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Biosensor for the evaluation of biochemical oxygen demand using photocatalytic pretreatment
Sensors and Actuators B 80 (2001) 15±20
Biosensor for the evaluation of biochemical oxygen demand using photocatalytic pretreatment Gab-Joo Chee, Yoko Nomura, Kazunori Ikebukuro, Isao Karube* Research Center for Advanced Science and Technology, University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904, Japan Received 15 May 2000; received in revised form 7 March 2001; accepted 8 May 2001
Abstract A highly sensitive biochemical oxygen demand (BOD) sensor using photocatalytic pretreatment was developed to evaluate low BOD levels in river waters. The photocatalytic oxidation was carried out in a commercial TiO2 as a semiconductor and a 6 W black-light tube as light sources. The photocatalytic oxidation was investigated about the effects of irradiation times, TiO2 concentrations, and pH on the sensor response. The optimal irradiation time was 4 min. TiO2 concentrations gave the optimum response to the BOD sensor at 1% (w/v). The sensor response was increased with increasing pH. The sensor responses obtained by photocatalysis to river samples were higher than those obtained without photocatalysis. A fairly good correlation between the values obtained by the photocatalytic pretreatment method and BOD5 was acquired. # 2001 Elsevier Science B.V. All rights reserved. Keywords: BOD sensor; Biosensor; Pseudomonas putida; Arti®cial wastewater; Photocatalysis; TiO2; UV; Pretreatment
1. Introduction The enzyme-based sensor for the determination of glucose levels was developed the ®rst by Clark and Lyons [1]. Since then, various biosensors using both biomaterials, such as microorganisms, enzymes and physico-chemical transducers, such as optical, electrochemical have been developed in many ®elds of analysis [2±7]. Biosensor in environmental analysis was remarkably developed, and a biochemical oxygen demand (BOD) sensor is one of the most typical sensors in this ®eld [8]. BOD is the amount of oxygen requirement of biodegrading organic compounds in aqueous solution. Up to date, the BOD sensors have employed glucose and glutamic acid (GGA) solutions which are used as standards for its calibration, according to the Japanese Industrial Standard (JIS) [9]. The GGA solutions should be suitable to evaluate industrial wastewaters, and not river waters which contain humic acid, lignin and tannic acid, etc. Therefore, as a standard solution for the estimation of BOD in river waters, arti®cial wastewater (AWW) was made from these recalcitrant organic compounds such as humic acid, lignin and tannic acid. The BOD sensor with Pseudomonas putida SG10 was developed to evaluate low BOD * Corresponding author. Tel.: 81-3-5452-5221; fax: 81-3-5452-5447. E-mail address: [email protected] (I. Karube).
values such as river waters [10]. In river waters analysis, however, this sensor often showed low values compared with BOD5 as well as other BOD sensors [11±15]. Such low BOD values would show that these recalcitrant organics in river waters are uneasily assimilable to the immobilized microorganism in such a short measuring times. These values would be increased by performing a pretreatment of the sample by using titanium dioxide-based photocatalysis. In 1972, Fujishima and Honda discovered the photocatalytic splitting of water which could be decomposed into hydrogen and oxygen over an illuminated titanium dioxide semiconductor electrode [16]. With the help of this event, photoelectrochemistry has expanded into a formidable ®eld encompassing solar energy conversion [17], photocatalysis [18±20], decomposition of agrochemical [21,22], air and water puri®cation [23±27]. In the photochemical oxidation method, short wavelength UV-C (<280 nm) light is commonly employed [28]. On the other hand, photocatalytic experiments with titanium dioxide usually use long wavelength UV-A (>315 nm) light [29±31]. Fig. 1 shows a general schematic representation of the degradation of organic compound in aqueous solution by electron-hole (e ±h) formation at the surface of an illuminated titanium dioxide particle [32]. When titanium dioxide is illuminated with band gap energy of greater than 3.2 eV (380 nm), a photon excites an electron from the
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G.-J. Chee et al. / Sensors and Actuators B 80 (2001) 15±20
Fig. 1. Schematic diagram of simplified mechanism for the photoactivation of a titanium dioxide particle.
