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Anal. Chem.

1994,66, 1841-1846
Optical Fiber Sensor for Biological Oxygen Demand
Claudia Preinlnger, Ingo Kllmant, and Otto S. Wolfbels'
Institute of Organic Chemistry, Analytical Division, Karl-Franzens University,
Heinrich Street 28, A-80 10 Graz, Austria
We describe the first fiber-optic microbial sensor for deter-
mination of biochemical oxygen demand (BOD). The sensing
membrane at the tip of the fiber consists of layers of (a) an
oxygen-sensitive fluorescent material, (b) Trichosporon cu-
faneum immobilized in poly(viny1 alcohol), and (c) a substrate-
permeable polycarbonate membrane to retain the yeast cells.
The layers are placed, in this order, on an optically transparent
gas-impermeable polyester support. Tris(4,7-diphenyl-l,lO-
phenanthroline)ruthenium(II) perchlorate is used as the oxygen
indicator. Typical response times are 5-10 min, and the
dynamic range is from 0 to 110 mg/L BOD when a glucose/
glutamate BOD standard is used. The fluorescent signal is
affected by various parameters, including the thickness of the
layers, the cell density of the yeast, and the rate at which the
substrate is passed through the flow-through cell. BOD values
estimated by this new biosensor correlate well with those
determined by the conventional BOD5 method. The main
advantages of this optical sensor are (a) a more rapid estimation
of BOD (in comparison to the BOD5 method which requires
5 days), (b) the fact that opticaloxygen sensorsdo not consume
oxygen, (c) the possibility of performing in situ monitoring
using fiber optics, and (d) the option of designing inexpensive
disposable sensor cells.
BOD5 is defined as the biochemical oxygen demand of
waste water measured over 5 days under specified standard
conditions. The parameter is based on the metabolic activity
of aerobic microorganisms and gives an estimation for the
amount of oxygen in waste-loaded water required for bio-
chemical degradation of organic matter. Although BOD5 is
a good indicator of the concentration of organic pollutants in
the water, biochemical oxidation is a slow process, and the
test, in its present form, takes 5 days until results areobtained.
Thus, the conventional test is not suitable for process control
and monitoring, where a rapid feedback is desirable. It is
therefore of considerable interest to develop alternative
methods that may replace this time-consuming test. This
was achieved by immobilizing microbes at the tip of an
amperometric electrode. Several kinds of microbial sensors
for BOD5 have been reported, some based on measurement
of a steady-state equilibrium,I4 others measuring in the kinetic
m~de. ~- ~ They consist of microorganisms immobilized on a
(1) Hikuma, M.; Suzuki, H.; Yasuda, T.; Karube, I.; Suzuki, S. Eur. J. Appl.
(2) Karube, I. Biorechnol. Bioeng. 1977, 19, 1535-1547.
(3) Kulys, J.; Kadziauskiene, K. Biofechnol. Bioeng. 1980, 22, 221-226.
(4) Tan, T. C.; Li, F.; Neoh, K. G. Se w. Acruarors 1992, B8, 167-172.
(5) Riedel, K.; Renneberg, R.; KOhn, M.; Scheller, F. Appl. Microbiol. Biorechnol.
Microbiol. Biorechnol. 1979, 8, 289-297.
1988, 28, 316-318.
(6) Tan, T. C. Sens. Acruutors 1993, BIO, 137-142.
(7) Riedel, K.; Lange, K.; Stein, H.; Kiihn, M.; Ott, P.; Scheller, F. Water Res.
1 9 9 0 , ~ 883-887.
0003-2700/94/ 0366- 184 1 $04.50/ 0
0 1994 American Chemical Society
porous membrane and an oxygen electrode. Various kinds of
microorganisms have been used. These include Trichosporon
cutaneum, 1.597,8 Bacillus s ~bt i l i s , ~J Hansenula a n ~ ma l a , ~ and
a mixed culture of B. subtilis and Bacillus l i cheni formi ~. ~~6
T. cutaneum is identical to Trichosporon beigeliiused in other
work.
