Fluid Phase Equilibria 407 (2016) 224235

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Fluid Phase Equilibria

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / fl u i d

Compatible solutes: Thermodynamic properties relevant for effective

protection against osmotic stress
Christoph Held* , Gabriele Sadowski
Laboratory of Thermodynamics, Department of Biochemical and Chemical Engineering, Technische Universitt Dortmund, Emil-Figge-Str. 70, 44227 Dortmund,
Germany

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

Article history: Organisms developed very different strategies to protect themselves against osmotic stress. To sustain
Received 16 March 2015 high salt concentrations of their surrounding some organisms accumulate so-called compatible solutes
Received in revised form 22 June 2015 (CSs), which increase the internal osmotic pressure without disturbing the organisms metabolism. At
Accepted 5 July 2015
constant temperature, osmotic pressure is mainly determined by the concentration of the compatible
Available online 8 July 2015
solute and the osmotic coefcient of the aqueous solution, and to a minor extent also by solution
densities.
Keywords:
Thus, osmotic coefcients and densities were measured for aqueous CS solutions in a broad range of
Aqueous solutions
Osmolality
concentration and at three temperatures (273 K, 310 K, 323 K) at atmospheric pressure. Further, the
Osmotic coefcient solubility of CSs in water was measured as function of temperature to determine the maximum CS
Activity coefcients concentration that can be applied in aqueous solutions. CSs under investigation were trimethylamine N-
Osmotic pressure oxide (TMAO), trehalose, citrulline, N,N-dimethylglycine, DMSO, glycerol, methylglycine, and ectoine.
Solubility The data was used to calculate real osmotic pressures induced by these CSs. PC-SAFT was applied to
Density model thermodynamic properties and phase equilibria of aqueous CS solutions in quantitative
PC-SAFT agreement to experimental data.
Modeling
Among the CSs investigated in this work, TMAO induced the highest osmotic pressure and thus can be
Measuring
considered the best protector against osmotic stress. The data was nally analyzed concerning the
inuence of CSs molecular size, charge, and hydrophobicity on osmotic pressure. This included also the
comparison to incompatible solutes (urea, glycine).
2015 Elsevier B.V. All rights reserved.

1. Introduction so-called organic osmolytes. These osmolytes are usually of low

Microorganisms living in extreme conditions (e.g., lakes with
high salt concentrations) have established different strategies to
protect themselves against environmental stresses. They have the
ability to pump in inorganic ions which, however, requires salt
adaptation by accumulation of large excess amounts of amino acids
with acidic side chains, e.g., aspartic acid or glutamic acid [1].
Alternatively, halophilic microorganisms accumulate or synthesize
Abbreviations: AO, Atlantic Ocean; ARD, absolute average relative deviation;
BaS, Baltic Sea; BlS, Black Sea; cit, L-citrulline; DMG, N,N-dimethylglycine; DMSO,
dimethyl sulfoxide; DSC, differential scanning calorimetry; E, ectoine; EOS,
equation of state; (e)PC-SAFT, (electrolyte) Perturbed-Chain Statistical Associating
Fluid Theory; FPO, freezing-point osmometer; M, method; MG, N-methylglycine,
sarcosine; MS, Mediterranean Sea; NaCl, sodium chloride; NS, North Sea; OP,
osmotic pressure; opt, optical; OP, Pacic Ocean; PS, Persian Sea; RS, Red Sea; TMAO,
trimethylamine N-oxide; TMG, N,N,N-trimethylglycine, glycine betaine; tre,
trehalose; VPO, vapor-pressure osmometer.
* Corresponding author.
E-mail address: christoph.held@bci.tu-dortmund.de (C. Held).
molecular weight and do not disturb metabolism mechanisms and
thus, they are usually known as compatible solutes (CSs).
CSs are used in nature against stresses due to heat [2], freezing
[3], and denaturants [4], against salt-induced osmotic stress [2,5],
against proteolysis [6] and other stresses (e.g., [2,7,8]), and they are
also used to promote biological processes (e.g., [9]). Several classes
of CSs are known: sugars, polyols, as well as amino acids and their
derivates [2]. CSs that are usually found in halophilic bacteria are,
e.g., ectoine or glycine betaine [2]. Cryobiologists have been using
CSs like dimethyl sulfoxide (DMSO) and glycerol [3,10] for decades.
Synthesis of CSs as protectants against osmotic stress requires a
lot of energy (in ATP equivalents) [1,11]. Therefore, the CSs applied
in nature have to be as effective as possible. The thermodynamic
strength of the protective effect of a CS against osmotic stress is
determined by the osmotic pressure the CS causes: the higher the
osmotic pressure induced by CSs, the more effective is the
protection against osmotic stress in a saline environment.
Although knowledge about the biodiversity in marine environ-
ments has been increased, the thermodynamic behavior of CS

http://dx.doi.org/10.1016/j.uid.2015.07.004
0378-3812/ 2015 Elsevier B.V. All rights reserved.
 C. Held, G. Sadowski / Fluid Phase Equilibria 407 (2016) 224235 225