valence band (VB) to the conduction band (CB) and leaves an electronic vacancy commonly referred to as a hole in the VB. The electron in the CB can be transferred to adsorbed H, O2 or the chlorinated pollutant initiating various reactions. The hole in the VB can react with surface-bound water, hydroxide groups, anions and organic substrate. Therefore, organic compounds in aqueous solution are decomposed to the formation of lower molecular weight compounds by photocatalytic oxidation. The results would increase the sensitivity of the BOD sensor, because organic compounds of lower molecular weight are more readily biodegradable through the bio®lm. This paper describes the comparison of the sensor response before and after photocatalytic oxidation, and the rising of the sensitivity of the BOD sensor. 2. Experimental 2.1. Preparation of biofilm and the BOD sensor Cell culture was carried out for P. putida in 200 ml of sterile culture medium containing nutrient broth (1.0 g l 1) and AWW (per liter: nitrohumic acid, 84.92 mg; gum arabic, 93.90 mg; sodium ligninsulfonate, 48.54 mg; tannic acid, 83.50 mg; linear alkylbenzene sulfonate, 18.84 mg) at pH 7.0. The culture broth was kept in a rotating shaker under aerobic conditions at 308C. The cells were harvested at the end of the exponential growth phase, about 24 h after seeding the culture medium. This was to ensure consistence in preparing the bio®lm for the BOD sensor fabrication. The cells were washed and centrifuged at 7000 rpm for 10 min, and were subsequently resuspended in 10 mM phosphate buffer (pH 7.0). The bio®lm for the BOD sensor was prepared using an aspirator connected to a syringe ®lter holder. A porous cellulose nitrate membrane (20 mm diameter, 0.45 mm pore size, Advantec, Japan) was carefully sandwiched in a syringe ®lter holder. Calculated amounts of the pure culture broth was dropped on the cellulose nitrate membrane. The amounts were calculated to give a maximal response to
the sensor [10]. The bio®lm was washed twice with 10 mM phosphate buffer solution using an aspirator. The bio®lm was placed on the top of the Te¯on gas membrane covering an oxygen probe (BO-U1, Able, Japan), and then was tightly secured using 200 mesh nylon and an O-ring. The remnant culture broth was stored at 48C in the refrigerator before preparing a new membrane. An oxygen probe was linked to both a digital multimeter (model TR6840, TakedaRiken, Japan) and an electronic recorder (model EPR-200A, TOA Electronics, Japan) as described in an early paper [10]. The BOD sensor was kept in 10 mM phosphate buffer solution at room temperature when not in use. 2.2. Experimental procedure As a standard solution for the calibration of the BOD sensor, AWW (16.485 mg l 1, 3.77 mg l 1 BOD) was employed to substitute for GGA as described in an early paper [10]. AWW solution was prepared using arbitrarily selected recalcitrant organic compounds in river waters and secondary ef¯uents [33,34]. The BOD sensor was inserted into the detection chamber containing 50 ml of 10 mM phosphate buffer (pH 7.0) solution. Agitation and oxygen saturation of the test solution was ensured by stirring continuously by a magnetic bar. The detection chamber was maintained at 308C by circulating water at constant temperature through the water jacket. The current response of the BOD sensor in 10 mM phosphate buffer solution was monitored on a digital multimeter and an electronic poly recorder. When the current reached a steadystate value, a calculated amounts of AWW were added to the phosphate buffer solution to give various BOD5 values. A commercial TiO2 (anatase ST-A01, Ishihara Sangyo Co., Japan) was used in the this study. The TiO2 granule diameter was 2 mm. The various amounts of TiO2 granule were added in 25 ml AWW solution and magnetically stirred. The TiO2±AWW solution was then illuminated with a 6 W black-light ¯uorescent tube (Ushio Co., Japan) as UV light sources. The illuminated AWW solution was injected to 50 ml phosphate buffer solution by micro-syringe pipette. The degradation of AWW solution by photocatalysis also was investigated as the total organic carbon (TOC) using a TOC analyzer (Shimadzu 5000, Japan). The pH value of the solutions was adjusted with NaOH. The conventional BOD5 was determined by the standard method according to the JIS [35]. 3. Results and discussion 3.1. Comparison of the sensor response with GGA and AWW Fig. 2 shows the difference of the sensor response between GGA and AWW solution without photocatalytic pretreatment.
