Journal of Bioscience and Bioengineering
VOL. 122 No. 3, 364e369, 2016www.elsevier.com/locate/jbiosc

Calorimetric studies of the growth of anaerobic microbes
Hideo Miyake,1, 2, * Yukiko Maeda,3 Takashi Ishikawa,1, x and Akiyoshi Tanaka1
Graduate School of Bioresources, Mie University, 1577 Kurimamachiya-cho, Tsu, Mie 514-8507, Japan,1 Life Science Research Center, Mie University, 1577 Kurimamachiya-cho, Tsu,
Mie 514-8507, Japan,2 and Faculty of Bioresources, Mie University, 1577 Kurimamachiya-cho, Tsu, Mie 514-8507, Japan3
Received 2 November 2015; accepted 12 February 2016
Available online 21 March 2016

This article aims to validate the use of calorimetry to measure the growth of anaerobic microbes. It has been difﬁcult
to monitor the growth of strict anaerobes while maintaining optimal growth conditions. Traditionally, optical density
and ATP concentration are usually used as measures of the growth of anaerobic microbes. However, to take thesemeasurements it is necessary to extract an aliquot of the culture, which can be difﬁcult while maintaining anaerobic
conditions. In this study, calorimetry was used to continuously and nondestructively measure the heat generated by thegrowth of anaerobic microbes as a function of time. Clostridium acetobutylicum, Clostridium beijerinckii, and Clostridium
cellulovorans were used as representative anaerobic microbes. Using a multiplex isothermal calorimeter, we observedthat peak time (tp) of C. acetobutylicum heat evolution increased as the inoculation rate decreased. This strong correlation between the inoculation rate and tp showed that it was possible to measure the growth rate of anaerobic microbes
by calorimetry. Overall, our results showed that there is a very good correlation between heat evolution and opticaldensity/ATP concentration, validating the use of the method.
Ó 2016, The Society for Biotechnology, Japan. All rights reserved.
[Key words: Calorimetry; Clostridium acetobutylicum; Clostridium beijerinckii; Clostridium cellulovorans; Anaerobic microbes; Growth processes]

It is necessary to understand the growth process of a microorganism to evaluate its activity. A growth curve is often generated
from measurements of the density of microbial cells at arbitrary
time points throughout growth, which are classiﬁed into lag phase,log phase, stationary phase, and death phase. The pattern of thegrowth process is basically conserved between different microbial
species, but the time spent in lag phase and the slope of the curve
during log phase growth differ depending on the type and numberof microorganisms and the growth environment. This information
is extremely important for the management of fermentation,
sampling inspection of microbial contamination of food and theenvironment, and for screening for useful microorganisms. Measurement of the optical density (OD) of a culture is commonly used
to assess the growth phase, and reﬂects the number of microorganisms in the culture at a given time (1). Measurement of integrated ATP concentration can also be used to assess the growth ofsome microorganisms (2). However, when a microorganism aggregates in a bioﬁlm, it is difﬁcult to measure growth. This isparticularly true for OD measurement, as microbial cells must be
uniformly dispersed in the culture medium to obtain an accurate
OD reading. In contrast, ATP can be measured with good sensitivity
from aggregated cells, but a sample of the culture medium muststill be removed for each measurement. However, because ATP ispresent not only in the microbial cell but also in components of the* Corresponding author at: Graduate School of Bioresources, Mie University, 1577
Kurimamachiya-cho, Tsu, Mie 514-8507, Japan. Tel./fax: þ81 59 231 9611.
E-mail address: miyake@bio.mie-u.ac.jp (H. Miyake).
x
Present address: National Research Institute of Aquaculture, Fisheries Research
Agency, Tsuiura, Kamiura, Saiki, Oita 879-2602, Japan.

