Biochemical Oxygen Demand Biosensor Research Articles

Acceptor properties of "carbon nanotubes-redox-active polymer based on bovine serum albumin modified with ferrocenecarboxaldehyde" composite for creating a BOD biosensor with Blastobotrys adeninivorans BKM Y-2677 yeast.

The possibility of using a composite material based on bovine serum albumin (BSA) covalently bonded with ferrocenecarboxaldehyde and containing carbon nanotubes (CNT) for the immobilization of Blastobotrys adeninivorans BKM Y-2677 (B. adeninivorans) yeast is discussed. The optimal ratio of ferrocenecarboxaldehyde to BSA for the redox-active polymer synthesis is 1:2, since the heterogeneous electron transfer constant is 0.45 ± 0.01s-1. When carbon nanotubes (CNTs) are added to this polymer, the heterogeneous electron transfer constant increases: at a CNT specific density of 2.5µg/mm2, it reaches a maximum value of 0.55 ± 0.01s-1. The addition of CNTs into the conducting system leads to increasing of the rate constant of interaction redox species with B. adeninivorans yeast by an order: the rate constant of interaction between B. adeninivorans yeast and electroactive particles in a redox-active polymer is 0.056 ± 0.005 dm3/g × s and in a composite material based on CNTs is 0.51 ± 0.02 dm3/g × s. The yeast specific density at the electrode of 0.1mg/mm2 and electrolyte pH of 6.2 was chosen as the working value for the receptor system operation. Immobilized in a composite material, yeast oxidizes a wider range of substrates compared with a similar receptor element based on the ferrocene mediator. The biosensors formed on the basis of hybrid polymers have a high sensitivity with a lower limit of determined concentrations of 1.5mg/dm3 with an assay time of 5min and a high correlation (R = 0.9945) with the results of the standard method for determining biochemical oxygen demand (BOD) in nine real surface water samples of the Tula region.

Design, optimization and application of a highly sensitive microbial electrolytic cell-based BOD biosensor

Biochemical oxygen demand (BOD) is an important biochemical indicator for determining the degree of water pollution and guiding the design of wastewater treatment processes. BOD sensors based on microbial electrochemical technology can conduct real-time online monitoring of organic matter and have attracted extensive attention. However, research on microbial electrolytic cell (MEC)-type BOD sensors is at the stage of theoretical exploration. Here, we designed and optimized a highly sensitive MEC-type BOD sensor by screening inoculants, comparing electrode materials, and optimizing the reactor configuration. The results showed that effective means to optimize a BOD sensor for fast activation and sensitive testing included the inoculation of the MEC reactor effluent with large amounts of biomass and highly active bacteria, selection of carbon felt electrodes with excellent adsorption and permeability, miniaturization of the reactor, regulation of suitable electrode spacing, and design of the penetrating fluid structure. Then, the optimized sensing system was applied to determine the BOD concentration in model solutions of sodium acetate in a laboratory environment, where it accurately measured BOD concentrations in the range of 10–500 mg/L and maintained good parallelism during long-term operation. Next, the MEC-type BOD sensors were put into practice in the field as an alarm for accidents at an actual sewage plant. The whole BOD sensing system was quickly assembled on site and started up, and it gave an early warning shortly after the concentration of organic matter in the water suddenly increased, thus showing a high potential for engineering applications. This study broadened the domains of application of MEC-type BOD sensors in environmental monitoring, and promoted the development of technological innovation in water ecology and environmental monitoring.

Energy savings with a biochemical oxygen demand (BOD)- and pH-based intermittent aeration control system using a BOD biosensor for swine wastewater treatment

Intermittently aerated activated sludge processes save energy by minimizing the total aeration time per day without deteriorating effluent water quality. Swine wastewater contains high, often widely fluctuating concentrations of biochemical oxygen demand (BOD) and nitrogen. BOD-based aeration control would therefore be a useful method to reduce the energy demands of aeration, but requires a reliable BOD sensor for swine wastewater. In this study we developed a novel approach for BOD- and pH-based intermittent aeration control (BAC) using a BOD biosensor based on exoelectrogenic bacteria and tested it at four swine wastewater treatment plants. This BAC system reduced aeration energy in the plants by 15–36% compared to fixed aeration cycle operations while maintaining low concentrations of BOD and total nitrogen in the effluent. The energy saved per wastewater volume was 477–1012 kWh/m3/yr, equivalent to a reduction of 2.2–7.0 t-CO2/m3/yr in greenhouse gas emissions. The biosensor was equipped with an internet connection to allow for remote monitoring of water quality data with a smartphone, saving labour in the operational management of the plants. Aquabacterium and the aerobic denitrifier Thauera were detected as the predominant genera in the activated sludge.