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TiO2±AWW solutions were illuminated from 0 to 5 min by black-light tube at room temperature. The current output showed the tendency to increase with increasing irradiation time. This tendency would resulted from the degradation of organic compounds by HO , R generated with hole trapped in VB [36], and these organic compounds would fast assimilate through the bio®lm. The slops at the irradiation time 3, 4 and 5 min at BOD of 4 mg l 1 were 0.209, 0.334 and 0.281, respectively. This gave the best result at irradiation time 4 min. Relative standard deviation of the sensor response at this irradiation time was 8.3%. The photocatalysis using TiO2 can be summarized by the following mechanism [28,36]. Fig. 2. Comparison of the sensor response with GGA and AWW ((&) GGA; (*) AWW).
GGA has been used as a standard solution for the calibration of the conventional BOD sensor and BOD5 method [9,35]. As shown in Fig. 2, current difference of the sensor of AWW and GGA at BOD of 0.5 mg l 1 was 0.06 and 0.22 mA, respectively. This result demonstrates that GGA may not be suitable as a standard solution for river waters, which contain recalcitrant organic compounds such as humic acid, lignin, etc. because recalcitrant organic compounds gave the low response to the sensor assimilating uneasily by microorganisms. The sensor responses with GGA were higher than those of AWW to each BOD value, because GGA was readily degraded by microorganisms. Therefore, recalcitrant organic compounds were pretreated by photocatalysis to be rapidly assimilated through microorganisms like GGA. 3.2. Effect of irradiation times
TiO2 hv ! e h e O2 ! O2 h OH ! HO ;
h R ! R
Hydroxyl radicals are formed on the surface of TiO2 by the reaction of h in VB, with adsorbed H2O, hydroxide groups, etc. The hydroxyl radicals decompose an organic compounds as very strong and unselective oxidants. TOC removal rates were also investigated against irradiation time. TOC removal rate was TOC with photocatalysis to TOC without. Although TOC removal rates were very low, the values increased with increasing irradiation time. TOC removal rate at irradiation time 4 min was about 4%. This behavior displayed the similar tendency like the sensor response which increased with increasing irradiation time. The response time of the BOD sensor was less than 10 min. 3.3. Effect of TiO2 concentration
All the BOD measurements in the following study were carried out using the AWW solutions. Fig. 3 shows the sensor response with irradiation times on TiO2 in AWW solutions. The amount of TiO2 granule was 1% (w/v). The
Fig. 4 shows the effect of the TiO2 concentration on the current output. The TiO2 concentration was investigated from 0 to 3% (w/v), and the utilized AWW solutions were adjusted to pH 7.0. The irradiation time of the test solutions was 4 min. The current output was hardly changed until
Fig. 3. Effect of irradiation times on the sensor response in AWW solutions. Amount of TiO2: 1% (w/v), pH: 7.0, T: room temperature (( ) irradiation time 0 min; (&) 1 min; (5) 2 min; (*) 3 min; (~) 4 min; ( ) 5 min).
Fig. 4. Effect of titanium dioxide concentration on the sensor response in AWW solutions. Irradiation times: 4 min, pH: 7.0, T: room temperature.
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0.5%, but increased to approximately double at 1% and then reached almost constant more than 1% TiO2. The TiO2 of 1% in investigated range would be quantity corresponding to the almost complete absorption of photons. UV absorbency spectra of AWW solutions before and after photocatalysis would be the similar tendency to that treated by ozone as described in a early paper [37]. 3.4. Effect of pH The effect of the initial pH on the photocatalytic degradation of the AWW solution was examined at four different pH values in range of pH 5.0±8.0. The photocatalized AWW solutions were injected into phosphate buffer (pH 7.0) with the BOD sensor. The effectivity of the photocatalytic degradation was characterized by the sensor response. The results of these were shown in Fig. 5. The sensor response is hardly changed to pH 6.0, but increased with increasing pH over pH 6.0. It is well known that the surfaces of metal oxides in aqueous solution are covered with hydroxyl groups [38]. Surface groups of a metal oxide are amphoteric and the surface acid±base equilibria are known as follows: BTiOH2 $ BTiOH H
Fig. 6. Comparison of BOD values evaluated by the sensor with those determined by the BOD5 method to various river waters ((&) BOD values evaluated by the sensor before photocatalysis; (*) BOD values evaluated by the sensor after photocatalysis; (- - -) calculated line of slope 1).