Conceivably, the amperometric measurement of oxygen
may be replaced by optical (fluorescent) measurement of
oxygen using an or706e (Greek; "theoptical way"). The major
advantage of optodes over electrodes in the context of BOD
is the fact that, unlike electrodes, they do not consume oxygen
during measurement, so that no depletion of oxygen can occur,
as occurs during electrochemical measurement. We therefore
perceived that the use of some of the oxygen sensors developed
by us in the past years would result in a sensor with improved
performance. Two sensing schemes were envisaged: (a)
placing an oxygen-sensitive membrane on the bottom of the
sample vessel and monitoring oxygen over 5 days (in an
instrument similar to a bacterial detection system using a
carbon dioxide optodeg) or (b) performing the test using a
biosensor arrangement using immobilized cells. We consid-
ered the latter to be advantageous over the former mainly for
the reason of being much faster and therefore providing a
rapid feedback signal.
In this work we show that BOD indeed can be measured
optically by using a microbial BOD biosensor membrane along
with a measuring scheme resembling flow injection. We also
show that this approach presents some attractive new features
and advantages over electrochemical detection. Although the
BOD measured with the biosensor (referred to as the BODS)
is not identical to the conventional BOD5, it is shown to be
a parameter that correlates acceptably well with the con-
ventional test and, hence, is a useful parameter for rapid
estimation of water quality.
EXPERIMENTAL SECTION
Microorganisms and Cell Growth. The yeast T. cufaneum
(now known to be identical with T. beigelii; DSM, Brunswick,
Germany) was grown under standard aerobic conditions in a
rotating shaker at 30 OC for 36 h in a medium containing
0.25% malt extract, 0.25% peptone, 0.25% yeast extract, and
1% glucose. The culture broth was centrifuged at room
temperature at 5000 rpm for 10 min, and the cell mass was
washed twice with a 0.1 M phosphate buffer of pH 6.8.
Immobilization. The washed cell mass was mixed with a
10% aqueous solution of poly(viny1 alcohol) (pva) (MW
(8) Riedel, K.; Alexiev, U.; Neumann, B.; Kahn, M.; Renneberg, R.; Scheller, F.
Biosensors: Applications in Medicine, Environmenral Protection and Process
Conrrol; GBF Monographs; VCH: Weinheim, Germany, 1989; Vol. 13, pp
71-74.
(9) Swenson, F. J . Sew. Acruarors 1993, Bl l , 315-321.
Analytjcal Chemisfty, Vol. 66, No. 17, June 1, 1994 1841
175vm l L - s
Figure 1. Cross-section of a sensing membrane for determination of
BODS. 1, polycarbonate cover; 2, layer of yeast immobilized in PVA;
3, ca. 1-pm layer of charcoal acting as an optical isolator; 4, oxygen-
sensltive fluorescent layer; 5, inert and gas-impermeable polyester
support. Excitation light (from the bottom) passes the polyester support
and excites fluorescence In the oxygen-sensltlve layer. Part of the
emitted light is collected by the fiber bundle (not shown) underneath
the polyester layer and guided to the photodetector.
100 000) in a ratio of 1:l (by weight) and spread in various
thicknesses onto an optical oxygen-sensing membrane. The
microbial membranes were dried at 4 O C for 24 h and stored
at 4 O C until used. Both the oxygen sensor and the immobilized
yeast were found still to work and to be useful for BOD
determination after a 1-year storage at 4 "C.
