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Cost Optical Sensor for Online Measurement of Biomass.

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Dev. Chem. Eng. Mineral Process. 13(1/2), pp. 63-70, 200.5.
Cost Optical Sensor for Online
Measurement of Biomass
M. He’, X.M. Li2, G.F. Qin’, S.K. Nguang’*
and X.D. Chen’
’Department of Electrical and Electronic Engineering
Department of Chemical and Materials Engineering
University of Auckland, Private Bag 92019, Auckland, New Zealand
~~
~~~
~
A low cost optical on-line biomass sensing system for fermentation processes is
presented in this paper. The calibration curve of the sensor obtained by using cell
cultures in tap water revealed a linear profile in the range 0.1-4.0 g/L. The sensor
was shown to have a fast response time and was insensitive to the natural light. A
good reproducibility was obtained between the optical sensor and cell dry-weight
method values.
Introduction
Modem bioprocesses are the most complex of all the fields of process engineering
due to their high dimension, non-linearity and dynamical characteristics. The ability
to control bioprocesses at their optimal states accurately and automatically is of
considerable interest to many fermentation industries. Thls enables them to reduce
their production costs and increase the yield, while at the same time maintaining the
quality of the metabolic product. However, it should be noted that designing a
control system for bioprocesses is not a straightforward task due to: (i) significant
model uncertainty; (ii) lack of reliable on-line sensors which can accurately detect
the important state variables; (iii) non-linear and time varying nature of the system;
and (iv) slow response of the process, in particular for cell and metabolic
concentrations. This work is mainly focused on the second problem. Over several
years, the key state variables (such as the concentration of biomass) in a
fermentation process are commonly determined using off-line laboratory assays
with a long measurement delay [I]. This limits the range of control algorithms that
can be applied to the process.
* Author for correspondence (sk.nguang@auckland.ac.nz).
63
M.He, X.M. Li. G.F. Qin, S.K. Nguang and X.D.Chen
Optical measuring technology is widely used not only in remote control and
microelectronic technology but also in biotechnology. Most of the online measuring
techniques for cell densities and concentrations are based on optical methods of
turbidimetry, fluorescence, capacitance or resistance of the microbiological culture.
With turbidimetric methods, light transmittance and/or scattering in the media can
be detected by different mechanisms, e.g. transmission (detecting lower turbidity
values), forward scattering, backscattering or lasers [2]. By controlling the cells
with various dyes injected in the media, the fluorescence detector sensor can be
used to monitor different cellular components. The sensors based on the capacitance
measurement use the principle of measuring the dielectric permittivity of microbial
suspensions [3].
In t h s paper, a low-cost infrared optical sensor was constructed for continuous
on-line measurement of biomass concentrations. The calibration curve was
obtained using cell cultures in tap water, and presented a linear profile in the range
0.1-4.0 g/l corresponding to 0.5-4.0 V output signal. The sensor was shown to have
a fast response time and was insensitive to the natural light. A good reproducibility
was obtained between the optical sensor and cell dry-weight method values.
Beer's Law
Beer's Law states that the absorbance of a solution is dependent on three factors: (a)
the molar absorptivity, the value of which depends on the absorbing species and on
the wavelength used; (b) the path length of the solution through which the light
must past; and (c) the concentration of the solution. Therefore:
A = Ebc
where A is the absorbance; & is the constant of proportionality (called the molar
absorptivity); b is the path length of the sample, i.e. the distance of a beam of
monochromatic radiation passing through the cuvette; c is the concentration of the
material in solution. Different molecules absorb different radiation wavelengths.
Transmittance is closely related to absorbance, and the efficiency of transmitting in
the materials depends on the physical characteristics of the material [4]. Although
the fermentation solution consists of different materials which absorb different
wavelengths, it is still possible to find a monochromatic radiation that is suitable for
the yeast with a particle size of about 1 pm.
Design Considerations
The main principle of this low-cost optical sensor design is to utilize Beer's Law
and suitable detector circuits for measuring the biomass on-line.
