Posted: October 27th, 2022

Unit 4 Assessment Research



Employ: Research Process

Evaluation Title: Research

You will have to complete a major research paper (8 to 10 pages) for the final assignment in this course.  In order to do this, you will need to write in a style appropriate for an academic discourse community and read numerous scholarly articles.  At least five of the articles must be located in the Herzing University Library.

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For this week’s assignment:

  • Identify 3 a minimum of primary resources and 2 secondary resources, for one of the following topics:

    3D Printing
    High speed rail
    Stem Cell Research

  • Complete a literature review for each of the resources identified
  • Following your literature review include the introduction to your paper which should include the claim statement and thesis statement.
  • Include a reference page

Your literary resources, and introduction should be in APA format. All citations and references should be in APA format.

Estimated time to complete: 6 hours

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A little acid can make a cell stemlike: mouse cells enter
primordial state capable of making any tissue
Author: Tina Hesman Saey
Date: Feb. 22, 2014
From: Science News(Vol. 185, Issue 4)
Publisher: Science News/Society for Science and the Public
Document Type: Article
Length: 732 words

Full Text:
Creating stem cells may be as simple as dunking cells into a mild acid bath.

Doing so turned cells from newborn mice into ultraflexible stem cells that could grow into any type of body tissue, researchers report
in the Jan. 30 Nature. Other stresses, such as squeezing cells through glass tubes, can also reprogram cells, Haruko Obokata of the
RIKEN Center for Developmental Biology in Kobe, Japan, and Brigham and Women’s Hospital in Boston and colleagues discovered.

If it works on human cells, the technique could provide replacement cells for diseased body parts, foster a better understanding of a
person’s disease risks and drug sensitivities and maybe serve as a fertility treatment.

The method has floored other researchers, who thought that creating stem cells required more complex operations: extracting cells
from embryos, transferring the nucleus of an adult cell to an egg cell or using viruses or other means to introduce factors that coax an
adult cell to behave like an embryonic stem cell.

“It’s fascinating. It’s perplexing. It’s potentially profound, but leaves lots of reasons to scratch my head,” says George Daley, a stem
cell researcher at Boston Children’s Hospital and Harvard Medical School. “It’s begging to be replicated,” he says, adding that his lab
will attempt to do just that.

In the new study, about 7 to 9 percent of cells from newborn mice survived the acid treatment and took just a week to form primordial
cells, dubbed STAP cells for stimulus-triggered acquisition of pluripotency. Pluripotent

cells can develop into cells of any tissue. Both embryonic stem cells and reprogrammed cells known as induced pluripotent stem
cells, or iPS cells, are pluripotent.

STAP cells may be even more flexible, Obokata says. When injected into mouse embryos, STAP cells not only incorporated into any
body tissue but could also form parts of the placenta. That’s a feat other pluripotent cells generally can’t accomplish, and it may
indicate that STAP cells are totipotent, or capable of forming a complete organism.

Obokata and her colleagues transformed skin, brain, muscle, fat, bone marrow, lung, liver and white blood cells from 1-week-old mice
into STAP cells. The technique worked, but not as well, on cells from young adult mice that were 6 weeks old, she says. The
researchers have begun testing the acid treatment on human cells.

Dieter Egli, a stem cell researcher at the New York Stem Cell Foundation, is skeptical. “If I were to describe this over a coffee break
to one of my colleagues, they’d say, ‘You must be kidding,'” he says. He knows of no mechanism that could explain how mild acid or
squeezing changes a cell’s fate so dramatically and consistently in one direction. Egli wonders why, for instance, blood cells became
stem cells instead of transforming into muscle or any other type of cell.

Cells undergo stress in daily life, Egli points out. If simple acid or mechanical stress causes cells to revert to an early developmental
state, he says, “it’s hard to imagine how our bodies would maintain integrity over a lifetime.”

But Qi-Long Ying, a stem cell biologist at the University of Southern California in Los Angeles, speculates that the body produces
inhibitory factors that prevent stress from reprogramming cells. Without those inhibitions, lab-grown cells can regress to an immature
state. Understanding how stress reverts mouse cells to the anything-goes state may teach researchers more about cancer, another
condition in which cells have no particular identity.

Ethical barriers may pop up on the road to using STAP cells. Because STAP cells may be totipotent, UCLA stem cell researcher

James Byrne worries that the new technology may raise old specters of human cloning. Acid-reprogrammed cells potentially could
grow into a fetus, placenta and all. If that’s true, the cells might treat infertility by creating an embryo from an adult’s cells, Byrne says.

Still unclear is whether researchers will choose STAP cells over other types of stem cells, says Louise Laurent, a stem cell biologist
at the University of California, San Diego. Regardless, she says, the work “will inspire people to explore less traditional ways of
changing a cell’s fate.”

Caption: By injecting a new type of stem cell into a mouse embryo, researchers showed that the cells could give rise to any type of
cell in the body. Fetal tissues derived from the stem cells glow green.


Please note: Illustration(s) are not available due to copyright restrictions.

Copyright: COPYRIGHT 2014 Science News/Society for Science and the Public
Source Citation (MLA 8th Edition)
Saey, Tina Hesman. “A little acid can make a cell stemlike: mouse cells enter primordial state capable of making any tissue.” Science

News, vol. 185, no. 4, 22 Feb. 2014, p. 6. Gale Academic OneFile, Accessed 6 Feb. 2021.

Gale Document Number: GALE|A359334166

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convenience and is in no way intended to replace original scanned PDF. Neither Cengage Learning nor its licensors make any
representations or warranties with respect to the machine generated PDF. The PDF is automatically generated “AS IS” and “AS
PURPOSE. Your use of the machine generated PDF is subject to all use restrictions contained in The Cengage Learning
Subscription and License Agreement and/or the Gale Academic OneFile Terms and Conditions and by using the machine
generated PDF functionality you agree to forgo any and all claims against Cengage Learning or its licensors for your use of the
machine generated PDF functionality and any output derived therefrom.

Chemists finally create elusive acid: textbook chemical
cyanoform was sought for over a century
Author: Beth Mole
Date: Oct. 31, 2015
From: Science News(Vol. 188, Issue 9)
Publisher: Science News/Society for Science and the Public
Document Type: Brief article
Length: 383 words

Full Text:
After more than a century of effort, chemists have nabbed a legendary acid.

Cyanoform, or tricyanomethane, appears widely in textbooks as one of the strongest carbon-based acids. Yet despite repeated
attempts to make the acid, cyanoform has evaded chemists until now. Researchers report September 18 in Angewandte Chemie
International Edition that they isolated the acid by figuring out crucial experimental conditions.

The main problem was temperature, says Andreas Kornath, an inorganic chemist at Ludwig Maximilian University of Munich.
Chemists had assumed that cyanoform is stable at room temperature. But using trial and error, Kornath and his team found that the
acid is stable only below-40[degrees] Celsius.


Cyanoform has a central carbon atom attached to a hydrogen atom and to three cyano groups, each consisting of a carbon
triplebonded to a nitrogen. The molecule easily loses its hydrogen, making it a strong acid and demonstrating a rule of carbon acids–
electron-loving groups (the cyano groups) attached to a central hydrogen-toting carbon pull on that carbon’s electrons. The
molecule’s electrons settle into a position close to the cyano groups, weakening the link to the hydrogen.

At room temperature, cyanoform decomposes, forming junk molecules, Kornath says. That probably happened when chemist
Hermann Schmidtmann tried to make cyanoform in 1896. He mixed sulfuric acid with a stable relative of cyanoform called sodium
tricyanomethanide. That molecule, a salt of cyanoform, has the same structure as the acid except it has lost the positive hydrogen
ion, resulting in a negative molecule that is paired with a positive sodium ion.

Schmidtmann expected that sulfuric acid would stick a hydrogen atom onto the negative tricyanomethanide, forming cyanoform.
Instead, he ended up with a concoction that probably contained only remnants of the unstable acid.

But at frigid temperatures, Kornath and colleagues made the acid. The team reacted a strong acid, hydrogen fluoride, with a salt of
cyanoform. Multiple chemical analyses showed that the resulting molecule matched cyanoform’s structure.

“It’s very noteworthy,” says physical chemist Daniel Kuroda of Louisiana State University in Baton Rouge. Theoretical chemistry
cannot predict the temperatures at which substances decompose, he says. But experimental information like this gives chemists new

Caption: Try, try again At last, researchers have isolated cyanoform (chemical structure shown), a strong carbon acid, by making it at
very cold temperatures.

Mole, Beth

Copyright: COPYRIGHT 2015 Science News/Society for Science and the Public
Source Citation (MLA 8th Edition)
Mole, Beth. “Chemists finally create elusive acid: textbook chemical cyanoform was sought for over a century.” Science News, vol.

188, no. 9, 31 Oct. 2015, p. 11. Gale Academic OneFile, Accessed 6 Feb. 2021.

Gale Document Number: GALE|A433481330

Guo et al. Biotechnol Biofuels (2018) 11:297


Changes in lipid metabolism convey acid
tolerance in Saccharomyces cerevisiae
Zhong‑peng Guo1,5, Sakda Khoomrung2,3, Jens Nielsen3,4 and Lisbeth Olsson1*

Background: The yeast Saccharomyces cerevisiae plays an essential role in the fermentation of lignocellulosic hydro‑
lysates. Weak organic acids in lignocellulosic hydrolysate can hamper the use of this renewable resource for fuel and
chemical production. Plasma‑membrane remodeling has recently been found to be involved in acquiring tolerance
to organic acids, but the mechanisms responsible remain largely unknown. Therefore, it is essential to understand the
underlying mechanisms of acid tolerance of S. cerevisiae for developing robust industrial strains.

Results: We have performed a comparative analysis of lipids and fatty acids in S. cerevisiae grown in the presence
of four different weak acids. The general response of the yeast to acid stress was found to be the accumulation of
triacylglycerols and the degradation of steryl esters. In addition, a decrease in phosphatidic acid, phosphatidylcholine,
phosphatidylserine and phosphatidylethanolamine, and an increase in phosphatidylinositol were observed. Loss of
cardiolipin in the mitochondria membrane may be responsible for the dysfunction of mitochondria and the dramatic
decrease in the rate of respiration of S. cerevisiae under acid stress. Interestingly, the accumulation of ergosterol was
found to be a protective mechanism of yeast exposed to organic acids, and the ERG1 gene in ergosterol biosynthe‑
sis played a key in ergosterol‑mediated acid tolerance, as perturbing the expression of this gene caused rapid loss
of viability. Interestingly, overexpressing OLE1 resulted in the increased levels of oleic acid (18:1n‑9) and an increase
in the unsaturation index of fatty acids in the plasma membrane, resulting in higher tolerance to acetic, formic and
levulinic acid, while this change was found to be detrimental to cells exposed to lipophilic cinnamic acid.

Conclusions: Comparison of lipid profiles revealed different remodeling of lipids, FAs and the unsaturation index of
the FAs in the cell membrane in response of S. cerevisiae to acetic, formic, levulinic and cinnamic acid, depending on
the properties of the acid. In future work, it will be necessary to combine lipidome and transcriptome analysis to gain
a better understanding of the underlying regulation network and interactions between central carbon metabolism
(e.g., glycolysis, TCA cycle) and lipid biosynthesis.

Keywords: Weak acids, Sustainable, Yeast physiology, S. cerevisiae, Oxidative stress

© The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License
(http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium,
provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license,
and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/
publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Weak organic acids such as acetic, formic and levulinic
acids are present in lignocellulosic hydrolysate as poten-
tial inhibitors that can hamper the use of this renewable
resource for fuel and chemical production [1]. The yeast
Saccharomyces cerevisiae plays an essential role in the
fermentation of lignocellulosic hydrolysates. However,

this yeast species is also a food spoilage agent when it
gains resistance against the currently used organic-acid
preservatives [2]. Therefore, it is essential to understand
the underlying mechanisms of acid tolerance of this yeast
either for developing robust industrial strains, or for con-
trolling spoiling yeasts.

The effects of weak acids on S. cerevisiae have been gen-
erally ascribed to acidification of the cytosol by the pro-
tons released and/or accumulation of the anions of the
acid, which can be toxic to essential metabolic functions
[3, 4]. Acetic acid in particular inhibits NADH dehydro-
genase and induces programmed cell death [5, 6]. Lipo-
philic weak acids, such as sorbate and benzoate which are

Open Access

Biotechnology for Biofuels

1 Department of Biology and Biological Engineering, Industrial
Biotechnology, Chalmers University of Technology, 412 96 Gothenburg,
Full list of author information is available at the end of the article

Page 2 of 15Guo et al. Biotechnol Biofuels (2018) 11:297

commonly used as preservatives in the food and beverage
industry, can damage the membrane and disrupt oxida-
tive phosphorylation [7, 8], influence the transportation
of nutrients [9], and trigger the endogenous production
of superoxide free radicals [10]. Responses to weak acids,
such as ATP-dependent efflux of the protons and ani-
ons, via plasma membrane H+-ATPase Pma1p and the
ATP-binding cassette transporter (Pdr12p), have been
suggested [11, 12]. The involvement of H+-ATPase and
Pdr12p at the expense of ATP compromises biomass for-
mation [13].

