Determination of Biomass Concentration by Capacitance Measurement

INTRODUCTION
Lack of development of measuring devices for application in fermentation and
biochemical processing has hindered proper and optimal control of such processes
t o achieve the maximum yield of products. This was enunciated recently at a workshop sponsored by the National Science Foundation of Biochemical Engineering
Fundamentals. One of the most important variables in biochemical processing involving microorganisms is the cell concentration. The concentration of microorganisms is related to substrate consumption rate and product formulation rate in several
fermentations. Consequently, cell concentration is an important state variable of the
system. In a control situation, availability of cell concentration data instantaneously
and continuously would enable closer monitoring and better operation of fermentation systems.
LITERATURE BACKGROUND

In general, methods available for cell measurement may be classified into direct
and indirect methods. Cell concentration is usually reported in mass of dry cells per
unit of volume of broth. The direct methods are determination of dry weight, wet
weight, and packed cell volume. All the three procedures involve filtering or centrifugation of the sample withdrawn from the fermentor. These procedures require a
great deal of time and are tedious. The most commonly used direct method, dry
weight, requires drying, which involves a significant amount of time. These procedures become even more difficult when suspended solids are present in the fermentor. The solids would have t o be dissolved or removed preferentially before any of
the direct methods can be employed.
Continuous cell concentration measurement is possible by measuring a system
property related to cell concentrations, such a s viscosity,*
fluorescence,8
and data based on yield of ATP.g Continuous turbidity measurement is done by
using flow cells. Its applicability is limited to nonmycelial, solid-free colorless broths.
Furthermore, turbidity measurement is limited to dilute systems. Another limitation
is that turbidity is a function of specific growth rate a s was pointed out by PirtO for
the organism Aerobactc,r chacac.. Hence, for proper evaluation of turbidity data,
specific growth data are needed. Consequently, the turbidity measurement is less
reliable. Formation of dirt and deposits on the flow cell walls will cause drift and
erroneous measurement. However, turbidity measurement enjoys wide industrial
applications. The chief disadvantage is that the method totally fails when suspended
solids are present in the fermentation broth.
In systems that contain suspended solids, the cell concentration is measured by
analyzing a component present in the cell that is not present in the suspended solids.
The usual components chosen for analysis are nitrogen, carbon, RNA, DNA, and
phosphorus. It is important that these components are present in a fixed quantity in
the cell. It is well known that the nitrogen content of Saccharomycrs cerevisiae
growing on ethanol varies with specific growth rate and that the RNA and DNA
content ofArrobacrer urrogcnos varies with population growth rate.* Consequently,
Biotechnology and Bioengineering, Vol. XXI, Pp. 1097-1 103 (1979)
0006-3592/79/0021-1097$01.OO

@ 1979 John Wiley & Sons, Inc.

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the assumption of constant cell composition may be applicable only under special
conditions.
Recently Harimas developed a method based on the relationship between specific
growth rate and specific ATP production rate (in mol ATP/g dry cell/hr). The basis
of this approach is that the dry weight of cells is proportional to the yield of ATP in
several anaerobic microorganisms. l3 Furthermore, the knowledge of specific carbon
dioxide production rates and specific oxygen consumption rates are needed. Also,
the initial value of cell concentration is needed for reliable estimates of cell concentrations. For systems in which not only carbon dioxide is produced. but hydrogen,
methane, and other gases are released, the approach of Harima will not be suitable
and some modification to take into account the above-mentioned complexities would
be needed.
The particular approach employed in this investigation for cell concentration is
based o n electrical impedance of fermentation broths. The idea that the impedance
change of a biological system is related to the biological activity is well known.
NASA has supported research activity in this area for biological experiments on the
Moon and on Mars. Several a ~ t h o r s ' ~have
- ~ ~reported that impedance changes of
microbial cultures provide a means of detecting biological activity of microorganisms.
CadyI6 reported that electrical impedance changes provide a means of detecting
microorganisms. He showed experimentally the features of the classical bacterial
batch growth curve can be identified by the measurement of impedance. Based on
the postulate that the impedance of the cell can be represented by a capacitor and
a resistor in series. CadyI6 reported that the capacitance component of impedance
change of an Eschrrichia coli culture was far greater than the resistance component.
It was suggested that the marked contribution of capacitance to the impedance
change and the high values of the capacitance obtained were not only due to changes
in dielectric constant of the medium but also due to deposition of a charged layer or
a microbial film on the electrode surface. Both of these phenomena increase the
capacitance between the electrodes. Hubert'? obtained results similar to those reported by earlier worker^'^,'^ with a stainless-steel electrode. Hubert presented
photographic evidence of the adsorption of E . co(i on the electrode surface and
attempted to quantify the capacitance changes due to changes in cell concentration.
Principles and techniques for the measurement of electrical impedances of biological materials such as tissue were reviewed by Schwan.'8 One of the major sources
of error in determinations of biological impedances is brought about by polarization,
which occurs at the boundary between the medium and the electrode. When a
metallic electrode is in contact with a solution or cellular suspension, a dc-boundary
potential is usually found to exist at the electrode-solution interface. At low current
densities, the polarization impedance is independent of the current. Platinum electrodes have the least polarization impedance. Furthermore, a porous coat of platinum
black when applied to a platinum electrode further reduces the polarization impedance by four orders of magnitude. The disadvantages of "platinum black" electrodes
are that they deteriorate with use.
Polarization impedance of the electrodes is usually considered in series with
biological impedance. 17m Procedures used by investigators in attempting to describe
impedance changes of bacterial cultures did not attempt to measure the polarization
impedance to obtain a measure of the impedance changes due to the culture only.
Impedance data reported by Cady,I6 Wheeler and G o l d ~ c h m i d t , Ur
' ~ and Brown,"
and others measured the total impedance of the culture.
It should be emphasized that previous work on impedance measurement has been