(0
Circuit design
The design of the low-cost optical sensor is illustrated in Figure 1. The emitter and
the detector are placed in the solution. The light falling on the detector decreases as
the biomass concentration increases. This leads to a drop in voltage at the negative
64
Cost Optical Sensor for Online Measurement of Biomass
input terminal of the operational amplifier. This drop in voltage forces the
operational amplifier to increase the current through the emitter and, hence, bring
the voltage at the negative input terminal back to the reference voltage. The final
output voltage of the circuit also increases. Hence, we refer to it as the Light
Feedback Balance Bridge (LFBB). The concentration of the biomass is converted
into an analogue signal through the LFBB circuit of the sensor. This analogue
signal is then converted into a digital signal by an A/D converter. A computer
receives the digital data via a serial port. All the data are displayed, analysed and
stored in the computer.
Balance Bridge
1
AID Converter
1
Computer
Displayer
Figure 1. Light Feedback Balance Bridge.
Figure 2. Laboratory probe.
65
M. He, X.M. Li, G.F. Qin, S.K.Nguang and X D. Chen
(ii) Probe design
With commercial applications in mind, probes are designed to fit the existing port of
a standard fermentor. The structures of the probes are illustrated in Figures 2 and 3.
The laboratory probe has a wider range of measurements, and the industry probe is
more robust.
Features of the Laboratory Probe (see Figure 2): The distance between the emitter
and the detector can be adjusted from both ends. The top-end adjustment enables
determination of the electrical sensitivity range for different mediums. The bottomend adjustment allows adjustment of the dynamic response range and the sensitivity
of the probe in the solution.
Figure 3. Industry probe.
Features of the Industry Probe (see Figure 3): The emitter and the detector are
tightly sealed so that they can be cleaned and disinfected together with the
fermentor. The light transmitting distance is fixed and only suited for a particular
biomass measurement.
Results and Discussion
The experiments were performed at the Biotechnology Laboratory, the Department
of Chemical and Materials Engineering, the University of Auckland, New Zealand.
A. Equipment, Materials and Weight Methods
Equipment: Laboratory probe, power supply (Topward 6303A), digital multimeter
(Agilent 34401A), Lambda 35 UVNIS Spectrometer (Perkin-Elmer Instruments),
scale (Mettler PL300), shaker, stirring machine, fermentation container.
Microorganism: Saccharomyces cerevisiae (dried baker's yeast packed for
Goodman Fielder Milling & Baking N.Z. Ltd).
66
Cost Optical Sensorfor Online Measurement of Biomass
Medium: YEPD medium used in the fermentation experiments is the composition
of yeast extract (10 gA), peptone (20 gA), dextrose (20 g/l) and commercial
antifoam ( 10 dropdl).
Pre-weight method: Weigh the dry yeast first and then pour it into the water. Use
shaker or stirring machine to make the solution uniform.
Dry-weight method: First centrihge the broth sample of 10 mL for 10 minutes at
4000 rpm. Then measure the dry weight of yeast after dehydrating in the centrifuge
tubes at 65°Cfor 48 hours [ 5 ] .
Figure 4. Experimental bench.
B. Caliiwation Curve
Figure 4 shows the experimental set-up. According to our requirements, different
concentrations of the solutions were manual mixed. The probe was placed in the
solution which was consequently stirred by a magnetic stirring machine. The data
was collected by the sensor. The following tests were performed.
Pure biomass solution test: Between l g and 8g of dried baker's yeast were added to
200 ml of tap water in the 250 ml flask, then well mixed by the magnetic stirrer. The
probe was placed in the solution and the sensor collected the data. The cell
concentration was determined by the dry weight method.
Nutrimental biomass solution test: YEPD medium (200 ml) was added to the
250 ml flask. After sterilizing at 110°C for 30 minutes, between Ig and 8g of dried
baker's yeast were added. The optical sensor and the dry weight method were used
to measure the cell concentration.
67
M. He, XM.Li, G.F. Qin, S.K.Nguang and X D. Chen
C. On-line Biomass Measurement
1. Shaker culture: A series of mediums in 250 ml flasks with volumes from
80 ml to 200 ml and glucose concentrations from 20 gfl to 50 gfl were
2.
prepared. After sterilizing at 110°C for 30 minutes, 1 g of dried baker's
yeast was added to each flask. They were cultured in the shaker at 30°C
and 200 rpm for 2 to 8 hours. Then 20 .ml of concentrated glucose solution
(300 g/l) were fed to each flask every hour, and one of the flasks was taken
out as a sample for determining its cell concentration by both the optical
sensor and the dry weight method.