culture medium, assessment of growth using ATP measurement iscomplicated and requires removal of the ATP from culture medium
components. Both OD and ATP measurements are thus laborious
work.
During the growth of microbial cells, heat is generated at around
1e100 pW per cell (3e6). Microcalorimetry can reliably measureheat evolution at values of approximately 1 mW. During microbial
cell culture, it is possible to measure the growth process bydetecting variations in heat evolution. A multiplex isothermal
calorimeter (7) can detect heat evolution of samples from differences in temperature between each sample cell and a reference cellby monitoring the thermoelectromotive force of each thermopile(7) (Fig. 1). The thermograms generated by the calorimetric measurement indicate time-dependent changes in heat evolution ofeach sample, and are independently obtained by outputting thedata from an acquisition unit. Using this method, it is not necessary
to remove samples from the culture as calorimetry can continuously and nondestructively measure heat evolution of the culture.
Previous studies have used a multiplex isothermal calorimeter tomeasure the growth of yeast under aerobic conditions and determine the effects of inhibitory substances on growth (8,9). The
growth of Escherichia coli under anaerobic conditions has also been
examined using this method (10). However, there is no research
into whether this method can be used to measure the growth ofobligate anaerobes quantitatively.
Obligate anaerobes cannot grow in the presence of oxygen.
When measuring the growth of these organisms, it is necessary tomaintain strict anaerobic conditions even when collecting samples
for measurement. This is often very difﬁcult to achieve and requires
an anaerobic chamber. Therefore, in the current study we investigated the use of calorimetry to measure the heat evolution of1389-1723/$ e see front matter Ó 2016, The Society for Biotechnology, Japan. All rights reserved.
http://dx.doi.org/10.1016/j.jbiosc.2016.02.006

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(B)

FIG. 1. Photograph of sample unit in the multiplex isothermal calorimeter (A) and the cross-sectional drawing of it (B). Each vial was placed on the thermopile and incubated in thesample unit. The temperature was maintained using a circulating water bath with a temperature control of 0.1 C.

obligate anaerobes and butanol-producing Clostridium strains
Clostridium acetobutylicum and C. beijerinckii, which are important
industrial microbes (11e13). The aggregating and cellulosomeproducing species Clostridium cellulovorans was also used
(14e16). Using this method we attempted to determine the growth
processes of each strain by measuring heat evolution by
calorimetry.
MATERIALS AND METHODS
Determination of calorimetric parameters
A multiplex isothermal calorimeter with 20 cells was used for calorimetry (7,17). A schematic diagram is shownin Fig. 1. Both sample cells and reference cells were included in the sampling unit ofthe device. The temperature was maintained using a circulating water bath with atemperature control of 0.1 C. The change in voltage corresponds to the heat
evolution. Eq. 1 was used to correct the heat loss to determine the heat evolution
q(t) assuming ideal adiabatic conditions (18,19),RqðtÞ ¼ gðtÞ þ K gðtÞdt

(1)

where g(t) is a raw data thermogram, which was obtained by subtracting the heat
evolution value of the reference from that of the sample. The thermal conductivity
constant (K) value of each cell was determined using the method described by
Takahashi (7).
The calorimetric parameters of the growth process were calculated using the
Richards model equation, which is a model of the growth curve (17,20) (Eq. 2).



 1
qðtÞ ¼ Q 1 þ ðd  1Þexp  B t  tp 1d(2)

where Q is the total amount of heat evolution, tp is the time taken to reach peak heat
evolution, d is a dimensionless adjustable parameter of the growth curve which
determines the asymmetry of the differential form q0 (t) of Eq. 2 with respect to tp(17), and B is a rate parameter (17). When t is inﬁnite, q becomes equal to Q. The
maximum heat evolution rate Vmax can be described using Q, B, and d by thefollowing equation:dVmax ¼ QBd1d(3)

The growth process of each anaerobic microbe was evaluated using these parameters on the basis of the differential form q0 (t) of Eq. 2 (17).
Bacterial strains and culture media
C. acetobutylicum (NBRC 13948) andC. beijerinckii (NCIMB 8052), representative butanol-producing strains, andC. cellulovorans 743B (ATCC 35296), a cellulosome-producing strain, were used.
Cultures were grown in 50-mL vials (Maruemu Co., Osaka, Japan), while precultures and dilutions were carried out in 16  125-mm Hungate tubes(Chemglass Life Sciences LLC, Vineland, NJ, USA). Thioglycollate medium was used
to culture C. acetobutylicum and C. beijerinckii. The medium contained per liter:
15 g hipolypeptone (Wako Pure Chemical Industries Ltd., Osaka, Japan), 5 g dried
yeast extract, 2.5 g NaCl, 0.5 g sodium thioglycolate, 0.5 g L-cysteine$HCl$H2O, and1 mg resazurin as an oxygen indicator. The culture medium was adjusted to pH