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Fig. I . Electrical circuit of the measurement system. A, function generator; B ,

decade resistor: C, inner electrodes; D. outer electrodes; E , rotary switch; F, digital
multimeter.

53
0

1 2 1 6
TIN,

2 0 2 4

~ 1 s

Fig. 2 . Impedance change of yeast culture growing on glucose. Temperature

31C; i = 25 pA;f = 10,000 Hz.

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BIOTECHNOLOGY AND BIOENGINEERING VOL. XXI (1979)

done to identify the qualitative relationship between impedance changes of microbial

cultures and the changes in cell concentration. Here. our prime motive is to develop
the available qualitative relationship into a quantitative one. with the idea being to
use such a method of measurement as a means of monitoring the cellular concentration in industrial fermentors.
EXPERIMENTAL METHOD
A sketch of the electrical circuit used in our experiments is given in Figure I . A
four-prong all-platinum electrode system was used in a modified fermentor vessel
(0.5 liter working volume) equipped with temperature and pH measurements. Samples of the cellular suspensions were taken using a hyperdermic syringe for determination of actual dry cell concentrations. Dry cell concentrations were determined
by centrifugation, washing, and then drying at I05"C until no change in weight
condition was observed. The organism used was Fleishmann's yeast obtained locally.
The medium consisted of pure glucose dissolved in distilled water. Constant current.
which was obtained by maintaining the potential i ' , (see Fig. 2) constant. was used.
The potential across the electrodes was measured from time to time in the frequency
range I to 20 kC. Since no potential is applied across the inner electrodes. the
measurement of the potential. between the inner electrodes I ' ~ .is independent of the
polarization impedance. The aeration was stopped when potential drop across the
electrodes was recorded. The reason for this procedure being that the presence of
air bubbles results in large fluctuations in the voltmeter reading. In industrial applications. impedance electrodes may be placed in a sampling line to prevent air bubbles
from interfering with the measurement.
RESULTS
Results of a typical run are given in Figure 2 in which it is noted that changes in
impedance is related to cell concentration changes. In the experiment shown in
Figure 2. the rate of growth of yeast was slow because nitrogen source and salts
were absent. The yeast was growing on the reserves. We note that for a less than
twofold increase in cell concentration, there is a 50% decrease in electrical impedance. Experiments were repeated to establish reproducibility of the results. The
resistance and capacitance parts of the impedance were calculated assuming that the
equivalent circuit may be represented by a series resistance capacitance circuit as
shown in Figure 3. The relationships for the resistance and capacitance are given by

where R , is the external resistance, 2, is the impedance across the outer electrodes.
and .f' is the frequency. Experimental measurements show a decrease in resistance
and an increase in capacitance with increasing time. In other words the capacitance

---1t-y-ikC

Fig. 3.

Equivalent circuit.