sat Saturated
Nomenclature
1 Innitely diluted

Roman symbols
a Helmholtz energy per number of molecules (J) solutions has only been rarely considered. So far, only a few CSs
Ai Constants of power series () were investigated with respect to their thermodynamic properties
ci Molarity (moles solute i per L solution) (mol/L) [10,12] and their interactions with biological molecules [13,14]. The
[348_TD$IF]kB Boltzmann constant, 1.38065
1023 J/K (J/K) availability of thermodynamic data of water/CS solutions is
kij Binary interaction parameter (1/K) however important to understand the thermodynamic phenome-
mi Molality (moles solute i per kg solvent) (mol/kg) na caused by CSs for protection against osmotic stress.
M Molecular weight (g/mol) One property that is an important factor in osmotic-stress
miseg Segment number () protection is the osmotic pressure induced by the CS. Estimating
n Number of moles () osmotic pressures requires osmotic coefcients and densities of
Niassoc Number of association sites of component i () water/CS solutions. Both properties depend on temperature as well
NP Number of data points () as on concentration. Further, the solubility of CSs in water has to be
R Ideal gas constant (J/mol/K) known as this is the maximum CS concentration that can be
DR Signal of the VPO () achieved without CS precipitation. These properties of CS/water
T Temperature (K) solutions (solution densities, osmotic coefcients, and CS solubili-
TSL Melting temperature (K) ty) are accessible experimentally and via thermodynamic models
u/kB Dispersion-energy parameter (K) [10,15,16].
wt% Weight percent (%) Modeling solutions containing biomolecules such as amino
x Mole fraction () acids or sugars has progressed considerably over the last years.
y Thermodynamic property () Equations of state such as the Perturbed-Chain Statistical
Associating Fluid Theory (PC-SAFT) are appropriate for modeling
Greek symbols densities, osmotic coefcients, and solubilities in aqueous
gi Activity coefcient of component i (related to pure biomolecule solutions. Using PC-SAFT, thermodynamic properties
component i) () of solutions containing amino acids and peptides [1517] and
g i* Rational activity coefcient of component i (related compatible and incompatible solutes [12,18] have been modeled
to innite dilution of i in the solvent) () accurately. Recently, the inuence of ectoine on osmotic coef-
Dr Difference in liquid density between seawater and cients and solution densities as well as the solubility of ectoine in
water (kg/m3) water was investigated [12]. Moreover, molecular dynamics
fi Fugacity coefcient of component i () simulations of aqueous ectoine solutions have been performed
eAiBi/kB Association-energy parameter (K) that show strong hydrogen bonding between ectoines and water
kAiBi Association-volume parameter () [14]. This prevents strong ectoineectoine interactions, which can
f Osmotic coefcient () be considered the key reason for the high osmotic pressures
r Density (kg/m3) induced by CSs [14].
p Osmotic pressure (bar) The goal of this work is to compare the effectivity of important
n Stoichiometric factor () CSs used in nature against osmotic stress. This comparison requires
si Temperature-independent segment diameter of a comprehensive data analysis for a broad variety of CSs in order to
molecule i () provide information for the understanding of organisms adaption
to their natural habitat. Such basic data is density and osmotic
Subscripts coefcients of CS/water solutions. Density should be as low as
cal Calibration possible in order to prevent cell sinking. Osmotic coefcients
cryo Cryoscopic should be as high as possible to maximize the protective effect of
i,j Component indexes CSs against osmotic stress.
k Control variable Besides this, solubility data gives the maximum concentration
T Function of temperature of CSs reachable in the organism. The availability of such basic data
seg Segment will allow revealing the inuence of various CSs on the strength of
sw Seawater the protective effect.
W Water In order to reach these goals, solution densities, osmotic
0 Pure substance coefcients, and CS solubilities in water have been measured in
this work. These thermodynamic properties were modeled with
Superscripts PC-SAFT. The data was analyzed in terms of molecular size, charge,
assoc Association and hydrophobicity of the CSs. This might allow estimating the
calc Calculated protection strength against osmotic stress based on the CS's
disp Dispersion architecture also for those CSs for which experimental data is not
exp Experimental yet available.
f Freezing
hc Hard chain 2. Thermodynamic properties relevant for CSs
ideal Ideal solution
m Based on molality Osmotic stress induced by high external osmotic pressure (e.g.,
max Maximum induced by high external salt concentration) can only be
real Real solution compensated by a constitutive osmotic pressure in the interior
res Residual of cells. The thermodynamic strength of the protective effect of a
CS against osmotic stress is determined by the osmotic pressure
 226 C. Held, G. Sadowski / Fluid Phase Equilibria 407 (2016) 224235
the CS causes. In ideal solutions at given temperature T, the osmotic
pressure depends on the CS molarity cCS only:
pideal RT nCS cCS (1)
here R and nCS are the universal gas constant and the
stoichiometric factor of the CSs (the number of species after
dissociating in water), respectively. The CSs under investigation
were treated with nCS = 1 throughout this work. Eq. (1) might
suggest that a high molarity is the only key for high intracellular
osmotic pressure. However, Eq. (1) is valid for diluted solutions
only (i.e., cCS ! 0) as the thermodynamic non-ideality of the
solution can be neglected only in that case. At nite CS molarity,
also the non-ideality of the solution has to be taken into account
and Eq. (1) becomes [19]
preal pideal fT; cCS (2)
where f is the osmotic coefcient of the CS solution. It describes
the molecular interactions in the solution and depends on
temperature as well as on kind and molarity of the CS. At zero
cCS,f becomes unity. The more CS is dissolved in the water, the
higher the molecular interactions among the components causing
f values which cannot be assumed to be unity any more. Eq. (2)
shows that the higher the osmotic coefcient, the higher the
osmotic pressure. Thus, the higher the f value, the more effective
becomes the CS against osmotic stress.
The unit molarity in Eq. (1) refers to one liter of solution (mol/L).
Due to simplicity, many researchers equalize one liter solution
with 1 kg of pure water. This is an appropriate assumption for very
low CS molarities, which, however, is not valid any more at
cCS > 0.5 mol/L. The conversion of molarity cCS (moles CS per liter
solution) and molality mCS (moles CS per kg pure water) is given
by:
0:001
rsolution kg=m3 
cCS mol=L (3)
1=mCS mol=kg 0:001
MCS g=mol
where rsolution is the solution density. Thus, solution densities have
to be known for calculating osmotic pressures in cases where not
the molarity but mole fractions, weight fractions, or molalities are
given.
Next to the impact of rsolution on Eq. (3) and thus on osmotic
pressures calculated using Eq. (1), this property has also a direct
impact on organisms living in seawater. Organisms containing high
concentrations of heavy CSs may reach intercellular rsolution values
that are higher than the density of their environment. This will lead
[(Fig._1)TD$IG]
HO
O HO
+ O
H3C N CH3
CH3 HO
OH
TMAO
O H
COO-
H2N N
H H3C
NH3
+ H
citrulline
Fig. 1. Solid CSs investigated in this work.
to sinking of these organisms to the ground of the sea. As a
consequence, these organisms will lose access to the resources
oxygen and light [11]. Thus, CSs have to be of low molecular weight,
or at least they have to cause a lower solution density compared to
their environment.
From Eq. (1) it becomes obvious that the CS molarity cCS is one
main factor determining the osmotic pressure. Osmotic pressure
can only be inuenced by solutes that are dissolved. Thus, the
maximum osmotic pressure that can be induced by CSs is limited
by the solubility of a CS in the considered solution. Concentrations
above CS solubility do not contribute to the protective effect
against osmotic stress. Thus, it is relevant to know the CS solubility
in water.
To sum up, f values, solution densities, and water solubilities
have to be known for calculating osmotic pressures of CS solutions.
Measuring and modeling these data will be presented in the next
sections.
3. Experimental work
3.1. Materials and reagents
The CSs under investigation in this work were glycerol, N,N,N-
trimethylglycine (TMG), ectoine, trehalose, trimethylamine N-
oxide (TMAO), and citrulline. To investigate the effect of
hydrophobic groups on osmotic pressure, also the organic
components glycine, N-methylglycine (MG) and N,N-dimethylgly-
cine (DMG) were considered. In contrast to these solid CSs, also
two liquid CSs, DMSO and glycerol, were investigated in this work.
The chemical structures of the solid CSs are shown in Fig. 1.
The CSs were used without further purication as they were
delivered with high purity (see Table 1). Experimental densities
and osmotic coefcients of solutions containing the other CSs
illustrated in Fig. 1 are available in the literature and thus have not
been re-measured in this work.
For calibration of the osmometers, sodium chloride solutions
and glycine solutions were prepared using NaCl (Merck KGaA
>99.5%) and glycine (SigmaAldrich Chemie GmbH, >99%). All
solutions were prepared gravimetrically by weighing with an
accuracy of 0.01 mg. Water from the Millipore purication
system was used for the preparation of all aqueous solutions.
The compounds that have been used in measurements are
specied in detail in Table 1.
OH
H3N + COO -
OH
O glycine
O OH H
OH
N+ COO -
trehalose H MG
N+ COO -
N
+ H DMG
N COO -
N+ COO -
ectoine
TMG
 C. Held, G. Sadowski / Fluid Phase Equilibria 407 (2016) 224235 227

Table 1
Sample provenance table.

Compound IUPAC name Suppliera Purity, mass Purication

based
NaCl Sodium chloride M >99.5% Used as
obtained
Glycine Aminoethanoic acid S >99% Used as
obtained
TMAO Trimethylamine oxide S >98% Used as
obtained
[329_TD$IF] -(+)-trehalose
D (2R,3S,4S,5R,6R)-2-(Hydroxymethyl)-6-[(2R,3R,4S,5S,6R)-3,4, 5-trihydroxy-6-(hydroxymethyl) M >98% Used as
oxan-2-yl]oxyoxane-3,4,5-triol obtained
L-citrulline 2-Amino-5-(carbamoylamino) pentanoic acid A >98% Used as
obtained
DMG 2-(Dimethylamino) acetic acid A >98% Used as
obtained
TMG 2-Trimethylammonioacetate S >99% Used as
obtained
a
S, SigmaAldrich Chemie GmbH; M, Merck KGaA; A, Alfa Aesar GmbH & Co. KG.