VB [36]. Therefore, the sensor responses would be high over pH 6 where many hydroxyl radicals was generated. The test solution, AWW was mixing compounds, which were humic acid, lignin, gum arabic, tannic acid, and LAS. Although AWW contained humic acid and tannic acid, this solution may be signi®cant photocatalytic activity at high pH.
BTiOH $ BTiO H
3.5. Analysis of river waters using photocatalysis
where BTiOH represents the ``titanol'' surface group. The neutral surface species, BTiOH is predominant over a broad range of pH 3±10. The pH of zero point of charge of TiO2, pHzpc, was known 6.1. At below the pHzpc, the TiO2 surface becomes a net positive charge because of the increasing fraction of total surface sites present as BTiOH2. On the other hand, at above pHzpc the surface has a net negative charge because of a significant fraction of total surface sites present as BTiO . Humic acid had high photocatalytic degradation rate at low pH, and tannic acid also would have the similar [39]. As shown in Fig. 5, however, the sensor responses increased with pH. The hydroxyl radicals which are very strong oxidant yield with hydroxide ions in the reaction of h of
The biosensor was used to evaluate the BOD of various river waters before and after photocatalysis. The results were compared with the BOD5 determined by the conventional method in Fig. 6. The pH of the samples pretreated by photocatalysis was adjusted to 7.0 in 10 mM phosphate buffer, the sensor response being the optimal at this pH [10]. The BOD values using the biosensor before photocatalysis gave less than those of the conventional BOD5. On the other hand, the values estimated after photocatalysis were almost the same to the BOD5. As shown in Fig. 6, fairly good correlation between the sensor method after photocatalysis and the conventional method was obtained for test samples. The BOD values (r 2 0:966) estimated after photocatalysis were higher than those (r 2 0:901) before photocatalysis. The results were roughly the same BOD values obtained by preozonation, as described in a early paper [37]. It showed that the sensor estimation for the BOD of river waters pretreated by photocatalysis was superior to that before photocatalysis. 4. Conclusions
Fig. 5. Effect of pH on the sensor response in AWW solutions. Amount of TiO2: 1% (w/v), irradiation times: 4 min, T: room temperature.
The photocatalytic pretreatment of the BOD sensor was used to determine low BOD level in river waters containing recalcitrant organic compounds. This pretreatment shows the advantages as following: (a) the determination of uneasily biodegradable recalcitrant organic compounds by microorganisms; (b) the estimation of low BOD levels like river waters; (c) a shorter determining time in comparison
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with the conventional sensor; (d) the prevention of choking with mold, etc. in tube due to sterilization of a test samples by illuminating UV; (e) semi-permanent utilization of titanium dioxide; (f) the degradation of a hazardous organic compounds by photocatalysis and no produce hazardous byproducts [39,40]. The free radicals (by reaction of h in VB) on TiO2 surface by photocatalysis worked as a very strong oxidant to destroy organic compounds. As shown in Fig. 3, the optimal irradiation time was 4 min. The sensor response at that irradiation time was similar to that of GGA in Fig. 2. This result demonstrated that AWW treated by photocatalysis was the same assimilability as GGA through the bio®lm. At 1% (w/v), TiO2 concentrations gave the optimal response to the sensor. The evaluation of BOD using photocatalytic pretreatment was improved in comparison with the conventional sensor method, and also shown fairly better results. The BOD sensor using TiO2 photocatalysis and the BOD5 method shown well agreement.
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1995. Her interests are bio- or chemical-sensors for monitoring environmental pollutants such as highly toxic compounds (e.g. cyanide) or endocrine disrupting.
Biographies
Kazunori Ikebukuro received PhD in Biosensors from the University of Tokyo in 1996. He is a lecturer at RCAST, the University of Tokyo and doing research on biosensors for environmental monitoring and DNA detection.
Gab-Joo Chee graduated with a BE in Chemical Engineering from Soong Sil University, Seoul, Korea and MS in Chemistry and Biotechnology from the University of Tokyo, Japan. He is currently studying for his PhD degree at the University of Tokyo. Yoko Nomura is a research associate of RCST, The University of Tokyo from 1998. She was received PhD from Japan Women's University in
Isao Karube obtained his PhD in Bioelectronics from Tokyo Institute of Technology in 1972. He is a Professor and Director in Center for Collaborative Research, the University of Tokyo. He works in the area of biosensors, bioelectronics and biotechnology.
