IMMOBILIZED ENZYME BIOSENSOR FOR BIODIESEL QUALITY TESTING
M.L. Templeton, B.J. Wilson, M.A. Yourek, D.S. Shrestha
Abstract. The purpose of this project was to design, fabricate, and test an immobilized enzyme biosensor for biodiesel
quality testing. The biosensor used an immobilized enzyme membrane attached to a pH probe to detect the amount of
free and total glycerol within the biodiesel sample. The premise of the biosensor was that as the enzyme reacted with
the glycerol in the sample, hydrogen ions would be released and measured by the pH probe, creating a change in the
pH and mV signal read by the meter. The change in pH could then be used to calculate the amount of free and total
glycerol within the sample using a linear relationship. During testing it was determined that the immobilized enzyme
membrane would not stay attached to the pH probe when submerged within a solution, whether in a buffer solution or
a sample of biodiesel. The experimental procedure was then altered so that the enzyme was added directly to the
biodiesel sample, and the pH probe was used to measure the release of hydrogen ions. The results gathered during
testing showed the opposite trend than what was expected; as the biodiesel reaction time increased so did the change
in pH measured. After analysis of the results it was determined that there was no relation between millivolt reading
and temperature, addition of soap, or amount of lipase, but there was a significant relation between the millivolt
reading and the feedstock tested.
Keywords. Biosensor, biodiesel, enzyme biosensor, lipase biosensor, biodiesel quality, immobilized enzyme.
INTRODUCTION
The transesterification process of making biodiesel
from oil creates an unwanted byproduct of unreacted
triglycerides, diglycerides, and monoglycerides. In
addition to these partially reacted species is glycerol, an
undesirable end product that is removed by extraction
with water. The level of free and total glycerol is an
indication of the quality of biodiesel. ASTM D 6751-09
sets the threshold total glycerol at 0.24% and free
glycerol at 0.02% as the maximum concentrations for
commercial-grade biodiesel. The current industry
standard method for testing the amount of total glycerol
in a sample of biodiesel is gas chromatography (GC),
which can be time consuming and expensive. The
purpose of this experiment was to design, fabricate, and
test a handheld biosensor that uses a modified pH meter
combined with an immobilized enzyme electrode to
detect the amount of free and total glycerol in a biodiesel
sample.
The design for the biosensor probe consisted of an
immobilized enzyme membrane covering the glass tip of
a combination pH electrode. The reaction that would
take place can be seen below would result in hydrogen
ions that give a change in pH (Houssain et al. 2010).
Lipase
Glycerides + H2O → Glycerol + Fatty acids + H+
The metering device featured an Arduino Uno
microcontroller with signal conditioning op-amps and
supporting circuitry encased in a rectangular housing
made from ABS plastic. A BNC connection was
installed in the housing to provide compatibility with the
pH probe. The goal was for the Arduino microcontroller
to convert the signal of millivolts (mV) from the probe
into a percentage of total glycerol using a calibration
curve. It was found that the ideal enzyme for the design
would be lipase because of its ability to hydrolyze the
tri- di- and monoglycerides in a biodiesel sample
(Houssain et al. 2010). The membrane chosen for
experimentation was a water in oil emulsion which used
docusate sodium salt as the emulsifying agent. This
method had been used in similar experiments. (Huang et
al. 2001).
METHODOLOGY
MEMBRANE FABRICATION
To create the membrane the first step was to mix
8.55 mL of 0.3 mol/L bis-(2-ethylhexyl) sulfosuccinate
sodium (AOT) in isooctane with 1.45 mL of distilled
water. Then 0.6 g of gelatin was added to the mixed
solution, and the mixture heated in a 50°C water bath
with vigorous stirring until cloudy and viscous, which
took an estimated time of 30 minutes. Next, the cloudy
mixture was cooled in an ice bath for about 10 minutes
in order to get a transparent fluid [non-viscous]. 20 mg
of lipase powder was added to the cold mixture with
stirring until lipase was fully dissolved. The dry
electrode was dipped into a mixture for 2-3 seconds,
then removed and rotated about its center axis for 2-3
minutes at room temperature. The process was repeated
three times at intervals of 15 minutes. An outer,
cellulose acetate membrane was applied to the probe by
dipping in 2% w/v cellulose acetate in acetone for 2-3
seconds at room temperature, and then drying under
vacuum for 12 hours (Huang et al. 2001). Once dry, the
probe with attached membrane was stored in buffer at
4°C.