3.2. Measurements and data reduction
In this work, solution densities and osmotic coefcients of
water/CS solutions, as well as CS solubility in water were
measured. Table 2 comprises the CSs considered in this work
distinguishing data already available in literature from those
measured in this work.
Concentration ranges for properties of the water/CSs solutions
measured in this work were chosen to make sure that the solutions
were undersaturated. Besides this, the concentration ranges was
chosen arbitrarily.
3.2.1. Solution densities
Solution densities were determined using an u-tube densim-
eter (e.g., [12]) Anton Paar DMA 602 (Anton Paar, Germany). It
was calibrated with pure water and air at 298 K prior to measuring
the densities of aqueous solutions containing TMAO, trehalose,
DMG, and citrulline, respectively. According to the manufacturer,
the uncertainty of the measured densities was 1.5
106 kg/m3.
In recent previous works with the apparatus we observed
deviations between 104 and 105 kg/m3 between own measure-
ments and literature data [21,22]. The results of the measurements
are listed in Table 3.
Solution densities for the systems with other CSs considered in
this work were taken from literature (glycerol [23], DMSO [24],
glycine [25], MG [26], TMG [27], urea [28], and ectoine [12]).
3.2.2. Osmotic coefcients and activity coefcients
In this work, the vapor-pressure osmometer (VPO O70) and the
freezing-point osmometer (FPO O10) by Gonotec (Berlin, Germany)
were applied to measure osmotic coefcients at temperatures
between 273 and 323 K.
3.2.2.1. Vapor-pressure osmometer (VPO). A vapor-pressure
osmometer measures the boiling-point elevation DTLV of an
aqueous solution compared to pure water. The apparatus
consists of a closed cell saturated with water vapor. Saturation
was achieved by lling the osmometer with approximately 30 mL
of Millipore water and tempering it at least 8 h prior to the
measurements. By covering both thermistors of the apparatus with
droplets of pure water, the baseline was set to zero. This was done
before and after each measurement.
For calibration, the signal DRcal was measured for NaCl/water
solutions at known NaCl molalities. These signals were ascribed to
osmotic-coefcient data of NaCl/water solutions (fcal) from
literature [29,30]. Both values, DRcal and fcal were used in Eq. (4).
Immediately after calibration, desired solutions of known CS
molality were placed on the tip of one thermistor until a stable
measuring signal DRCS could be observed. The results of more than
three stable signals DRCS were averaged and converted into
osmotic coefcients f of the considered CS solution by
mcal ncal DRCS
fCS=water fcal (4)
mCS DRcal
where mCS is the molality of the CS. The subscript cal (calibration)
is related to NaCl/water solutions, which were used for calibration.
In this work, ncal was set to two for NaCl/water solutions. Based on
our experience with the used apparatus [31], the uncertainty of the
VPO method can be estimated to be 1% with respect to osmotic
coefcients. Experimental data measured in this work by VPO are
listed in Tables 4 and 5.
3.2.2.2. Freezing-point osmometer (FPO). In contrast to VPO
measurements, the application of FPO yields the freezing-point
depression DTSL of an aqueous solution compared to pure water.
Prior to the measurements, the baseline was adjusted using
Millipore water. Glycine/water solutions were used to calibrate the
apparatus with osmolality values taken from literature [32].
In this work, 50 mL solution samples were used. The super-
cooling speed was set to the 100% value, and the samples were
supercooled to 266.15 K.
The measured freezing-point depression DTSL can directly be
converted into osmotic coefcients by

Table 2
Considered properties of water/CS solutions in this work and origin of experimental data (TW = this work).

CS TMAO MG TMG DMG Ectoine Glycerol DMSO Cit Tre Glycine Urea
Density TW, [20] [30_TD$IF][26] [27] TW [12] [23] [24] TW TW [16] [12]
Osmotic coefcient TW 31_TD$IF][[ 36] [37] TW [12] [23] [10] TW TW [16] [12]
Solubility TW n.a. TW TW [32_TD$IF][12] TW [44] [17] [42]
228 C. Held, G. Sadowski / Fluid Phase Equilibria 407 (2016) 224235

Table 3
Experimental liquid densities of aqueous CS solutions of TMAO, trehalose (tre), DMG, and citrulline (cit) at 298 K and 1 bar.a

mTMAO (mol/kg) rsolution (kg/m3) mDMG (mol/kg) rsolution (kg/m3) mtre (mol/kg) rsolution (kg/m3) mcit (mol/kg) rsolution (kg/m3)
0.4060 998.235 0.4001 1005.638 0.1997 1022.607 0.1989 1008.220
0.7971 999.133 1.0031 1016.883 0.3989 1046.201 0.3842 1018.594
1.1987 1000.206 1.4007 1024.010 0.5995 1068.335 0.6007 1028.997
1.5901 1001.540 1.9925 1033.693 0.7914 1087.593
2.0018 1002.010
2.4449 1003.030
a
Standard uncertainties u are u(T) = 0.1 K and u(m) = 0.001 mol=kgH2 O, and the combined expanded uncertainty Uc is Uc(r) = 0.8 kg/m3 (for 0.95 level of condence).

Table 4
Experimental osmotic coefcients and rational CS activity coefcients in aqueous TMAO, DMG, trehalose (tre), and citrulline (cit) solutions at 310.15 K. CS activity coefcients
were obtained by Eq. (8).a

mTMAO (mol kg1) fT;AO g *,mTMAO mDMG (mol kg1) fDMG g *,mDMG mtre (mol kg1) ftre g *,mtre mcit (mol kg1) fcit g *,mcit
0.4060 1.0336 1.1064 0.2005 1.0351 1.0864 0.0998 1.0365 1.0076 0.1989 1.0029 1.0159
0.6200 1.0617 1.1673 0.4001 1.0720 1.1725 0.1997 1.0197 1.0152 0.3942 0.9986 1.0184
0.7971 1.1520 1.2203 0.8024 1.0759 1.2490 0.2995 0.9990 1.0229 0.4915 0.9973 1.0148
0.9921 1.1326 1.2816 1.0031 1.1257 1.3416 0.3989 1.0097 1.0306 0.6892 1.0017 0.9975
1.1987 1.1496 1.3501 1.1926 1.1407 1.3852 0.4992 1.0224 1.0385
1.3916 1.1919 1.4175 1.4007 1.1469 1.4160 0.5995 0.9904 1.0464
1.5901 1.3148 1.4907 1.5855 1.1537 1.4438 0.6932 1.0189 1.0538
1.7970 1.1690 1.4862 1.4691 1.0634 1.1175
1.9925 1.2060 1.5626 1.9824 1.0799 1.1617
a
Standard uncertainties u are u(Tcell) = 0.3 K, u(Tthermistor) = 105 K and u(m) = 0.001 mol=kgH2 O, and the combined expanded uncertainty Uc is Uc(f) = 0.008 (for 0.95 level of
condence).

Table 5
Experimental osmotic coefcients and rational CS activity coefcients in aqueous DMG, TMAO, and citrulline (cit) solutions at 323.15 K. CS activity coefcients were obtained
by Eq. (8).a

mTMAO (mol kg1) fTMAO g *,m TMAO mDMG (mol kg1) fDMG g *,m DMG mcit (mol kg1) fcit g *,mcit
0.5008 1.0759 1.0593 0.2056 0.9810 0.9860 0.2016 1.0067 1.0095
1.0151 1.1714 1.1416 0.2889 0.9890 0.9816 0.4020 1.0007 1.0137
1.5000 1.2722 1.2430 0.4998 1.0078 0.9741 0.6001 0.9931 1.0127
1.0151 1.0214 0.9758
1.5000 1.1140 1.0038
a
Standard uncertainties u are u(Tcell) = 0.3 K, u(Tthermistor) = 105 K and u(m) = 0.001 mol=kgH2 O, and the combined expanded uncertainty Uc is Uc(f) = 0.008 (for 0.95 level of
condence).