Biosensor for the evaluation of biochemical oxygen demand using photocatalytic pretreatment Gab-Joo Chee, Yoko Nomura, Kazunori Ikebukuro, Isao Karube* Research Center for Advanced Science and Technology, University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904, Japan Received 15 May 2000; received in revised form 7 March 2001; accepted 8 May 2001
Abstract A highly sensitive biochemical oxygen demand (BOD) sensor using photocatalytic pretreatment was developed to evaluate low BOD levels in river waters. The photocatalytic oxidation was carried out in a commercial TiO2 as a semiconductor and a 6 W black-light tube as light sources. The photocatalytic oxidation was investigated about the effects of irradiation times, TiO2 concentrations, and pH on the sensor response. The optimal irradiation time was 4 min. TiO2 concentrations gave the optimum response to the BOD sensor at 1% (w/v). The sensor response was increased with increasing pH. The sensor responses obtained by photocatalysis to river samples were higher than those obtained without photocatalysis. A fairly good correlation between the values obtained by the photocatalytic pretreatment method and BOD5 was acquired. # 2001 Elsevier Science B.V. All rights reserved. Keywords: BOD sensor; Biosensor; Pseudomonas putida; Arti®cial wastewater; Photocatalysis; TiO2; UV; Pretreatment
1. Introduction The enzyme-based sensor for the determination of glucose levels was developed the ®rst by Clark and Lyons [1]. Since then, various biosensors using both biomaterials, such as microorganisms, enzymes and physico-chemical transducers, such as optical, electrochemical have been developed in many ®elds of analysis [2±7]. Biosensor in environmental analysis was remarkably developed, and a biochemical oxygen demand (BOD) sensor is one of the most typical sensors in this ®eld [8]. BOD is the amount of oxygen requirement of biodegrading organic compounds in aqueous solution. Up to date, the BOD sensors have employed glucose and glutamic acid (GGA) solutions which are used as standards for its calibration, according to the Japanese Industrial Standard (JIS) [9]. The GGA solutions should be suitable to evaluate industrial wastewaters, and not river waters which contain humic acid, lignin and tannic acid, etc. Therefore, as a standard solution for the estimation of BOD in river waters, arti®cial wastewater (AWW) was made from these recalcitrant organic compounds such as humic acid, lignin and tannic acid. The BOD sensor with Pseudomonas putida SG10 was developed to evaluate low BOD * Corresponding author. Tel.: 81-3-5452-5221; fax: 81-3-5452-5447. E-mail address: [email protected] (I. Karube).
values such as river waters [10]. In river waters analysis, however, this sensor often showed low values compared with BOD5 as well as other BOD sensors [11±15]. Such low BOD values would show that these recalcitrant organics in river waters are uneasily assimilable to the immobilized microorganism in such a short measuring times. These values would be increased by performing a pretreatment of the sample by using titanium dioxide-based photocatalysis. In 1972, Fujishima and Honda discovered the photocatalytic splitting of water which could be decomposed into hydrogen and oxygen over an illuminated titanium dioxide semiconductor electrode [16]. With the help of this event, photoelectrochemistry has expanded into a formidable ®eld encompassing solar energy conversion [17], photocatalysis [18±20], decomposition of agrochemical [21,22], air and water puri®cation [23±27]. In the photochemical oxidation method, short wavelength UV-C (<280 nm) light is commonly employed [28]. On the other hand, photocatalytic experiments with titanium dioxide usually use long wavelength UV-A (>315 nm) light [29±31]. Fig. 1 shows a general schematic representation of the degradation of organic compound in aqueous solution by electron-hole (e ±h) formation at the surface of an illuminated titanium dioxide particle [32]. When titanium dioxide is illuminated with band gap energy of greater than 3.2 eV (380 nm), a photon excites an electron from the
0925-4005/01/$ ± see front matter # 2001 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 5 - 4 0 0 5 ( 0 1 ) 0 0 8 8 3 - 8
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G.-J. Chee et al. / Sensors and Actuators B 80 (2001) 15±20
Fig. 1. Schematic diagram of simplified mechanism for the photoactivation of a titanium dioxide particle.