Oxygen Sensor Membrane. In 5 mL of tetrahydrofuran
(THF) were dissolved 13.5 mg of tris(4,7-diphenyl-l,10-
phenanthroline)ruthenium(II) perchlorate [ (Ru(dpp)], pre-
pared by a modification of a published method,IO 0.5 g of
poly(viny1 chloride) (pvc) (Fluka, Buchs, Switzerland), and
0.5 g of 2-nitrophenyl octyl ether (NPOE) (Fluka). This
solution was spread onto a 175-pm polyester film (Mylar,
DuPont) acting as an optically transparent solid support. After
solvent evaporation, the resulting clear oxygen-sensitive layer
on the polyester film had a calculated thickness of around 10
pm. The concentration of the dye in the plasticized pvc film
was approximately 12 mM. The red fluorescence of the
ruthenium complex, which was reversibly quenched by oxygen,
was the analytical information of this ~ystem.~I -'~
Assembling the Sensor. A cross-section of the microbial
optode is shown in Figure 1. A polyester support (Mylar,
type GA-10, DuPont, Vienna), being impermeable to oxygen,
served as a mechanical support onto which a 10-pm oxygen-
sensitive fluorescent layer was spread. The polyester support
enables a much easier handling of the sensing layers. The
fluorescent layer was covered with a layer of commercial
charcoal, which served as an optical isolator. The charcoal
was spread evenly onto the pvc layer while still slightly wet,
using a thin sieve. The optical isolation prevents ambient
light from entering the optical system and blue excitation
light from exciting fluorescence in the sample and makes the
sensor insensitive to changes in the refractive index of the
sample. The black layer was covered by a layer of
immobilized yeast by spreading the suspension of yeast in a
10% pva solution onto the membrane, using a home-made
spreading device. The preferred thickness of the yeast layer
(IO) Watts, R. J.; Crosby, G. A. J. Am. Chem. SOC. 1971, 93, 3184-3188.
(1 1) Wolfbeis, 0. S.; Leiner, M. J . P.; Posch, H. E. Mikrochim. Acta (Vienna)
(12) Bacon, J . R.; Demas, J . N. Anal. Chem. 1987, 59, 2780-2784.
( 13) Carraway, E. R.; Demas, J . N.; DeGraff, B. A.; Bacon, J . R. Anal. Chem.
(14) Moreno-Bondi, M . C.; Wolfbeis, 0. S.; Leiner, M. J . P.; Schaffar, B. P. H.
1986, 3, 359-366.
1991, 63, 337-341.
Anal. Chem. 1990, 62, 2377-2380.
after drying on ambient air was 10 pm. Layer thicknesses
were calculated from the volume spread and the amount of
water that evaporated during drying. On this layer was placed
a porous polycarbonate membrane of pore diameter 0.4 pm
(Bio-Rad, Vienna), which was permeable to dissolved organic
matter but retained the microorganisms.
Apparatus. The continuous flow system has been described
in some detail previ0usly.1~I t consists of an optical sensor
membrane, a peristaltic pump (Gilson Minipuls 3, Villiers-
le-Bel, France), an automatic sampler (ND 12, Besta,
Germany), a fiber-optic photometer (Oriel 3090, Chelsea
Instruments, London, U.K.), a 150-W pulsed xenon lamp as
a light source, and an R 928 photomultiplier (PMT)
(Hamamatsu, Munich) as detector. A 480-nm interference
filter was placed in front of the Xe lamp to isolate the
appropriate excitation light, and a 560-nm long-pass filter
was placed in front of the PMT to block scattered 480-nm light
but to allow the red fluorescence, which has a maximum at
610 nm, to pass. Fluorescence intensity data were transferred
to a data acquisition unit (Keithley 575) controlled by an
Standard Solution and Determination of the 5-Day BOD.
A solution containing 150 mg/L glucose and 150 mg/L
glutamate (resulting in a BOD of 220 mg/L) was employed
as a standard solution (referred to as GGA solution) for
calibration of the BOD sensor according to Riedel et al.' The
BOD5 of waste water was determined by the standardized
dilution method (DI N 38 409).
Sensor Conditioning. Microbial membranes stored at 4
"C were taken out of the refrigerator 2-3 days before
measurement to allow the immobilized yeast to adapt to room
temperatureconditions. A 3-mm-diameter spot was punched
out of the large sensing membrane and covered with the
polycarbonate membrane, and the sensing membrane was
placed in the flow cell. Standard solutions of various
concentrations and pH were injected into the system and passed
over the sensing membrane until a constant signal was attained.
