:98
Fig. 4  Effect of urea or ammonium on different biochemical and physiological parameters of S. cerevisiae in ethanol fermentation. Data pre-
sented are the average of three independent cultivations and error bars represent standard deviations
ammonium decreased the pHi by 14%. For the aspect
of energy, the values of ATP/DCW were 0.16 mg/g and
0.37 mg/g respectively with urea and ammonium (Fig. 4b).
Therefore, urease converting urea into ammonia resulted
in a more than 50% lower of ATP when comparing with
ammonium, indicating higher respiratory capacity of S.
cerevisiae in the presence of urea (Azmat et al. 2013).
The significant differences prompted us to determine the
level of redox for each nitrogen source. Figure 4c showed
the values of NADH/DCW were 0.21 mg/g and 0.14 mg/g
respectively with urea and ammonium, suggesting a 33%
lower NADH. Clearly, in batch cultures with ammonium
the NADH influences glycerol generation, whereas the
glycerol affected ethanol generation. Glycerol dehyde-
3-phosphate dehydrogenase (G3PDH) was determined to
confirm that reduced glycerol formation in medium pre-
pared with ammonium is due to elevated enzyme activity
of G3PDH. Figure 4d showed that the enzyme activity was
0.0225 and 0.0275 U/mg protein respectively with urea
and ammonium. The increase in the enzyme activity of
G3PDH suggested that the G3PDH were consistent with
the yields of glycerol (Fig. 4d).
Page 5 of 7 98
Discussion
TerSchure et al.(2000) reported that growth on good nitrogen
sources such as ammonia, glutamine and asparagine seemed
to yield relatively higher growth rates than on poor ones such
as proline and urea. However, Wang et al.(Wang et al. 2012)
reported that urea could lead to a relatively higher growth
rate and ethanol production rate than ammonium, which was
consistent with the results of the present study. Urea is trans-
ported into the cell by the inducible urea permease encoded
by DUR3, which is an active transport system working
mainly below 25 mM extracellular urea (Elberry et al. 1993).
Another way of getting urea into the cell was by facilitated
diffusion which predominantly occurs at extracellular con-
centrations of above 0.5 mM (Cooper and Sumrada, 1975;
Sumrada et al. 1976). In this study, the nitrogen concentra-
tion of urea was 0.5 g/L (the concentration of urea was 17.86
mM). Therefore, the transport system of urea was to com-
bine both approaches. Although central carbon metabolism
can also reduce the pH of the medium, the degradation of the
pH is mainly due to the excretion of organic acid which are
formed as by-products of metabolism. But for ammonium
13
98 Page 6 of 7
using as nitrogen source, a proton must be exported in order
to maintain homoeostasis of the proton motive force (Kok
et al. 2012), which caused the pH of the medium decreasing
to 2.2–2.3 after 15 h (Fig. 3e). In urea-sufficient cultures of
S. cerevisiae, urea uptake was not proton-coupled, which
did not cause medium acidification (Hensing et al. 1995).
Medium prepared with ammonium inhibited cell growth and
the yield of ethanol from glucose, which altering the flow of
carbon when comparing with urea.
The effect of each nitrogen source on pHi was studied
in this paper. Though the range of pH values that can be
accurately measured was limited, as the resolution of the
calibration curve deteriorates below 5.5 and above 8.0, but
it was sufficient to measure pH values currently reported
in living cells (Orij et al. 2009). The present of ammonium
acidized the medium, which caused the pHi of S. cerevi-
siae significantly decrease when comparing with urea. The
changes in intracellular pH affected the ionization state of
all organic weak acids and bases, including the essential
building blocks of life, thus contributing to an extensive
array of biological processes (Brett et al. 2006). Urease
converts urea into ammonia and ­CO2 in a two-step reaction
that involves ATP hydrolysis (Mobley et al. 1995), resulting
in a cost of 0.5 ATP per mol N ­ H3 assimilated into product
(Milne et al. 2015). Therefore, the energy generated in the
medium prepared with urea was consumed by urease, caus-
ing the decrease of ATP. Under anaerobic fermentations, net
formation of NADH produced during synthesis of biomass
and organic acids, i.e., acetic acid, and pyruvic acid, must
be reoxidized to ­NAD+ by formation of glycerol in order to
avoid a serious imbalance in the ­NAD+/NADH ratio. Syn-
thesis of 1 mol glycerol from glucose leads to reoxidation of
1 mol NADH (Zhang & Chen, 2008). The result of NADH
was consistent with glycerol, suggesting the decrease of
NADH was due to the increase of glycerol. Metabolic shift
in microorganisms can be induced by various environmen-
tal changes (Sunya et al. 2012; Zhu & Yang, 2004). The
decrease of G3PDH activity in urea suggested that the cell
occurred a metabolic shift at logarithmic phase, thus more
carbon was transferred into ethanol rather than for glycerol.
Zhu and coworkers (Zhu & Yang, 2004) found that the meta-
bolic shift was closely related to the activities of several key
enzymes. Similarly, the activity of G3PDH was increased
when ammonium was used as nitrogen source, suggesting
that the metabolic shift may be induced by a pH decrease.
Conclusions
Ammonium and urea were two cheap nitrogen sources
commonly used in large scale fermentation processes. The
results in this paper showed that urea was the suitable nitro-
gen source for ethanol product by S. cerevisiae. The study
13
World Journal of Microbiology and Biotechnology (2021) 37:98
will have a certain guiding significance for the using of
nitrogen source in industrial ethanol production.
Acknowledgements This study was financially supported by the fol-
lowing projects: (1) Shandong postdoctoral innovation project. (2) Doc-
tor’s fund of University of Jinan.
Declarations
Conflict of interest The authors declared that they have no conflicts of
interest to this work.
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