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Methods 76 (2015) 3–10

Contents lists available at ScienceDirect

Methods
journal homepage: www.elsevier.com/locate/ymeth

What does calorimetry and thermodynamics of living cells tell us?


Thomas Maskow ⇑, Sven Paufler
UFZ, Helmholtz Centre for Environmental Research, Dept. Environmental Microbiology, Permoserstr. 15, D-04318 Leipzig, Germany

a r t i c l e i n f o a b s t r a c t

Article history: This article presents and compares several thermodynamic methods for the quantitative interpretation of
Received 28 August 2014 data from calorimetric measurements. Heat generation and absorption are universal features of microbial
Received in revised form 23 October 2014 growth and product formation as well as of cell cultures from animals, plants and insects. The heat pro-
Accepted 28 October 2014
duction rate reflects metabolic changes in real time and is measurable on-line. The detection limit of
Available online 13 November 2014
commercially available calorimetric instruments can be low enough to measure the heat of 100,000 aer-
obically growing bacteria or of 100 myocardial cells. Heat can be monitored in reaction vessels ranging
Keywords:
from a few nanoliters up to many cubic meters. Most important the heat flux measurement does not
Calorimetry
Biothermodynamic
interfere with the biological process under investigation. The practical advantages of calorimetry include
Bioprocess control the waiver of labeling and reactants. It is further possible to assemble the thermal transducer in a pro-
Enthalpy balances tected way that reduces aging and thereby signal drifts. Calorimetry works with optically opaque solu-
Oxycaloric equivalent tions.
Law of Hess All of these advantages make calorimetry an interesting method for many applications in medicine,
environmental sciences, ecology, biochemistry and biotechnology, just to mention a few. However, in
many cases the heat signal is merely used to monitor biological processes but only rarely to quantita-
tively interpret the data. Therefore, a significant proportion of the information potential of calorimetry
remains unutilized. To fill this information gap and to motivate the reader using the full information
potential of calorimetry, various methods for quantitative data interpretations are presented, evaluated
and compared with each other. Possible errors of interpretation and limitations of quantitative data anal-
ysis are also discussed.
Ó 2014 Elsevier Inc. All rights reserved.

1. Introduction elaborated optimization and design of primer, probes and hybrid-


ization conditions as known for molecular biology-based methods
Heat is an inevitable by-product of any microbial growth and [3] are not required in calorimetry. Finally, thermal transducers
product formation processes. Heat is directly related to growth may be, different to many other biochemical sensors, mounted in
stoichiometry and heat production rate to the metabolic fluxes a protected location. This keeps the transducer stable for long time
via Law of Hess. Calorimetry measures the metabolic activity of a and reduces the signal drift. All of these advantages increase the
population of cells. By this, any metabolic change can be monitored scientific and technical demand for calorimetric measurement
on-line and in real time. These days, heat production rate can be methods and data interpretation. As a result commercially avail-
measured with a specific limit of detection of 105 W L1 which able highly sensitive, high throughput calorimeters, able to mea-
corresponds to a microbial oxygen depletion of air saturated med- sure up to 48 channels in parallel have been developed. Fields of
ium (approx. 6.7 mg L1 at 37 °C) within 44 days. In case of aerobic application ranges from medical research [4], environmental
glucose combustion it relates to the tiny consumption rate of microbiology [5–7] to the food industry [8]. Due to the develop-
0.03 lM h1 or 5 lg L1 h1 [1]. These data show the potential of ment of highly sensitive calorimeters with a high throughput and
calorimetry. A good overview about the dependency of the limit their validation in different application fields the quantitative data
of detection on the considered reaction volume and on the applied interpretation becomes more and more important. First reports on
instrument is given by Zogg et al. [2]. Calorimetry works in opaque enthalpy balances around systems of different scales which are
media and doesn’t need any labeling agents. Long-lasting and also applicable to calorimeters, bioreactors or even ecosystems
were published more than two decades ago [9,10]. The authors
⇑ Corresponding author. Fax: +49 341 235 1351. correlated metabolic fluxes and growth stoichiometry with heat
E-mail addresses: Thomas.maskow@ufz.de (T. Maskow), Sven.paufler@ufz.de flows via Law of Hess. However, advanced chemical analytics are
(S. Paufler). indispensable for such a data analysis. Quite often comprehensive

http://dx.doi.org/10.1016/j.ymeth.2014.10.035
1046-2023/Ó 2014 Elsevier Inc. All rights reserved.
4 T. Maskow, S. Paufler / Methods 76 (2015) 3–10