DT SL 3.2.2.3. Conversion of osmotic coefcients into activity

f P (5)
K cryo mCS coefcients. The osmotic coefcients measured by VPO (Eq. (4))
where Kcryo is the cryoscopic constant of water (1.86 K kg mol1). and FPO (Eq. (5)) can be converted into water activity coefcients
The maximum osmolality (osm = f m) measurable with the FPO g w by
010 from Gonotec (Gonotec, Berlin, Germany) was 3 osm. ( )
1 X
According to the manufacturer (Gonotec, Berlin, Germany) g w xw ; T exp f
0:001
Mw mCS (6)
xw
the temperature difference is captured with an accuracy of CS

1.86
103 K. This yields an uncertainty of the osmotic coef-
cients of 0.6% (without accounting for the uncertainty in
molality due to weighing) [18]. This value agrees with measure-
ments from a recent previous work [22]. Every measurement was
repeated at least three times and the average values are reported.
Experimental data measured in this work by FPO are listed in
Table 6.
where xw and Mw are the mole fraction of water and the molecular
weight of water in [g/mol], respectively.
Besides osmotic coefcients or water activity coefcients, the
rational CS activity coefcients g *CS are also of interest in many
applications, e.g., for the calculation of Gibbs energies of reaction
[33,34]. In binary solutions, the GibbsDuhem relation allows for
the converting osmotic-coefcient data into rational CS activity

Table 6
Experimental osmotic coefcients of aqueous DMG, TMG, and citrulline (cit) solutions at the solutions respective freezing temperatures TSL determined by FPO. CS activity
coefcients were obtained by Eq. (8).a

mDMG (mol kg1) f g *,m DMG TSL (K) mTMG (mol kg1) f g *,m TMG TSL (K) mcit (mol kg1) f g*,m cit TSL (K)
0.21 1.0617 1.0026 272.735 0.50 1.0451 1.0513 272.178 0.20 0.9660 0.9603 272.791
0.96 1.1309 1.0569 271.131 0.95 1.1258 1.1091 271.161 0.40 0.9618 0.9358 272.434
1.02 1.1614 1.0644 270.947 1.49 1.2321 1.1954 269.735 0.60 0.9520 0.9193 272.088
1.49 1.2328 1.1425 269.733 1.95 1.3234 1.2860 268.350
a
Standard uncertainties u are u(m) = 0.001 mol=kgH2 O, and combined expanded uncertainty Uc is Uc(f) = 0.006 (for 0.95 level of condence).
 C. Held, G. Sadowski / Fluid Phase Equilibria 407 (2016) 224235 229

coefcients. For that purpose, the required osmotic coefcients are In a second experiment, a dened mass of pure CS was given
usually approximated by a power series: into a 2 mL glass vial, which was thermostated to a desired
temperature. Afterwards, water was added drop by drop with the
X
n
f1 Ai miCS (7) help of a 1 mL syringe. The solution was allowed to equilibrate after
i1 shaking. In the case that any particles were observed optically,
The Ai values in Eq. (7) are constants of the power series and n another droplet of water was added and the solution was shaken
refers to the number of parameters needed to represent the and allowed to equilibrate again. This procedure was repeated until
experimental osmotic coefcients. solid particles could not be observed optically any more. The
Eq. (7) was used to convert osmotic coefcients into rational CS weight of added water was determined from weighing the nal vial
activity coefcients on molality basis g *,mCS by: and the mass of the initial amount of CS + vial. The results of the
two optical experiments were averaged.
ZmCS To check the accuracy of the optical methods, the DSC-
f1
lng ;m
CS f  1 dmCS (8) measured solubility data of TMAO and L-citrulline in water were
mCS
0 re-measured optically. Both, DSC and optical method, yielded
Rational CS activity coefcients obtained by Eq. (8) are listed in results which were in the same order of magnitude. The maximum
Tables 46. Experimental data for solutions with other CSs deviation between the two methods was 7 K for TMAO and less
considered in this work were taken from literature (glycerol than 1 K for L-citrulline, respectively.
[23] glycine [35], DMSO [10], MG [36], TMG [37], urea [38], and Using the two methods (DSC and optical method), the
ectoine [12]). solubilities of DMG, TMG, TMAO, and L-citrulline in water were
measured at temperatures between 298.15 and 368.15 K. The
3.2.3. Solubility in water results are summarized in Table 7 and illustrated in Fig. 2.
In this work, differential scanning calorimetry (DSC) has been The solubility of the other solutes considered in this work is
applied to measure the solubility of TMAO in water and of L- already well-known even in a broad temperature range (trehalose
citrulline in water. For the DSC measurements a DSC Q100 from TA [40], ectoine [12], glycine [41], urea [42]; glycerol and DMSO are
Instruments (Eschborn, Germany) was used. A known amount of completely soluble in water in the considered temperature range).
CS was dissolved in water yielding roughly 15 mg homogeneous
solution. The solution was lled with a syringe into a hermetically [(Fig._2)TD$IG]
closed aluminum crucible with a volume of 110 mL. To guarantee
tightness, the crucible was sealed by a collet chuck and weighed
before and after each measurement. The samples were rst
equilibrated at 280.15 K. After that, the temperature was raised at a
constant heating rate of 1 K/min. This led to dissolution of the CS
accompanied by a change of enthalpy. The solubility temperature
at known composition was determined as the offset temperature
of the heat-ow curve. The procedure was adopted from and is
explained in more detail in [39].
Unfortunately, the DSC measurements failed for the DMG and
TMG due to decomposition. Thus, for DMG and TMG an optical
method was used to estimate the solubility in water. First, a dened
supersaturated CS/water solution was lled into 2 mL glass vials.
The vials were tempered in a mini thermostat provided with
double-jacket glass walls. With the help of a PT100 temperature
sensor, the temperature was recorded with an accuracy of 0.01 K.
Starting from room temperature, the solution was heated (Lauda
RE 304, Lauda-Knigshofen, Germany) with a rate of 0.5 K/min. The
Fig. 2. Solution densities of binary CS/water systems at 298.15 K and 1 bar. Symbols
temperature at which all the solid particles were completely
represent experimental data (circles: TMAO, stars: TMG, upside-down triangles:
dissolved and a particle-free solution was obtained (observed glycerol, diamonds: ectoine, triangles: citrulline (cit), squares: trehalose(tre)), lines
optically) was determined to be the solubility temperature represent PC-SAFT modeling. The dashed-dotted line is the solution density of
corresponding to the known composition in the vial. seawater containing 4 wt% NaCl at 298.15 K and 1 bar.

Table 7
Experimental solubility msat of TMAO, L-citrulline (cit), DMG, and TMG in water between 296.35 and 368.04 K, respectively. The two methods (M)optical (Opt)a and DSCb
were applied.