Bacterial Cellulose Immobilized S. cerevisiae as Microbial Sensor for Rapid BOD Detection

As one of the most important parameters to characterize the organic pollution of water, biochemical oxygen demand (BOD) determined through a rapid, in situ and on line method is very attractive. In this paper, a new BOD biosensor has been developed, which are composed of the ecofriendly precursor of Saccharomyces cerevisiae (S. cerevisiae) embedded in bacterial cellulose (BC) matrix and the Ketjen Black (KB) modified glassy-carbon electrode. Furthermore, a doublemediator system is constructed using potassium ferricyanide and menadione in reaction cell to transfer electron from the microbe to electrode. The responses of glucose glutamic acid (GGA) standard solutions are amperomertrically measured with an applied potential of 0.25 V versus Hg/Hg2Cl2 in a three-electrode system. Under the optimum conditions, the (BC/S. cerevisiae-menadione)/KB modified electrode shows a high operational stability with relative standard deviation (RSD) of 4.16% (fourteen assays), a good repeatability (RSD=3.10%), a fast response time (in 20 minutes) and a wide linear range (from 10–220 mg O2l−1). The BOD values measured by this method have been highly correlated with the standard BOD 5-day method for wastewater samples (R2=0.9859, n=3), indicating that it can meet the requirement of BOD rapid measurement.

Performance of mediator-less double chamber microbial fuel cell-based biosensor for measuring biological chemical oxygen

Recently, the microbial fuel cell-based biosensor has been considered as an attractive technology for measuring wastewater quality such as biochemical oxygen demand (BOD). In this study, a mediator-less double compartment MFC based biosensor utilizing carbon felt as an anode electrode and inoculated with mixed culture was developed to improve the real application of a rapid BOD detection. This study aims to: (i) establish the effect of the operating conditions (i.e., pH, external resistance, fuel feeding rate) on MFC performance; (ii) investigate the correlation between biochemical oxygen demand (BOD) and signal output, and (iii) evaluate the operational stability of the biosensor. The presented result reveals that the maximum current and power production was obtained while 100 mM NaCl and 50 mM Phosphate buffer saline was used as a catholyte solution, neutral pH condition of media and fuel feeding rate at 0.3 mL min−1. Notably, a wider range of BOD concentration up to 300 mg L −1 can be obtained with the voltage output (R2 > 0.9901). Stable and steady power was produced by running MFC in 30 days when cells operated at 1000 Ω external resistance. Our research has some competition with the previous double chamber MFC in the upper limit of BOD detection. This results might help to increase the real application of MFC based BOD biosensor in real-time measurement.

Simple approach for the rapid estimation of BOD5 in food processing wastewater.

A simple approach was developed for the rapid and accurate estimation of 5-day biochemical oxygen demand (BOD5) in food processing wastewater. Immobilization of the natural microbial consortium that was collected from an aerobic compartment of a food processing wastewater treatment plant was simply performed by adhesion using a low-cost porous carrier. Pseudomonas aeruginosa, Bacillus cereus, and Streptomyces, whose salt-tolerance and ability to break down organic compounds have been widely reported, were found to be predominant. These microorganisms may cause an enhancement of the bioreactor response in the presence of sodium chloride. Consequently, a modified glucose-glutamic acid (GGA) calibration standard was proposed in which an appropriate amount of NaCl was added; this solution was found to be more effective in terms of accuracy and practicality than both conventional GGA and the synthetic wastewater recipe from the Organisation for Economic Cooperation and Development (OECD). The calibrated self-built packed-bed bioreactor exhibited good precision of 3% or less in predicting BOD5 in influent, which is similar to the performance of the most common commercial biochemical oxygen demand (BOD) bioreactors. There was a statistical agreement between the results obtained from this rapid BOD biosensor and the conventional methods, even when testing treated wastewater samples.