7.0. A 20% (w/v) glucose solution was prepared separately and added to themedium after autoclaving. Both the culture medium and the glucose solution
were ﬂushed with CO2 gas for 20 min prior to autoclaving (40 min at 121 C). The
culture medium for C. cellulovorans was prepared as described by Sleat et al. (14).
Cellobiose (5 g/L) was added to the culture medium. When OD or ATP
measurements were performed, a resazurin-minus culture medium was used.
Inoculation of culture media
A 1-mL aliquot of pre-culture was inoculated
into vials containing 20 mL of culture medium for use in calorimetry and OD and ATP
assays. To vary the inoculation rate, pre-culture was diluted 10e108-fold in sterileculture medium in an anaerobic chamber. A 1-mL aliquot of each dilution wasthen individually inoculated into a vial containing 20 mL of culture medium
containing 0.5% (w/v) glucose and used as a sample for calorimetry. The samples
were immediately placed within the sample unit of the multiplex isothermal
calorimeter, and the heat evolution of each sample was measured until noevolution was observed at 35 C. Uninoculated culture medium was used as areference.
When carrying out calorimetry and OD measurements of samples with varying
glucose concentrations, a one-way valve was mounted in the butyl rubber stopper ofthe vial. Thus, gas generated by microbial metabolism was released from the culture
medium.
Measurement of optical density and ATP concentration
As it is notpossible to remove samples from the culture medium during calorimetry measurements, duplicate cultures were prepared in an incubator maintained at the same
temperature as the calorimetry assay. ODs and ATP concentrations were determinedby sampling these cultures across the growth period. The OD of each sample wasmeasured at 590 nm using a WPA CO7500 colorimeter (Biochrom Ltd., Cambridge,
UK). The ODs of samples with varying glucose concentrations were measured at thecompletion of the calorimetry. For ATP measurement, 10 mL of ATP eliminating reagent (Kikkoman Biochemifa Co., Tokyo, Japan) were added to 100 mL of culture
solution and incubated for 30 min to eliminate extracellular ATP. The intracellular
ATP concentration was then determined using a LuciPac Pen (Kikkoman Biochemifa
Co.) and Lumitester PD-20 (Kikkoman Biochemifa Co.). The integrated ATPconcentration was determined by approximating the integral value using the
integration function of Origin software (OriginLab Co., Northampton, MA, USA).

RESULTS
Previous calorimetric studies using yeast under aerobic conditions have shown that the time taken to reach logarithmic growthphase and the time until peak growth rate change depends on the
inoculation rate (8). Therefore, we ﬁrst examined the effects of the
inoculation rate on the growth rate of the anaerobic microbes using
a multiplex isothermal calorimeter. The thermograms and calorimetric parameters for various inoculation rates of C. acetobutylicum
are shown in Fig. 2 and Table 1, respectively. Fig. 2C shows anexample of comparison of the observed q0 (t) values obtained on thebasis of Eq. 1 with the theoretical q0 (t) values obtained using the366

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(B)

(C)

FIG. 2. The growth thermograms generated for cultures with various inoculation rates. Each sample was used the medium containing 0.5% glucose. The inoculation rate indicates
dilution rate of pre-culture (diluted 10e100,000,000-fold and undiluted). (A) Thermogram for C. acetobutylicum. (B) Plot of tp versus the converted log2 value from the inoculation
rate. (C) Comparison of the observed (closed circles) and the theoretical (solid line) values of q0 (t) of the undiluted experiment in Fig. 2A. The former values were obtained from g(t)using Eq. 1, and by numerical differentiation with respect to the time. The solid line is drawn using the values listed in Table 1 and the differential form of Eq. 2 (17).

values listed in Table 1. Good agreement between these values indicates that the evaluated values of the parameters are reliable. The
tp (time taken to reach peak heat evolution) for this bacterium
changed between 22 and 49 h. When the inoculation rate wassmall, the value of tp increased, indicating a highly negative correlation between the inoculation rate and tp (Fig. 2B). Conversely,
no signiﬁcant change in the Vmax and Q values were observed when
the inoculation rate was altered (Table 1). No heat evolution wasobserved at dilutions of the inoculum of greater than 106-fold.
The heat evolution recorded by the multiplex isothermal calorimeter was then analyzed to determine whether it reﬂected thegrowth processes of C. acetobutylicum and C. beijerinckii. The thermograms generated for each of the strains were compared with the