T (K) msatTMAO (mol kg1) M T (K) msatcit (mol kg1) M T (K) msatDMG (mol kg1) M T (K) msatTMG (mol kg1) M
297.95 7.28 Opt 296.35 0.44 Opt 299.45 8.28 Opt 298.55 11.28 Opt
336.15 18.39 Opt 298.95 0.71 Opt
351.15 21.55 Opt 301.35 0.588 DSC
354.03 21.548 DSC 302.15 0.59 Opt
357.32 21.548 DSC 318.05 1.42 Opt
361.15 38.65 Opt 322.03 1.388 DSC
368.04 38.508 DSC 323.75 1.39 Opt
324.15 1.90 Opt
325.64 1.899 DSC
333.15 2.39 Opt
a
Standard uncertainty u is u(T) = 0.3 K. Combined expanded uncertainty Uc is Uc msat 0:4mol=kgH2 O (for 0.95 level of condence).
b
Standard uncertainty u is u(T) = 0.3 K Combined expanded uncertainty Uc is Uc(Tsat) = 4 K (for 0.95 level of condence).
230 C. Held, G. Sadowski / Fluid Phase Equilibria 407 (2016) 224235

4. PC-SAFT modeling and parameters
Modeling thermodynamic properties of CS solutions requires
explicitly accounting for hydrogen bonding. PC-SAFT appears to be
an appropriate model as hydrogen bonding is considered by
association sites that are provided with a certain binding volume
and energy. In PC-SAFT, thermodynamic properties are calculated
based on the residual Helmholtz energy of a system ares that is
composed of various Helmholtz energy contributions yielding:
ares ahc adisp aassoc
hc
whereas the rst contribution a describes the repulsion of hard
chains, the other contributions account for attractive forces due to
van-der-Waals interactions (adisp) and hydrogen bonding (aassoc).
The exact denitions of each of them can be found in the original
PC-SAFT publication [43]. Derivations of ares with respect to mole
fraction and system density allows for the determination of other
thermodynamic properties such as pressure p or fugacity
coefcients [12]. The activity coefcient of water g w is
determined by
w T; p; xw
gw
0w T; p; xw 1
here 0w denotes the reference state (pure water) at the same
conditions (T, p) as the considered aqueous solution.
In contrast, the rational CS activity coefcient g *CS is related to
the innitely diluted state. It is expressed as the fugacity coefcient
of CS in the mixture divided by the fugacity coefcient of CS
innitely diluted in water:
T;p;x
lng ;m
CS 1
CS
T;p;xw 1(11)Osmotic coefcients f were calculated
CS
CS
based on the modeled g w (Eq. (10)) and using Eq. (6).
Modeling non-polar components with PC-SAFT requires three
parameters, the segment number mseg, the segment diameter s,
and the dispersion-energy parameter u/kB. According to our
previous work [16], the CSs were modeled as associating
components, i.e., the amino group and the carboxyl group are
both assigned one association site. In the case of citrulline, four
association sites were applied due to the additional functional
groups (NH2 and NHCO). For trehalose, 16 association sites were
assumed according to [44]. A preliminary sensitivity analysis
within this work showed that the kind of association scheme only
marginally inuences the modeling results of water/glycerol
systems, and thus the 2B scheme was applied for glycerol.
(9) Considering the association-energy contribution aassoc in Eq. (9)
requires two additional parameters: the association-energy
parameter eAiBi/kB and the association-volume parameter kAiBi.
In contrast to other associative substances considered in this work,
TMAO and DMSO molecules are not able to build hydrogen bonds
as pure components, but solely with water. This was accounted for
by applying the induced-association modeling strategy [45]. In this
case eAiBi/kB was set to zero and kAiBi was set to the value of water.
For the dispersive interactions between two unlike molecules i
and j, a binary interaction parameter kij was introduced correcting
for deviations from the geometric-mean rule:
(10) p
uij 1  kij ui uj (12)
According to our previous works [12,16], the pure-component
parameters of the CSs and the kij between CS and water were tted
simultaneously to solution densities and osmotic coefcients of
CS/water solutions.
The pure-component parameters of the considered compo-
nents and the binary interaction parameters between water and CS
are given in Table 8. The absolute relative deviations (ARD)
between PC-SAFT correlations and experimental data are calculat-
ed by:

Table 8
Purecomponent PC-SAFT parameters for components considered in this work and binary interaction parameters kij between CS and water. ARD values between PC-SAFT and
experimental solution densities and osmotic coefcients are given as well.

Parameter Unit Abbr. TMAO L- MG DMG TMG DMSO Glycerol Water Urea Ectoine Glycine Trehalose
citrulline
Parameter reference [a] [a] [a] [a] [a] [46] [a] [47] [12] [12] [16] [a]e
Segment number () mseg 8.928 5.473 5.315 4.001 8.466 2.2922 [3_TD$IF]2.007 1.2047 4.244 1.250 4.8495 13.692
Segment diameter () s 2.248 3.158 2.563 3.085 2.547 3.278 [34_TD$IF]3.815 c
2.446 5.050 2.3270 2.856
Dispersion energy (K) u/kB 245.44 309.70 194.07 256.76 266.59 355.69 [35_TD$IF]430.82 353.95 368.23 530.00 216.96 319.85
Association sites () Niassoc 2 4 2 2 2 2 2 2 2 2 2 16
Association energy (K) eAiBi/ [36_TD$IF] 0 2569.41 2570.36 2567.98 2541.62 0 [37_TD$IF]4633.47 2425.70 3068.67 3500.00 2598.06 5000.00
kB
Association volume () kAiBi 0.0451 0.0385 0.0390 0.0389 0.0384 0.0451 38_TD$IF]0
[ .00189 0.0451 0.001 0.090 0.0393 0.100
Interaction () kij 0.1489 0.0314 0.1098 0.0796 0.0922 0.065 39_TD$IF][ 0.005 0.0438 b 0.0612 d
parameter with
water|

Solution density
T (K) 298.15a 298.15a 298.15[340_TD$IF] 298.15a 298.15 298.15 298.15[23] 298-303 288 298 298.15a
[26] [27] [24] 318 318
ARD (%) 0.17 0.05 0.12 0.05 0.05 1.51 [341_TD$IF]0.97 0.11 0.08 0.05 0.03
(0 < xgly < 0.28)

Osmotic coefcient
T (K) 310a 273/310/ 298.15[342_TD$IF] 273/ 298[34_TD$IF] 273 298.15[23] 288 273/ 298.15 273[34_TD$IF][48]/
323a [36] 310/ [37]/ [10] 323 310/323 310a
323a 273a
ARD (%) 4.47 0.61 0.17 2.00 0.44 2.07 [345_TD$IF]0.58 0.07 1.91 1.20 0.59
(0 < xgly < 0.28)f
a
[346_TD$IF]PC-SAFT parameters from this work.
b
kij = 2.933
103 + (T/K  298.15 K)
5.787
104.
c
The expression s = 2.7927 + 10.11 exp(0.01775 T)  1.417 exp(0.01146 T) was used [47].
d
kij = 2.74
102 + (T/K  298.15 K)
2.19
104.
e
Fitted to osmotic coefcients and densities [this work, [44][347_TD$IF]].
f
ARD with respect to water activity coefcient.
 C. Held, G. Sadowski / Fluid Phase Equilibria 407 (2016) 224235
!
XNP
yPCSAFT
ARD 100 j 1  k exp j (13)
k1
yk
ARD values for each property y (in this work solution density and
osmotic coefcient) of the CS/water mixtures are also summarized
in Table 8. It can be concluded from these values that PC-SAFT is
highly appropriate for modeling properties and phase equilibria in
CS systems.
5. Discussion
5.1. Densities
In order to avoid sinking of the cell to the ground of a sea the CS
should cause low mass solution densities. Especially CSs of low
molecular weight fulll this criterion. At least the CS solutions have
to possess a lower mass density compared to the environment the
organisms live in. Therefore, the density of the environment (e.g.,
seawater) has to be known in a rst step. Thereafter, densities of
water/CS solutions might be investigated; by this, it will become
obvious which (if any) of the CSs cause high solution densities
causing high cellular specic masses.
5.1.1. Density of high-salinity environments
An organism living in saline water has to be lighter than its
environment. Taking the Dead Sea as example, a total salt amount
of approximately 30 wt% (corresponds to a molality of about 5 mol/
kg) with the main salts being MgCl2 (50.8%), NaCl (30.4%), CaCl2
(14.4%), and KCl (4.4%) is dissolved in water. The corresponding
solution density at 298.15 K can be estimated using ePC-SAFT
which was earlier shown to be able to precisely model salt/water
densities [49]. Using ePC-SAFT and the ion parameters from [49], a
Dead Sea water density of 1233 kg/m3 is obtained at 298.15 K and [349_TD$IF]is
listed in Table 9 as rsw value. This is the maximum solution density
allowed for cells living in the Dead Sea to avoid sinking.
5.1.2. Density of environments of moderate salinity
For other seas, the total salt molality is (1) much lower compared
to Dead Sea and (2) the salt can be reduced to only NaCl (85% of the
total salt content). Doing this, the solution densities of the Baltic Sea
(salt weight percent of 0.8wt%), Black Sea (1.7wt%), North Sea,
Atlantic Ocean, Pacic Ocean, and Indian Ocean (all 3.5wt%),
Mediterranean Sea (3.8wt%), as well as Red Sea and Persian Sea
(both 4wt%) were calculated with ePC-SAFT (using the Na+ and Cl
parameters from [49]) and are summarized in Table 9 as rsw values.
231
The liquid density of pure water r0w(T) is temperature-dependent
according to Eq. (15) [50]:
     