Enhancement of biological oxygen demand detection with a microbial fuel cell using potassium permanganate as cathodic electron acceptor

When dual-chamber microbial fuel cell (MFC) is used to detect biochemical oxygen demand (BOD), dissolved oxygen is traditionally used as cathodic electron acceptor. The detection limit of this MFC-based BOD biosensor is usually lower than 200 mg/L. In this paper, the startup of MFC-based BOD biosensor was researched and the external resistor of MFC was optimized. Results showed that the MFC started up with the dissolved oxygen as cathodic electron acceptor within 10 d, and the external resistor was optimized as 500 Ω to ensure the maximum output power of MFC. Dissolved oxygen and potassium permanganate (KMnO4) were used as cathodic electron acceptor to run MFC for detection of wastewater BOD, and the performances of two kinds of BOD biosensors were compared. The MFC-based BOD biosensor using KMnO4 (10 mmol/L) as cathodic electron acceptor exhibited an excellent performance, compared with that using dissolved oxygen. The upper limit of BOD detection was greatly broadened to 500 mg/L, the response time was shortened by 50% for artificial wastewater with a BOD of 100 mg/L, and the relative error of BOD detection was reduced to less than 10%. The MFC-based BOD biosensor using KMnO4 as cathodic electron acceptor showed a better linear relationship (R2 > 0.992) between the electric charge and BOD concentration within a BOD range of 25–500 mg/L. The MFC-based BOD biosensor using the KMnO4 as cathodic electron acceptor is promising with a better application prospect.

A Mediated BOD Biosensor Based on Immobilized B. Subtilis on Three-Dimensional Porous Graphene-Polypyrrole Composite.

We have developed a novel mediated biochemical oxygen demand (BOD) biosensor based on immobilized Bacillus subtilis (B. subtilis) on three-dimensional (3D) porous graphene-polypyrrole (rGO-PPy) composite. The 3D porous rGO-PPy composite was prepared using hydrothermal method following with electropolymerization. Then the 3D porous rGO-PPy composite was used as a support for immobilizing negatively charged B. subtilis denoted as rGO-PPy-B through coordination and electrostatic interaction. Further, the prepared rGO-PPy-B was used as a microbial biofilm for establishing a mediated BOD biosensor with ferricyanide as an electronic acceptor. The indirect determination of BOD was performed by electrochemical measuring ferrocyanide generated from a reduced ferricyanide mediator using interdigited ultramicroelectrode array (IUDA) as the working electrode. The experimental results suggested a good linear relationship between the amperometric responses and BOD standard concentrations from 4 to 60 mg/L, with a limit detection of 1.8 mg/L (S/N ≥ 3). The electrochemical measurement of real water samples showed a good agreement with the conventional BOD5 method, and the good anti-interference as well as the long-term stability were well demonstrated, indicating that the proposed mediated BOD biosensor in this study holds a potential practical application of real water monitoring.

Development and Long-Term Stability of a Novel Microbial Fuel Cell BOD Sensor with MnO₂ Catalyst.

A novel microbial fuel cell (MFC)-based biosensor was designed for continuous monitoring of biochemical oxygen demand (BOD) in real wastewater. To lower the material cost, manganese dioxide (MnO2) was tested as an innovative cathode catalyst for oxygen reduction in a single chamber air-cathode MFC, and two different crystalline structures obtained during synthesis of MnO2 (namely β- and γ-MnO2) were compared. The BOD sensor was studied in a comprehensive way, using both sodium acetate solution and real domestic wastewater (DWW). The optimal performance of the sensor was obtained with a β-MnO2 catalyst, with R2 values of 0.99 and 0.98 using sodium acetate solution and DWW, respectively. The BOD values predicted by the β-MnO2 biosensor for DWW were in agreement with the BOD5 values, determined according to standard methods, with slight variations in the range from 3% to 12%. Finally, the long-term stability of the BOD biosensor was evaluated over 1.5 years. To the best of our knowledge, this is the first report of an MFC BOD sensor using an MnO2 catalyst at the cathode; the feasibility of using a low-cost catalyst in an MFC for online measurement of BOD in real wastewater broadens the scope of applications for such devices.