metabolic information are not available for interpretation of calo- organisms is designated in the following as biomass. The elemental
rimetric experiments. For this, the applicability of more simple composition of X1 = 1.70; X2 = 0.42 and X3 = 0.25 is typical for bac-
growth models assuming a constant heat production rate per cell terial biomass [14].
or per converted electron were successfully tested just recently
rS CHS1 OS2 þ rN NH3 þ rO2 O2 ! r X CHX1 OX2 NX3 þ r CO2 CO2
[11]. Finally, the recent tendency to simplify and to standardize
calorimetric measurements leads to potential misinterpretations þ r H2 O H 2 O ð2Þ
of calorimetric experiments [12,13]. ri
This review relates different types of calorimetric data interpre- with Y i=X ¼ rX

tation to the level of available information. It explains weaknesses follows Y S=X CHS1 OS2 þ Y N=X NH3 þ Y O2 =X O2 ! CHX1 OX2 NX3
and advantages of the evaluation methods. Also, causes of various ð3Þ
misinterpretations of calorimetric measurements are going to be
þ Y CO2 =X CO2 þ Y H2 O=X H2 O
discussed. Finally, the compromise between modern trends in The five unknown stoichiometric/yield coefficients Yi/X are not
respect to miniaturization and high-throughput measurements independent of each other. They have to fulfill four elemental bal-
and the requirement for more analytical information for a correct ances for C, H, O and N. This means the yield coefficients can be cal-
calorimetric data interpretation will be discussed. culated if one more conditional relation is known. This additional
relation can be the enthalpy balance which is also called the Law
2. Extracting quantitative information from the calorimetric of Hess (Eq. (4)).
signal
X
n X
n
DR H X ¼ ðY i=X Df Hi Þ ¼  ðY i=X DC Hi Þ ð4Þ
2.1. Data interpretations using the first law of thermodynamics i¼1 i¼1

The energy balance/Law of Hess correlates the measured


Every open system, independent of its size and the ability to
growth reaction enthalpy DRHX with the energy contributions of
monitor the heat production, can be considered as a type of calo-
each chemical species i involved in the growth reaction. DRHX is
rimeter. A ‘‘system’’ can be a part of an ecosystem, a bioreactor,
the enthalpy released (negative sign) or consumed (positive sign)
an animal or a plant but it could also be a simple medical or food
during the formation of one mole biomass. The energy contribution
sample containing active cells. Clearly defining the systems bound-
of each species can be described as enthalpies of formation DfHi or
aries is essential for designing the experiments and later for data
of combustion DCHi (see Eq. (4)). However, the correct standard
evaluation. Fig. 1 shows a simplified chart of such an open system
and reference state of the enthalpy of all species have to be
exchanging energy and matter with the environment.
selected and to be corrected for temperature. Von Stockar and
The general balance of such a system has the structure of:
co-workers discussed consequences of wrong usage of reference
Accumulation in the system ¼ Input  Output states and neglecting temperature corrections [10]. Note, the usage
 Chemical; biological conversions of combustion enthalpies simplify the enthalpy calculation (in this
specific case) because the combustion enthalpies of CO2 and H2O
ð1Þ are zero (see Eq. (5))
Every conserved quantity (e.g., elements, electrons, individual DR HX ¼ ðDC HX  Y S=X DC HS  Y N=X DC HN Þ ð5Þ
chemical and biological species as well as enthalpy) can be bal-
anced. The usage of enthalpy balances for evaluation of calorimet- The calorimetrically determined heat production rate Q_ con-
ric results makes most sense if information about the metabolic tains additional kinetic information[15,16]. Eq. (6) shows it at the
fluxes ri (Eq. (2)) or about the growth stoichiometric coefficients example of the growth rate rX.
Yi/X (Eq. (3)) is available or aspired. The difference between heat
Q_ ¼ r X DR HX ð6Þ
and enthalpy is discussed in a later section of the article. However,
R
the main barrier to application of fully balanced systems is the The growth reaction heat (DR Q ¼ Q_ dt) is equal to the growth
requirement of an exhaustive chemical analysis of the calorimetri- reaction enthalpy DRH for open systems with a constant pressure
cally monitored process. The following example shows the sim- (dp = 0). In case of calorimetric measurements in the often used
R
plest case of aerobic biomass formation (CHX1OX2NX3) from any closed ampoules, a contribution of work ( V dp) has to be added.
organic carbon source (CHS1OS2). The cell dry mass of biological In case of a typical ampoule of 4 mL with 2 mL cellular suspension
and 2 mL air space a pressure increase of 1 atm (101,325 Pa) pro-
vides only 0.2 J, which can often be neglected in comparison to
the growth heat.
Calorimetry measure the average metabolic activity of a popu-
lation of cells. An in-depth data interpretation can only be done
if enough analytical information is available. The calorimetric sys-
tems must be large enough for extensive sampling or to include
online sensors. This is the case for experiments in a reaction calo-
rimeter, in bioreactors combined with a flow through calorimeter
[17–20], or by performing a calorimetric experiment in parallel
to an equivalent experiment in a bioreactor. The latter two are
error-prone due to metabolic processes in the flow through line
or wall growth [21,22]. Differences between the physical environ-
ment in the calorimeter and in the bioreactor may distort the calo-
rimetric result. An experiment must be carefully designed to
Fig. 1. Calorimeter as an open system exchanging energy and matter with the minimize distortions of the calorimetric signal due to (i) the addi-
environment. The calorimeter can incorporate parts of ecosystems, bioreactors or
simple samples with living matter. The system can be in steady state or in a transient.
tion of substances with deviating temperatures, (ii) the heat of
It has to be adapted to the specific experimental conditions. Convective heat flows neutralization by keeping the pH constant, (iii) heat of evaporation
due to exchange of matter is not considered. The figure is derived from [9]. due to aeration, (iv) heat effects of gas adsorption and release, and
T. Maskow, S. Paufler / Methods 76 (2015) 3–10 5