1 1 kg
r0w T 0:0055 2 T 2 3:0437 T 580:03 3 (15)
K K m
The difference between the density of the seawater and pure (salt-
free) water, denoted by Drsw depends on the salt concentration. It
is assumed to be independent of temperature (reasonable
assumption, e.g., [49]).
The Drsw values in Table 9 were calculated with ePC-SAFT.
These values were further used to t an easy-to-use correlation for
seawater density as function of salt weight percent according to:
kg
Drsw 6:0212
wNaCl % (16)
m3
Using Eq. (14) together with Eqs. (16) and (15) allows calculating
seawater densities at various salt concentrations and temper-
atures. As example, the rsw value at 4 wt% NaCl at 298.15 K
(1021 kg/m3) is given as a reference line in Fig. 2. This line describes
the mass density of a CS solution that must not be exceeded in
order to avoid sinking.
5.1.4. Density of CS/water solutions
Fig. 2 illustrates experimental and PC-SAFT modeled densities
of some CS/water solutions considered in this work. As expected,
the CS of lowest molar mass considered in this work (TMAO)
causes the lowest solution density. Water/TMAO densities (even at
high TMAO molalities) do not deviate much from the density of
pure water. Assuming that an organism living in the Dead Sea only
consists of CS and water, it can be observed from Fig. 2 that the
densities of all considered CS solutions are below the critical
external solution density (1233.00 kg/m3, see Table 9). For other
sea waters however, the use of too large (i.e., too heavy) CSs (e.g.,
trehalose) is possible only at very small CS molalities (mtrehalose
< 0.2 mol/kg).
Applying the PC-SAFT allows modeling the CS/water densities
in quantitative agreement with the experimental data. This is
illustrated in Fig. 2 and becomes further obvious from the ARD
values listed in Table 8. (DMSO causes solution densities that are
very close to the ones of TMG/water and therefore was omitted in
Fig. 2 for a better view.)
5.1.5. Density of CS/water solutions at high pressure
Halophilic bacteria might live at conditions where non-
atmospheric pressures are present, e.g., in deep seas. Thus, it

5.1.3. General density correlation for habitats environments [(Fig._3)TD$IG]

A general correlation is proposed here for calculating seawater
densities without using ePC-SAFT. Solution density depends on
temperature and on salt concentration and for simplicity be
considered as the sum of two terms:

rsw T; wt%NaCl r0w T Drsw wt%NaCl (14)