AT-PC.
RESULTS
The sensing scheme is based on the measurement of oxygen
consumed by yeast, using an oxygen-sensitive fluorescent
membrane similar to previous oxygen-sensitive materials based
on luminescent ruthenium compl exe~~~- ~~ but using plasticized
pvc as a matrix, which has advantages in terms of strong
optical signal, efficient quenching by oxygen (the signal is
quenched by 50% in going from nitrogen to air), rapid response,
and ease of manufacturing. We therefore first characterized
the oxygen sensor. Table 1 gives figures of merit. They show
the sensor to have a dynamic range that covers the range of
interest, a response time fast enough to monitor the bacterial
metabolism, and an excellent long-term stability (except when
in contact with samples containing detergent).
The change in the optical signal of this particular sensing
material is shown in Figure 2 for the 0-1 50 Torr range, which
is the one of interest in this context, along with the respective
Stern-Volmer plot. The Stern-Volmer equation (eq 1) relates
fluorescence intensity in the absence ( l o ) and presence (I) of
oxygen, respectively, to the concentration of oxygen ([02]):
(1 5) Weigl, B. I n Chemical, Biochemical, and Environmenral Sensors III;
Liekerman, R. A., Ed.; Proc. SPIE-Int. SOC. Opt. Eng. 1991,1587,288-295.
1042 Analytical Chemistty, Vol. 66, No. 11, June 1, 1994
Table 1. Figures of MerH for the Oxygen Sensor Made from
Plasilclzed Poly(viny1 chiorlde)
Stern-Volmer constanta
quenching on going
from nitrogen to air
oxygen sensitivity
fluorescence intensity
response time
photostability
storage stability
operational lifetime
leaching of the dye
leaching of the plasticizer
reproducibility of K,
0.0073 Torr'
0-200 Torr
very high
<1 s to gases, <1 min to liquids
no bleaching detectable with a
6+ months
1 week minimum
not detectable when stored in
buffer at roomtemperature
negligible in water, strong in
water containing detergent
h2% (n =6)
-52 %
blue LED after 2 weeks
nd interferences by
interferences by SOz (20 ppm)
0 Initial slope.
COZ (IO00 ppm), HzS (100 ppm),
NH3 (50 ppm)
loll re1 intensity I I arb. units
2.5 1100
I
I
2 -
1
1,5
1
0 50 100 150
pO2 / torr
+ Stern-Volmer plot + intensity plot
Figure 2. Stern-Volmer plot and intensity plot of the quenching of the
fluorescence of Ru(dpp) by oxygen.
I 1
0 10 20 30 40 50 60 70 80
time (miid
Figure 3. Response time, relative signal change, and reversibillty of
the microbial sensor when exposed to a standard BOD solution (GOA)
having a BOD of 11 0 mg/L.
K, is the so-called Stern-Volmer constant. Most importantly,
the resulting plot (Figure 2) is virtually linear, a fact that
greatly facilitates calibration and, in principle, makes possible
a 1-point calibration.
Figure 4 shows typical response curves of the optical
biosensor using immobilized T. cutaneum. Initially, the Ru-
(dpp) fluorescence is partially quenched by the oxygen
dissolved in the buffer. This signal represents the fluorescence
in absence of substrate and to some extent also reflects the
endogenous respiration of the immobilized microorganisms.
After approximately 10 min, a synthetic sample solution
containing glucose and glutamate was injected into the system.
The organic compounds permeate through the porous mem-
brane and are metabolized by the microbial cells, which thereby
consume oxygen. As a result, the oxygen partial pressure
inside the oxygen-sensitive pvc layer decreases until a steady-
state oxygen concentration is reached. The change in signal
can be related to BOD. When pure buffer is passed over the
sensor system again (after ca. 25 min in Figure 3), fluorescence
returns to the initial level. Response times are on the order
of 5-10 min.