To develop more robust biocatalysts with high acetic-
acid tolerance, metabolic engineering [14–17], genome
shuffling [18], evolutionary engineering [19] and
genome-wide gene screening [20, 21] have been used.
Despite these efforts, there is still a need to develop
strains of S. cerevisiae tolerant to acetic acid and/or other
acids. Plasma-membrane remodeling has recently been
suggested to play a role in the acetic-acid adaptation of S.
cerevisiae and Zygosaccharomyces bailii [22–26]. Particu-
larly, sphingolipids have been shown to play an important
role in acetic-acid resistance in Z. bailii [24, 25]. How-
ever, the mechanism responsible and the physiological
significance of cell-membrane remodeling in response to
acid stress remain largely unexplored.

The main components of the cell membrane of S. cer-
evisiae are glycerophospholipids, sterols and intra-mem-
brane proteins [27, 28]. In addition, yeast cells have a pool
of neutral lipids consisting of triacylglycerols (TAGs) and
steryl esters (STEs), stored as lipid droplets that serve
as reservoirs of cellular energy and building blocks for
membrane lipids. The most abundant fatty acid (FA) spe-
cies of the yeast cells are oleic acid (C18:1n-9) and palmi-
toleic acid (C16:1n-7), followed by palmitic acid (C16:0)
and stearic acid (18:0), and small amounts of myristic
acid (C14:0) and arachidic acid (C20:0) [27]. Quantitative
studies of the response of neutral lipids and cellular FAs
under conditions of acid stress may help to increase our

knowledge on lipid metabolism under specific growth
conditions. In the present study, we have analyzed both
lipids and fatty acids in S. cerevisiae exposed to stress
from different acids, i.e., hydrophilic acetic, formic, lev-
ulinic acid and lipophilic cinnamic acid. The aim of this
study was to map the changes in the lipid profile of the
yeast cells when exposed to weak acids with different
properties, and to guide the genetic engineering of yeast
to control its robustness in acid stress.

Physiological response of S. cerevisiae to weak acids
Under the reference condition (without addition of acid),
yeast started to grow on glucose without a lag phase,
at μmax reaching 0.41  h−1 followed by a second growth
phase on the ethanol produced during the glucose growth
phase (Table  1). Yeast growth stopped immediately fol-
lowing addition of the acids. It was noted that 0.17 mM
undissociated cinnamic acid, a much smaller amount
than the other acids, led to a 50% reduction in the bio-
mass yield (Table 1), which indicates that the hydropho-
bicity of the acid governs the toxicity of the acid. Less
undissociated formic acid (10.0 mM) was required to give
the same level of biomass reduction as 68.7  mM acetic
acid and 79.0 mM levulinic acid. Formic acid has a lower
hydrophobicity than acetic and levulinic acids, but its
higher toxicity has been ascribed to its smaller molecular
size [29, 30].

The growth of yeast on glucose and ethanol in the pres-
ence of the organic acids was greatly impaired, as can
be seen from the long lag phases and low growth rates
(Table  1, Additional file  1: Fig. S1). In addition, it was
noted that glucose and ethanol were continuously con-
sumed by acid-stressed cells during the adaptation phase
on either of the carbon sources, and that this was not
accompanied by any accumulation of biomass. No obvi-
ous decrease in acid concentration was observed in any
of the cultures during the adaptation phase on glucose

Table 1 Effects of weak acids on the growth of S. cerevisiae under aerobic conditions

N/A not available, Glc glucose, EtOH ethanol
a LogP, the lipophilic tendency given by the partition coefficient octanol–water (P)

Control Acetic acid Cinnamic acid Formic acid Levulinic acid

pKa N/A 4.79 4.44 3.75 4.66

LogPa N/A − 0.17 2.13 − 0.54 − 0.49
Concentration (mM) 0 180 0.7 180 260

Undissociated acid (mM) 0 68.7 0.15 10.0 79.0

Adaptation phase Glc. (h) 0 32 4 24 48

μmax‑glc (h
−1) 0.41 ± 0.01 0.18 ± 0.01 0.12 ± 0.01 0.20 ± 0.01 0.22 ± 0.01

Yx/s (g‑DCW/g‑glc) 0.15 ± 0.01 0.07 ± 0.01 0.07 ± 0.01 0.07 ± 0.00 0.07 ± 0.01
Adaptation phase EtOH. (h) 0 60 N/A 32 54

Page 3 of 15Guo et al. Biotechnol Biofuels (2018) 11:297

(Additional file  1: Fig. S1). Strikingly, growth was not
resumed for the yeast exposed to cinnamic acid. More-
over, yeast cells exposed to acid stress exhibited a sig-
nificant decrease in specific rates of O2 consumption,
compared to the control (Table 2). In addition, increases
were observed in the specific rates of glucose consump-
tion, and ethanol and CO2 production in response to
formic, acetic and levulinic acids stress. However, these
specific rates were lower in cells exposed to cinnamic
acid than the control, reflecting the acid-dependent inhi-
bition of glycolysis and respiration. After the adaptation
on ethanol, yeast started to grow on acetic acid only
after ethanol depletion. It is unclear why the presence of
ethanol represses the consumption of acetic acid. As for
formic acid-stressed cells, formic acid was co-consumed
with ethanol. In S. cerevisiae, formic-acid consumption is
catalyzed by NAD+-dependent formate dehydrogenases,
which oxidize formate to carbon dioxide and H2O, with-
out energy generation [31]. In this case, the biomass was
mainly produced from ethanol. By contrast, yeast was
unable to consume levulinic acid (Additional file  1: Fig.

Comparison of neutral lipid storage
The amount of neutral lipid storage in lipid droplets
is generally relatively low in S. cerevisiae (< 15%), but is probably highly dynamic as yeast is readily and rapidly able to adjust its internal metabolism according to the growth conditions [32]. The cellular content of STEs decreased by 18% in cells exposed to formic, levulinic and acetic acids, and by 25% in cells under the stress of cinnamic acid during the adaptation phase on glucose (phase 1). Thereafter, a continuous decrease in STEs was observed in the cells grown on glucose (phase 2) and dur- ing adaptation on ethanol (phase 3), especially the cells exposed to levulinic and cinnamic acids, for which the decrease in STEs was up to 40% and 50%, respectively, in the stationary phase (phase 5). However, the con- trol adapted on ethanol (phase 3) showed about a 20% increase in cellular STE content compared with cells in the exponential phase (Fig. 1a).

In contrast, the cellular TAG content increased from
18 to 23% in cells under acid stress compared with the
control in the adaption phases on glucose and ethanol.

During the growth phase on glucose, TAGs were rap-
idly mobilized in yeast cells exposed to formic, acetic
and levulinic acids, but not cinnamic acid. However, a
30% increase in TAG content was observed for the cells
exposed to acetic acid in the stationary phase. In com-
parison, in the stationary phase, with the depletion of
the carbon sources in media, the presence of levulinic
and cinnamic acids imposed a continuous requirement
for ATP generation. As one way to supply energy, degra-
dation of FAs from TAGs to β-oxidation led to a further
decrease in the cellular TAG content of those cells [33]
(Fig. 1b).

Comparison of the cellular ergosterol content
The ergosterol content in the cell membrane of S. cerevi-
siae changed considerably under acid stress. While the
control showed a gradual decrease in ergosterol from the
exponential phase to the stationary phase, exposure of the
cells to acids led to continuous accumulation of the sterol
content during different growth phases. Specifically,
yeast cells exposed to cinnamic acid showed a continuous
increase in cellular ergosterol content, ranging from 28 to
70% throughout the cultivation process, followed by cells
subjected to levulinic-acid stress, for which an increase
from 20 to 60% was observed. Similarly, an increase in
cellular ergosterol content was observed in cells exposed
to formic acid and acetic acid before glucose depletion.
However, none of these three acids resulted in a signifi-
cant increase in ergosterol in cells grown on ethanol, and
the cells contained similar contents of ergosterol to the
control during the stationary phase (Fig. 1c).

Mapping of the cellular phospholipid profile
Exposure of the yeast cells to formic, levulinic and ace-
tic acids did not lead to a significant change in the cel-
lular content of phosphatidic acid (PA) compared to the
control at the different growth phases. Remarkably, more
than a 30% decrease in cellular PA content was observed
in yeast cells exposed to cinnamic acid throughout culti-
vation (Fig. 1d).

Yeast cells exposed to acetic, formic and levulinic acids
showed a 10% increase in the cellular content of cardi-
olipin, and after acid adaptation, the growing cells con-
tained slightly less cellular cardiolipin than the control.

Table 2 Metabolic flux analysis of S. cerevisiae in the presence of weak acids at pH 5 under aerobic conditions

Control Acetic acid Cinnamic acid Formic acid Levulinic acid

O2 (mmol/g/h) 11.6 ± 0.2 7.3 ± 0.2 6.0 ± 0.0 8.3 ± 0.2 7.8 ± 0.2
Glucose (mmol/g/h) 18.1 ± 0.2 18.6 ± 0.2 15.9 ± 0.1 18.9 ± 0.6 19.4 ± 0.3
CO2 (mmol/g/h) 29.0 ± 0.4 29.4 ± 0.1 20.0 ± 0.2 29.3 ± 0.5 30.3 ± 0.4
Ethanol (mmol/g/h) 26.5 ± 0.2 28.8 ± 0.2 17.9 ± 0.3 29.6 ± 0.4 30.1 ± 0.3

Page 4 of 15Guo et al. Biotechnol Biofuels (2018) 11:297

However, a continuous decrease in cardiolipin content,
up to 60%, was observed in cells stressed by cinnamic
acid throughout cultivation. Less decrease (up to 30%)
was observed in the cells exposed to levulinic acid, and
only after depletion of the glucose or ethanol (Fig.  1e).
The cellular contents of phosphatidylethanolamine (PE),
phosphatidylcholine (PC) and phosphatidylserine (PS)
decreased in yeast cells under acid stress during the two
adaptation phases, and the exponential growth phase on
glucose (phase 2), compared to the control (Fig.  1f–h).
However, the cellular content of phosphatidylinositol
(PI) in yeast cells subjected to acid stress increased dur-
ing the adaptation phase on glucose (phase 1) and the
two growth phases (phase 2 and 4), and decreased during

the adaption phase on ethanol (phase 3), compared to the
control (Fig. 1i).

FAs and the unsaturation index of FAs in response to weak
Interestingly, in contrast to the relatively small change
in the cellular content of phospholipids, exposure of
the yeast cells to different acids triggered a significant
rearrangement of FA composition of the phospholipids.
Concerning the profile of the FAs obtained from polar
lipids, mainly phospholipids, in yeast cells under acid
stress, the amount of C14:0 was around 5% of the total
FAs, similar to that of the control. However, a decrease
in C16:0 and C16:1n-7, and an increase in C18:1n-9

Fig. 1 Comparison of the lipidome profiles of the yeast strains during aerobic culture without and with the addition of acetic, formic, levulinic and
cinnamic acids, at five different growth phases, at pH 5.0. a STEs, b TAGs, c ES, d PA, e CL, f PE, g PC, h PS and i PI. The growth phases are defined as:
phase 0, the exponential growth phase before acid addition; phase 1, the adaptation phase on glucose after acid addition; phase 2, the exponential
growth phase on glucose; phase 3, the adaptation phase on ethanol; phase 4, the exponential growth phase on ethanol; and Phase 5, the stationary
phase. Level change = (lipid content of phase 1–5—lipid content of phase 0)/lipid content of phase 0

Page 5 of 15Guo et al. Biotechnol Biofuels (2018) 11:297

and C18:0 were observed for all the acid-stressed cells
throughout cultivation. It was noticed in particular that
yeast cells exposed to cinnamic acid showed a smaller
increase in C18:1n-9 (up to 30%) and a smaller decrease
in C16:0 (up to 13%), but a greater increase in C18:0
(up to 90%) and a greater decrease in C16:1n-7 (up to
55%), than the cells stressed by the other acids (Fig. 2).
Moreover, about 2% lignoceric acid (C24:0) was found
in yeast cells exposed to cinnamic acid, while this FA
was negligible in the cells exposed to the other acids
and in the control.

Although the FA composition showed significant dif-
ferences, comparing the content of unsaturated FAs illus-
trated that the unsaturation index was largely unaffected
in the cells exposed to cinnamic acid, compared with the
control. However, exposure of the cells to the other acids
resulted in an continuous increase in the unsaturation

index of FAs compared with the control throughout cul-
tivation (Table 3).

