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 determinationof 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 cufaneumimmobilized in poly(viny1alcohol), and (c) a substratepermeable 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,lOphenanthroline)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 biochemical 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 ~ d e . ~They - ~ consist of microorganisms immobilized on a (1) Hikuma, M.; Suzuki, H.; Yasuda, T.; Karube, I.; Suzuki, S . Eur. J . Appl. Microbiol. Biorechnol. 1979, 8 , 289-297. (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. S e w . Acruarors 1992, B8, 167-172. (5) Riedel, K.; Renneberg, R.; KOhn, M.; Scheller, F. Appl. Microbiol.Biorechnol. 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
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1994 American Chemical Society
porous membrane and an oxygen electrode. Various kinds of microorganisms have been used. These include Trichosporon cutaneum,1.597,8Bacillus s ~ b t i l i s , Hansenula ~J a n ~ m a l aand ,~ a mixed culture of B. subtilis and Bacillus licheniformi~.~~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 considered 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 conventional test and, hence, is a useful parameter for rapid estimation of water quality.
EXPERIMENTAL SECTION Microorganismsand 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, B l l , 315-321.
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Figure 1. Cross-section of a sensing membrane for determination of BODS. 1, polycarbonatecover; 2, layer of yeast immobilized in PVA; 3, ca. 1-pm layer of charcoal acting as an optical isolator;4, oxygensensltive fluorescent layer; 5, inert and gas-impermeable polyester support. Excitation light(fromthe bottom) passesthe 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,10phenanthroline)ruthenium(II) perchlorate [ (Ru(dpp)], prepared 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 ~ y s t e m . ~ 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 oxygensensitive 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) 1986, 3, 359-366. (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. 1991, 63, 337-341. (14) Moreno-Bondi, M . C.; Wolfbeis, 0. S.;Leiner, M. J. P.; Schaffar, B. P. H. Anal. Chem. 1990, 62, 2377-2380.
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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~ It consists of an optical sensor membrane, a peristaltic pump (Gilson Minipuls 3, Villiersle-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 AT-PC. 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 (DIN 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.
RESULTS The sensing scheme is based on the measurement of oxygen consumed by yeast, using an oxygen-sensitive fluorescent membrane similar to previous oxygen-sensitivematerials based on luminescent ruthenium c o m p l e x e ~ 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. In Chemical, Biochemical, and Environmenral Sensors III; Liekerman, R. A., Ed.; Proc. SPIE-Int. SOC.Opt. Eng. 1991,1587,288-295.
Table 1. Figures of MerH for the Oxygen Sensor Made from Plasilclzed Poly(viny1 chiorlde)
0.0073 Torr' -52 %
Stern-Volmer constanta quenching on going from nitrogen to air oxygen sensitivity fluorescence intensity response time photostability
0-200 Torr very high