Small-scale bioreactor culture: Two litres of medium was added to the
3-litre bioreactor (New Brunswick Scientific Co., Inc.) with a working
volume of 2.5 litres. The complete assembly with the optical probe was
sterilized by autoclaving at 1 10°C for 30 minutes. Then 4 g of dried baker's
yeast were inoculated into the bioreactor. The fermentation process was
operated for 4 or 5 hours. The fermentation broth was sampled every half
hour, and the cell concentration was also determined by the dry weight
method.
Results and Discussion
From the experiments, it was found that different monochromatic radiation
wavelengths possessed similar features after passing through the solution. Thus, a
near-infrared phototransistor was chosen that is less sensitive to the natural light.
Further experiments showed that the emitting diode SE5455 with the
phototransistor SD5443 were the most robust pair. They can work at 935 nm
wavelength and 125°C temperature, and are also insensitive to the natural light and
the colour of the solution. As shown in Figure 5, the longer the distance between
the emitter and detector then the larger the range of the output voltage. The best
distance between the emitter and the detector in our experiments was 10 c m
,.....
c
6 0.5
0
50
+ Series1
8 Series2
100
Distance between emitter and
detector (mm)
Figure 5. Transmission distance effects on circuit output.
68
Cost Optical Sensorfor Online Measurement of Biomass
calibration
6 2
-Linear
0
0.0 1.0 2.0 3.0 4.0 5.0
concentration (g/L)
Figure 6. Pure biomass test results.
Figure 6 shows the calibration curve for the pure biomass solution. The output
voltage from the sensor shows a linear relationship with the concentration of the
biomass. Figure 7 shows the test results for the nutrimental biomass solution, it also
shows a linear relationship between the output voltage of the sensor and the
biomass concentration. The three series depicted in Figure 7 represent three
different experiments completed at the three different times. The reproducibility of
the sensor was shown to be very good.
Series2
3.5
0.5-
+ Series 1
n
’.
31
A
- 0
0
1
2
3
4
5
concentration
Figure 7. Nutrimental biomass test results.
69
M.He, X.M. Li, G.F. Qin, S.K.Nguang andX.D. Chen
In Figure 8, the on-line biomass concentration measured by the sensor was
compared to the biomass concentration obtained by the dry-cell weight method. The
voltages from the sensor are converted to biomass concentrations according to the
calibration curve. A linear relationship was also obtained in thls test.
Dynamic experiment
0
0.5
1
1.5
2
Biomass, Dry cell weight (glL)
Figure 8. Online biomass measurements.
Conclusions
This paper has proposed a novel way of designing a low-cost infrared sensor for online measurement of biomass concentrations. This infrared sensor has been
successfully used for measuring biomass concentration ranges from 0.1 to 4.0 gA
with good reliability. The sensor has been shown to have a fast response time and is
insensitive to the natural light. The cost of the sensor is less than US$SO. This
robust and low-cost infrared sensor may provide an attractive solution for the
fermentation industries and dairy industries.
References
1.
2.
3.
4.
5.
70
Olsson, L., and Nielsen, J. 1997. On-line and in situ monitoring of biomass in submerged
cultivations, Trends in Biotech, 15(12), 517-522.
MacMichael, G.,Armiger, W.B., Lee, J.F., and Mutharasan, R. 1987. On-line measurement of
hybridoma growth by culture fluorescence, Biorechnol. Technol., 1,213-21 8 .
Salgado, A.M., Folly, R.O.M., and Valdman, B. 2001. Biomass monitoring by use of a continuous
on-line optical sensor. Sensors and Acruators. 75(1/2), 24-28.
van Holde, K.E.1985. Physical Biochemistry, Prentice-Hall, New Jersey, USA.
Atkinson, B., and Mavituna, F. 1991. Biochemical Engineering and Biotechnology Handbook,
Second edition, Stockton Press, New York.
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