Overexpression and repression of OLE1 in S. cerevisiae
The OLE1 gene encodes the only ∆-9 fatty acid desatu-
rase in S. cerevisiae and it is required for the production
of monounsaturated FAs [34]. To investigate whether
the increase in the unsaturation index of FAs is a pro-
tective mechanism of yeast cells in response to acid
stress, FA desaturase was overexpressed or repressed
in S. cerevisiae CEN.PK 113-5D. Under normal growth
conditions, the recombinant S. cerevisiae CEN-RO1
(PTEF-OLE1-reverse) in which OLE1 is repressed, and
S. cerevisiae CEN-O1 (PTEF-OLE1) in which OLE1
is overexpressed, showed similar growth patterns to
the control strain CEN.PK 113-5D harboring plasmid
p426TEF (data not shown). In addition, comparing the

Fig. 2 Comparison of the profiles of the abundant fatty acids in polar lipids of yeast strains during aerobic culture without and with the addition of
acetic, formic, levulinic and cinnamic acids, at six different growth phases, at pH 5.0. a Oleic acid, b stearic acid, c palmitic acid and d palmitoleic acid

Page 6 of 15Guo et al. Biotechnol Biofuels (2018) 11:297

FA composition of the phospholipids showed that the
amount of C14:0 was largely unchanged in yeast cells in
which OLE1 was overexpressed or repressed, compared
with the control. Interestingly, a significant increase in
C18:1n-9 and a considerable decrease in C16:0 were
observed in yeast cells overexpressing OLE1, while
yeast cells in which the expression of OLE1 had been
repressed showed a dramatic increase in C16:0 and a
significant decrease in C18:1n-9 (Fig.  3a). The unsatu-
ration index of FAs in yeast cells overexpressing OLE1
increased by 26%, 20%, 9% and 8% in the exponential
phase, ethanol adaption phase, ethanol growth phase
and stationary phase, respectively, compared with the
control. In contrast, the repression of OLE1 led to a
50% decrease in the unsaturation index of FAs in yeast
cells growing on glucose, compared to the control.
However, a smaller decrease in the unsaturation index
of FAs was seen in this yeast during the other growth
phases (Fig.  3b). Therefore, the increase/decrease
in the unsaturation index of cells in which OLE1 was

overexpressed or repressed was mainly due to the
increase/decrease in cellular content of C18:1n-9.

The composition and unsaturation index of FAs and acid
tolerance of the yeast
Yeast cells in which OLE1 is overexpressed or repressed
were inoculated into cultures in which formic, acetic,
levulinic and cinnamic acids had been added. Increasing
the unsaturation index of FAs had a beneficial effect in
that it reduced the lag phase and improved the survival
rate of the yeast cells exposed to formic, acetic and lev-
ulinic acids (Fig.  4, Additional file  1: Fig. S2). However,
a significant change in μmax was observed, compared to
the control. In contrast, yeast cells with a lower unsatura-
tion index of FAs under the stress of formic, acetic and
levulinic acids showed a longer lag phase and lower sur-
vival rate than the control (Fig.  4, Additional file  1: Fig.
S2). When the unsaturation index of FAs was reduced,
yeast was unable to grow in the presence of 175 mM for-
mic acid, 175 mM acetic acid and 300 mM levulinic acid.

Table 3 Effects of weak acids on the unsaturation index of fatty acids under aerobic conditions

Control Acetic acid Cinnamic acid Formic acid Levulinic acid

Initial growth phase 62.5 ± 0.8 62.5 ± 0.8 62.5 ± 0.8 62.5 ± 0.8 62.5 ± 0.8
Adaptation glucose N/A 66.0 ± 1.0 64.0 ± 1.2 65.6 ± 1.0 66.5 ± 0.9
Growth phase on glucose 63.5 ± 1.0 67.0 ± 0.6 65.2 ± 0.6 67.0 ± 0.3 66.8 ± 1.0
Adaptation ethanol 66.0 ± 0.7 69.1 ± 0.0 65.3 ± 1.0 69.0 ± 0.6 67.0 ± 0.3
Growth phase on ethanol 66.0 ± 1.0 70.0 ± 0.4 N/A 70.0 ± 1.1 N/A
Stationary phase 67.0 ± 1.6 69.0 ± 0.8 66.0 ± 2.0 70.0 ± 0.6 68.0 ± 1.0

Fig. 3 Fatty acid profile (a) and unsaturation index (b) of the control and recombinant S. cerevisiae strains CEN‑RO1 (PTEF‑OLE1‑reverse) and CEN‑O1
(PTEF‑OLE1) during the exponential growth phase on glucose (phase 1), lag phase on ethanol (phase 2), ethanol growth phase (phase 3) and
stationary phase (phase 4). The unsaturation index was calculated as sum of weight of FA multiplied by the number of unsaturated bonds for each
FA in the mixture

Page 7 of 15Guo et al. Biotechnol Biofuels (2018) 11:297

Fig. 4 The lag phase and μmax of the control and recombinant S. cerevisiae strains CEN‑RO1 (PTEF‑OLE1‑reverse) and CEN‑O1 (PTEF‑OLE1) during
aerobic growth with the addition of 100–200 mM acetic acid (a, b), 75–175 mM formic acid (c, d), 100–300 mM levulinic acid (e, f) and 0.3–0.7 mM
cinnamic acid (g, h), at pH 5.0

Page 8 of 15Guo et al. Biotechnol Biofuels (2018) 11:297

Interestingly, yeast cells in which the unsaturation index
of FAs decreased showed a shorter lag phase and higher
survival rate than the control under cinnamic acid stress
(Fig. 4g, h, Additional file 1: Fig. S2).

Ergosterol and acid tolerance of the yeast
To investigate whether the accumulation of ergosterol
is a protective mechanism in yeast cells in response to
acid stress, the ergosterol biosynthesis pathway was
perturbed by repressing the expression of squalene
epoxidase (ERG1), which plays an essential role in the
ergosterol biosynthesis pathway. The cellular content of
ergosterol in recombinant S. cerevisiae CEN-RE1 (PTEF-
ERG1-reverse), in which ERG1 was repressed, decreased
by 50% during exponential growth on glucose, compared
to the control. In addition, knocking down the expression
of this enzyme catalyzing the epoxidation of squalene to
2, 3-oxidosqualene impaired the growth of the yeast on
glucose (data not shown). Moreover, S. cerevisiae CEN-
RE1 was more sensitive to acid stress than the control, as
the exposure of S. cerevisiae CEN-RE1 to different acids
resulted in rapid loss of viability (Fig. 5).

Despite the presence of 10.0 μg/ml ergosterol, the cel-
lular content of ergosterol in cells under the non-stressed
condition was largely unaffected. However, the accu-
mulation of ergosterol, up to 10.0  mg/g dry cell weight
(DCW), was observed for cells subjected to acid stress,
i.e., a 30% increase compared with the control under the
stress conditions. In addition, yeast cells with a higher
cellular ergosterol content were more resistant to acid
stress than the control as they showed a higher survival
rate under 24-h acid stress (Fig. 5).

The lipid remodeling in S. cerevisiae during acid adap-
tation is summarized in Fig.  6. The biosynthesis and
hydrolysis of nonpolar lipids (TAGs and STEs) play an
important role in cellular FA composition and sterol
homeostasis [35]. Indeed, enhanced biosynthesis and
the accumulation of neutral lipids have been observed in
yeast exposed to environmental stress and starvation [36,
37]. As acid causes dysfunction of the mitochondria and
impairs respiration [33], the high specific rate of glucose
uptake accompanied by extremely low rate of respiration
(almost negligible during acid adaptation as determined
by oxygen consumption in this study) causes the accu-
mulation of the intermediates of glycolysis and the TCA
cycle. For instance, accumulation of acetyl-CoA, glyc-
erol-3-phosphate and dihydroxyacetone phosphate was
observed in acid-stressed cells (data not shown), which
may have contributed to the storage of TAGs. Another
important contribution to the increase in TAG synthesis
lies in the transcriptional regulation of lipid metabolism.
For instance, yeast cells exposed to acetic-acid stress
showed up-regulation of the PAH1 gene, which favored
the conversion of PA into TAG, and down-regulation
of the genes involved in the synthesis of PE, PS and PC
(PSS1, PSD1, EKI1 and CKI1), which indirectly supports
TAG accumulation, as their synthesis could compete for
the intermittent PA (Fig. 1, Additional file 1: Fig. S3).

Interestingly, yeast cells exposed to weak acids
showed down-regulation of FA synthase 1 (Fas1p),
which plays a key role in acyl-CoA production (the pre-
cursor for PA and TAG biosynthesis) (Additional file 1:
Fig. S3). It is unclear how the PA and TAG biosynthe-
sis was favored when the expression of FAS1 decreased
at regulation level. It has been shown that exposure of
yeast cells to H2O2 stress induced a decrease in both Fas
expression and activity in the evolved cells. In addition,
deletion of one of the FAS alleles, which caused a 50%
reduction in Fas activity, led to an increase in the resist-
ance of yeast to H2O2 [38]. As a follow-up to this obser-
vation, the cell-membrane composition was explored to
investigate the relation between the reduction of FAS
activity and H2O2 resistance, and the accumulation
of very-long-chain fatty acids (VLC-FAs) lignoceric
acid (C24:0) (40%) and cerotic acid (C26:0) (50%) was
found in the plasma membrane of the mutant cells. The
authors, therefore, ascribed the H2O2 resistance to the
fact that a high content of VLC-FAs reduces the over-
all or localized plasma-membrane permeability to H2O2
through interdigitation or by modulating the formation
of lipid rafts [38]. Yeast cells exposed to weak acids suf-
fered from oxidative stress induced by acids (Additional
file  1: Fig. S4); the increase in VLC-FA content of the
plasma membrane is probably a defense response of

Fig. 5 Viable fractions of the S. cerevisiae control, the control
supplemented with 10.0 μg/ml ergosterol and the recombinant strain
CEN‑RE1 (PTEF‑ERG1‑reverse) under the stress of: (a) 150 mM acetic
acid, (b) 150 mM formic acid, (c) 200 mM levulinic acid and (d) 0.8 mM
cinnamic acid, at pH 5.0

Page 9 of 15Guo et al. Biotechnol Biofuels (2018) 11:297

yeast to acid stress [39]. Indeed, VLC-FAs are the pre-
cursors of sphingolipid biosynthesis, and the accumu-
lation of VLC-FAs is expected to increase the cellular
sphingolipid content and its complexity [26]. Sphin-
golipids are essential structural components of cellular

membranes, in particular the plasma membrane [40].
Recent studies have suggested a link between high lev-
els of complex sphingolipids and the intrinsic tolerance
of Z. bailii species to acetic acid [23–25]. However, the
correlation between the decrease in the expression of

Fig. 6 Overview of lipid remodeling in yeast during acid adaptation, where heavy arrows indicate enhanced biosynthesis. Model created
from the data available for S. cerevisiae. Abbreviations used for major metabolic intermediates are: G3P glycerol‑3‑phosphate, CDP-DAG
cytidine diphosphate‑diacylglycerol, TAGs triacylglycerols, STEs steryl esters, PA phosphatidic acid, PC phosphatidylcholine, CL cardiolipin, PE
phosphatidylethanolamine, PI phosphatidylinositol, PS phosphatidylserine, ES ergosterol, FFA free fatty acids, LC-FAs long‑chain fatty acids, MC-FAs
medium‑chain fatty acids. Key gene names refer to the following encoded enzymatic activities: SCT glycerol‑3‑phosphate acyltransferase, SLC LPA
acyltransferase, ACC acetyl‑CoA carboxylase, ARE acyl‑CoA:cholesterol acyltransferase, DGA acyl‑CoA:DAG acyltransferase, FAS fatty acid synthetase,
LRO phospholipid:diacylglycerol acyltransferase, MFE multifunctional enzyme, PAP phosphatidate phosphatase, FAA fatty acyl‑CoA synthetase, PIS
phosphatidylinositol synthase, PSS phosphatidylserine synthase, PSD phosphatidylserine decarboxylase, EKI ethanolamine kinase, CKI choline kinase,
ERG1 squalene epoxidase, ERG6 squalene reductase, POT thiolase, POX acyl‑CoA oxidase, PXA peroxisomal acyl‑CoA transporter, TGL triacylglycerol
lipase. The changes in the expression levels of several key genes (inside the dark blue box) were verified by qPCR. The arrows with dashed lines
indicate that multiple reactions are involved in the corresponding synthetic pathway

Page 10 of 15Guo et al. Biotechnol Biofuels (2018) 11:297

Fas and the increase in VLC-FA content needs to be
further elucidated.

Using comparative functional genomics analysis, it has
been found in a previous study that yeast with a higher
tolerance to acetic acid has more oleic acid in the plasma
membrane [15]. Our findings confirmed that a higher
level of cellular oleic acid contributes to the tolerance of
S. cerevisiae to acetic, formic and levulinic acids (more
hydrophilic), but was detrimental in cells exposed to cin-
namic acid (which is more lipophilic). Given the similar
property of cinnamic acid to those used as preservatives,
such as sorbate and benzoate, in the food and beverage
industry, reducing oleic acid content and/or unsaturation
index of fatty acids in cell membrane is expected to be
useful strategies to impair the survivability of the spoil-
age yeasts. It remains to be elucidated on the molecular
and structural levels, how membrane remodeling influ-
ences the FA composition, the degree of saturation and
unsaturation. Further work is needed to study how these
changes influence the properties of the cell membrane in
terms of permeability, integrity and rigidity, either indi-
vidually or collectively. It is unclear how the change in the
expression level of fatty acid desaturase (OLE1) was able
to significantly influence the cellular content of C18:1n-9
and C16:0.

Cardiolipin is an important phospholipid, known to
maintain membrane potential and the architecture of
the mitochondria, and provides essential structural and
functional support to several proteins involved in mito-
chondrial bioenergetics [41]. Cardiolipin is particularly
susceptible to peroxidation due to the abundance of dou-
ble bonds in its structure [42], and its close association
with respiratory chain proteins, which are known to be
a major source of ROS in the mitochondria [43]. Acid
stress induces oxidative stress, and lipid peroxidation
could cause the loss of cardiolipin content in the mito-
chondria. Therefore, the enhancement of cardiolipin
biosynthesis may partially compensate for the loss of car-
diolipin and stabilize the mitochondria in cells stressed
by acetic, formic and levulinic acids. However, peroxida-
tion and loss of cardiolipin cannot be avoided in the case
of cinnamic acid, due to its ability to cause cell-mem-
brane disruption and oxidative stress. Therefore, prevent-
ing cardiolipin loss is probably important in maintaining
the normal function of the mitochondria in cells under
acid stress.