C O M M U N I C A T I O N S TO T H E EDITOR

1101

o f the culture was found to increase with increasing number o f yeast cells. Hanai et
a1.20have shown that the limiting value o f dielectric constant o f a biological medium
i s strongly dependent on the c e l l volume fraction. They also showed that the dielectric constant o f a medium containing yeast cells i s much larger than that of the
suspending medium at low frequencies. Thus the capacitance o f medium between
the electrodes i s expected to change depending on the number o f yeast cells. Each
yeast cell acts as a capacitor with a low capacitance value.
I n Figures 4 and 5 the variation o f capacitance per unit surface area of the electrode
i s given as a function o f dry cell concentration. The slope o f the line relates to the
sensitivity of the measurement. I n the range o f concentrations studied, the slope of
er,
i s reproducible
the lines in Figures 4 and 5 are about 6.67 x 1 0 - 3 ~ F I ~ m 2 / g l l i t which
within 10% error. However. the experimental data show a fair amount o f dispersion.
which may be due to nonreproducible hydrodynamic conditions at the electrodefluid interface. I t i s belie~ed'~.''
that the primary factor affecting impedance changes
i s due to the surface adsorbed microbes. I t seems plausible that the surface concentration o f adsorbed cells bear a constant relationship with the concentration o f cells
i n the fermentor bulk. provided that the hydrodynamic conditions are constant.
Sparging o f air. which i s needed for the respiration o f microbes. may cause a nonsteady-state condition at the electrode-broth interface region. Cady16has shown that
the rate of desorption i s a slow process. A constant hydrodynamic condition near
the electrode-broth interface can be experimentally obtained in a flow system. where
a sample i s drawn continuously through a pipe that contains the electrodes for
measurement of impedance. The flow in the sampling line should be laminar.
A decrease in resistance or an increase i n conductivity i s also noted from the
experimental results. The increase i n conductivity may be due to leakage o f ionizable
chemicals from the interior o f cells to the suspending medium. Also. yeast cells are
known to behave as conductive particles and an increase in the overall conductivity
o f the medium i s due to an increase i n the number o f yeast cells.
When ionizable salt nutrients were included in the medium. impedance measurements discussed earlier give a constant value for capacitance in the range o f 17 to

2
z

10
i.5

1.6

1.7

1.8

1.9

hY

2.0

2.1

hX"ATlctc,

2.2

2.3

2.4

2.5

&LITER

Fig. 4. Relationship between dry cell concentration and capacitance. Temperature = 31C: i = 25 p A : f ' = 10.000 Hz.

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BIOTECHNOLOGY A N D BIOENGINEERING VOL. XXI (1979)

10
1.5

1.6

1.7

13

1.9

2.0

2.1

2.2

2.3

2.4

29C: i

DRY h L b X W T l W &LITER

Fig. 5. Capacitance vs. yeast concentration. Temperature

10,000 Hz.

25 pA:f

18 pF/cm*, which was independent of cell concentration. This is because the medium
is very conductive and the capacitance measured is essentially the double-layer
capacitance. Experiments were carried out to obtain cell constants using 0. IN KCI
solution and 0.01N KCI solution. Using the cell constants so obtained. the conductance of the fermentation medium was calculated and found to be in the range of 0.51
to 0.61 O-/cm. In the experiments where large amounts of salts are present, the
double-layer capacitance becomes significant in comparison with the solution capacitance. Therefore the measurement of capacitance due to the microbial cells
becomes difficult. We are currently developing an electrode system that can measure
the capacitance of microbial cultures in a medium that contains ionizable salts.

CONCLUSIONS
We have described a cell concentration measurement technique based on capacitance measurement. that gives reliable and reproducible results when conductance
of the fermentation broth is low. However, when the conductivity of the suspending
medium is high or when ionizable salts are present, the method fails to detect
capacitance changes corresponding t o changes in yeast cell concentration.
References
1 . A . E. Humphrey, Verbal summary of session chairman on fundamentals in
bio-instrumentation and control, Workshop on the Fundamentals of Biochemical
Engineering Sponsored by NSF, University of Maryland, December 12- 14, 1977.
2. B. W. Shimmons, W. Y. Svreck. and J . E. Zajic, BiotcJc,hno/.BiocJng.. 18,
1793 (1976).
3. G. G . Meynell and E. Meynell. Theory and Pruc,tic.iJin Expc,rimc,nta/ Bacteriology (Cambridge U. P.. London, 1970).
4. P. J . Wyatt, Differential light scattering techniques, in Mothods i n Microbiology (Academic, New York, 1973), Vol. 2.
5 . P. J . Wyatt, Automation of differential light scattering for antibiotic suscep-

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tibility testing." in Autofnation in Microbiology uiid Immunology (Wiley, New York,

1975).
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~
New York, 1975).
8. J . Fazel-Madjlessi, D. Sincic, and J . E. Bailey, "Multiparameter observations
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10. S . J . Pirt. Proc. R . Soc. Lorid., Scr. B , 163, 224 (1965).
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.
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16. P. Cady. "Rapid automated identification by impedance measurements," in
Nrrt, Approuclws t o t h e IdrtrtiJicutio,i ~ fM' i c r 0 [ ~ r g u i i i s (Wiley,
~s
New York, 1975),
pp. 73-99.
17. 0. F. Hubert. "Detection of bacterial growth by electrochemical method,"
M. S. thesis, University of Pennsylvania, 1976.
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ch
New York, 1963).
19. H. P. Schwan, Z . No/rrrfi,rsc~/7..66, 121 (1951).
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MEHMETA. GENCER
RAJAKKANNU
MUTHARASAN
Department of Chemical Engineering
Drexel University
Philadelphia, Pennsylvania 19104
Accepted for Publication November 19, 1978