Table 9
Solution densities rsw of seawater (represented by NaCl/water solutions) at 298.15 K
and 1 bar as obtained from ePC-SAFT for the Baltic Sea (BaS), Black Sea (BS), and
North Sea (NS), the Atlantic Ocean (AO), Pacic Ocean (PO), and Indian Ocean (IO),
and the Mediterranean Sea (MS), Red Sea (RS), Dead Sea (DS), and Persian Sea (PS).
Drsw values according to Eq. (16).
Sea BaS BS NS, AO, PO, IO MS RS, PS DS
wtNaCl % 0.8 1.7 3.5 3.8 4.0 30.0
rsw [kg/m3] 1001.81 1007.27 1018.06 1020.06 1021.06 1233.00 Fig. 3. Solution densities of binary TMAO/water solutions at 100 bar and
Drsw 4.77 10.23 21.02 23.02 24.02 235.96 temperatures between 278 and 323 K. Symbols represent experimental data from
[kg/m3] [20] (black stars: 278.15 K, triangles: 288.15 K, diamonds: 298.15 K, grey stars:
308.15 K, squares: 323.15 K), lines represent PC-SAFT modeling.
 232 C. Held, G. Sadowski / Fluid Phase Equilibria 407 (2016) 224235
might be of interest whether the conclusions drawn from Fig. 2 and
in Section 5.1.4 are transferable to high-pressure conditions.
Recently, new data on TMAO/water densities in a broad tempera-
ture and pressure range were measured by Makarov et al. [20]
(data at 100 bar shown in Fig. 3).
In this work, PC-SAFT was used to describe the experimental
density data from Makarov et al. at 100 bar at temperatures
between 278.15 and 323.15 K. The pure-component parameters for
TMAO as well as the binary parameter kij between water and TMAO
were taken from Table 8. It should be noted, that these parameters
were tted to densities and osmotic coefcients of TMAO/water
solutions until 2.4 mol/kg at 1 bar and temperatures of 298.15 K
and 310.15 K, respectively. They allow for modeling the TMAO/
water densities in good agreement to experimental data, which is
also shown in Fig. 3. That is, PC-SAFT allows for extrapolating CS
molality, system pressure as well as temperature in good
agreement with experimental data.
5.2. Osmotic coefcients of CS/water solutions
A thermodynamically effective CS should cause high w values
and thus high osmotic pressures for a good protective effect against
osmotic stress. This chapter analyzes the dependence of osmotic
coefcients on the chemical structure of the CSs using the
experimental data obtained within this work but also other data
obtained from literature. In order to understand the effect of solute
functional groups on osmotic coefcients, osmotic coefcients of
amino-acid solutions will be investigated in Section 5.2.1. These
observations will be extended to CS/water solutions in Sec-
tion 5.2.2.
5.2.1. Inuence of solute functional groups on osmotic coefcients
Due to the availability of experimental data and the broad
variety in the chemical composition, discussing osmotic coef-
cients of amino-acid solutions can particularly contribute to a
basic understanding of functional groups on osmotic coefcients.
There exist series of amino acids which only differ in one functional
group and therewith allow for determining the inuence of adding
nonpolar (e.g., CH3) or polar (e.g., OH) groups on osmotic
coefcients. Fig. 4 illustrates osmotic coefcients of different
amino-acid solutions.
It can be observed from Fig. 4 that alanine causes osmotic
coefcients around unity independent of CS molality. Obviously,
adding a polar functional group to alanine causes lower f values
[(Fig._4)TD$IG]
Fig. 4. Osmotic coefcients of aqueous solutions of alanine (leftright triangles,
298.15 K [51]), serine (circles, 298.15 K [52]) glycine (squares, 298.15 K [35]), MG
(triangles, 298.15 K [36]), DMG (diamonds, 310.15 K [this work]), and TMG (stars,
298.15 K [36]). Symbols represent experimental data, lines represent modeling
results with PC-SAFT.
(alanine ! serine) which, according to Eq. (2), leads to lower
osmotic pressures. The opposite behavior is observed for an
increasing number of nonpolar groups (glycine ! alanine) which
yields increased osmotic coefcients at constant temperature and
concentration.
These results drawn from aqueous amino-acid solutions can be
transferred to solutions containing glycine-based CS. Considering
aqueous solutions of methyl glycines, the sequence of osmotic
coefcients is glycine < MG < DMG < TMG. The more hydrophobic
the CS, the higher the osmotic coefcients, the higher the osmotic
pressures and the more effective the protection against osmotic
stress.
5.2.2. Osmotic coefcients of CS/water solutions
The conclusions drawn from Section 5.2.1 are not only valid for
amino-acid based CSs like TMG. Fig. 5 compares the inuence of
different CSs on osmotic coefcients of aqueous solutions.
Obviously, all substances being known as CSs induce f values
higher than unity making them effective protectants against
osmotic stress in contrast to very polar solutes not known as CSs
(e.g., urea), which cause f values smaller than unity. It has already
been discussed that obviously incompatible solutes (urea in Fig. 5)
cause osmotic coefcients which are lower than unity [12].
It can also be observed from Fig. 5 that the dependence of the f
values on CS molality is qualitatively equal for all considered CS.
That is, the higher the CS molality the higher the osmotic
coefcients. This further increases the CS effect against osmotic
stress as the osmotic pressure is proportional to the product of CS
molarity cCS and osmotic coefcient f. However, a distinct
quantitative difference among the CS can be observed. Regardless
of CS molality, the osmotic coefcients caused by the most
important CS are sequenced in the order fectoine < fTMG < fTMAO.
This sequence also reects the order of the CS polarities and
supports the conclusions drawn from Fig. 4.
PC-SAFT can model the experimental data almost quantitative-
ly. The deviations between experimental and modeled osmotic
coefcients are given in Table 8.
5.3. Temperature dependence of osmotic coefcients
There are several organisms, animals, or plants, which use the
CSs considered in this work. However, only some particular CSs are
applied by organisms that live at non-ambient temperatures.
TMAO is used as CS by arctic shes, i.e., TMAO has to be an effective
[(Fig._5)TD$IG]
Fig. 5. Osmotic coefcients [328_TD$IF]of aqueous solutions of ectoine (diamonds, 298.15 K
[12]), TMAO (circles, 310.15 K [this work]), glycerol (black triangles, 298.15 K [53]),
DMSO (squares, 273.15 K [10]) and urea (grey triangles, 298.15 K [38]). Symbols
represent experimental data, lines represent modeling results with PC-SAFT.
 C. Held, G. Sadowski / Fluid Phase Equilibria 407 (2016) 224235
CS (high f values) especially at low temperatures. In contrast,
citrulline is present in water melons, i.e., osmotic coefcients have
to be high also at elevated temperatures.
The FPO method was applied to determine osmotic coefcients
at low temperatures, whereas VPO was used for measuring
osmotic coefcients at temperatures above 298.15 K. Comparing
experimental f values between 273.15 K and 323.15 K (Tables 46)
it can be stated that the temperature dependence of osmotic
coefcients is not very pronounced. This is an important result
meaning that the high osmotic coefcients observed in CS/water
solutions (Fig. 5) are also high in the biologically relevant
temperature range (278310 K). However, slight dependencies
on temperature can be observed. The osmotic coefcients of DMG/
water increase with decreasing temperature. In contrast, f values
decrease with decreasing temperature for citrulline/water sol-
utions. This is of relevance as citrulline is applied at elevated
temperatures, where osmotic coefcients are higher than at low
temperature.
PC-SAFT can be used to model the temperature dependence of
the osmotic coefcients in CS/water systems. In the case of
citrulline/water, PC-SAFT was used to predict the temperature
effects as the PC-SAFT parameters for citrulline were adjusted to
thermodynamic data at 298.15 K (solution densities, Table 3) and
310.15 K (osmotic coefcients, Table 4), only. Using these
parameters, osmotic coefcients of citrulline/water solutions
between 273 and 310 K were predicted. These predictions are
shown in Fig. 6 for a solution of 0.6 mol citrulline/kg water. It can be
observed that data and predictions are in qualitative agreement,
although PC-SAFT overestimates the absolute values of the osmotic
coefcients and slightly underestimates the slope of the tempera-
ture dependence. The deviations between experimental and
predicted osmotic coefcients are given in Table 8.
5.4. Solubility in water
CS precipitation has to be avoided in organisms, even at high CS
concentrations. Thus, CSs which are used by very halophilic
organisms (high external salt concentrations (e.g., in the Dead
Sea)) have to be well-soluble at the prevalent temperature. Among
the considered CS, the low-molecular-weight molecules which are
moreover very polar should be best soluble in water.
This is the case for the very well-soluble TMAO. Glycerol and
DMSO are even completely soluble in water at ambient conditions.
All other CSs considered in this work have a limited solubility in
water, even at very high temperatures.
However, compared to other biomolecules, the water solubility
of the most CSs considered in this work is indeed very high, even at
[(Fig._6)TD$IG]
Fig. 6. Osmotic coefcients of a binary solution of 0.6 mol citrulline/kg water
between 273.15 K and 323.15 K. Symbols represent experimental data (Tables 46),
line represents modeling result with PC-SAFT.
233
low temperature, which can be observed from Table 7. The reason
is the low size of most of the molecules, a high amount of OH-
groups which can form hydrogen bonds with water, or the charged
(or even zwitterionic) character. However, some of the considered
CSs (trehalose and citrulline) are rather weakly soluble in water.
That is, they can be applied as CSs only at low concentrations and
thus are not used by halophilic organisms.
5.5. Impact of thermodynamic properties on osmotic-stress protection
Table 10 shows the osmotic pressures for CS/water solutions at
a xed CS molality of 3 mol/kg, which was chosen as typical
concentration in the environment of a halophilic organism.
Required osmotic coefcients were calculated by PC-SAFT using
parameters from Table 8.
It can be seen from Table 10 that TMAO causes the highest
osmotic pressure; furthermore, the difference between the most
and the least effective CS is about 55 bars (corresponds to a
difference of >200%) at the given conditions.
Table 10 also lists the osmotic pressure of a 3 mol urea/kg water
solution and a 3 mol glycine/kg water solution at 298.15 K. Only the
CSs citrulline and trehalose cause lower osmotic pressures
compared to the two incompatible solutes urea and glycine.
Eqs. (1) and (2) allow calculating the molarity of a CS required
for the establishing a certain osmotic pressure. Assuming that a
marine organism lives in an environment with a very high salinity
of 3 mol NaCl/kg water, the external osmotic pressure can be
calculated to be 142.97 bar (see Eq. (2) with r (298.15 K, mNaCl =
3 mol/kg) = 1105 kg/m3 and f (298.15 K, mNaCl = 3 mol/
kg) = 1.045 and n = 2). Table 11 lists the molalities of the CSs that
are required to obtain a pressure that allows compensating the
chemical-potential-induced loss of water.
It can be observed that very high CS concentrations are required
at an external 3 mol/kg NaCl environment. Moreover, some of these
concentrations exceed the solubility limit of the considered CSs in
water at 298.15 K (see e.g., Table 7). Thus, it can be concluded from
Table 11 that only DMG, TMG, TMAO, DMSO allow compensating
the high external osmotic pressure of 142.97 bar caused by a 3 mol/
kg NaCl medium at 298.15 K.
The considerations from Tables 10 and 11 further allow
calculating the CS molality that can be saved by using a certain
CS instead of another one. At 298.15 K and 3 mol/kg NaCl external
salinity, a cell that accumulates TMAO (the CS causing the highest f
values) can save 9.5 moles solute per kg of water (or 9.5 mmol per g
of water) compared to cells that accumulate citrulline as their main
CS for stress protection. As it is known that accumulation/synthesis
of CSs requires a very high amount of energy (up to 2 ATP
equivalents e.g., for ectoine synthesis [54]), it becomes clear that
savings of only a few mmol of the respective solute (or the energy
equivalents) may then be used for cell growth. This might be one
reason why the CSs TMAO, TMG and ectoine are used very often by
halophilic organisms in highly-concentrated salt media.
5.6. Maximum osmotic pressures
In chapter 4.2 it was shown that the most hydrophobic CSs
cause the highest osmotic coefcients. Unfortunately, this nding
is opposed to the CS solubility in water: the lower the CS polarity
the lower is usually its water solubility. Thus, a certain compromise
has to be found to nd a CS that is well-soluble and at the same
time very effective against osmotic stress. That is, CSs have to be as
hydrophobic as possible for high f values but they also have to
possess polar patches to be enough soluble in water.
Organisms living in environments with extreme salinity (e.g.,
Dead Sea) have to accumulate CSs that cause very high osmotic
pressures. Based on the solubility and on the osmotic-coefcient
234 C. Held, G. Sadowski / Fluid Phase Equilibria 407 (2016) 224235