concept [27]. The potential of reaction calorimetry in this context


was demonstrated at the example of Saccharomyces cerevisiae
shifting from the respiratory to the respire-fermentative metabo-
lism [28] and at the example of the cleavage of aromatic ring struc-
tures in ortho- or meta position [29].
Other applications are the usage to quantify the energetic costs
of metabolic adaptations. This was demonstrated at the examples
of adaptation to a shortage of nutrients [30] or to stress exerted
by high salt concentrations [31–34]. For instance it was found that
the adaptation to salt stress using ectoine as protection molecule
does not burden the metabolism (for details see [33]). Also the
more complex haloadaptation process of the non-conventional
yeast Debaryomyces hansenii was revealed using enthalpy balances
[35].
How valuable the calorimetry in combination with enthalpy
balances is, shows the following very early and simple example
(Fig. 2). The energy distribution, during the growth of yeast, is
quantified in this work. Important changes in the assimilation
pathway from the usage of glucose to usage of ethanol are imme-
diately indicated by two main maxima in the heat production rate.
In order to solve the describing equation system for a calorim-
eter at least as many measured entities have to be available as the
number of degrees of freedom of the system. However, sometimes
even more analytical information are available than the degrees of
freedom of the system. In such cases, it is said that the system is
over-determined or contains redundant information. The redun-
dancy can be used for detection of gross and systematic errors
and data reconciliation. The bases for these applications were
founded more as two decades ago [36–39]. Taking the simple aer-
obic growth (see Eq. (3)) as an example, the five independent
growth yields are accompanied by four conservation equations.
Therefore, the degree of freedom is one. With the measurement
Fig. 2. Heat production rate (top) and relative energy distribution between the of the reaction heat is the system completely described and with
substrate (glucose) and the products (ethanol, biomass, intermediates and heat) the additional measurement of CO2 evolution, or oxygen consump-
during the time course of growth of Saccharomyces cerevisiae (bottom). The energy
tion over-determined. Even first application of pure calorimetric
input (i.e., in form of glucose) was set to 100%. The dotted lines indicate sudden
changes in the heat production rate due to the consumption of glucose or ethanol,
monitoring of bioreactors was reported very early [40] and
respectively. The data were taken from [17]. 14 years later demonstrated at industrial scale [41]. The applica-
tion of redundancy analysis to calorimetrically monitored bioreac-
tors is discussed by van der Heijden et al. [37].
In contrast to systems with redundant information is the case of
(v) heat input by stirring of media with a potentially changing vis- the biodegradation of trace pollutants (in the lg/L range). Here it is
cosity have also to be taken into account. This can be done as for often difficult or even impossible to follow the degradation kinetics
instance described by von Stockar et al. and others (see for exam- of pollutants or the formation of biomass or intermediates by
ple [10,23–26]). All these are unwanted energetic flows resulting in chemical analysis due to the extreme small changes in concentra-
increased noise, baseline offset and signal drift. They have a nega- tion. The following example measured by isothermal titration cal-
tive impact on the methods overall limit of detection and limit of orimetry will illustrate how calorimetry can, in combination with
quantification that are closely related to the systems signal to noise enthalpy balances, yield new insights. The first step during the bio-
ratio. degradation of the herbicide 2,4-Dichlorphenoxybutyrate (2,4-DB)

