**3.3 Subsequent development of BOD biosensors and a new insight into the future perspectives of the BOD biosensor fields**

Even after our studies on the BOD biosensors have been reported, the studies on next-generation BOD biosensors are ongoing. In this section, the progress of our studies on the BOD biosensors performed by other groups is introduced.

In our studies that the author principally conducted, the most practical BOD biosensor or measurement method was the BODRCI-*Sc* measurement method. Unfortunately, we did not achieve the instrumentation. However, in 2021, Azevedo et al. developed an automated *S. cerevisiae*-based spectrophotometric instrument using methylene blue (MB) dye as a RCI, although this instrument was not for BOD measurement, but also possible to measure BOD [86]. The instrument consists of a sequential injection analysis (SIA) flow system, which enables high throughput measurements with small quantities of reagent consumption. On the other hand, DCIP has been used not only as a RCI but also as a redox mediator for electrochemical BODSM biosensors. For example, Reshetilov et al. developed several types of electrochemical microbial biosensors using DCIP as the redox mediator [87]. In 2017, Niyomdecha et al. fabricated a practical electrochemical FIA type microbial biosensor system for highly precise BOD measurements [88]. They used MB dye as a redox mediator and successfully obtained highly sensitive results of the detection limit at 0.1 mg O2/L. This study demonstrated that highly reproducible results provided highly precise results, enabling highly sensitive measurements.

As recent progress of the DM-type microbial biosensors, several groups were reported. For example, in 2017, Gao et al. reported a DM-type *S. cerevisiae* biosensor for toxicity of wastewater and co-immobilized *S. cerevisiae*, menadione, and ferricyanide onto a glassy carbon electrode by electrodeposition of a chitosan hydrogel and nanocrystalline diamond particles [89]. In this study, it seems that *S. cerevisiae* and two mediators were successfully immobilized into the matrix without leakage and made it possible to distribute toxic matters into the matrix. However, this biosensor has a complication of the fabrication, therefore reproducible fabrication of the biosensor electrodes might be difficult. In 2017, Zaitseva et al. developed a batch-type BODDM biosensor employing yeast *Debaryomyces hansenii* (*Dh*) and ferrocene-MB mediator system [90]. The BODDM-*Dh* biosensor had practical characteristics of a short measurement time of 10 min, a minimum limit of determination of 2.5 mg O2/L, and long-time

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

stability of 43 days. Then, they assumed as a new insight that lipophilic mediators, such as MB and DCIP, are capable of penetrating inside the microbial cell through the lipid membrane, taking electrons, and passing them on to the electrode immobilized ferrocene. If their assumption is true, new future perspectives in microbial electron transfer techniques can be opened.

As recent progress of the BODCL-*Sc* biosensor, in 2018, Costa et al. developed a practical SIA microfluidic system of a BODCL-*Sc* biosensor [91]. The SIA system was programmed to precisely control the flow of multiple channels in an accurate time, making it easier to obtain highly reproducible results. In addition, the incubation time under constant temperature for *S. cerevisiae* to assimilate the organic matters in the sample solution was also precisely controlled by setting the stopped period. If hydrogen peroxide is not used, highly sensitive measurements might be obtained by reduction of the background signal, and if the mixed solution could be agitated during incubation, both reaction efficiency and reproducibility might simultaneously be improved.

As the other progress, MFC biosensors have many potentials not only as BOD biosensors but also as self-powered devices for biosensors [92]. As a new insight into the future perspectives of the BOD biosensor fields, the author have one idea that is our past application study on a damped glycolytic oscillation induced in living yeast cells for toxicity assays (**Figure 4**) [93, 94]. We extracted six indexes from the wave shape and observed that these indexes were changed depending on both toxicity and the concentration of each toxic matter. By applying this principle, wave changes depending on both bioavailability and the concentration of the organic matters dissolved in a sample solution might be determined. In fact, excellent correlation was obtained between one of the indexes and the concentration of glucose (*r* = 0.9989, 2.5 μM–0.25 M, 12 points).

#### **Figure 4.**

*(a) Typical damped glycolytic oscillation induced in living yeast cells. (b) Six indexes in an oscillation wave shape. (c) Response to glucose. These were permitted from Springer Nature [93].*

Glycolysis is an important system that plays a central role in metabolism [14]. If this metabolic oscillation is applied to environmental monitoring, it may be possible to continuously sense the state of the aquatic environment perceived by organisms living in aquatic ecosystems in real-time. By doing so, for example, not only the index of BOD concentration but also the nutritional status of water quality and the substantial effect of heavy metals and antibiotics can be understood through the metabolic status of microbes employed for biosensing.