Table 10
Osmotic pressures in aqueous solutions of TMAO, DMG, TMG, ectoine, citrulline (cit), trehalose (tre), glycerol, DMSO, glycine, and urea at solute molalities of 3 mol/kg and at
298.15 K.

Solute TMAO DMG TMG Ectoine Cit Tre Glycerol DMSO Glycine Urea
Osmotic pressure [bar] 104.7 79.4 84.7 69.8 50.4 46.6 63.4 74.3 58.2 59.9

Table 11
CS molalities of TMAO, DMG, TMG, ectoine, citrulline (cit), trehalose (tre), glycerol, DMSO, glycine, and urea required for establishing an osmotic pressure of 142.97 bar at
298.15 K. Solubilities msat,298.15K are experimental values at 298.5 K.

Solute TMAO DMG TMG Ectoine Cit Tre Glycerol DMSO Glycine Urea
sat,298.15K
m [mol/kg] 7.3 8.3 11.3 6.5 0.7 2.7 completley miscible completley miscible 3.0 19.5
Molality 4.5 7.7 5.1 7.4a 14.0a 6.8a 12.8 11.1 10.4a 22.0a
[mol/kg]
a
Supersaturated (i.e., molality > solubility limit at 298.15 K).

Table 12
Maximal osmotic pressures at 298.15 K in saturated CS solutions containing TMAO, DMG, TMG, ectoine, citrulline (cit), trehalose (tre), glycine, and urea. Solubilities msat,298.15K
are experimental values, solution densities rsolutionsat,298.15K and osmotic coefcients fsat,298.15K were calculated with PC-SAFT.

Solute TMAO DMG TMG Ectoine Cit Tre Glycine Urea

preal,max [bar] 290.6 161.7 269.0 123.7 16.0 42.7 58.1 212.6
msat,298.15K [mol/kg] 7.3 8.3 11.3 6.5 0.7 2.7 3.0 19.5
fsat,298.15K 2.439 1.326 2.012 1.274 0.989 1.007 0.887 0.793
rsolutionsat,298.15K [kg/m3] 1021.3 1100.7 1110.0 1155.7 1033.7 1230.8 1079.2 1204.0

measurements provided in Tables 46 the maximal osmotic
pressure induced by a CS can be calculated by
preal;max 298K RTcsat 298K
fsat csat ; 298K
For the conversion of the experimental msat values (Table 7) to the
csat values, saturated-solution densities were calculated with PC-
SAFT and used in Eq. (3). Saturated-solution osmotic coefcients
fsat were also calculated with PC-SAFT. The so-obtained maximal
osmotic pressures at 298.15 K are listed in Table 12 for the
considered CSs in this work.
It can be observed from Table 12 that TMAO and TMG are by far
the best CSs against osmotic stress at very high environmental
salinities. That is, organisms can only protect themselves against
very high osmotic stress by using TMAO or TMG. Comparing the
maximum solubility msat,298.15K and the maximum osmotic
pressure preal,max of the solutions, only a rough correlation can
be observed between the two properties. The correlation is mainly
disrupted by density and osmotic coefcients, which again points
to the importance of available experimental thermodynamic data
in CS solutions.
The availability of density, osmotic-coefcient and solubility
data will thus be helpful to design CSs that cause high osmotic
pressures. The key properties for this are a low molecular weight
(good water solubility and low solution density) with hydrophobic
as well as hydrophilic patches to nd the best balance between a
high-enough water solubility, low-enough solution densities, and
osmotic coefcients as high as possible. Many CSs indeed have
these properties. Among the considered CSs, TMAO, TMG and
ectoine best fullled these criteria. Indeed, these CSs are used by
most of the halophilic organisms against osmotic stress
6. Conclusion
Several preconditions have to be fullled by an organic
molecule to act as CS in order to protect organisms against the
external osmotic stress in concentrated salt media. Once the
pathway for CS synthesis (or the transporter system for their
accumulation) exists, thermodynamic properties determine the
(17) strength of the protection against osmotic stress. First, CSs should
not cause high solution densities to avoid cell sinking. Secondly,
CSs must be highly-soluble in water to assure the full protective
effect especially when present in external media of high salt
concentrations. Thirdly, the strength of the protective effect is
inuenced by osmotic coefcients f the CS causes. The higher the
f values, the higher osmotic pressures and the better the
protective effect of CS against osmotic stress.
In this work, solution densities, solubilities, and f values of
aqueous CS solutions containing TMAO, TMG, DMG, trehalose, and
citrulline have been measured and compared to each other with
regard to their inuence on osmotic pressure. It turned out that the
best CS among the solutes considered in this work is TMAO. It (1) is
of low molecular weight (good water solubility and low solution
density), (2) possesses polar patches for good water solubility, and
simultaneously (3) contains very hydrophobic groups which are
required for causing high osmotic coefcients.
Interestingly, all CSs considered in this work cause high osmotic
coefcients, and among them TMAO causes the highest one. Osmotic
coefcients follow the order TMAO > TMG > DMG > DMSO > ectoine >
glycerol > trehalose > citrulline. The cryoptrotectants DMSO and
glycerol cause also osmotic coefcients higher than unity, and
DMSO is effective (causes higher osmotic coefcients). Osmotic
coefcients mainly determine the thermodynamic strength of the
protective effect against osmotic stress, thus TMAO was found to be
the strongest agent against osmotic stress.
TMG and ectoine, powerful CSs considered in this work, are
used as main CSs by the most salt-tolerant cyanobacteria. TMAO is
used in arctic shes as it is of low mass density and provides high f
values even at cold temperatures. Sugars (e.g., trehalose) are next
to their weaker osmotic force certainly also due to their lower
solubility used only by slightly halotolerant organisms. The
 C. Held, G. Sadowski / Fluid Phase Equilibria 407 (2016) 224235
results of this work give one possible explanation for the different
use of CS in the different organisms.
The PC-SAFT equation of state was used to describe thermody-
namic properties of aqueous CS solutions. Solution densities and
osmotic coefcients were modeled quantitatively. PC-SAFT was
successfully used to extrapolate concentration and temperature
ranges not considered in the parameter estimation.
[350_TD$IF]Acknowledgements
The authors gratefully acknowledge the nancial support by the
German Society of Industrial Research (AiF) with Grant 14778N/3.
We thank our students Eric Ritter and Jihyun Yoo for VPO
measurements.
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