ð7Þ

In fundamental research fully balanced calorimetric experi- is an enzymatically catalyzed cleavage of the ether bond (Eq. (7)).
ments can be applied to detect shifts in metabolic pathways, The question arises what happens with the cleavage products.
because a given pathway is always connected to a certain growth For clarifying that question a tiny amount of 2,4-DB was
stoichiometry. The growth stoichiometry in turn can be calculated added to a bacterial suspension and the degradation monitored
in advance by knowledge of the respective pathway using the YATP calorimetrically resulting in a reaction heat of 1149 ± 16 kJ/mol
6 T. Maskow, S. Paufler / Methods 76 (2015) 3–10

2,4-DB. This measured reaction heat was compared with calculated called oxycaloric equivalent and usually attains values between
reaction heats from enthalpy balances assuming different fates of 430 and 480 kJ/mol-O2 with an average value of
2,4-DB (Fig. 3). The comparison suggested that the metabolic end (455 ± 15) kJ/mol-O2 [49].
products are dichlorophenol (DCP) and biomass [42]. The accumu- Since the calorimetry in this simple picture reflects the cata-
lation of DCP during biodegradation of 2,4-DB has already been bolic side of metabolism, it is interesting to compare the calorim-
reported [43], thus supporting this interpretation. Taking this as etry with methods, which show the other sides of the
an example the potential fate of biodegradation of other pollutants metabolism. For instance, the comparison with microscopy (mea-
can also be revealed. sure of biovolume), the counting of colony forming units (measure
of fertility), fluorescence microscopy (live/dead distinction), ATP
2.2. Data interpretations using a constant energy per converted content (energy charge of cells) has the potential to deliver mech-
electron anistic information about the metabolic target of biocides or anti-
biotics [7,50,51]. This can be applied for instance to characterize
In the most common form of calorimetry (i.e., microcalorime- toxic properties of chemicals, the action of antibiotics or of biolog-
try) but also with the newly emerging miniaturized or chip-calo- ical agents.
rimeter it is difficult to fully characterize the biological process. Even more interesting for calorimetric data interpretation are
Furthermore, for many applications of calorimetry in medicine, deviations in Q_ =r O2 from the oxycaloric equivalent. Three different
pharmacy, food industry, and environmental sciences is the real cases can be distinguished. First, if peroxides or other reactive oxy-
time detection of metabolic activity sufficient. If the calorimetric gen species (ROS) are formed, the oxycaloric equivalent is violated.
signal has to be interpreted without additional information, it is For example, Oroszi measured the photo-synthetically absorbed
a good idea to assume a constant heat production per converted heat of the diatom Phaeodactylum tricornutum and compared it
electron. If this assumption is correct, then the heat signal reflects with oxygen production at different irradiances. He observed
mainly the catabolic part of the metabolism because most elec- strong deviations from oxycaloric equivalent and assigned this to
trons are converted via electron transport phosphorylation. The photosynthetic protection mechanisms that may be associated
concept of a constant heat per converted electron goes back to with the synthesis of ROS or peroxides [52].
Thornton [44] who first described a linear relation of the combus- Second, and of more practical importance are deviations that
tion enthalpy of multiple organic compounds DCHi versus degree of are caused by either partially anaerobic metabolism or anaerobic
reductance ci (Eq. (8)). Such a relation (in the following called zones in the considered bioreactor or ecosystem. Fig. 4 illustrates
Thornton-Rule) was confirmed by many other authors [9,45–48]. the effect of anaerobiosis taking glucose as substrate, ignoring ana-
bolic reactions and assuming three different types of anaerobic cat-
DC Hi ¼ ci DC He þ di with ci ¼ 4 þ S1  S2 ð8Þ abolic reactions (A – alcoholic fermentation, B – lactic acid
fermentation, C – homoacetic acid fermentation).
DCHe, di describe the heat related with the conversion of one
Fig. 4 shows clearly that a small percentage of anaerobiosis
mole electrons and the deviation between the linear relation and
(caused by anaerobic zones in a bioreactor or ecosystem or by
the actual combustion enthalpy of the chemical compound i
mixed fermentative metabolism) has only a little influence on
respectively. ci in Eq. (8) is related to one carbon mole (C-mol) of
the Q_ =rO2 . This influence increases with increasing ratio of anaero-
the compound i having the composition of CHS1OS2NS3. For
biosis and approaches theoretically to infinity for 100% anaerobio-
instance one mole of ethanol (C2H6O) corresponds to two carbon
sis. Indeed such deviations are reported by a few publications (e.g.,
mole of ethanol (CH3O0.5). From this linear relation and depending
[17]) reaching values of 11,000 kJ/mol-O2, which have been
on the applied data base a heat per mole converted electrons DCHe
attributed to lactic acid formation under anoxic conditions by the
between (107  120) kJ e-mol1 can be derived [14,44].
authors [53].
Therefore, another exciting question can be answered. The heat
The third reason for potential deviations from the oxycaloric
Q released per mol oxygen respired r O2 is ((107  120) kJ/e-
equivalent of the total system could be deviations in
mol  4 e-mol/mol-O2 = (428  480) kJ/mol-O2). That value is