valence band (VB) to the conduction band (CB) and leaves an electronic vacancy commonly referred to as a hole in the VB. The electron in the CB can be transferred to adsorbed H, O2 or the chlorinated pollutant initiating various reactions. The hole in the VB can react with surface-bound water, hydroxide groups, anions and organic substrate. Therefore, organic compounds in aqueous solution are decomposed to the formation of lower molecular weight compounds by photocatalytic oxidation. The results would increase the sensitivity of the BOD sensor, because organic compounds of lower molecular weight are more readily biodegradable through the bio®lm. This paper describes the comparison of the sensor response before and after photocatalytic oxidation, and the rising of the sensitivity of the BOD sensor. 2. Experimental 2.1. Preparation of biofilm and the BOD sensor Cell culture was carried out for P. putida in 200 ml of sterile culture medium containing nutrient broth (1.0 g l 1) and AWW (per liter: nitrohumic acid, 84.92 mg; gum arabic, 93.90 mg; sodium ligninsulfonate, 48.54 mg; tannic acid, 83.50 mg; linear alkylbenzene sulfonate, 18.84 mg) at pH 7.0. The culture broth was kept in a rotating shaker under aerobic conditions at 308C. The cells were harvested at the end of the exponential growth phase, about 24 h after seeding the culture medium. This was to ensure consistence in preparing the bio®lm for the BOD sensor fabrication. The cells were washed and centrifuged at 7000 rpm for 10 min, and were subsequently resuspended in 10 mM phosphate buffer (pH 7.0). The bio®lm for the BOD sensor was prepared using an aspirator connected to a syringe ®lter holder. A porous cellulose nitrate membrane (20 mm diameter, 0.45 mm pore size, Advantec, Japan) was carefully sandwiched in a syringe ®lter holder. Calculated amounts of the pure culture broth was dropped on the cellulose nitrate membrane. The amounts were calculated to give a maximal response to
the sensor [10]. The bio®lm was washed twice with 10 mM phosphate buffer solution using an aspirator. The bio®lm was placed on the top of the Te¯on gas membrane covering an oxygen probe (BO-U1, Able, Japan), and then was tightly secured using 200 mesh nylon and an O-ring. The remnant culture broth was stored at 48C in the refrigerator before preparing a new membrane. An oxygen probe was linked to both a digital multimeter (model TR6840, TakedaRiken, Japan) and an electronic recorder (model EPR-200A, TOA Electronics, Japan) as described in an early paper [10]. The BOD sensor was kept in 10 mM phosphate buffer solution at room temperature when not in use. 2.2. Experimental procedure As a standard solution for the calibration of the BOD sensor, AWW (16.485 mg l 1, 3.77 mg l 1 BOD) was employed to substitute for GGA as described in an early paper [10]. AWW solution was prepared using arbitrarily selected recalcitrant organic compounds in river waters and secondary ef¯uents [33,34]. The BOD sensor was inserted into the detection chamber containing 50 ml of 10 mM phosphate buffer (pH 7.0) solution. Agitation and oxygen saturation of the test solution was ensured by stirring continuously by a magnetic bar. The detection chamber was maintained at 308C by circulating water at constant temperature through the water jacket. The current response of the BOD sensor in 10 mM phosphate buffer solution was monitored on a digital multimeter and an electronic poly recorder. When the current reached a steadystate value, a calculated amounts of AWW were added to the phosphate buffer solution to give various BOD5 values. A commercial TiO2 (anatase ST-A01, Ishihara Sangyo Co., Japan) was used in the this study. The TiO2 granule diameter was 2 mm. The various amounts of TiO2 granule were added in 25 ml AWW solution and magnetically stirred. The TiO2±AWW solution was then illuminated with a 6 W black-light ¯uorescent tube (Ushio Co., Japan) as UV light sources. The illuminated AWW solution was injected to 50 ml phosphate buffer solution by micro-syringe pipette. The degradation of AWW solution by photocatalysis also was investigated as the total organic carbon (TOC) using a TOC analyzer (Shimadzu 5000, Japan). The pH value of the solutions was adjusted with NaOH. The conventional BOD5 was determined by the standard method according to the JIS [35]. 3. Results and discussion 3.1. Comparison of the sensor response with GGA and AWW Fig. 2 shows the difference of the sensor response between GGA and AWW solution without photocatalytic pretreatment.