EXPERIMENTATION
Initial experiments were conducted with the
membrane attached, but the observation of diminished
probe sensitivity as a result of the membrane covering
the glass electrode prompted a different set of
experiments which utilized free lipase and a bare pH
probe. The purpose of these trials was to determine the
important variables involved in the measured signal
from the pH electrode. A total of 32 experiments were
performed with different combinations of temperature
(20C or 40 C), lipase (10 mg or 20 mg), soap (1 mL or
absent), and biodiesel or canola oil for the feedstock.
The tests measured change in pH and mV from a
solution that would include 1 mL of water, 20 mL of
biodiesel or canola oil, and sometimes 1 mL of soap.
This would be mixed together using a stir rod and when
testing at 40 C in a hot water bath. The initial reading
of pH and mV would be taken using a Thermo Scientific
Orion Star A329 Portable Meter, and the continuous
measurements used a Vernier LabQuest meter. After 2
minutes of reading the standard mixture, the lipase
would be added to the solution and measurements taken
for another 15 minutes to ensure adequate reaction time.
SIGNAL CONDITIONING
The voltage signal was conditioned using a circuit
consisting of two different operational amplifiers and a
simple low pass filter (Fig. 1).
was -142.5 mV, and the largest positive value recorded
was 143 mV.
The adjusted signal was amplified using an
AD623AN operational amplifier. This operational
amplifier has an internal gain resistor of 100 kΩ, so the
overall gain through the resistor can be altered by
changing the value of the resistor (Rg) across pins 1 and
8 as follows:
𝐺 = 1 + 100/𝑅𝑔 .
A resistor with a value of 24 kΩ was chosen to give
a final amplification of 5.17.
Once the signal was amplified it was passed through
a simple RC low pass filter to remove any electronic
noise added to the signal from amplification. A 0.04 μF
capacitor and a 35 kΩ resistor were used to create a
cutoff frequency of 113 Hz, as determined by the
equation:
𝑓𝑐 =
1
2𝜋𝑅𝐶
.
The final step of the signal conditioning takes place
in the microcontroller. The voltage signal read by the
microcontroller was converted into the input voltage
value from the pH probe using the calibration data and
equation found in figure 2. The input voltage for the
calibration was the voltage before amplification, and the
output voltage was the voltage after amplification.
Figure 1. Diagram of signal conditioning circuit
Figure 2. Signal calibration curve and fit line
When a measurement was taken with the pH probe
the signal would first encounter a simple voltage
addition using a LM741CN operational amplifier.
During testing, negative voltage signals were recorded
for some samples of biodiesel. Since the amplification
section of the circuit required a positive voltage, a
constant positive voltage was added to the signal to meet
that requirement. The constant voltage added to the
signal was about 500 mV. The 500 mV constant voltage
was chosen because the largest negative value recorded
The microcontroller used was an ATMega328
mounted on an Arduino UNO project board. The
ATMega328 microcontroller has a 10 bit resolution, a
clock speed of 16 MHz, 32 kB of flash memory, and 14
Digital I/O pins [?].
RESULTS AND DISCUSSION
IMMOBILIZED LIPASE MEMBRANE
The water in oil emulsion membrane impeded the
probe’s sensitivity to changes in pH. A control test was
run in order to determine the amount of time taken for
the probe to adjust to a pH 10.0 buffer having previously
been immersed in a pH 4.0 buffer. The probe was rinsed
between measurements. The sensor was found to have a
strong tendency for adhering to the pH of the first buffer
(pH 4.0). In fact, it took at least 20 minutes to record the
change. It was hypothesized that membrane thickness
would have a significant impact on probe sensitivity, but
trials conducted with a single layer of cellulose acetate
exhibited similar restrictions. Failure at this baseline
level casts significant doubt over whether the data
collected from the probe with a membrane attached can
give a reliable measure of the pH change taking place in
biodiesel as a result of glyceride hydrolysis.
It has been observed that the pH measured for a
biodiesel or oil sample most nearly resembles that of the
immediately prior sample. It is suggested that this
behavior can be ameliorated by thoroughly rinsing the
probe with soapy water before each use, but the data is
lacking to support this hypothesis. It appears that a
purely DI water rinse is unable to remove the
hydrophobic biodiesel from the membrane. As a result,
the residual biodiesel may be interfering significantly
with movement of new sample media into the
membrane.
FREE LIPASE
Having been unsuccessful with the attached
membrane, tests were conducted using free lipase to
catalyze the hydrolysis of residual glycerides. By using
free lipase in a stirred mixture, the conditions of the
enzymatic hydrolysis were altered dramatically. Instead
of substrate binding at fixed sites on the membrane
surface, the reaction was disseminated throughout the
turbulent lipase mixture. An example of the change in
millivolt of an experiment run at 40 °C, with 20 mL of
biodiesel, 1 mL of water, 1 mL of soap, and 20 mg of
lipase where the lipase is added after 2 minutes can be
seen in figure 3.