Fig. 3. Comparison of the measured reaction enthalpy of the biodegradation of the


herbicide 2,4-DB by Rhodococcus erythropolis K2–3 with enthalpy balance calcula-
tions assuming different fate of 2,4-DB. Complete oxidation (A); Incomplete Fig. 4. Influence of anaerobiosis on deviation from the oxycaloric equivalent.
oxidation (B: DCP is remaining and succinate is combusted; C: DCP is remaining 460 kJ mol1 was assumed as value for the oxycaloric equivalent. The graph
and succinate is used to produce biomass; D: DCP and succinate is remaining). The shows the percentage deviations from this value. The thermodynamic base data
data were taken from [42]. were taken from [10].
T. Maskow, S. Paufler / Methods 76 (2015) 3–10 7

Thornton-Rule for educts consumed or products formed during the Table 1


bioconversion. As an example, the ratio Q_ =r O2 provides theoreti- Limits of detection for the cell concentration as a function of the cell specific heat
production rate Q_ C . A typical Q_ D ¼ 100 nW (limit of detection of the applied
cally a real time measure for the biomass related yield coefficient calorimeter) and V = 2 mL (sample size) were assumed. Note, these values provide
YX/S (Eq. (9)). just a raw picture as they obviously depend on the respective growth conditions. Data
were taken from [1,87].
4½dS  Y X=S ðdX  X 3 dN Þ
Q_ =r O2 ¼ 4DC He þ ð9Þ Cell type Condition Q_ C Limit of detection
cS  Y X=S ðcX  3X 3 Þ
(pW cell1) (103 cells mL1)
dX, dS and dN describe the deviation of the biomass, substrate Bacteria
and ammonia as nitrogen source from the Thornton-Rule. The Escherichia coli Endogenous 0.05 1000
calculated typical dX and dS are less than 10% based on published Escherichia coli Anaerobic 0.2 250
values [10]. dN is approximately 2% taking the data of [54]. The Escherichia coli Aerobic 0.8 62
Staphylococcus Aerobic 2.5 20
total effect of these deviations on Q_ =rO2 is less than 10%. Indeed, aureus
the observed Q_ =r O2 ratio for complete aerobic growth without Mycoplasma hominis Aerobic 11 4.5
product formation vary between 385 and 495 kJ/mol-O2 [55]. Yeast
This means that for exploiting the relation in Eq. (9), the Q_ as well Schizosaccharomyces Aerobic 63 0.79
as rO2 have to be measured very accurately. However, Regestein pombe
and co-worker overcame that problem and showed in 2013 that Protozoa
deviations from the oxycaloric equivalent can be applied to moni- Tetrahymena Aerobic 3,300 0.015
tor biotechnological lysine formation in real time in a bioreactor pyriformis
[56]. Mammalian cell lines
Hepatozytena Aerobic 250 ± 6 0.2
Myocardial cells Aerobic 2,000 0.025
2.3. Data interpretations using a constant cell specific heat production
a
rate Own measurements.

of biomass concentration X with the cell specific growth rate l (Eq.