TABLE 1. Summary of calorimetric parameters of the growth of C. acetobutylicum
when cultured using varying inoculation rates.
The inoculation ratea

tp/h

Vmax/mV h1

Q/mV

B/h1

d

1.0 (undiluted)
1  101
1  102
1  103
1  104
1  105

21.9
26.7
31.8
39.2
42.0
48.5

0.480
0.486
0.510
0.505
0.468
0.525

2.43
2.55
2.64
2.79
2.60
2.75

0.98
0.80
0.82
0.85
0.82
0.90

2.80
2.17
2.21
2.60
2.48
2.61

a

Inoculation rate indicates dilution of pre-culture.

growth curves obtained by OD measurements under the same
conditions (Fig. 3A and B, respectively). C. acetobutylicum enteredlogarithmic phase after 18 h, and reached stationary phase 40 hlater. C. beijerinckii reached logarithmic phase at 10 h, and reachedstationary phase 30 h later. The thermograms of both strains werehighly reproducible, with the time taken to shift from lag phase tolog phase and the growth rate in log phase being almost identicalacross the replicates. It is thus evident that the curves generated by
the thermograms corresponded with OD measurements.
When C. cellulovorans is cultured in medium containing a soluble carbon source, the cells aggregate (14). Therefore, the thermogram generated for C. cellulovorans was compared with thegrowth curve generated from integrated ATP concentration measurements instead of OD. Heat evolution by this strain wasobserved after 10 h, and lasted for 40 h (Fig. 3C). Again, the q(t)thermograms and the integrated ATP concentration growth curves
corresponded based on time.
Butanol-producing Clostridium strains grow by metabolizing acarbon source such as glucose (13). By varying the glucose concentration in the culture medium, the cell concentration at stationary phase also changes. Therefore, the growth of C. beijerinckii
was analyzed under different glucose concentrations by calorimetry to determine whether there were observable changes in thethermograms and the calorimetric parameters (Fig. 4A and B).

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FIG. 3. Time courses of heat evolution by C. acetobutylicum (A), C. beijerinckii (B), and C. cellulovorans (C). C. acetobutylicum (A) and C. beijerinckii (B) were grown with 5% glucose.
C. cellulovorans (C) was grown with 0.5% cellobiose. The q(t) of each strain was calculated from g(t) obtained using the multiplex isothermal calorimeter (line). Circles and squares
indicate optical density and integrated ATP concentration, respectively.

When the glucose concentration in the culture medium wasincreased, heat evolution was detected for a longer period. No
signiﬁcant differences in the peak heat evolution were observed for
glucose concentrations of >0.1%. The calorimetric parameters andOD measurements for the glucose concentration assays are shownin Fig. 5. Q, tp, and OD all increased when the glucose concentration
was increased. Vmax remained similar at glucose concentrations of
0.1% or greater.
DISCUSSION
Calorimetric analysis of the growth of the obligate anaerobe
C. acetobutylicum showed that the tp increased as the inoculation
rate decreased (Fig. 2A, Table 1), indicating a strong correlation
between the inoculation rate and tp (Fig. 2B). Similar results havebeen observed for yeast under aerobic conditions (8). Therefore, thetp value accurately reﬂects the characteristics of microbial growthfor both aerobic and anaerobic microbes. Additionally, the slope ofthe relationship between the log2 of the inoculation rate and tp is ameasure of the doubling time, which in this case was 1.67 h(Fig. 2B). When q1 at time t1 reaches q2 at t2, the heat evolution rate(v) is expressed by the equation v ¼ lnðq2 =q1 Þ=ðt2  t1 Þ. Here,when the heat evolution is assumed to correspond to the concentration of microbial cells, the heat evolution should double when
the number of microbial cells doubles; that is q2/q1 ¼ 2. Doublingtime (td) of microbial cells at the maximum growth rate (Vmax) isrepresented by td ¼ ln 2=Vmax. The average doubling time calculated from Vmax in Table 1 was 1.4 h, indicating that Vmax determined by calorimetry is a reliable parameter. These results also
conﬁrm that it is possible to determine the growth rate of obligate
anaerobes using calorimetry in the same way as aerobic bacteria,
and that tp and Vmax are useful parameters for characterizing the
growth rate.
The current study also showed that there is a good correlation
between the q(t) thermograms and growth curves generated from
OD measurements in C. acetobutylicum and C. beijerinckii (Fig. 3A
and B). Additionally, there was good correlation between the q(t)thermogram of C. cellulovorans and the growth curve generated
from integrated ATP concentration analysis (Fig. 3C). Therefore,
we concluded that the heat evolution of these strains obtainedby calorimetry reﬂects the number of anaerobic microbial cells
present in a culture at a given time point. Using a multiplex
isothermal calorimeter, it is possible to continuously measure heat
evolution while maintaining anaerobic conditions. Furthermore,