Free ergosterol is mainly incorporated into the plasma
membrane and is responsible for structural properties of
the membrane such as fluidity and permeability [44]. Ear-
lier studies have reported a positive correlation between
heat sensitivity and ergosterol levels, and that ergosterol
contributes to the ethanol tolerance of S. cerevisiae [45,
46]. In addition, changes in sterol composition from

ergosterol to ergosta5, 8-diene-3-ol have been suggested
to contribute to the HCl tolerance of the evolved strains
[47]. The ERG1 gene, encoding squalene epoxidase
which catalyzes the epoxidation of squalene to 2, 3-oxi-
dosqualene, has been suggested to be the rate-limiting
enzyme in ergosterol biosynthesis [48]. In the present
study, we demonstrated for the first time that higher cel-
lular levels of ergosterol improve the viability of yeast
cells under acid stress, and repressing the expression
level of ERG1 suggested that the ERG1 gene played a key
role in ergosterol-mediated acid tolerance. The disrup-
tive effect of weak acids on the cell membrane has been
known for a long time [4]. The presence of ethanol can
exhibit a synergistic inhibitory effect on yeast cells, as a
consequence of the effect both the acid and ethanol have
on the cell membrane [49]. Therefore, the accumulation
of ergosterol may protect the cell membrane against acid
stress, as a high level of ergosterol prevents interdigita-
tion and maintains an optimal membrane thickness, as
has already been described under ethanol stress [50].
Ergosterol can be produced either by the degradation of
STEs, which liberates ergosterol and sterol precursors,
or by de novo ergosterol synthesis [51, 52]. Although the
sterol intermediates released by the hydrolysis of STEs
may be converted into ergosterol much faster than de
novo sterol synthesis [53], given the fact that the STE
pool is very small when ergosterol is needed for mem-
brane formation during exponential growth, the decrease
in the STE pool alone can hardly contribute to the high
accumulation of ergosterol in yeast cells under acid
stress. In addition, the idea that acid stress enhances de
novo ergosterol synthesis is in agreement with our obser-
vations that the ERG1 and ERG6 genes involved in the
ergosterol biosynthetic pathway were up-regulated, and
the TGL1 gene for STE degradation was slightly down-
regulated, which further confirmed the important role of
ERG1 in ergosterol-mediated acid tolerance. The ARE1
gene-encoding sterol esterase was also down-regulated
(Additional file  1: Fig. S3). A recent study has revealed
that yeast cells under acetic-acid stress contained less
ergosterol in the mid-exponential growth phase than
non-stressed cells [23]. As yeast physiology is highly
dependent on the environmental conditions, the physio-
logical responses obtained in current study may be differ-
ent from those generated under other growth conditions
in the previous study [23]. Differences seen include acid
addition from the beginning of culture, cell samples at a
different growth phase, and different acid concentration,
which determines the toxicity of the acid. Given the com-
plex nature of sterol metabolism, a better understanding
of the mechanisms underlying ergosterol biosynthesis
is required to design suitable engineering strategies to
improve the acid tolerance of yeast.

Page 11 of 15Guo et al. Biotechnol Biofuels (2018) 11:297

Materials and methods
Yeast strains and media
The haploid, prototrophic S. cerevisiae strain CEN.
PK 113-7D (MATa) was grown in a defined medium
containing vitamins, trace elements and salts includ-
ing: 7.5  g/l (NH4)2SO4, 3.5  g/l KH2PO4 and 0.7  g/l
Mg2SO4·7H2O with 30  g/l glucose [54]. S. cerevisiae
CEN.PK 113-5D (MATa, SUC2, MAL2-8 c, ura3-52)
was cultured in YPD medium containing 20  g/l pep-
tone, 10 g/l yeast extract and 20 g/l glucose.

Growth conditions and acid pulse
The yeast was pre-cultured in defined medium (as
described above) until the exponential growth phase.
Batch cultures were carried out in a 3-l DASGIP biore-
actor (DASGIP Biotools LLC, Shrewsbury, MA) with a
working volume of 2 l. The temperature was set to 30 °C
and the pH was maintained at 5.0 by the automatic
addition of 2.0 M KOH. To prevent excessive foaming,
0.30  ml silicone antifoam (Sigma A8311) was added.
Aeration was set to 0.5 vvm, and the stirring speed to
600  rpm to give a dissolved oxygen tension of at least
60% of air saturation throughout fermentation. Yeast
cells were first cultivated until the optical density at
600  nm (OD) reached 1.0 (exponential growth phase),
after which, acetic, formic, levulinic or cinnamic acid
was added to the medium. The concentration of each
acid added was intended to result in half the biomass
yield obtained on glucose under aerobic conditions, as
determined by preliminary experiments (Table 1). Dur-
ing the cultivation process, CO2 production or O2 con-
sumption was measured continuously using an off-gas

Determination of substrate and extracellular metabolites
Cell suspensions (two of 1.5 ml each) were rapidly trans-
ferred from the culture into liquid nitrogen. The frozen
suspension was thawed on ice. Samples were centrifuged
at 3000×g for 5 min at 4  °C, and the supernatants were
subsequently subjected to high-performance liquid chro-
matography (HPLC). The measurement conditions used
for glucose, glycerol and ethanol, and acetic, formic and
levulinic acids were the same as in our previous work
[29]. Cinnamic acid was measured using GC–MS, as
described previously [55].

Dry weight determination
Two 10  ml culture samples were filtered through pre-
weighed polyethersulfone filters (0.45 μm, Sartorius Bio-
lab, Germany). The biomass retained by the filters was

washed, dried in a microwave oven at 150 W for 15 min,
and then placed in a desiccator before being weighed.

Calculation of physiological parameters
All data are presented as the mean ± standard deviation
(SD) of biological replicates (N ≥ 3). Lag phase was esti-
mated using DMFIT ( y/DMfit
), as described previously [56]. The biomass yield was
obtained as the slope of the linear curve when plotting
the biomass concentration versus the glucose concentra-
tion during exponential growth on glucose. The specific
rates of substrate consumption and product formation
were calculated as described previously [57]. The evap-
oration rate of ethanol was determined in a separate
cell-free experiment, and all data were corrected for the
evaporation of ethanol (1% of the ethanol at each point).

Lipid extraction
Yeast cells were harvested at different growth phases and
centrifuged at 3000×g for 5 min at 4 °C to collect the bio-
mass. The samples were then immediately frozen in liquid
nitrogen and placed in a freeze-dryer at − 40 °C overnight
before analysis. Lipids were extracted from the yeast cells
using a microwave-assisted method as described previ-
ously [58]. Briefly, freeze-dried cells (~ 10 mg) were sus-
pended in 7 ml of a mixture of chloroform and methanol
(2:1, v/v) containing 50 μg cholesterol as internal stand-
ard in a Pyrex borosilicate glass tube (16 × 100  mm).
The samples were flushed with nitrogen gas for 30 s and
sealed with a Teflon screw cap. After vigorous vortexing,
the samples were placed in the microwave reaction vessel
(12 cm × 3 cm I.D., 0.5 cm thickness; Milestone Stard D,
Sorisole Bergamo, Italy) containing 30 ml Milli-Q water.
The vessels were heated from 25 to 60 °C (800 W for 24
vessels) within 6  min, and maintained at this tempera-
ture for 10 min. The samples were then cooled to room
temperature, and 1.7 ml NaCl (0.73% w/v) was added to
the samples. The samples were then vortexed and centri-
fuged at 3000×g for 10 min, and the organic phase (lower
phase) was transferred into a clean tube. Finally, the lipid
extracts were dried under vacuum and re-suspended in
a chloroform–methanol solution (2:1, v/v) to a final vol-
ume of 200 μl, ready for total lipid analysis. The measure-
ment conditions used for the analysis of phospholipids,
ergosterol, triacylglycerols and steryl esters with HPLC-
CAD were the same as in our previous work [58]. For
lipid nomenclature, see Additional file 1: Table S1.

Separation of neutral and polar lipids
The protocol used in this study was adapted from the
protocol of Löfgren et  al. [59]. The lipids obtained
from microwave-assisted extraction were dried under
vacuum, and the samples then re-suspended in a

Page 12 of 15Guo et al. Biotechnol Biofuels (2018) 11:297

heptane–methanol mixture (98:2, v/v) to a final volume
of 200 μl. After vortexing for 10 min, 1 volume of metha-
nol–water (with 0.23% NH3) was added to the solution.
The sample was vortexed for a further 10  min at room
temperature, after which the upper phase (the heptane
phase) was transferred to a clean tube. The lower phase
(the methanol phase) was re-extracted twice with hep-
tane (200  μl), and the heptane phases containing the
neutral lipids were pooled together. The methanol phase,
containing the polar lipids, was transferred to a clean
tube. Finally, the solvents (methanol and heptane) were
removed by vacuum evaporation, and the dried extracts
remaining were used for total FA analysis with GC–MS.

Analysis of total FAs by GC–MS
The total FAs from the neutral and polar fractions were
converted into fatty acid methyl esters (FAMEs), and
analyzed using GC–MS, as described in our previous
work [60]. Briefly, the dried fractions of neutral and polar
lipids were mixed with 800 μl hexane, 400 μl 14% BF3 (in
methanol) and 20  μg of an internal standard (C17:0) in
an extraction tube. The FAs were derivatized to FAMEs
using a microwave-assisted method, as described previ-
ously [38]. The upper phase (hexane phase) containing
FAMEs was analyzed using GC–MS (Focus GC ISQ sin-
gle quadrupole, Thermo Fisher scientific, Austin, TX).
Unknown FAMEs were identified by comparing their
retention times and mass spectrum profiles with authen-
tic standards. The unsaturation index was calculated as
the sum of the percentage of each unsaturated FA (w/w)
multiplied by its number of unsaturated bonds in the
mixture [61].

Plasmid construction
OLE1 encoding ∆ (9) FA desaturase (GenBank Accession
Number: NC_001139.9) was amplified from genomic
DNA of S. cerevisiae CEN.PK 113-7D using high-fidelity
DNA polymerase (Thermo Fisher Scientific) with the
fragment including the entire coding region was obtained
and then inserted into the 2-micron plasmid p426TEF
[62] under the TEF promoter with BamHI/HindIII to
yield the plasmid p426TEF-OLE1.

The expression levels of ∆ (9) FA desaturase and
squalene epoxidase (ERG1) were knocked down using
the antisense oligonucleotide method, as described
previously [63]. The antisense oligonucleotide of the
conserved catalytic domain of OLE1 was created by
annealing the primer pair ELOF (5′-CCC AAG CTT TGG

GTG AGA GTG GCC CCA-3′). The antisense oligonu-
cleotide of the conserved catalytic domain of ERG1 was
created similarly using the annealing primer pair ELOF
The two 33-bp antisense DNA fragments, thus, obtained
were then inserted into the plasmid p426TEF under the
TEF promoter with BamHI/HindIII, separately, forming
the plasmids p426TEF-ROLE1 and p426TEF-RERG1.

Strain construction
Yeast transformations were performed as described by
Gietz and Schiestl [64]. S. cerevisiae CEN.PK 113-5D
was transformed with p426TEF-OLE1, p426TEF-ROLE1
and p426TEF-RERG1, separately. S. cerevisiae CEN.PK
113-5D harboring empty p426TEF was used as the con-
trol. Transformants were then selected on yeast nitrogen
base plates with the addition of amino acids. Transfor-
mants harboring the relevant plasmid were confirmed
by plasmid extraction and PCR. The depression of OLE1
and ERG1 expression was confirmed using qPCR. The S.
cerevisiae strains harboring p426TEF-OLE1, p426TEF-
ROLE1 or p426TEF-RERG1 were designated CEN-O1
(PTEF-OLE1), CEN-RO1 (PTEF-OLE1-reverse) and CEN-
RE1 (PTEF-ERG1-reverse), respectively.

Acid‑tolerance test using high‑throughput screening
High-throughput toxicity screening was performed using
Bioscreen C MBR (Oy Growth Curves AB Ltd, Helsinki,
Finland) to determine the appropriate range of each
acid to better illustrate the tolerance of the engineered
yeast. Recombinant strains harboring p426TEF-OLE1,
p426TEF-ROLE1 or p426TEF-RERG1 and the con-
trol were pre-cultured in defined medium (as described
above) until the exponential growth phase, and then
transferred into a 100-well plate containing 120 μl defined
medium per well at pH 5.0, with the addition of each acid.
The initial OD was about 0.1. Different concentrations of
formic (25–250 mM), acetic (25–250 mM), levulinic (50–
400 mM) and cinnamic (0.2–1 mM) acid were tested. The
100-well plate was incubated at 30  °C with continuous
shaking. The duration of the lag phase and the maximum
specific growth rate (μmax) are presented as mean values
of at least five biological replicates ± SD.