In medicine, pharmacy, food industry and water management,
(13)).
the number of microorganisms is often more important than the
exact knowledge of their metabolic activities. This is also the case Q_ ¼ rX DR HX ¼ lX DR HX ð13Þ
for many toxicological studies in environmental sciences. The sim-
plest and most applied assumption in these cases is a constant cell The comparison of Eq. (13) with Eq. (12) shows a linear relation
specific heat production rate Q_ C . We will later discuss how to get between the cell specific heat production rate Q_ C and the specific
and how to apply a more realistic estimation of Q_ C . But, related growth rate l (Eq. (14)).
to the number of cells, how sensitive can calorimetric measure- Q_ C ¼ lQ C with Q C ¼ C C DR HX ð14Þ
ments be? A modern commercially available isothermal calorime-
ter achieves a detection limit of Q_ D ¼ 100 nW (see for instance: The correlation factor QC can be understood as growth reaction
www.tainstruments.com or [4]). With Q_ C a relation between the heat (in J) per cell. For estimation of QC the knowledge of the
measured heat signal Q_ , the cell concentration X and the sample enthalpy of growth reaction DRHX and the mean carbon content
volume V can be written (Eq. (10)). (in mole) of a single cell CC is required. A simple expression for
the enthalpy change of growth reaction DRHX as function of degree
Q_ ¼ X Q_ C V ð10Þ of reductance of the substrate cS and the biomass cX (Eq. (14))
Putting Q_ D in Eq. (10) allows the calculation of the detection results from applying Eq. (14) to growth stoichiometry.
limit for the cell concentration for a typical sample volume in DR HX ¼ 115kJ=C-molðcX  Y S=X cS Þ ð15Þ
dependency on Q_ C (Table 1).
The cell concentration X in Eq. (10) is not constant. Its increase Many authors (for instance, [60–64]) recognized that the yield
can be described using the SKIP (sum kinetics with interaction coefficient YS/X depends on the energy content of the substrate.
parameter) model [57–59] (Eq. (11)). The energy content of the substrate depends on the relative degree
of reductance cS as known from the Rule of Thornton. The simplest
dX X
M
lmax;i Si relation between the relative degree of reductance cS of the sub-
¼ lX with l¼ XM ð11Þ strate and the yield coefficient YS/X (Eq. (16)) is described by [65].
dt i¼1 K S;i þ Si þ I S
j¼1 j;i j
7:69
Y S=X ¼ for cS  4:67
Here are M the number of all nutrients considered as essential, Si cS ð16Þ
the concentration of these nutrients, and KS,i, lmax,i, Ij,i growth Y S=X ¼ 1:67 for cS > 4:67
parameters. At the beginning of the calorimetric experiment all
P
nutrients are sufficiently available and Si  K s;i þ Mj¼1 I j;i Sj . This
The enthalpy change of the growth reaction DRHX can be calcu-
means, that l can be considered constant for a certain time period lated based there upon (Eq. (17)).
(Eq. (12)).
DR HX ¼ 115kJ=C-molðcX  7:69Þ for cS  4:67
ð17Þ
Q_ ¼ X 0 Q_ C V expðltÞ ð12Þ DR HX ¼ 115kJ=C-molðcX  1:67cS Þ for cS > 4:67
But during this period of time calorimetry can be applied to This means that for many naturally occurring substrates (e.g.,
determine the maximum specific heat production rate of a given carbohydrates, approx. 70% of natural amino acids, proteins, DNA
cell type in a defined medium simply by plotting the logarithm and RNA) the reaction enthalpy per C-mol formed bacterial mass
of Q_ versus time t. is independent from the applied carbon-substrate. Describing
Of course a constant cell specific heat production rate can be the elemental composition of biomass by CH1.70O0.42N0.25
called into question. Based on Eq. (6), this controversy can be over- (M = 23.99 g/C-mol) [14] approx. 440.5 kJ/C-mol results for
come by considering the definition of the growth rate rX as product carbon sources with a degree of reduction 64.67.
8 T. Maskow, S. Paufler / Methods 76 (2015) 3–10

The second unknown information in correlation factor QC is the much larger than reaction enthalpy of the conversion. However it
carbon content of a single cell CC. This carbon content depends could be argued that YEtOH/S and Y CO2 =S are not independent of each
obviously on the size of the considered cell. This value can be esti- other. They have to fulfill balances of the elements and redox
mated from the cell specific weight (in g) and the cell composition. states. Indeed, for the alcoholic fermentation is YEtOH/S = Y CO2 =S = 2
Taking Escherichia coli as a typical bacterial example a mean carbon fixed. What happens in a more complex reaction with analytical
content per cell of 15  1015 mol was estimated by this way [66]. errors which fulfill elemental and redox balances? A good example
Thereof the mean cell specific growth enthalpy for bacteria of a might be the mixed acid fermentation of glucose to acetic (AC) and
similar size as E. coli can be estimated to be 6.6 nJ/cell. With spe- butyric (BU) acid. This reaction can be conceptually separated into
cific growth rates between 0.1 and 1 h1 cell specific heat produc- two sub-reactions (Eqs. (19) and (20)).
tion rate for aerobe growing ‘‘typical’’ bacteria between 0.18 and
1.8 pW/cell can be estimated according to literature values (see
C6 H12 O6 þ YH2 0=S H2 O ! YAC=S C 2 H3 O2 þ YCO2 =S CO2
Table 1).
þ YH2 =S H2 þ YH=S Hþ ð19Þ