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TiO2±AWW solutions were illuminated from 0 to 5 min by black-light tube at room temperature. The current output showed the tendency to increase with increasing irradiation time. This tendency would resulted from the degradation of organic compounds by HO , R generated with hole trapped in VB [36], and these organic compounds would fast assimilate through the bio®lm. The slops at the irradiation time 3, 4 and 5 min at BOD of 4 mg l 1 were 0.209, 0.334 and 0.281, respectively. This gave the best result at irradiation time 4 min. Relative standard deviation of the sensor response at this irradiation time was 8.3%. The photocatalysis using TiO2 can be summarized by the following mechanism [28,36]. Fig. 2. Comparison of the sensor response with GGA and AWW ((&) GGA; (*) AWW).
GGA has been used as a standard solution for the calibration of the conventional BOD sensor and BOD5 method [9,35]. As shown in Fig. 2, current difference of the sensor of AWW and GGA at BOD of 0.5 mg l 1 was 0.06 and 0.22 mA, respectively. This result demonstrates that GGA may not be suitable as a standard solution for river waters, which contain recalcitrant organic compounds such as humic acid, lignin, etc. because recalcitrant organic compounds gave the low response to the sensor assimilating uneasily by microorganisms. The sensor responses with GGA were higher than those of AWW to each BOD value, because GGA was readily degraded by microorganisms. Therefore, recalcitrant organic compounds were pretreated by photocatalysis to be rapidly assimilated through microorganisms like GGA. 3.2. Effect of irradiation times
TiO2 hv ! e h e O2 ! O2 h OH ! HO ;
h R ! R
Hydroxyl radicals are formed on the surface of TiO2 by the reaction of h in VB, with adsorbed H2O, hydroxide groups, etc. The hydroxyl radicals decompose an organic compounds as very strong and unselective oxidants. TOC removal rates were also investigated against irradiation time. TOC removal rate was TOC with photocatalysis to TOC without. Although TOC removal rates were very low, the values increased with increasing irradiation time. TOC removal rate at irradiation time 4 min was about 4%. This behavior displayed the similar tendency like the sensor response which increased with increasing irradiation time. The response time of the BOD sensor was less than 10 min. 3.3. Effect of TiO2 concentration
All the BOD measurements in the following study were carried out using the AWW solutions. Fig. 3 shows the sensor response with irradiation times on TiO2 in AWW solutions. The amount of TiO2 granule was 1% (w/v). The
Fig. 4 shows the effect of the TiO2 concentration on the current output. The TiO2 concentration was investigated from 0 to 3% (w/v), and the utilized AWW solutions were adjusted to pH 7.0. The irradiation time of the test solutions was 4 min. The current output was hardly changed until
Fig. 3. Effect of irradiation times on the sensor response in AWW solutions. Amount of TiO2: 1% (w/v), pH: 7.0, T: room temperature (( ) irradiation time 0 min; (&) 1 min; (5) 2 min; (*) 3 min; (~) 4 min; ( ) 5 min).
Fig. 4. Effect of titanium dioxide concentration on the sensor response in AWW solutions. Irradiation times: 4 min, pH: 7.0, T: room temperature.
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0.5%, but increased to approximately double at 1% and then reached almost constant more than 1% TiO2. The TiO2 of 1% in investigated range would be quantity corresponding to the almost complete absorption of photons. UV absorbency spectra of AWW solutions before and after photocatalysis would be the similar tendency to that treated by ozone as described in a early paper [37]. 3.4. Effect of pH The effect of the initial pH on the photocatalytic degradation of the AWW solution was examined at four different pH values in range of pH 5.0±8.0. The photocatalized AWW solutions were injected into phosphate buffer (pH 7.0) with the BOD sensor. The effectivity of the photocatalytic degradation was characterized by the sensor response. The results of these were shown in Fig. 5. The sensor response is hardly changed to pH 6.0, but increased with increasing pH over pH 6.0. It is well known that the surfaces of metal oxides in aqueous solution are covered with hydroxyl groups [38]. Surface groups of a metal oxide are amphoteric and the surface acid±base equilibria are known as follows: BTiOH2 $ BTiOH H
Fig. 6. Comparison of BOD values evaluated by the sensor with those determined by the BOD5 method to various river waters ((&) BOD values evaluated by the sensor before photocatalysis; (*) BOD values evaluated by the sensor after photocatalysis; (- - -) calculated line of slope 1).