Viability of the yeast cells under acid stress
The yeast transformants in which OLE1 was overex-
pressed or OLE1 or ERG1 was knocked down, as well as
the control, were cultivated until the exponential growth
phase. Yeast cells were then recovered and washed twice
with sterile water, and re-suspended in defined medium
without glucose. These cell suspensions were transferred

Page 13 of 15Guo et al. Biotechnol Biofuels (2018) 11:297

into defined medium without glucose containing
150 mM formic acid, 150 mM acetic acid, 200 mM lev-
ulinic acid or 0.8 mM cinnamic acid at pH 5.0, to yield an
OD of 1.0.

To investigate the effect of cellular ergosterol content
on acid tolerance, S. cerevisiae CEN.PK 113-7D (wild
type) was grown on defined medium until the exponen-
tial growth phase. The cells were recovered and washed
twice with sterile water, and re-suspended in defined
medium without glucose. These cell suspensions were
transferred into defined medium containing 5  g/l glu-
cose and 10.0 μg/ml ergosterol [44] with the addition of
150 mM formic acid, 150 mM acetic acid, 200 mM lev-
ulinic acid or 0.8 mM cinnamic acid at pH 5.0, under oxy-
gen-limited conditions, to yield an OD of 1.0. Yeast cells
cultivated under the same stress conditions but without
the addition of ergosterol were used as controls. Samples
were taken from acid-stressed cultures at various times
over a 24-h period. Cell viability was determined by col-
ony counts on YPD plates. Colonies were counted after
2 days’ incubation at 30  °C, and the viability of the cells
is reported as the percentage of surviving yeast cells over

Additional file

Additional file 1: Fig S1. Comparison of the ethanol and biomass
production, and glucose consumption of the yeast strain during aerobic
culture without acid (a), and with the addition of 180 mM acetic acid (b),
180 mM formic acid (c), 260 mM levulinic acid (d) and 0.7 mM cinnamic
acid (e), at pH 5.0. The first dashed line on the left shows the time at which
the acid was pulsed into the culture. Typically, growth phases are defined
as: phase 0 (P0), the exponential growth phase before acid addition; phase
1 (P1), the adaptation phase on glucose after acid addition; phase 2 (P2),
the exponential growth phase on glucose; phase 3 (P3), the adaptation
phase on ethanol; phase 4 (P4), the exponential growth phase on ethanol;
and phase 5 (P5), the stationary phase, as has been indicated (b). Fig
S2. Viable fractions of the S. cerevisiae control strain and recombinant
strains CEN‑RO1 (PTEF‑OLE1‑reverse) and CEN‑O1 (PTEF‑OLE1), under stress
resulting from (a) 150 mM acetic acid, (b) 150 mM formic acid, (c) 200 mM
levulinic acid and (d) 0.8 mM cinnamic acid, at pH 5.0. Fig S3. Expression
levels of the key genes in lipid metabolism of S. cerevisiae CEN.PK 113‑7D
in aerobic cultures before (CT, control condition, exponential growth
phase) and after the addition of 180 mM acetic acid (AC), 180 mM formic
acid (FA), 260 mM levulinic acid (LA) and 0.7 mM cinnamic acid (CA), at
pH5.0 (samples were taken 1 h after the addition of the acid). The qPCR
results were normalized to TAF10 and compared with the expression level
of each target gene under non‑stressed condition. Fig S4. Intracellular
oxidation level of S. cerevisiae CEN.PK 113‑7D in aerobic cultures without
acid and with the addition of 180 mM acetic acid, 180 mM formic acid,
260 mM levulinic acid and 0.7 mM cinnamic acid, at pH5.0. (a) Adapta‑
tion phase on glucose, (b) glucose growth phase, (c) adaptation phase
on ethanol, (d) ethanol growth phase and (e) stationary phase. Table S1.
Lipid classes in S. cerevisiae

Authors’ contributions
ZG and LO conceived the study and participated in its design. ZG designed
the constructs, carried out all the experiments and drafted the manuscript. SK

has been involved in lipids and fatty acid analysis. ZG, SK, JN and LO revised
the manuscript. All authors read and approved the final manuscript.

Author details
1 Department of Biology and Biological Engineering, Industrial Biotechnology,
Chalmers University of Technology, 412 96 Gothenburg, Sweden. 2 Depart‑
ment of Biochemistry and Siriraj Metabolomics and Phenomics Center, Faculty
of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand. 3 Depart‑
ment of Biology and Biological Engineering, Systems and Synthetic Biology,
Chalmers University of Technology, 412 96 Gothenburg, Sweden. 4 Novo Nor‑
disk Foundation Center for Biosustainability, Technical University of Denmark,
Building 220, 2800 Kongens Lyngby, Denmark. 5 Present Address: LISBP, INSA,
INRA, CNRS, Université de Toulouse, Toulouse, France.

This work was financed by Vinnova Grant No. 2012‑02597, Biovacsafe Grant
No. FP7, 115308 (http://www.biova csafe .eu/) and the Novo Nordisk Founda‑
tion. We are grateful to Suwanee Jansa‑Ard (Systems and Synthetic Biology,
Chalmers University of Technology) for her help with lipid analysis. We
would also like to thank Michael Gossing for his comments and constructive

Competing interests
The authors declare that they have no competing interests.

Availability of supporting data
All data generated or analyzed in the present study are included in this pub‑
lished article and in additional information.

Consent for publication
All authors consent for publication.

Ethics approval and consent to participate
Not applicable.

Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in pub‑
lished maps and institutional affiliations.

Received: 9 August 2018 Accepted: 15 October 2018

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“Acid spike” formation in the fast neutron radiolysis of
supercritical water at 400 °C studied by Monte Carlo track
chemistry simulations
Md Mohsin Patwary, Sunuchakan Sanguanmith, Jintana Meesungnoen, and Jean-Paul Jay-Gerin

Abstract: A reliable understanding of radiolysis processes in supercritical water (SCW) cooled reactors is required to ensure
optimal water chemistry control. In this perspective, Monte Carlo track chemistry simulations of the radiolysis of pure,
deaerated SCW at 400 °C by 2 MeV mono-energetic neutrons were carried out as a function of water density between 0.15


0.6 g/cm3. The yields of hydronium ions (H3O+) formed at early time were obtained based on the G values calculated for the first
three generated recoil protons. Combining our calculated G(H3O+) values with a cylindrical track model allowed us to estimate
the concentrations of H3O+ and the corresponding pH values. An abrupt, transient, and highly acidic pH response (“acid spikes”)
was observed at early times around the “native” fast neutron and recoil proton trajectories. This intra-track acidity was found to
be strongest at times of less than a few tens to a hundred of picoseconds, depending on the value of the density considered
(pH � 1). At longer times, the pH gradually increased for all densities, finally reaching a constant value corresponding to the
non-radiolytic, pre-irradiation concentration of H3O+, due to the autoprotolysis of water. Interestingly, the lower the density of
the water, the longer the time required to reach this constant value. Because many in-core processes in nuclear reactors critically
depend on the pH, the present work raises the question whether such highly acidic pH fluctuations, though local and transitory, could
promote or contribute to corrosion and degradation of materials under proposed SCW-cooled reactor operating conditions.

Key words: supercritical water (SCW), fast neutron and recoil protons, radiolysis, acid spike, Monte Carlo track chemistry
simulations, generation IV SCW-cooled reactor.

Résumé : Il importe d’avoir une compréhension fiable des processus de radiolyse qui ont cours dans les réacteurs refroidis à l’eau
supercritique afin d’assurer un contrôle optimal de la chimie de l’eau. Dans cette optique, nous avons effectué des simulations
Monte Carlo de la trajectoire des particules pour décrire la radiolyse à 400 °C de l’eau supercritique pure, désaérée, par des
neutrons monoénergétiques de 2 MeV en fonction de la densité de l’eau dont les valeurs sont comprises entre 0,15 et 0,6 g/cm3.
Les rendements d’ions hydronium (H3O+) formés dans les premiers instants ont été obtenus à l’aide des valeurs G calculées pour
les trois premiers protons de recul générés. En intégrant nos valeurs calculées de G(H3O+) à un modèle de trajectoires cylin-
driques, nous avons été en mesure d’estimer les concentrations de H3O+ et les valeurs de pH correspondantes. Nous avons
observé une réponse caractérisée par une variation brève et abrupte du pH vers des valeurs très basses (« pics d’acidité ») aux
premiers instants de la trajectoire des neutrons rapides/protons de recul. Cette acidité intratrajectorielle s’est révélée être la plus
forte à des instants correspondant à moins de quelques dizaines à une centaine de picosecondes, selon la valeur de densité
considérée (pH � 1). À des temps plus longs, le pH augmentait graduellement à toutes les valeurs de densité, pour finalement
atteindre une valeur constante correspondant à la concentration de H3O+ non radiolytique prévalant avant l’irradiation, en
raison de l’autoprotolyse de l’eau. Fait intéressant, plus la densité de l’eau est faible, plus le temps nécessaire pour atteindre cette
valeur constante est long. Comme de nombreux processus au cœur des réacteurs nucléaires dépendent dans une large mesure
du pH, les présents travaux soulèvent la question à savoir si de telles fluctuations de pH fortement acide, bien qu’elles soient
localisées et transitoires, pourraient favoriser la corrosion et la dégradation des matériaux dans les conditions proposées de
fonctionnement d’un réacteur refroidi à l’eau supercritique. [Traduit par la Rédaction]

Mots-clés : eau supercritique, radiolyse par neutrons rapides/protons de recul, pic d’acidité, simulations Monte Carlo de la
trajectoire des particules, réacteur de 4e génération refroidi à l’eau supercritique.

Supercritical water (SCW) (i.e., water at temperatures and pres-

sures above its thermodynamic critical point in the P–V–T diagram; for
light water, H2O: Tc = 373.95 °C, Pc = 22.06 MPa, and �c = 0.322 g/cm3)
has been a subject of growing interest in recent decades. Besides its
importance for fundamental scientific research, SCW has at-
tracted attention for its important role in a variety of innovative
technological and industrial applications.1−5 Most of this attention

is driven by the nature of SCW whose density can be varied contin-
uously at constant temperature over a wide range from liquid-like to
gas-like values with only small changes in applied pressure. This
tunability of SCW densities with pressure provides access to a wide
range of density-dependent water properties while avoiding the oth-
erwise perturbing gas-to-liquid phase transition.

Among the most attractive applications in this area is the pro-
posed next generation (Gen IV) SCW-cooled reactor (SCWR) con-

Received 27 November 2018. Accepted 10 January 2019.

Md M. Patwary, S. Sanguanmith, J. Meesungnoen, and J.-P. Jay-Gerin. Département de Médecine Nucléaire et de Radiobiologie, Faculté de Médecine
et des Sciences de la Santé, Université de Sherbrooke, 3001, 12e Avenue Nord, Sherbrooke, QC J1H 5N4, Canada.
Corresponding author: Jean-Paul Jay-Gerin (email:
Copyright remains with the author(s) or their institution(s). Permission for reuse (free in most cases) can be obtained from RightsLink.



Can. J. Chem. 97: 366–372 (2019) Published at on 23 January 2019.


cept to meet future global demand for electricity, hydrogen, and
other products.6−8 The future Gen IV SCWR is a promising ad-
vanced nuclear reactor system5,9 with �45% increased efficiency
compared with �28%–32% for current conventional pressurized
water reactors. The homogeneous supercritical phase also allows
for more simple plant design and operation.

Before such technologies as the SCWR can be fully utilized,
however, a thorough understanding of the SCW chemistry is re-
quired. In particular, one of the most significant challenges for
water chemistry in SCWR designs is to predict and, if possible,
mitigate the effects of water radiolysis on material performance
and corrosion, as the reactors under consideration operate at core
inlet and outlet temperatures of �350 and 625 °C, respectively, at
a pressure of 25 MPa.5 Under such “extreme” irradiation condi-
tions of high temperatures and pressures, the effect of an intense,
mixed fast neutron and �-radiation field passing through the re-
actor core results in the radiolytic formation of oxidizing species
at high concentrations such as •OH, H2O2, O2 (produced by decom-
position of H2O2), and O2•− (or HO2•, depending on the pH).10,1


These species are highly reactive and can significantly increase
the corrosion and degradation of structural materials both in the
core and in the associated piping components of the reactor. Proper
control of water chemistry, e.g., adding a small concentration of
excess H2 to the reactor coolant, as with current pressurized high
temperature water reactors, may be the key to maintaining the
integrity of the reactors, although it is still unclear whether this
strategy to suppress water radiolysis would also be effective under
SCWR conditions.5,11,1


Direct measurements at very high temperatures and pressures,
especially beyond the critical point of water, are difficult to per-
form. Moreover, as Gen IV SCWRs are currently at the conceptual
design stage, studies on water radiolysis in a SCWR have been
laboratory based rather than reactor based. Consequently, exper-
imental data on radiation chemistry and reaction kinetics of tran-
sients under the proposed SCWR operating conditions are very
limited and significant gaps still exist.5,13,14 Under these condi-
tions, theoretical modeling and computer simulations are an
important route of investigation for predicting the detailed radi-
ation chemistry in a SCWR and the consequences for materials.
Although a large body of data relevant to the radiolysis of water by
�-rays or high-energy (�1 MeV) electrons is readily available in the
literature, the fast neutron induced water chemistry remains
largely unknown for the proposed SCWR operating conditions.