3. Reasons for possible misinformation


C6 H12 O6 ! YBU=S C4 H2 O þ YCO2 =S CO2 þ YH2 =S H2 þ YH=S Hþ ð20Þ
Sometimes the interpretability of the calorimetric measure-
ment reaches their limit due to peculiarities of the investigated
The yield coefficients of each sub-reaction are fixed by the bal-
biological system or by characteristics of the design of the calori-
ances of the elements and redox states to YH2 O=S = YAC/
metric experiment. Two respective examples will be discussed in
S = YCO2 =S = YH/S = 2; YH2 =S = 4 (Eq. (18)) and YBU/S = YH/S = 1;
the following.
YCO2 =S = YH2 =S = 2 (Eq. (19)). The respective reaction enthalpies
are 76.6 kJ/mol (Eq. (18)) and 57.6 kJ/mol (Eq. (19)). The advan-
3.1. Apparent discrepancies between enthalpy values measured with tage of this approach is that analytical measurement error can be
high performance calorimetry and calculated from chemical analysis understood as a shift of the ratio of the two reactions and that
thereby the elementary and redox balances are not violated.
Nowadays, high performance reaction calorimeter can measure Fig. 5 shows the influence of analytical errors (5% acetate rate)
with a high accuracy and resolution. Depending on the experimen- on the theoretical error of the reaction enthalpy.
tal setup and being able to minimize undesired heat flows (see Sec- Depending on the ratio of the formation of acetate and buty-
tion 2.1) reaction calorimetry can achieve a limit of detection of rate, a large range of enthalpies swept. Even a change from exo-
better than 5 mW/L and good long term baseline stability thermic to an endothermic reaction happens at the ratio of 0.43.
(0.2 mW/(L h)). Such performance parameters allow to monitor This switching point is a discontinuity with the consequences
even low enthalpy yielding reactions (e.g., anaerobic fermenta- that a small error in chemical analysis results in infinite error
tions) and to analyze them based on full enthalpy balances. How- in reaction enthalpy. The upper part of Fig. 5 show the error
ever, new challenges of data interpretation may arise thereof. in reaction enthalpy for experimentally observed reaction ratios
There is no doubt that biological growth processes have to fulfill [67]. Even in this range causes an error of chemical analysis of
the first law of thermodynamics. Any discrepancy between mea- 5% an error in reaction enthalpy between 7% and 20%. The cho-
sured and calculated heat production rate based on enthalpy bal- sen example demonstrates clearly that in case of anaerobic
ances should indicate some not considered metabolic events. The growth, high performance calorimetry may provide more accu-
applied metabolic model might be over-simplified. However, rate process information as the chemical reference analysis.
errors in chemical analysis of educts or metabolic products may The chosen example certainly simplifies the realities. But even
bias the result. In case of aerobic growth the substrate related if biomass formation is taken into account, for example, the
growth reaction enthalpy is often in the magnitude of the combus- main message of the error estimation changes with little respect
tion enthalpy of the substrate. For instance, the pure biological (unpublished results).
combustion of glucose delivers 2813 kJ/mol whereas growth on
glucose (assuming a typical yield coefficient of 0.4 g/g) provides
1398.1 kJ/mol. The values where calculated applying the Law of
Hess (Eq. (5)), taking the already explained typical biomass compo-
sition and estimating the energy content of biomass using the
Thornton-Rule. Assuming an error in the growth yield coefficient
of 10% (maintaining the balances of redox and elements) caused
by chemical analysis an error in growth reaction enthalpy of
141 kJ/mol or 10.1% can be calculated. This corresponds with the
general experience that the heat production rate can be well pre-
dicted after knowing the relevant conversion rates. However the
situation changes for anaerobic systems. The simplest example is
the fermentation of glucose (Gluc) to ethanol (EtOH) and carbon
dioxide (Eq. (18)).

C6 H12 O6 ! Y EtOH=S C2 H6 O þ Y CO2 =S CO2ðgÞ with Y EtOH=S ¼ Y CO2 =S ¼ 2


ð18Þ
The reaction enthalpy is 100 kJ/mol of glucose. Already small
errors in chemical analysis e.g., in EtOH production (rate) measure- Fig. 5. Influence of 5% analytical error in acetate formation rate on the error in
reaction enthalpy balance. The total reaction enthalpy as function of the ratio of
ment, would translate into relatively large errors in calculated formation rates (acetate or butyrate) is also shown. For better resolution the part of
reaction enthalpy (e.g., if the rate is 3.7% too big then DRHgluc = 0) the graph where the most experiments are located is shown magnified in the top
since combustion enthalpy values of the educts and products are right corner.
T. Maskow, S. Paufler / Methods 76 (2015) 3–10 9

