*3.2.1 Development of early time BOD biosensors and accomplishment by Dr. Isao Karube in this field*

As described above, the BOD5 method is time-consuming and requires tedious operations. For example, it is not possible to detect the abnormality of wastewater before and after the treatment because it takes 5 days to obtain the measurement result. In other words, even if abnormal wastewater flows into the treatment facility or is not sufficiently treated at the treatment facility, it can be detected only after the wastewater has flowed out to the environmental water. In addition, the tedious operations of the BOD5 method also make it difficult to make accurate and accurate measurements.

In 1977, Dr. Karube studied a practical microbial biosensor for BOD [58]. The key technique was the immobilization of microbes to a thin collagen membrane. The microbial membrane was put onto the surface of a DO electrode. By the addition of a sample solution into a batch system, microbial respiration was activated by the decomposition of organic matters, and the degree of DO consumption by the microbes was determined by the DO electrode. The microbial BOD biosensor indicating DO consumption (BODDO) could successfully determine the BOD value at drastically shortened incubation and measurement times (ca. 30 minutes). By the study, the possibilities for solving the problems of wastewater control were enhanced.

In 1979, Hikuma and Karube et al. developed a flow system of the BODDO biosensor [59]. In the study, omnivorous yeast *Tricbosporon cutaneum* (*Tc*), as a practical microbe, was used in this BODDO-*Tc* biosensor. The microbes were immobilized onto a microporous membrane and attached to the surface of the DO electrode. Based on the study, a flow-type BODDO-*Tc* biosensor was available for practical applications.

Both the desktop-type for rapid measurements and the installation-type for continuous monitoring were sold from the Central Kagaku Co. in 1983. These BODDO-*Tc* biosensor instruments enabled wastewater control by both real-time measurement and continuous monitoring at sewage plants and factories. The BODDO-*Tc* biosensor was established as one of the JIS methods (JIS K 3602) in 1990 [60]. Since then, the BODDO-*Tc* biosensor instrument has also been used in educational settings as a science teaching material [61].

Since the first microbial biosensor was reported, many kinds of microbial biosensors have been studied for not only environmental applications but also food applications, including fermentation. These studies on both environments and foods by microbial biosensing methods were reviewed [3, 10, 15, 62].

Dr. I. Karube was widely studied all-fields of biosensor development as one of the leading scientists in the world. His notable study on biosensor development was summarized in the review [10] and the detailed history of his study on microbial biosensor development was described in one chapter of Encyclopedia [57].

The notable studies on the microbial BOD biosensor development performed by Karube et al. are briefly described as follows. As the study on the BOD biosensors, a microbial fuel cell (MFC) type biosensor has also been developed. However, the MFC biosensor at that time used expensive materials, easily deteriorated electrodes, anaerobes, etc., and had low cathode reaction efficiency due to low electron transfer from the anaerobes to the cathode. In addition, a flow-type cathode chamber of the MFC biosensor has a low exchange efficiency of sample solution, making it difficult to repeat and rapid BODMFC measurements.

After two types of BODDO-*Tc* biosensor instruments were practically used, the other types of BOD biosensors have been widely studied and developed. One of the practical studies was the development of a bioluminescence BOD biosensor using a luminous bacterium *Photobacterium phosphoreum* (*Pph*) [63]. In 1993, Hyun et al. studied a BODBL-*Pph* biosensor; however, the emission intensity of BL released by *P. phosphoreum* decreased with each measurement. Thus, a reagent-type BODBL-*Pph* biosensor instrument was practically used by Tamiya et al.

Another practical study was the development of portable type instruments for on-site monitoring. To realize the on-site monitoring, it was required to stop using air-supply equipment, to reduce the size of measurement devices, to miniaturize and single-use biosensors, to employ omnivore and vital microbes, etc. Dr. Hiroaki Suzuki has been studying the miniaturization of the biosensors to be used as disposable sensor chips [64]. In 1996, Suzuki and Yang et al. studied BODDO-*Tc* biosensors for on-site monitoring and developed a low-cost paper-based DO electrode [65] and a disposable BODDO-*Tc* biosensor chip [66]. However, in general, the BODDO-*Tc* biosensors were difficult to accurately measure the high BOD value of a sample solution, because the solubility of oxygen into water is limited (8.11 mg O2/L at 25°C and 1 atm).