Recently, Monte Carlo track chemistry simulations were used
to calculate the yields (or G values) of hydronium ions (H3O+) at
ambient and elevated temperatures, which formed in spurs or
tracks of the low or high linear energy transfer (LET) radiolysis of
pure, deaerated water during and shortly after irradiation.15−1


Using simple, LET-dependent, spatiotemporal models of a spur or
track, we found that the in situ, highly nonhomogeneous radio-
lytic formation of H3O+ temporarily renders the “native” spur or
track regions more acidic than the surrounding medium. At 25 °C,
an abrupt transient acidic pH effect (which we termed an “acid
spike”) was observed to be greatest for times shorter than �1 ns in
an isolated “spherical” spur (characteristic of low-LET radiation,
such as 60Co � and fast electron irradiation, LET � 0.3 keV/�m). In
this time range, the pH remained almost constant at �3.3. For
an axially homogeneous “cylindrical” track (characteristic of high-
LET radiation), the acid-spike response to ionizing radiation was
much more intense than that for the spherical spur geometry. For
example, for a 2.4 MeV incident helium ion (LET � 150 keV/�m),
the pH was found to be about 0.5 on a time scale of �100 ps. At
longer times, the pH gradually increased for both low- and high-
LET radiation types, ultimately reaching a constant value of seven
(neutral pH at 25 °C) at �1 �s for the spur model and �0.1 ms for
the track model.15

Interestingly, this early generation of a transient acid pH re-
sponse around charged particle tracks was first highlighted in the

late 1940s.18,19 Although several authors have shown evidence for
this intra-track acidity experimentally,20 these acid-spike effects
have been largely ignored in water or in aqueous environments so
far.21 From a chemical point of view, this may be somewhat sur-
prising in view of the potential implications of a local, albeit
temporary increase in acidity on damage induction and corrosion
in water-cooled reactors.

In this work, we extend our previous calculations to determine
the yields of H3O+ resulting from the radiolysis of pure, deaerated
SCW (H2O) by mono-energetic 2 MeV incident neutrons at 400 °C
as a function of water density (pressure) over the range of
0.15–0.6 g/cm3 (�24–56 MPa). Our goal is to investigate whether
these early acid-spike effects persist under SCW irradiation con-
ditions and then to determine their magnitude and time depen-
dence. The 2 MeV energy of neutrons was considered to be
representative of the average initial energy of a fast neutron flux
in a reactor.22 The chosen density range mimics the coolant con-
ditions in the heat transport system of the SCWR. SCW acts like a
“dense fluid” whose density can vary continuously with tempera-
ture and (or) pressure from �0.1 to 0.2 g/cm3 (low-density or “gas-
like” regime at the reactor core outlet temperature) to higher
values (�0.6–0.7 g/cm3) similar to those of liquid water below the
critical point (high-density or “liquid-like” regime near the reac-
tor core inlet).5

Fast neutron interaction with water
“Fast” neutrons (i.e., those with kinetic energies ranging from

�0.5 to 10 MeV), which concern us in this work, deposit their
energy in the water through ion recoils; in H2O, proton recoils
absorb �88% of the neutron energy and the remainder is absorbed
by oxygen ions.23 In this work, only the proton recoil component
will be considered, as oxygen ion recoils are of minor importance
for the fast neutron radiolysis of water due to their low average
energies.24,25 Moreover, these proton recoils have maximum ranges
(i.e., penetration depths) that are much smaller than the average
distance between two successive neutron interactions. For exam-
ple, the mean free path of a 2 MeV incident neutron in water at
25 °C is about 4 cm, whereas the maximum range of the proton
recoil at this energy is �75 �m.24,26 Therefore, they can be con-
sidered as behaving independently of each other, and under nor-
mal irradiation conditions, their energy is deposited locally in
isolated, dense tracks in the water, near the incident neutron
collision sites (i.e., the generation points of the recoils).

For the estimation of the radiation chemical yields due to 2 MeV
neutrons, only the contributions of the first three recoil protons
was considered in the present calculations, because further recoil
protons generated by the neutron as it is further moderated do
not contribute significantly to radiolysis due to their low average
energies.25,27,28 The initial proton energies (Epi, i = 1–3) are 1.264,
0.465, and 0.171 MeV.25 The fast neutron yields were then calcu-
lated by summing the G values associated with each recoil proton
considered (as determined by our Monte Carlo simulations; see below),
weighted by its fraction of total neutron energy deposited:22,24,25

(1) G(X) �






where G�X�pi is the yield of species X associated with the recoil
proton pi and

(2) ET � �


is the sum of all recoil proton energies.

Patwary et al. 367

Published by NRC Research Press

Monte Carlo track chemistry simulations
The entire sequence of events generated in the radiolysis of

SCW at 400 °C by incident protons of various initial energies
was modeled using an extended version24,25,29 of our Monte Carlo
track chemistry simulation code called IONLYS-IRT.30 In short, the
IONLYS step-by-step program is used to cover all the events of the
early physical and physicochemical stages of radiation action up
to �1 ps in the track development in a three-dimensional geomet-
rical environment. The complex, highly nonhomogeneous spatial
distribution of the reactants formed at the end of the physico-
chemical stage [eaq

� (hydrated electron), H+ (or H3O+), OH−, H•, H2,
•OH, H2O2, O2•− (or HO2•), •O•, O•−, etc.]20,30,31 is then used directly
as the starting point for the subsequent nonhomogeneous chem-
ical stage. This third stage, in which the various radiolytic species
diffuse randomly (at rates determined by their diffusion coeffi-
cients) and react with each other (or competitively with any dis-
solved solutes present in sufficient concentrations) until all spur
or track processes are complete, is covered by our independent reac-
tion times (IRT) program.32 This program uses the IRT method,32,33

a computationally efficient stochastic simulation technique that
is used to simulate reaction times without having to follow the
trajectories of the diffusing species. Its implementation has been
reported previously32 and its ability to provide accurate time-
dependent chemical yields under different irradiation conditions
has been well validated by comparison with complete random
flights Monte Carlo simulations, which follow the reactant trajec-
tories in detail.34 In addition, this IRT program can be used to
efficiently describe the reactions that occur in the bulk solution
during the homogeneous chemical stage, i.e., in the time domain
typically beyond some microseconds after the first ionization

The current version of IONLYS-IRT has made various updates
and modifications in the description of certain key parameters
involved in the physicochemical and chemical stages of radiolysis.
These changes are summarized as follows:

(i) We assumed that at 400 °C the thermalization distance (rth)
of “subexcitation-energy electrons” (esub

� ) (those with ki-
netic energies lower than �7.3 eV, the first-electronic ex-
citation threshold in liquid water) is only affected by
changes in the water density (�) and we scaled it according
to a (1/�)1/3 law,36 namely

(3) rth(400 °C, �) � rth(400 °C, 0.6 g/cm
3)�0.6 g/cm3


with rth (400 °C, 0.6 g/cm3) ≈ 3.2 nm.37 This means that
decreasing density further separates the water molecules but
does not change their ability to interact with the energetic

� , resulting in an increase of rth. The density dependence
of rth used in this work is shown in Fig. 1.

(ii) We included in the simulations a prompt geminate electron–
cation (H2O•+) recombination (i.e., prior to thermalization
of the esub

� ) that decreased in irradiated SCW at 400 °C as
the water density decreased from �0.6 to 0.15 g/cm3.



(iii) We used the rate constants recently predicted by Liu et al.,39,


based on their so-called cage effect model that accounts for
the non-Arrhenius temperature dependence of many reac-
tions in water, for a number of reactions involved in the
radiolysis of SCW at 400 °C. Given the lack of experimental
data, this new database is important in providing us with
recommendations for the best rate constant values to use
at this time in modeling the radiolysis of SCW near and
above the critical point. In some cases, we also used the
chemical kinetic data compiled by Elliot and Bartels,22 sim-
ply extrapolated above their experimentally measured
temperature ranges (mostly 20–350 °C), as well as the re-

cent pulse radiolysis measurements by Muroya et al.41 for
the rate constant of the radiation-induced reaction:

(4) H • � H2O ¡ H2 �

that is a key reaction in high-temperature water radiolysis.
In the absence of any other information, we chose to
neglect any dependence of the reaction rate constants on
water density for the 400 °C isotherm of interest. In the
0.15–0.6 g/cm3 range studied here, this approximation
does not appear to have a large impact, considering the
relatively slowly varying k values for the few reactions
whose rates have been measured as a function of SCW


(iv) We have taken into account that due to their limited
ranges, all the recoil protons are completely stopped in the
water. The chemistry measured under these conditions is
an average over the proton energies from the initial proton
energy to zero. To avoid complexity arising from the result-
ing variations in the energy of the moving protons, simula-
tions were performed with the simplifying approximation
that the energies of the three considered recoil protons
remained constant when passing through the water me-
dium. These constant track average energy values Ēpi (i = 1–3)
were obtained according to a procedure described previ-
ously by Islam et al.17 using the SRIM software45 and our
own Monte Carlo track structure simulations. They were
found to be �0.6, 0.3, and 0.17 MeV, respectively. Interest-
ingly, these values varied only slightly (at most �5%) as a
function of water density over the studied range of 0.15–
0.6 g/cm3 and were therefore kept constant in all our chem-
ical yield calculations.

The density dependences of the viscosity, static dielectric con-
stant, and molar concentration of SCW at 400 °C used in this work
were taken from the NIST Chemistry WebBook.46 The values for
the ionic product of water (Kw) were obtained from Bandura and
Lvov.47 From a microscopic perspective, we ignored the heteroge-
neous character of the molecular structure of SCW,48 which is due
to the presence of density fluctuations (or water “clustering”) as-
sociated with criticality. In this study, we assumed that the overall

Fig. 1. Variation of the thermalization distance rth (in Å) of
subexcitation electrons in pure, deaerated SCW at 400 °C as a
function of water density in the range of �0.1–0.7 g/cm3 used in this

0.1 0.2 0.3 0.4 0.5 0.6









Water density (g/cm3)

SCW, 400 oC

368 Can. J. Chem. Vol. 97, 201


Published by NRC Research Press

instantaneous picture of SCW at 400 °C could simply be viewed as
a continuum medium with a mean density equal to the density of
bulk water (�).

All our calculations were performed by simulating short (typi-
cally, �15–150 �m) proton track segments, over which the energy
and LET of the recoil protons are well defined and remain nearly
constant. Such model calculations thus gave “track segment” yields
for a well-defined LET as a function of time. For the three recoil
protons under consideration, whose track average energies are
�0.6, 0.3, and 0.17 MeV, respectively, the corresponding mean LET
values increase from �5.5, 8.2, and 10.4 keV/�m to �22, 33, and
42 keV/�m, respectively, when the SCW density is increased from
0.15 to 0.6 g/cm3. With this LET range, the proton’s track can be
modeled as a cylinder, characteristic of high-LET radiation15,17 (see
below). The number of individual proton “histories” (usually �10–150,
depending on the proton energy) was chosen to ensure only small
statistical fluctuations in the computed averages of chemical
yields, while meeting acceptable computer time limits.

Throughout this paper, G values are quoted in units of molecules
formed or consumed per 100 eV of radiation energy absorbed. For
conversion into SI units, 1 molecule/100 eV ≈ 0.10364 �mol/J.

Results and discussion
Figure 2 shows the variations of G(H3O+) and G(OH−) calculated

from our simulations of the radiolysis of pure, deaerated SCW
(H2O) at 400 °C by 2 MeV incident neutrons as a function of time
from �1 ps to 10 �s, for different water densities in the range of
0.15–0.6 g/cm3. Obviously, the hydroxide ion OH−, which is formed
largely by the reaction:

(5) eaq
� � •OH ¡ OH�

during the track stage of the radiolysis, contributes to an alkaline
track and consequently counteracts the acid-spike effect discussed
in this work. However, as can be seen from Fig. 2, G(OH−) remains

much smaller than G(H3O+) over the time range of interest, inde-
pendent of the considered density. As a result, its effect modifies
the quantitative features of the pH only slightly and can be ig-
nored to a good approximation. To our knowledge, there are no
experimental data in the literature at 400 °C with which to com-
pare these temporal variations of G(H3O+) or G(OH−) shown in
Fig. 2.

The in situ formation of H3O+ by the generated recoil protons
renders the “native” track regions acidic. A qualitative physical
image, based on the spatial distribution of the various initial prod-
ucts formed across an ionizing track,17−19 can be offered to explain
the origin of this local and transitory acidity. Interestingly, there
is indeed a charge separation that develops quickly between the
more concentrated positive-ion (mainly H3O+ and •OH) core of the
track and the negative ions (mainly OH− and H•) in the surround-
ing medium, which are somewhat removed from the track. This
charge separation is due to the faster motion and long penetration
(or thermalization) range of the ejected secondary electrons,37

which, once hydrated, are captured by the slowly moving •OH and
H3O+ to form OH− and H•. As a result, it is easy to see that this
charge separation, and its associated local acidity will last until
the diffusion of •OH and H3O+ has brought these species to the
remote positions then occupied by the eaq

� .
As discussed previously,15 the observed decrease of G(H3O+) is

predominantly due to H3O+ reacting with eaq
� and with OH−, ac-

cording to

(6) H3O
� � eaq

� ¡ H • � H2O


(7) H3O
� � OH� ¡ 2H2O

Other reactions such as H3O+ + O•− ¡ •OH + H2O and H3O+ + HO2− ¡
H2O2 + H2O also contribute to the decay of G(H3O+) but only very
weakly. This can be seen clearly in Figs. 3a and 3b, in which we
show the time dependence of the cumulative yield variations
�G(H3O+) for each of the reactions that contribute to the decay of
G(H3O+), calculated from our Monte Carlo simulations at 400 °C,
for � = 0.15 and 0.6 g/cm3, respectively, in the interval �1 ps–10 �s.