Calibration. A calibration curve was established for the
microbial biosensor membrane using diluted GGA stan-
dard solutions. As can be seen from Figure 4, a linear
relationship was observed at below 110 mg/L BOD of the
standard. The minimum detectable BOD is around 2-3 mg/
L. The signal was reproducible to within f4% of the mean
value in a series of 10 samples of 5 5 mg/L BOD standard
GGA solutions.
Effect of pH. The physiological state of microorganisms,
in particular their respiratory activity, strongly depends on
pH. Consequently, its effect on the optical biosensor was
examined. The 55 mg/L glucose/glutamate standard was
employed for these experiments. Figure 5 demonstrates that
the maximum signal change is obtained at pH 6.8.
Effect of Thickness and Cell Concentration of the Microbial
Layer. Clearly, one would wish to have a microbial layer as
thick as possible in order to achieve a large signal change even
at low BOD. However, increasing the thickness of the
microbial pva layers to above 100 pm causes the response
time to increase to up to 30 min, the relative signal change
to decrease by up to 25%, and the conditioning of the sensing
membrane to take much more time, typically 5-1 2 h. Figure
6 shows the effect of varying cell density of T. cutaneum in
the pva layer on the response. It is obvious that sensing
membranes containing high cell concentrations are more
sensitive but have a smaller dynamic range. Thus, by varying
the cell density, membranes with dynamic ranges up to 110
mg/L BOD may be produced. This does not come unexpected
because (a) substrate diffusion is slower in highly loaded layers
and (b) more oxygen is consumed by the cells through
endogenous respiration.
Sensor Lifetime. Sensors were reactivated after various
times of storage. Typically, there was a 30% drop out after
4 weeks of storage in a lab refrigerator at 4 "C. Of those
sensors that could be reactivated, all had an operational lifetime
of 1-2 weeks, sometimes even 30 days. Similar stabilities
have been reported by A major problem is
reconditioning. Even of those made in the same batch, some
sensors could not be reactivated after storage, while others
had no problem. As a result, all sensors had to be recalibrated
after reconditioning.
Variation of Substrates. The response of the biosensor to
various organic substrates was tested, and the resulting
response curves are shown in Figure 7. The shape of all work
functions is similar, but there are distinct differences between
glucose and maltose on the one side and pyruvate on the other.
The slope, and hence the sensitivity of the optode, is bigger
for the former two than for L-glutamate, ethanol, and pyruvate.
Analytlcal Chemistry, Vol. 66, No. 11, June 1, 1994 1043
0
Figure 4. Optical signal changes of the BOD sensor when contacted with standard (GGA) solutions of increasing BOD, and the respective work
function.
0 15 30 45 60 75
time (mid
Flgure 5. Response of the microbial sensor to GGA standard solutions
(55 mg/L BOD) of various pH.
Figure 8. Effect of cell densityon the measured change in fluorescence.
Membranes with 16 (curve 2) and 32 mg (curve 1) of dry yeast per
milliliter of pva suspension were employed.
A distinct change in slope occurs at substrate concentrations
of around 0.1 mM, but at this point wecan only speculate as
to the reason. Possibly, the effect is related to the endogeneous
respiration of the cells, Le., consumption of oxygen in the
absence of digestable substrate as a result of the normal
(endogeneous) respiration of cells.
Application. The microbial optode sensor was applied for
the estimation of BOD of untreated waste waters from sewage
plant effluents and municipal sewage. The resulting oxygen
demand is referred to as BODS in order to differentiate it
from the standard BODS. The latter was determined by the
conventional dilution method. As shown in Figure 8, a fairly
good correlation between BODS and BOD5 was obtained ( r
=0.9704; n =12). The ratios between optodes BODS and
BOD5 ranged from 0.72 to 1.66. There is good reason to
assume that this variation is caused by the different components
- 1
1
2 50 3
a 4
5 40 5
v, b
30
&20
$10
8! ' 0 2 ' o k 06 ' 0'8 ' i o
concentration (mM)
Figure 7. Effect of varying substrates on the respiratory activity and
response of pva-immobilized T. cufaneum in the BOD biosensor. 1,
ffilucose; 2, maltose; 3, o,L-lactate; 4, ethanol; 5, L-glutamate; 6,
pyruvate.