VB [36]. Therefore, the sensor responses would be high over pH 6 where many hydroxyl radicals was generated. The test solution, AWW was mixing compounds, which were humic acid, lignin, gum arabic, tannic acid, and LAS. Although AWW contained humic acid and tannic acid, this solution may be signi®cant photocatalytic activity at high pH.
BTiOH $ BTiO H
3.5. Analysis of river waters using photocatalysis
where BTiOH represents the ``titanol'' surface group. The neutral surface species, BTiOH is predominant over a broad range of pH 3±10. The pH of zero point of charge of TiO2, pHzpc, was known 6.1. At below the pHzpc, the TiO2 surface becomes a net positive charge because of the increasing fraction of total surface sites present as BTiOH2. On the other hand, at above pHzpc the surface has a net negative charge because of a significant fraction of total surface sites present as BTiO . Humic acid had high photocatalytic degradation rate at low pH, and tannic acid also would have the similar [39]. As shown in Fig. 5, however, the sensor responses increased with pH. The hydroxyl radicals which are very strong oxidant yield with hydroxide ions in the reaction of h of
The biosensor was used to evaluate the BOD of various river waters before and after photocatalysis. The results were compared with the BOD5 determined by the conventional method in Fig. 6. The pH of the samples pretreated by photocatalysis was adjusted to 7.0 in 10 mM phosphate buffer, the sensor response being the optimal at this pH [10]. The BOD values using the biosensor before photocatalysis gave less than those of the conventional BOD5. On the other hand, the values estimated after photocatalysis were almost the same to the BOD5. As shown in Fig. 6, fairly good correlation between the sensor method after photocatalysis and the conventional method was obtained for test samples. The BOD values (r 2 0:966) estimated after photocatalysis were higher than those (r 2 0:901) before photocatalysis. The results were roughly the same BOD values obtained by preozonation, as described in a early paper [37]. It showed that the sensor estimation for the BOD of river waters pretreated by photocatalysis was superior to that before photocatalysis. 4. Conclusions
Fig. 5. Effect of pH on the sensor response in AWW solutions. Amount of TiO2: 1% (w/v), irradiation times: 4 min, T: room temperature.
The photocatalytic pretreatment of the BOD sensor was used to determine low BOD level in river waters containing recalcitrant organic compounds. This pretreatment shows the advantages as following: (a) the determination of uneasily biodegradable recalcitrant organic compounds by microorganisms; (b) the estimation of low BOD levels like river waters; (c) a shorter determining time in comparison
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with the conventional sensor; (d) the prevention of choking with mold, etc. in tube due to sterilization of a test samples by illuminating UV; (e) semi-permanent utilization of titanium dioxide; (f) the degradation of a hazardous organic compounds by photocatalysis and no produce hazardous byproducts [39,40]. The free radicals (by reaction of h in VB) on TiO2 surface by photocatalysis worked as a very strong oxidant to destroy organic compounds. As shown in Fig. 3, the optimal irradiation time was 4 min. The sensor response at that irradiation time was similar to that of GGA in Fig. 2. This result demonstrated that AWW treated by photocatalysis was the same assimilability as GGA through the bio®lm. At 1% (w/v), TiO2 concentrations gave the optimal response to the sensor. The evaluation of BOD using photocatalytic pretreatment was improved in comparison with the conventional sensor method, and also shown fairly better results. The BOD sensor using TiO2 photocatalysis and the BOD5 method shown well agreement.
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1995. Her interests are bio- or chemical-sensors for monitoring environmental pollutants such as highly toxic compounds (e.g. cyanide) or endocrine disrupting.
Biographies
Kazunori Ikebukuro received PhD in Biosensors from the University of Tokyo in 1996. He is a lecturer at RCAST, the University of Tokyo and doing research on biosensors for environmental monitoring and DNA detection.
Gab-Joo Chee graduated with a BE in Chemical Engineering from Soong Sil University, Seoul, Korea and MS in Chemistry and Biotechnology from the University of Tokyo, Japan. He is currently studying for his PhD degree at the University of Tokyo. Yoko Nomura is a research associate of RCST, The University of Tokyo from 1998. She was received PhD from Japan Women's University in
Isao Karube obtained his PhD in Bioelectronics from Tokyo Institute of Technology in 1972. He is a Professor and Director in Center for Collaborative Research, the University of Tokyo. He works in the area of biosensors, bioelectronics and biotechnology.