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FIG. 4. Growth thermograms (A, B) of C. beijerinckii generated for cultures with various glucose concentrations. C. beijerinckii was grown with between 0% and 0.75% glucose. (A)From the bottom, 0%, 0.05%, 0.10%, 0.15%, 0.20%, 0.25%, 0.3% and 0.35% glucose. (B) From the bottom, 0.40%, 0.45%, 0.50%, 0.55%, 0.60%, 0.65%, 0.70% and 0.75% glucose. (C) Plot of Q
versus OD.

the growth process of anaerobic microbes can still be measured
using this method even if the cells are aggregated. Additionally, thismethod is effective in the presence of insoluble sugars and biomass
given that calorimetry is not affected by the OD of the sample. This
technique may therefore be particularly useful in industrial
applications.
When C. beijerinckii was cultured with various glucose concentrations, OD was measured at the stationary phase, which was thencompared with total heat evolution (Q) (Fig. 4C). A relatively highcorrelation was observed between OD and Q, suggesting that thevalue of Q obtained by calorimetry reﬂects the cell density in theculture. However, when the OD increased, that is, when the glucose
concentration was high, the correlation weakened. It is likely thatheat evolution from the metabolic products contributed to an
overestimation of the actual value of Q. In future studies, the heat
evolution from the metabolic products should be considered so as
to improve the sensitivity of the thermopile and to accurately
perform calorimetry under high glucose concentrations.
The value of tp increased in accordance with increasing glucose
concentrations (Fig. 5A). Although the initiation time of the heat
evolution was approximately the same based on the shape of thethermograms (Fig. 4A and B), we inferred that this was because theheat evolution time became longer in accordance with increasing

glucose concentration. There was no signiﬁcant difference in Vmax
under any of the conditions except at glucose concentrations of 0%
and 0.05% (Fig. 5B). As described above, Vmax corresponds to the
doubling time as td ¼ ln 2=Vmax . When there is no carbon source,
or a very low concentration of the carbon source in the culture
medium, microbial growth is minimal. However, when there are
sufﬁcient amounts of carbon in the culture medium, regardless ofthe microbial cell density, the doubling time is approximately
constant.
Calorimetry using the multiplex isothermal calorimeter can
automatically collect data without the need to remove samples
from the culture medium. Therefore, it is possible to accurately andsimply observe the growth of anaerobic microbes while maintaining anaerobic conditions. While the OD cannot be accurately
measured when cells are aggregated, calorimetry is a promising
method of measuring microbial growth even when cells areaggregated. This study also showed that tp, Vmax, and Q are usefulparameters for characterizing the growth rate and growth processes of microbes, even under anaerobic growth conditions. Thismethod may be useful for high-throughput measurements of thegrowth of novel biotechnological strains, and to screen and breed
mutant strains for tolerance to inhibitors such as alcohol andsolvents.

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FIG. 5. Plot of calorimetric parameters and OD of C. beijerinckii versus glucose concentration.

ACKNOWLEDGMENTS
This research was supported in part by a grant from the Programfor Promotion of Basic and Applied Researches for Innovations in
Bio-oriented Industry, and the Science and Technology Research
Promotion Program for Agriculture, Forestry, Fisheries, and Food
Industry.

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