80 ,
Figure 8. Correlation of BODS data as determined by the microbial
optode sensor (measurements in triplicate) with BOD5 values (obtained
by the conventional 5day method). (- - -) Calculated line of slope 1;
(-) regression line ( r =0.9704).
contained in waste water, all of which are degraded at different
rates. I t is known, for example, that large molecules such as
starch and cellulose are slowly degraded by the metabolic
enzymes of immobilized yeast. Very large molecules do not
even pass the polycarbonate cover membrane of the sensor
presented here. Consequently, they escape BODS detection.
The BODS data, therefore, are generally lower than the BOD5
data in case of high polysaccharide levels. A high BODS, in
contrast, may be caused by the higher respiratory rate of T.
cutaneum for certain substrates contained in sewage.
1844 Analytical Chemistry, Vol. 66, No. 11, June 1, 1994
Table 2. Flgureo of MerH for Varlow BODS Teeto
upper limit of detection
ref microorganism sensing scheme response tim (mg/L BOD)
1 T. cutaneum amperometric, steady state 18 min 60
2 C. butyricum amperometric, steady state 15 min 300
3 H. anomala amperometric, steady state 15-20 min
5 B. subtilis amperometric, kinetic 15-30 20
5 T. cutaneum amperometric, kinetic 15-30 8 100
this work T. cutaneum fluorescence quenching 3-10 min 110
6 B. subtilis plus B. lichenijormis 7B amperometric, kinetic 15-30 s 80
DISCUSSION
The results show that optical sensing of BOD is an attractive
alternative method to the conventional BOD5 test. At pH 6.8
and 30 O C , the BODS compares favorably with the BOD5 in
terms of relative signal change and response times. The optode
sensor is comparable in performance with electrochemical
steady-state BOD sensors (Table 2). The time required for
an assay is comparable with the steady-state amperometric
methods (entries 1-3 in Table 2). BODS sensors using kinetic
signal evaluation (i.e., by relating the first derivation of the
current/time curve to the increase in yeast metabolism) are
inherently faster because there is no need for establishing an
equilibrium. Although not done so far, we have all reasons
to assume that the optode sensor can be submitted to kinetic
data evaluation as well. In each instance, BOD sensors give
results in much shorter times than the conventional 5-day
assay. Table 2 compares figures of merit of the various types
of amperometric BOD sensors.
Steady-state electrochemical sensors are comparable in
response time and upper limits of detection, while kinetic
electrochemical methods are clearly faster and, in one instance,
cover the low BOD range. Optical biosensing of BOD may
have advantages over amperometric methods because no
oxygen is being consumed by the oxygen transducer. Elec-
trochemical reduction of oxygen can prevent BOD assay in
cases of samples with low oxygen loading because this limits
the amount of oxygen supposedly consumed by the microbes.
The BOD optode sensor spots described here can be
manufactured at very low costs from standard plastic materials
and minute amounts of a fluorophore. In our case, they are
fabricated in standard-size sheets (A4 or B4), and 3-mm
diameter spots are punched out of the sheet. Although not
intended when starting the work, we now think that-in view
of the costs-one application of the microbial biosensors is in
low-cost disposable sensors.
The yeast T. cutaneum, although pathogenic,I6 was shown
to be an efficient biodegrading agent for organics in aqueous
solution, with good kinetics, sensitivity, stability, and repro-
ducibility. Notwithstanding this, other microbes (some given
in Table 2) may be used as well. Both bacterial and yeast
species have been used previously, including Clostridiums
(B.) and Trichosporon- and Hansenula-type yeast. More
recently, a bacterium/ yeast combination (Rhodococcus eryth-
ropolis/Zssatchenkia orientalis) has been implemented in a
commercially available amperometric BOD meter. Two-
bacilli systems (such as a B. subtilisllicheniformis combina-
tion) have been applied as well. A highly practical sensor
results from the useof activated sludge containing the kind of
(16) Leblond, V.; Bellefiqh, S. Cancer 1986, 58, 2399-2405.
microbes found in sewage and therefore being identical to the
species that create a BOD. Soil bacteria have been used as
well.