3.2. Distortions of calorimetrically derived growth parameter total sequence of lab processes to perform biochemical and micro-
biological analysis. Indeed several thermal micro-sensors which
Distortions of the calorimetric signal due to overlapping physi- have the potential to be integrated into lTAS have already been
cal signals (e.g., stirring of the sample, evaporation, neutralization, developed during the past two decades [75–77]. They achieve res-
etc.) or instrumental errors (e.g., undefined heat flows, baseline olutions of a few nW and are able to manipulate samples of pL [78].
variations) can complicate the calorimetric data interpretation Even arrays of thermal LOCs are possible [79,80]. However, for bet-
but will be not discussed in the following. Here we will focus on ter data interpretation thermal LOC’s have to be combined with
distortions of the metabolic activity caused by changing physical other methods (e.g., micro-sensors for different substances or
environments. For simplicity, calorimetric measurements are often impedance spectroscopy for biomass) to get real calorimetrically
performed (semi)automatically in sealed ampoules. This measur- based lTAS.
ing technique is in the following discussed. In closed ampoules The other two promising ways to combine calorimetric signals
any concentration is expected to change over time as result of met- with elevated process analysis is performing the experiments in
abolic activity. Especially, relatively large cells (e.g., fungi, yeasts or optimized bioreactors where an energy balance is drawn around
micro algae) have the tendency to sediment and to cause heteroge- the complete reactor or inserting a calorimetric immersion probe.
neity. The latter can be avoided by adjusting the density of the The first development was already initiated 25 years ago by von
measurement solution to the density of the biological entity or Stockar [81] and led to the calorimetric monitoring of a bioreactor
by enhancing the viscosity of the measurement solution by adding in the m3 scale [82]. Recent developments achieve resolutions of
metabolically inactive additives [12]. More serious problems arise up to 20 mW/L [24] and are applicable to reactors of elevated pres-
from the oxygen consumption due to metabolic activities. In case sure [83]. However, this sensitivity is not good enough if, for exam-
of heat produced by aerobic metabolic activity above certain ple, anaerobic bioprocesses have to be analyzed or even controlled.
threshold (e.g., 100 J/L at 37 °C) oxygen diffusion and not the Thus, methods have to be developed to increase sensitivity, accu-
metabolism alone starts to govern the heat trace. The threshold racy and stability of calorimetrically monitored bioreactors. A first
is caused by oxygen solubility in the given medium and tempera- step in this direction using an improved thermal shield was just
ture and the oxycaloric equivalent [68]. Potential consequences of recently published [84].
ignoring the threshold are that important parameters like the spe- A calorimetric immersion probe was suggested for the first time
cific growth rate or the growth reaction enthalpy might be deter- in 1975 [85] and in 2012 developed on the basis of chip-calorime-
mined incorrectly [13]. Although the heat trace suggests a try [86]. The latter was sensitive enough for low cell concentrations
sequence of metabolic events (first aerobic later anaerobic) in real- but failed at high cell densities due to technical limitations. The
ity a mixed respiro-fermentative metabolism occurs. The specific development of a calorimetric immersion probes for microbial sus-
growth reaction rate and growth reaction enthalpy differ signifi- pensions of large activity range is still an open but also rewarding
cantly depending on the type of metabolism. More details are task. Numerous applications in bioprocess engineering are
given in [13]. possible.
In general, problems also arise from the growing importance of
fed-batch, continuous and perfusion cultures in biotechnology.
4. Conclusions and outlook Here, R & D work is required to reduce distortions of the calorimet-
ric signal due to convective heat flow and to mathematically model
Essentially, three types of calorimetric data interpretation the remaining effects (e.g., via multiphysics simulation of whole
(enthalpy balances, constant heat per exchanged electron, and con- calorimetric systems).
stant heat per cell) have been established during the long history of Beside the development of calorimetric instruments which pro-
biocalorimetric research. What is applied at the end depends on vide more data the developments of mathematical models that
the scientific question but also on the available non-calorimetric actually process those data is rewarding. For applications of calo-
data. Beyond the formal modeling or hypothesis testing task of rimetry in medicine or food industries which often trust on mea-
calorimetric experiments, exploratory data analysis should be surement of simple closed ampoules an automated parameter fit
developed to extract more information from the data. As a possible would be helpful. These parameters would provide the number
example, calorimetric data of contaminated sites could be related of bacterial cells at the beginning of the experiment and their
to soil microbiome data to better understand the relation between growth kinetics depending on biocide or antibiotic concentration,
degradation rate, degree of mineralization and the chemical prop- physical parameters etc. First steps of such developments are
erties of the pollutants, habitat characteristics and microbiome recently published [11] and deserve further development.
composition. As statistical tools factor analysis (FA) principle com-
partment analysis (PCA), correspondence analysis (CA), and cluster
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