Figure 3. Change in mV compared to the time of the experiment
ANOVA was run on data representing variables of
possible significance using MATLAB. The outcome
showed that only feedstock has a significant impact on
the change in millivolt output. Linear regression analysis
on each combination of the three variables: temperature,
lipase, and soap demonstrated that 20 °C, 20 mg lipase,
and 1 ml of soap yielded the clearest partition among
biodiesel and oil data.
Six additional tests were conducted using these
optimal conditions. A total of three samples were tested,
with one replicate for each sample. The three samples
were canola oil, 30 minute biodiesel, and 60 minute
biodiesel. Time values preceding “biodiesel” in this
report refer to the reaction time in making the batch of
biodiesel, thus “30 min. biodiesel” indicates that canola
oil underwent the conversion to biodiesel via
transesterification reaction with methanol under
controlled conditions at 60°C for a total of 30 minutes
Results from these six trials are shown in the following
table.
Table 1: Free lipase trials using three feedstocks
The dimensionless value is an assigned variable for
relative likeness to finished biodiesel. The canola oil is
therefore assigned a value of zero because it is has not
undergone any reaction, whereas the 60 minute biodiesel
sample has reacted the longest, and is therefore regarded
as the most complete and given a value of 1.
CONCLUSION
Figure 4: Difference in millivolt output in relation to feedstock
Whether the linear relationship would hold over a
wider and more varied range was not possible to
determine due to time constraints. The plot is simply
meant to show an upward trend in millivolt change as
the completeness of the biodiesel increases. The trend
line was expected to slope in the opposite direction, that
is, canola oil was predicted to show the greatest
response owing to its high glyceride levels. The opposite
was observed. The higher the impurity of the sample,
which would have a higher glyceride concentration, had
a smaller millivolt change. This is counterintuitive to the
idea that higher substrate concentrations speed up
enzymatic reactions. There are apparently competing
processes in the unreacted oil of low purity biodiesels
that dampens the response, which warrants further
investigation. Also, it appears that additional variables
beyond those tested with ANOVA may be significant
because considerable variation was observed between
replicate samples. There is an inherent disadvantage to
using pH as a surrogate measurement of concentration in
non-aqueous solutions like biodiesel; the probe requires
substantial time stabilize.
It might be possible to construct a calibration curve,
in a like manner to how the linear trend was derived, if a
definite correlation could be found between purity of the
biodiesel and the signal read from the probe. The
concentration might then be related to total change in
millivolts over the course of the lipase-catalyzed
reaction. For any calibration model, GC analysis would
be the source of all true concentration values as it is the
current laboratory standard. The calibration curve would
plot concentration (%wt) vs. change in millivolts. Once
enough biodiesel samples had been prepared and GC
analyzed for concentration, validation of the curve
would consist of interpolating %wt from the curve based
on measured millivolt change. The accuracy of the
prediction would then be determined by comparing it
with the true GC concentration. A confidence interval
could then be established based on the standard error of
the predicted values.
The immobilization of lipase using water in oil
emulsion and subsequent attachment to a pH probe was
carried out successfully; however, the membrane
performance could not be evaluated due to its hindrance
of probe sensitivity. A study using free lipase mixed into
a biodiesel sample demonstrated no direct correlations
between change in millivolt output from the sensor and
either temperature, mass of lipase, or whether soap was
present in the sample. However, there was a moderately
high correlation (R2 = .91) between millivolt change and
the feedstock used. The response to addition of lipase
was found to be greatest for the most complete biodiesel
and smallest for canola oil. The future testing will focus
on identifying a membrane type and thickness
compatible with the pH sensor, and obtaining timely
consistent pH measurements of the biodiesel sample.
ACKNOWLEDGMENTS
We wish to thank the Biological and Agricultural
Department of Engineering at University of Idaho for
funding this project. We also want to express our
gratitude to Dr. Dev Shrestha, Dr. Joe Thompson, Dr.
Tom Hess, and Sam Wos for their support and guidance
on this project.
REFERENCES
Huang, X.R., Y.Z. Li,G.L. Yong, L.L. Liu, Y.B.
Qu, and W.J. Zhang. 2001. A Novel method for
fabrication of a glass-electrode-based lipase sensor.
Chinese Chemical Letters. 12(5): 453-456.
Hossain, M.Z. 2008. Biosensor applications in
biological and agricultural engineering
and design of a novel potentiometric lipase
biosensor for biodiesel quality
sensing. M.E. design project. Moscow, Id.:
University of Idaho, Department of Biological and
Agricultural Engineering.