The relation between BODS and the measured fluorescence
signal (I) can be deriyed from the Stern-Volmer equation.
The situation resembles that of enzyme-based biosensors with
oxygen optode transducers.14J 7 The fluorescence intensity
obtained with the sample in the absence of any oxygen
consumption (la) is a measure for the initial oxygen concen-
tration which can be calculated from la and IO via the Stern-
Volmer equation (eq 1). Let us set the signal obtained before
microbial action, i.e., (l0/Za - l ), equal to a:
a =~S,[O,l (2)
Following microbial action, oxygen will be partially or totally
consumed. An additional term [-A(O2)] has to be added to
eq 2 to account for this. Under steady-state conditions, a new
Z value will then be measurable, which we may call I b. The
respective signal is (ZO/Ib - l), and weset it equal to P:
P =~S,[O,l - KS,[A(O2)1 (3)
The difference in oxygen concentration [A(O2)] caused by
microbial oxidation of organic matter can be calculated by
subtracting the two equations:
a - P =- ~s v[ A( 02) l (4)
The relation between [A(O,)] and BODS can be described
by
[A(O,)If= BODS ( 5 )
where f represents a conversion factor that accounts for the
unknown rate of consumption of oxygen by bacteria. Factor
f is governed by type and activity of the microbes, pH,
temperature, and other parameters. If measurements are to
be performed in a kinetic way, f also depends on the time
period between the first (a) and the second measurement ( P) .
By combining (4) with ( 5 ) , we end up with a fairly simple
relation between BODS and the two optical signals:
(6)
This equation allows calculation of BODS from two fluo-
rescence intensity data. I t is independent of the initial oxygen
concentration and, hence, allows determination of BODS even
in cases of samples which are not air-equilibrated but already
have suffered from oxygen depletion, a situation frequently
encountered in practice. It is applicable, though, only over
the linear part of the Stern-Volmer equation and under the
BODS =( P - a) / f K, ,
(17) Wolfbeis, 0. S. Ed. Fiber Opfic Chemical Sensors and Biosensors; CRC
Press: BocaRaton, FL, 1991; Vol. I, pp 93 ff.
Analytical Chemistry, Voi. 66, No. 17, June 1, 1994 1845
provision that enough oxygen is present so as not to end up
with complete oxygen consumption.
Figure 8 demonstrates that the BODS is similar but not
identical to the BODS. This has been known for quite some
time and is due to the fact that immobilized microbes are slow
in responding to large molecules such as starch and cellulose
derivatives, which are species frequently encountered in
domestic sewage and industrial effluents.
In conclusion, we think we have shown that optical
biosensing of BOD is possible and has certain advantages
over amperometric methods in that no oxygen is being
consumed by the oxygen transducer, a fact that can prevent
BOD assay in cases of samples with low oxygen loading because
the amperometric sensor consumes the oxygen supposedly
consumed by the microbes. Another interesting feature of
BOD optodes is their cost. In contrast to conventional
amperometric oxygen electrodes, the oxygen sensor spots
described here can be manufactured at very low costs from
standard plastic materials and minute amounts of a fluoro-
phore. Hence, low-cost disposable sensors for single use may
be envisioned.
ACKNOWLEDGMENT
Financial support by the FWF, project S 5702, is gratefully
acknowledged. The authors would also like to thank Mr. J .
Rosmann (Styrian Government Laboratories) and Dr. Stuhl-
bacher (Institute of Waste Technology, Graz University of
Technology) for supplying sewage waste water samples and
performing standard BOD measurements.
Received for review J anuary 3, 1994. Accepted March 8, 1994.'
*Abstract published in Advance ACS Abstracts. April 15, 1994.
1846 Analytical Chemistry. Vol. 66, No. 11, June 1, 1994