To solve the problem, in 2000, Yoshida et al. studied two types of BOD biosensor principles for on-site monitoring. One was a single mediator (SM) type of an electrochemical BOD biosensor [67]. In the study, omnivorous bacteria *Pseudomonas fluorescens* (*Pf*) was isolated from a sewage plant. By using potassium ferricyanide as a highly soluble mediator in water (460 g/L), a BODSM-*Pf* biosensor that does not require air-supply equipment was developed. The principle was applied to a portable type instrument and a disposable BODSM-*Pf* biosensor chip for on-site monitoring [68]. As a result, a wide linear range of calibration (15 and 260 mg O2/L) was obtained. Another type was an optical BOD biosensor using the redox electron acceptor 2,6-dichlorophenolindophenol (DCIP) sodium salt as a redox color indicator

### *Developmental Studies on Practical Enzymatic Phosphate Ion Biosensors and Microbial BOD… DOI: http://dx.doi.org/10.5772/intechopen.104377*

(RCI). Before the development of a portable type optical instrument, an absorptiometric high-throughput BODRCI-*Pf* measurement method was studied using a microplate reader, which is able to measure 96 samples simultaneously [69]. Based on the principle, the portable type optical BODRCI-*Pf* biosensor instrument was constructed using three pairs of light-emitting diodes (LEDs; 600 nm) and silicon photodiodes (Si-PDs), and a transparent disposable chip containing three biosensing spots [70]. As a result, a linear relationship was observed below 176 mg O2/L, and the detection limit was 14 mg O2/L (*n* = 6, RSD = 10.3%). An excellent correlation coefficient (*r* 2 = 0.989) was obtained after 600 seconds incubation time. By the study of Yoshida et al., two portable types of the practical BOD*Pf* biosensor instruments were developed using two types of redox electron acceptors.

On the other hand, in 1999, Chee et al. studied highly sensitive BODDO biosensors for low BOD measurements [71]. The background of this study was that the spread of sewage treatment facilities improved the water quality of rivers at that time, and it became necessary to measure low BOD including persistent organic matters in the sample solution. To properly measure the river water quality at that time, isolation of the microbe that biodegrades persistent organic matters was needed, and they isolated *Pseudomonas putida* (*Ppu*) from a sewage plant. To enhance the biodegradability of the persistent organic matters by *P. putida*, they examined pretreatment methods of the sample solution and finally established the photocatalytic UV-TiO2 method [72]. As the result, a practical linear range of calibration (0.5 and 8 mg O2/L) was obtained using artificial wastewater containing persistent organic matters.

The studies on the practical BOD biosensor were successfully performed by Dr. Karube et al. and the practical instruments including the potable instruments for on-site monitoring were developed.

#### *3.2.2 Development of BOD biosensors that the author principally conducted*

After these excellent studies on the practical BOD biosensors by Dr. Karube et al., the author explored other possibilities of BOD biosensors with better functionality, for example, improvements of (1) detection limit, (2) signal repeatability of the microbial biosensor, and (3) suitability of microbe used.


Then, the author tried to satisfy these requirements by employing the absorptiometric BODRCI measurement method and a temperature-controlled three-cuvette-stir system [73, 74]. As the usable (easily available, omnivorous, and vital) microbe, Baker's dry yeast *Saccharomyces cerevisiae* (*Sc*) was used after liquid culturing. *S. cerevisiae* is budding yeast; therefore, it was suitable for forming uniform suspension in the cuvette. On the other hand, *T. cutaneum*, which is conventionally used for the BOD biosensors and makes flocks by sticking together, was not suitable for this study because it was filamentous fungi. In this method, DCIP was used as RCI of a high absorption coefficient (*epsilon* = 1.45 × 105 cm−1 M−1) to enhance the sensitivity of the absorptiometry. Then, the absorbance of DCIP decreased due to degradation of organic matters by *S. cerevisiae* in the measurement suspensions, and the absorbance change between before and after incubation was measured as the time difference method (**Figure 3**).

In general, the suspension is not suitable for absorptiometry due to the occurrence of light scattering. However, by a combination of the cuvette-stir system and the time difference method, the influences of the light scattering were efficiently canceled and only absorbance change of DCIP was accurately determined. Further, by repeating the exact same measurement operation three times, the fluctuation of the measured value became small, and the reproducibility was improved, so that highly precise measurement became possible. As a result, the significant difference from the blank value became large, and highly sensitive measurement became possible.