The effect of density (pressure) on the yield of H3O+ shown in
Fig. 2 can be understood as follows. As we lower the density in
SCW, there are fewer water molecules to present a “barrier” or,
in other words, a solvent cage effect.49 This results in the in-
creased cage escape of the various species originating from water
dissociation, including H3O+, as the proximity condition that
would allow them to combine or recombine is not favored. In con-
trast, these density effects work in the opposite direction in the
high-density liquid-like region, where a large barrier of solvent is
present. In this case, the caged radiolytic products are forced to
remain as colliding neighbors within the proton track


they are formed, thus increasing the likelihood of combination–
recombination reactions38 and hence leading to a fast decrease of
G(H3O+). This is in agreement with what we see in Fig. 2 (see also
Fig. 3).

To calculate the pH values prevailing in the fast neutron and
recoil proton track regions, we estimated the radiolytically gener-
ated concentrations of H3O+ in these regions as a function of time
using a cylindrical track model, characteristic of high-LET radia-
tion.15 For each of the three considered recoil protons, we assumed
the proton’s track as an axially homogeneous cylinder, with a
length L = 1 �m and initial radius rc equal to the radius of the
physical track “core” (corresponding to the tiny radial region
within the first few nanometers around the impacting proton
path, at �10−13 s).15,17,50,51 In this case, for the generated recoil

Fig. 2. Temporal evolution of the yields (in molecule per 100 eV) of
radiolytically produced H3O+ (solid lines) and OH− (dashed lines) ions
obtained from our Monte Carlo simulations of the radiolysis of pure,
deaerated SCW by 2 MeV incident neutrons in the interval of �1 ps to
10 �s, for six different water densities: 0.15 (black), 0.2 (orange),
0.3 (olive), 0.4 (blue), 0.5 (green), and 0.6 (red) g/cm3 at 400 °C.
Calculations are based on the radiation effects in 0.6, 0.3, and
0.17 MeV recoil proton tracks (see text).

10-12 10-11 10-10 10-9 10-8 10-7 10-6 10-5



+) SCW, 400 oC







Time (s)


= 0.15 g/cm3


0.3 g/cm3


Patwary et al. 369

Published by NRC Research Press

proton pi (i = 1–3), the track concentration of radiolytically gener-
ated H3O+ can be derived from15,30

(8) �H3O��pi,radiolytic(t) � G(H3O
�)pi(t)�(LET)pi r(t)2 �


(9) r(t)2 � rc
2 � 4D(H3O


is the change with time of rc due to the two-dimensional diffusive
expansion of the track. Here, t is time, D is the diffusion coeffi-
cient for H3O+ in water, and rc was estimated directly from our
simulations. We assumed rc = 2 nm for all recoil protons and all
considered densities. For D(H3O+), which is essentially unknown

at 400 °C, we first extrapolated the data reported by Elliot and
Bartels22 over the 20–350 °C temperature range and then assumed
that its dependence on density equaled that of the self-diffusion
coefficient of compressed SCW at 400 °C.52 The variation of
D(H3O+) in SCW at 400 °C as a function of density used in this work
is shown in Fig. 4.

Finally, the total concentration of H3O+ is the sum of [H3O+]radiolytic,
which simply results from the average of �H3O

��pi,radiolytic for the three
generated recoil protons, and the non-radiolytic, pre-irradiation
concentration [H3O+]autoprotolysis that results from the autopro-
tolysis of water:16,17,47

(10) �H3O��total(t) � �H3O��radiolytic(t) � �H3O��autoprotolysis

The pH in the corresponding track regions is then given by the
negative logarithm (to the base 10) of [H3O+]total:

(11) pH(t) � �log �H3O��total(t)

The temporal evolution of the pH values calculated from eqs.
8–11 for 2-MeV irradiating neutrons in pure, deaerated SCW (H2O)
at 400 °C is shown in Fig. 5 for different water densities ranging
from 0.15 to 0.6 g/cm3. As shown, for all densities considered,
there is an abrupt, temporary, and highly acidic pH effect at the
beginning of the chemical stage. This “acid-spike” effect is stron-
gest at times of less than a few tens to a hundred of picoseconds,
depending on the value of the density considered. In this time
range, the pH remains nearly constant, around unity. Over
�100 ps, the pH gradually increases over time. Ultimately, it
reaches a constant value (pH of the body of the solution) equal to
–log([H3O+]autoprotolysis), which depends on the density.47 As can be
seen from Fig. 5, the lower the density of the water, the longer the
time required to reach this constant value, ranging from �0.1 �s
at 0.6 g/cm3 (pH � 5.6) to more than 100 �s at 0.15 g/cm3 (pH � 8.6).

Most interestingly, regardless of the SCW density considered,
we see from Fig. 5 that the early acid pH conditions surrounding
the “native” fast neutron and recoil proton trajectories persist for
a period of more than six orders of magnitude. Rather surpris-
ingly, the generation of such an early acid response around
charged particle tracks has largely gone unnoticed in water or in

Fig. 3. Time dependence of the extents �G(H3O+) (in molecule per
100 eV) of the different reactions that are involved in the decay of
H3O+, obtained from our Monte Carlo simulations of the radiolysis
of pure, deaerated SCW by 2 MeV incident neutrons in the interval
of �1 ps to 10 �s, for � = 0.15 (panel a) and � = 0.6 (panel b) g/cm3 at
400 °C.

Fig. 4. Variation of the diffusion coefficient (in m2/s) for the
hydronium ion, D(H3O+), in SCW at 400 °C as a function of water
density in the range of �0.1–0.7 g/cm3 used in this work (see text).

0.1 0.2 0.3 0.4 0.5 0.6 0.7






2 /

Water density (g/cm3)
SCW, 400 oC

370 Can. J. Chem. Vol. 97, 2019

Published by NRC Research Press

aqueous environments subject to high-LET radiation, either at
ambient or at elevated temperatures. Because many in-core pro-
cesses in nuclear reactors, and in particular in proposed SCWRs,
critically depend on the pH, a key water chemistry parameter,5

the present work raises the question whether such abrupt, highly
acidic pH variations, which extend spatially about tens of nano-
meters, could promote or contribute to material corrosion and
damage. Because corrosion is a surface phenomenon, this can
easily be envisioned, for example, when fast neutron and recoil
proton tracks are formed in the immediate vicinity of a metal–
water interface. The presence of H3O+ in contact with structural
materials may readily induce spontaneous electrochemical reac-
tions, which may release positive metal ions at the metal surface,
thus creating a corrosive environment.5,53,54 The continuous re-
lease of these ions from a certain location may actually cause a
“stress corrosion cracking” (SCC) site after years.55 Perhaps more
importantly, once the crack is developed, radiolysis in the crack
and the resulting “acid spikes” could greatly speed up the SCC

In this regard, this work should stimulate novel predictive mod-
eling of corrosion driven by these local, density-dependent acid-
spike effects, which can then be tested with new measurements
under SCWR conditions.

Summary and conclusion
In this work, Monte Carlo track chemistry simulations were

used to calculate the yields of H3O+ formed early in the radiolysis
of pure, deaerated SCW by 2 MeV incident neutrons at 400 °C for
different water densities in the range of 0.15–0.6 g/cm3, chosen to
mimic the coolant conditions in the heat transport system of
proposed SCWRs. The fast neutron G(H3O+) values were obtained
by assuming that the most significant contribution to radiolysis

comes from the first three recoil protons generated by the pas-
sage of the irradiating neutron. The concentrations of H3O+ and
the corresponding pH values for these three recoil protons were
then obtained from our calculated G(H3O+) values using an ax-
ially homogeneous cylindrical track model. An abrupt, tran-
sient, highly acidic pH response was observed at early times
around the “native” fast neutron and recoil proton trajectories.
The magnitude and duration of this in situ “acid-spike” effect were
found to be sensitive functions of the water density. At 400 °C and at
times less than �10 ps, the pH for the highest (“liquid-like”) and
lowest (“gas-like”) densities considered was around 0.8 and 1.3, re-
spectively. At longer times, the pH gradually increased for all den-
sities and finally reached a constant value corresponding to the
non-radiolytic, pre-irradiation concentration of H3O+, due to the
autoprotolysis of water at �0.1–100 �s following irradiation.

In conclusion, the question arises whether the strong intra-track
acidity described here, although local and transitory, can trigger
chemically aggressive conditions on metal surfaces, promoting the
corrosion and degradation of materials in water-cooled nuclear re-
actors, as well as in proposed SCWRs. As far as we know, the
generation of such acidic pH spikes in water subject to the action
of high-LET radiation, at both ambient and elevated temperatures
including under SCW conditions, has never been mentioned

  • Acknowledgements
  • Md M.P. is the recipient of a scholarship from the Faculty of

    Medicine and Health Sciences of the Université de Sherbrooke.
    Thanks are due to Dr. David Guzonas and Dr. Craig R. Stuart
    (Canadian Nuclear Laboratories, Chalk River, Ontario) for stimu-
    lating correspondence and for their continued encouragement.
    J.-P.J.-G. is grateful to the Natural Sciences and Engineering Re-
    search Council of Canada (NSERC) for its financial support (Grant
    No. RGPIN-2015-06100).

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    Fig. 5. Temporal evolution of the pH prevailing in the track regions
    of 2 MeV irradiating neutrons calculated for pure, deaerated SCW at
    400 °C in the interval of �1 ps to 10 �s for the same six water densities
    as in Fig. 2 (see text). For the sake of comparison, the dashed lines
    show, for � = 0.15 (black) and 0.6 (red) g/cm3, the variation of pH with
    time in an isolated spherical “spur” (characteristic of low-LET
    radiation) (see Kanike et al.15,16) as calculated for irradiating
    300 MeV protons (which mimic 60Co �/fast electron irradiation;
    LET � 0.3 keV/�m) using an initial spur radius (taken here as equal
    to rth; see Fig. 1) of 42.2 and 32 Å for the two water densities

    10-12 10-11 10-10 10-9 10-8 10-7 10-6 10-5

    = 0.15 g/cm3 = 0.6 g/cm3p

    Time (s)
    SCW, 400 oC

    Patwary et al. 371

    Published by NRC Research Press

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    Published by NRC Research Press

    • Article
    • Introduction
      Fast neutron interaction with water
      Monte Carlo track chemistry simulations
      Results and discussion
      Summary and conclusion

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    Modular Process Design:Chemical and Thermal Recycling of
    Date: Apr. 20, 2020
    From: Chemical Engineering
    Publisher: Access Intelligence, LLC
    Document Type: Article
    Length: 2,687 words

    Full Text:
    In order to recycle spent acids from different applications, a design approach based on different process modules gives operators the
    flexibility needed to handle different contaminants

    Spent or waste acids (SA) containing sulfuric or nitric acid are a byproduct of various processes involving nitration or sulfonation
    reactions or of processes, in which these acids are used for leaching or as a catalyst. These reactions and processes are used in the
    industrial production of polymers, drugs, pigments, other specialty chemicals, explosives, battery chemicals or metallurgical products.
    The SA from such industries is diluted by reaction water and contaminated with organic and inorganic compounds.

    Stricter environmental regulations and rising raw-material prices in recent years have led to considerable cost and organizational
    effort for the procurement of fresh acid and the disposal of SA. An effective acid-recycling system can contribute to a more cost-
    efficient, independent and environmentally friendly production process. A challenge for recycling systems, however, is the wide
    variety of production processes and the resulting SA with partly unknown contaminants.

    Chemical and thermal recycling of SA consists of various steps and the respective unit operations. The applicability of these unit
    operations has to be determined individually, considering factors such as volatility, solubility and reactivity of the known and unknown
    contaminants, the required quality and concentration of the recycled acid and the available materials of construction for the treatment
    of the highly corrosive acids. For an efficient process design, pre-defined process modules can be combined, comprising unit
    operations for specific cases. With such a modular approach, the thermal SA recycling is divided into three steps: (1) removal of
    contaminants; (2) separation of acid mixtures; and (3) acid concentration increase and absorption. The used modules include
    oxidation reactors, precipitation, filtration, distillation and rectification, as well as absorption and stripping equipment.

    Composition of spent acids

    Spent acids result from a variety of processes in which nitric or sulfuric acids are used as reactants, leaching solutions or as a
    catalyst. Depending on the production process, the composition of the SA varies considerably in terms of acid concentration and
    impurities. Figure 1 illustrates the classification of SA resulting from nitration or sulfonation reactions (Figure 2) and acid leaching
    based on the containing acid (nitric or sulfuric acid or both) and the type of contaminants (organic or inorganic). While hydrochloric
    acid is also used in various industrial processes, chemical or thermal recycling of spent hydrochloric acid is applied less often as
    compared to spent nitric and sulfuric acids and therefore will not be discussed further here.