BODRCI-*Sc* measurement method: As a result, a calibration curve between 1.1 and 22 mg O2/L (*r* = 0.988, six points, *n* = 3, RSD = 1.77%) was obtained by this highly sensitive BODRCI-*Sc* measurement method when the incubation mixture was incubated for exact 10 minutes at 30°C. Employing salt-tolerant yeast *S. cerevisiae*

#### **Figure 3.**

*A key principle for the simple absorptiometric measurement method using redox color indicator [73].*

*Developmental Studies on Practical Enzymatic Phosphate Ion Biosensors and Microbial BOD… DOI: http://dx.doi.org/10.5772/intechopen.104377*

ARIF KD-003 (*Sc*II), a BODRCI-*Sc*II measurement method was also studied for seawater [75]. In these studies, the highly sensitive BODRCI-*Sc* measurement method was developed by enhancing the repeatability of the measurements and by employing the usable yeast. From the future perspective of this study, the BODRCI-*Sc* measurement method might be improved by applying it to an absorptiometric-FIA system.

BODDM-*Sc* biosensor: As the other application of *S. cerevisiae* to a BOD biosensor, the author also studied a mediator type BOD biosensor. By applying a double mediator (DM) system coupled with ferricyanide and a lipophilic mediator, menadione, electrochemical signals were obtained from the eukaryote *S. cerevisiae* cells [76]. For practical use, the author designed a package-free disposable microbial biosensor chip containing living microbial cells [77] and the biosensor chip was applied to a DM-type BODDM-*Sc* biosensor [78]. Under the optimal conditions, a calibration curve was obtained with a practical range of 6.6–220 mg O2/L (five points, *n* = 3, RSD = 6.6%). Thus, the BODDM-*Sc* biosensor was developed as the third generation of the BOD biosensor [3, 10].

BODCL-*Sc* measurement method: By applying the principle of the BODDM biosensor, the author next studied a chemiluminescence BOD (BODCL-*Sc*) measurement method. Because Yamashoji et al. in 2004 already established luminol CL assay for the viable microbial detection method using ferricyanide and menadione [79]. Their principle was based on hydrogen peroxide (H2O2) determination produced by the reaction of the viable microbes and menadione. After optimization of the measurement conditions, a practical correlation between BODCL-*Sc* value and amount of organic matters assimilated by *S. cerevisiae* was observed with a range of 11–220 mg O2/L (six points, *n* = 3, RSD = 3.71%) at the incubation time of only 5 minutes [80, 81]. Then, the detection limit was 5.5 mg O2/L.

BODDM:Trinder-*Sc* measurement method: As one of the reagents for H2O2 measurement, there is a modified Trinder's reagent. It is an absorptiometric H2O2 measurement method using peroxidase (POD), 4-aminoantipyrine (4-AA), and *N*-ethyl-*N*-(2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline sodium salt (MAOS). Applying this reagent, we already developed a surface plasmon resonance (SPR) biosensor for H2O2 measurement using a modified Trinder's reagent as a color indicator [82]. This reagent had high selectivity, however, POD was unsuitable for applying to the microbial biosensor due to take cost. On the other hand, H2O2 reacted as the reactant in BODCL-*Sc* method. This meant that H2O2 or active oxygen species might also react as the reactant in BODDM-*Sc* method. To verify the possibility, MAOS and 4-AA, which were used in the modified Trinder's reagent, were added to the principle of the BODDM-*Sc* method. As the result obtained without the use of POD as the biocatalyst, correlations between BODDM:Trinder-*Sc* value and amount of organic matters assimilated by *S. cerevisiae* were observed in several measurement conditions, although further practical examinations are required [83].

In the studies that the author principally conducted, excellent functions in the BOD biosensor were achieved in (1) the practical detection limit, (2) the signal reproducibility, and (3) the suitability of the microbe used. In addition, some principles of both microbial BOD biosensors and measurement methods have been studied for practical use, but none of the studies the author have principally conducted has reached practical use. The most practical BOD measurement method in the studies might be the BODRCI-*Sc* measurement method. The reasons are as follows:


For future study, automatic instrumentation of the BOD biosensor would be required having the features that we obtained. Then, further suitability of the microbe might be needed to be considered, for example, use of thermally killed microbes [84] or cell crushed microbes, or direct use of available dry yeast [85].