    In industrial production processes involving nitration and sulfonation reactions (for example, in the production of polymers, drugs,
    explosives or pigments), the SA is contaminated with organic compounds. These contaminants include at least traces of the
    reactants and product (for instance, nitrocellulose, nitrobenzenes, benzenesulfonic acid, glycolnitrates, picric acid, nitrotoluenes) but
    also byproducts. For recycling of these SA, in some cases, a complete removal of certain contaminants is required for safety reasons
    (such as for explosive compounds) while some contaminants can be recycled to the originating process. Depending on the acid used
    for the nitration, the SA is either a nitric acid or a mixture of nitric and sulfuric acid with organic impurities.

    Spent acids from inorganic industries like the production of inorganic pigments or battery chemicals (for example, titanium dioxide via
    the sulfate route or lithium refining) or metallurgical industries (such as copper electrolysis, or demister acid from smelter sulfuric acid
    plants) contain inorganic impurities that could precipitate in the production process and/or affect the product quality. These inorganic
    impurities must be depleted or removed entirely.

    The concentration levels of the acid in the SA range from diluted solutions (below 10 wt.% acid) to high concentrations with partly
    anhydrous acids.

    Process modules

    The treatment of SA applying process modules consists of unit operations for mechanical separation (filtration), thermophysical
    separation (evaporation, distillation/rectification) as well as chemical (oxidation) and physicochemical operations (precipitation,
    stripping and absorption). Together with the auxiliary machines (pumps, vacuum pumps and compressors) and static equipment
    (heat exchangers, evaporators, tray columns, packed-bed columns, reaction vessels) made of corrosion- and temperature-resistant
    materials these unit operations form process modules that can be combined to treat various SA. In general, each process module
    serves one of the three above-described steps in the SA recycling process as is further described below. Table 1 shows a summary
    of the process modules.

    Step 1: Removal of contaminants. The contaminants or impurities found in the SA can be classified as volatile or non-volatile.
    Modules for the removal of these contaminants are shown in Figure 3. Volatile compounds can be removed from a high-boiling SA by
    stripping with air or steam (module A). In general, the vapor pressure of many organic compounds (for example, nitrobenzene) and
    some inorganic compounds (such as hydrogen fluoride or oxides of nitrogen) increases with temperature. Therefore steam stripping
    promotes the separation. For an optimal gas-liquid contact, stripping is performed in packed bed columns. These columns are made
    of either glass-/polymer-lined steel, fiber-reinforced polymer or stainless steel. By stripping these impurities from the SA, it is possible
    to recover them in a condensation or absorption step downstream of the stripping unit. However, in some cases, recovery is not
    possible due to a severe safety hazard being posed by some organic nitrates.

    Non-volatile or hazardous volatile organic compounds can be destroyed by thermal oxidation (module B). The oxidation must destroy
    all organics non-selectively to achieve a purified acid and protect downstream equipment from explosive compounds. In order to
    achieve a complete oxidation, the SA must be heated up to temperatures between 120 and 200[degrees]C. For diluted acids whose
    boiling point is lower than the required decomposition temperature, sulfuric acid can be added to achieve an oxidation in the liquid
    phase, resulting in smaller equipment sizes. Nitric or mixed acids already contain nitric acid as oxidizing agent, while for SA without
    nitric acid, an oxidizing agent consisting, of hydrogen peroxide or nitric acid, for example, must be added. An example of a reaction of
    an organic impurity (benzene) with hydrogen peroxide or nitric acid is shown in Equations (1) and (2). Figure 2 depicts a sulfonation
    SA before and after the thermal oxidation step.

    C 6 H 6 + 15H 2 O 2 a 6CO 2 + 18H 2 O


    2C 6 H 6 + 30HNO 3 a

    12CO 2 + 21H 2 O + 15NO + 15NO 2


    Precipitation and subsequent filtration removes non-volatile inorganic compounds (module C). The precipitation / crystallization is
    facilitated by increasing the concentration of the inorganic impurities beyond the solubility, which depends on the temperature and
    acid concentration. This can be achieved by evaporation of excess water and/or cooling of a hot SA to decrease the solubility. A
    higher acid concentration usually also decreases the solubility of inorganic compounds and therefore has a positive influence on
    remaining impurities in the system.

    Step 2: Separation of acid mixtures. The separation of acid mixtures is done by rectification, as shown in module D (Figure 4a). The
    higher boiling sulfuric acid is discharged at the column bottom and the nitric acid as distillate from the column overhead. Due to the
    hygroscopic nature of sulfuric acid, the water from the feed SA is discharged with the sulfuric acid whereby high concentrations of
    nitric acid can be achieved. The highly corrosive nature of the boiling sulfuric acid-nitric acid-mixture requires glass or glass-lined
    packed bed columns for the rectification.

    Step 3: Acid concentration increase and absorption. An increase of the acid concentration is achieved by evaporation (Figure 4b).
    Sulfuric acid concentrations near the azeotropic point at approximately 98.5 wt.% are achievable by evaporation of excess water.
    Depending on the concentration of the feed SA and the targeted concentration of the product acid, evaporators operated under
    atmospheric pressure (module E) or vacuum (module F) can be used. By combining evaporators at two different pressure levels, it is
    possible to reuse the evaporated water by vapor compression for the heating of vacuum falling-film evaporators. In vacuum
    evaporators with an absolute pressure as low as 40 mbar the sulfuric acid concentrations close to the azeotropic point can be
    achieved at significantly lower temperatures (approximately 225[degrees]C) compared to the atmospheric boiling point
    (approximately 335[degrees]C).

    Precursors of nitric and sulfuric acid (NOx and SO 3 ) that are formed during oxidation (as described in Step 1), rectification and
    evaporation can be absorbed to produce moderate or high concentrated nitric or sulfuric acid (Figure 4c). Absorption of NOx
    produces nitric acid with concentrations close to 70 wt.%. Absorption of NOx to produce nitric acid involves a gas-phase oxidation of
    nitrogen monoxide and absorption of nitrogen dioxide in water [see Equations (3) and (4)]. The gas-phase oxidation as well as the
    dissolution of nitrogen dioxide in aqueous solution is favored by increased gas pressure. The absorption is performed in stainless-
    steel tray columns.

    2NO + O 2 a 2NO 2 (3)

    2NO 2 + H 2 O a HNO 3 + NO (4)

    Evaporation of sulfuric acid above 90 wt.% produces a SO 3 -rich gas. The absorption of this SO 3 increases the concentration of
    sulfuric acid. The absorption does not necessarily require an increased gas pressure and can be performed in the same pressure
    system as the initial evaporation step.

    Design criteria

    The selection of the process modules is determined by the composition of the SA and the required concentration and quality of the
    product acids. For the removal of contaminants, modules A to C are selected depending on the type of contaminant. To determine
    the adequate design parameters for these process modules, depending on the SA to be treated, laboratory tests or even pilot plant
    tests may be required prior to a commercial design. If a separation of a SA consisting of nitric and sulfuric acid is necessary, the
    process module D is used. Depending on the target concentration of the sulfuric acid product, the evaporation modules E or F are
    applied or combined. With the aim of achieving high energy efficiency within the system, vacuum evaporation (module F) is used
    when very low (<15%) concentrated acid must be recycled or high sulfuric acid concentrations shall be achieved. The absorption modules are used for internal recovery of NOx (module G) or SO 3 (module H) whenever large quantities of these gases are produced. An efficient energy recovery is achieved by using the energy provided in the previous process step (for example, thermal oxidation) in the subsequent steps (for example, evaporation). The sequential design of process steps also protects sensitive downstream equipment by initially removing contaminants.

    Example: Recycling of nitration acid

    As an example, a recycling process for the treatment of nitration SA is shown in the following. It combines various process modules
    to produce concentrated nitric acid of 98.5 wt.% and sulfuric acid of 96 wt.% from a SA. The SA originates from nitroglycerine
    production with the composition shown in Table 2. The process design shown in Figure 5 considers a mass flowrate of 1,000 kg/h
    SA. All product acids are exported with 40[degrees]C.

    The illustrated exemplary process comprises the decomposition of organic contaminants by thermal oxidation and a subsequent
    rectification to separate the acid mixture. The nitric acid is separated as distillate and cleaned from volatile NOx by stripping to
    produce a clear acid. The NOx are recovered as nitric acid in a pressure absorption. The sulfuric acid from the rectification column
    bottom is concentrated to 96% by vacuum evaporation while emerging SO 3 is recovered as sulfuric acid.

    Heating, evaporation, condensation and cooling in the thermal recycling process requires heating and cooling energy. This exemplary
    process requires approximately 460 kW heat and 480 kW cooling energy to produce 125 kg/h of concentrated nitric acid and 675 kg/h
    of sulfuric acid. Besides the positive environmental effect of recycling, the feasibility of recycling SA rather than disposing it and
    buying fresh nitric and sulfuric acids from the market has to be evaluated by plant operators who are considering a financial
    investment into a SA recycling plant. Due to the high costs for SA disposal and fresh acid procurement, recycling can significantly
    reduce the operating expenses (opex) for any production process where great quantities of SA arise.

    In order to determine the financial viability of such a capital investment project, the plant operators will have to perform a
    comprehensive financial assessment study that considers the initial capital expenses (capex) and location-specific opex including
    energy cost, disposal cost for hazardous waste, product acid pricing.

    Final remarks

    In order to recycle spent acids from various applications, a flexible design approach, applying process modules, enables operators to
    deal with spent acids containing different contaminants. The overall process design is combining independent process modules to an
    overall recycling process that can recycle different SA with varying acid concentrations and containing organic or inorganic
    contaminants to achieve different acid concentrations and purities depending on the specific requirements. An efficient energy
    recovery within the process can be achieved, thus enabling significantly reduced opex as compared to the disposal of SA as a
    hazardous waste and buying of fresh acid from the market. With increasing prices for fresh acids and more stringent environmental
    regulations applying to the disposal of SA, chemical and thermal recycling of SA becomes increasingly beneficial for plant operators.


    Kevin Schnabel is process/project engineer for Plinke GmbH (Kaiser-Friedrich-Promenade 24, 61348 Bad Homburg, Germany;
    Phone: +49-6172-126-156; Email:, where he works on the basic and detailed engineering and commissioning
    of treatment and concentration plants for sulfuric, nitric and mixed acids. He is also responsible for Plinke’s CO 2 -related R&D
    projects. Schnabel earned two masters degrees in environmental technology and energy systems from the University of Applied
    Sciences Mittelhessen (THM) and is currently also a lecturer at THM for waste treatment and CO 2 -abatement technologies.

    Sebastian Bialek is head of Laboratory for Plinke GmbH (same address as above; Phone: +49-6172-126-137; Email: He has more than 10 years of experience in industrial and research laboratories. Initially he started his career as a
    laboratory technician for Evonik Stockhausen GmbH, Germany. After that he earned a bachelor and master of science degree in
    business chemistry from the Heinrich-Heine-University in DA1/4sseldorf, with main focus on crystallization of arene complexes of
    main group metals. Since then, he has also worked as a laboratory engineer for GEA Messo GmbH, Germany. He joined Plinke
    GmbH in 2018 in his current position.

    Peter Pataky is director of Technology for Plinke GmbH (same address as above; Phone: +49-6172-126-315: Email: In his current position, he is responsible for R&D, process design and technical proposals. He has more than 34
    years of international experience with technologies for treatment and concentration of sulfuric, nitric, hydrochloric and mixed waste
    acids, as well as adiabatic nitration of benzene while working for Plinke GmbH in basic and detailed engineering, project
    management, procurement, erection supervision and commissioning. From 2005 to 2019, he was director of Engineering and Project
    Execution and took over his current position in 2019. He has an engineering diploma (masters degree) in process engineering from
    the University of Applied Sciences, Frankfurt am Main, and is inventor/co-inventor in various international patents and patent

    Max Heinritz-Adrian is managing director for Plinke GmbH (same address as above; Phone: +49-6172-126-134; Email: and for KBR Ecoplanning Oy (Pori, Finland). He has more than 20 years of international experience in petroleum
    refining, petrochemical and chemical technologies. He started his professional career with Uhde GmbH, Germany (now thyssenkrupp
    Industrial Solutions AG), where he held various leadership positions, including head of the Process Department, head of the Gas
    Technology Division and Member of the Board of Directors for KEPCO-Uhde Inc., a joint venture of thyssenkrupp Industrial Solutions
    and Korea Electric Power Generation. Heinritz-Adrian joined KBR as director Technology, Olefins in 2016 and took over his current
    position in 2018. He has an engineering diploma (masters degree) in process engineering from the Technical University in Clausthal-
    Zellerfeld, Germany, and is inventor/co-inventor in various international patents and patent applications.

    Note: For more on acid recovery, see part 2, “Acid Recovery and Recycle Technologies,” on pp. 46-51, as well as the Newsfront,
    “Acid Recovery Becomes the Norm,” Chem. Eng., October 2018, pp. 14-19).

    Copyright: COPYRIGHT 2020 Access Intelligence, LLC
    Source Citation (MLA 8th Edition)
    “Modular Process Design:Chemical and Thermal Recycling of Acids.” Chemical Engineering, 20 Apr. 2020. Gale Academic OneFile, Accessed 6 Feb. 2021.
    Gale Document Number: GALE|A625737399

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