Chromobacterium Violaceum for Rapid Measurement of Biochemical Oxygen Demand

Abstract: Biochemical Oxygen demand (BOD) is an important parameter indicating the total biodegradable organic pollutants in waters. Fast BOD determination could be achieved using BOD biosensor. However, most of the developed BOD biosensors are dependent on dissolved oxygen concentrations. Low solubility of oxygen in water decreases the reliability of oxygen-dependent BOD biosensor. However, replacement of oxygen with a mediator solution solves this problem. In the present study, an effective ferricyanide-mediated approach was modified from ferricyanide-mediated BOD assay and used for BOD determination in a water system. Several different types of microorganisms were isolated from different organic-rich environmental sources and their ability to use ferricyanide during organic (standard glucose-glutamic acid solution) degradation were effectively assessed using ferricyanide-mediated BOD assay. Around 90% of the GGA was degraded by Chromobacterium violaceum after 1 hour of incubation period. Therefore, C. violaceum has been found to be a potential microorganism to be used as a biosensing element in the BOD biosensor. This assay is not only effective in selecting the suitable microorganisms for ferricyanide-mediated BOD detection; it also could be applied to select the suitable microorganisms for other mediated microbial biosensor and bioremediation by changing the substrate and conditions.

BOD biosensor based on the yeast Debaryomyces hansenii immobilized in poly(vinyl alcohol) modified by N-vinylpyrrolidone

An amperometric biosensor for assessing the biochemical oxygen demand (BOD) was formed by immobilizing Debaryomyces hansenii VKM Y-2482 yeast cells in poly(vinyl alcohol) modified by N-vinylpyrrolidone. Modification provided for a high sensitivity and stability of the bioreceptor. A high oxidative activity of the receptor element and the absence of any toxic effect of assayed compounds were shown for 34 substrates (alcohols, carbohydrates, carboxylic acids, amino acids, nitrophenols and surfactants) that may occur in wastewaters. Estimates of the measurement range and region of the linear dependence of signals on the BOD level, pH and temperature sensitivities, dependences of signals on concentrations of salts, stability, Michaelis kinetic constants and assay rates were obtained. The BOD values determined by the biosensor in assayed wastewater samples were shown to have a high correlation with those obtained by the standard dilution method.

A co-immobilized mediator and microorganism mediated method combined pretreatment by TiO2 nanotubes used for BOD measurement

In this paper, we proposed a method by using co-immobilized Escherichia coli (E. coli) as a biocatalyst and neutral red (NR) as an artificial electronic acceptor to modify glassy carbon electrode (GCE) for biochemical oxygen demand (BOD) measurement. Two different modification approaches of GCE were utilized and compared. In one approach, NR was electropolymerized on the surface of GCE, and E. coli cells were mixed with grafting copolymer PVA-g-PVP (briefly gPVP) and covered on NR polymer film to obtain a (gPVP/E. coli)/PNR/GCE. In the second approach, both NR and E. coli cells were mixed with the copolymer gPVP and modified GCE, after drying, which was electrochemically treated similar as above for obtaining a (gPVP/E. coli/NR)p/GCE. Based on the electrochemical evaluation, the performance of the latter was better, which may be caused by that the NR deposited on the surface of E. coli resulting in a good electron transport and permeability of cells membrane. To develop the results obtained at (gPVP/E. coli/NR)p/GCE further, the pretreatment by TiO2 nanotubes arrays (TNTs) was employed, and different effects on samples of GGA, OECD, urea and real wastewater were evaluated. These results suggest that the present method holds a potential application for rapid BOD biosensor.

Non-steady response of BOD biosensor for the determination of biochemical oxygen demand in wastewater

A biochemical oxygen demand (BOD) biosensor for effective and expeditious BOD(7) estimations was constructed and the non-steady phase of the output signal was extensively studied. The modelling approach introduced allows response curve reconstruction and a curve fitting procedure of good quality, resulting in parameters indicating the relationship between response and organic substrate concentration and stability properties of the BOD biosensor. Also, the immobilization matrixes of different thicknesses were characterized to determine their suitability for bio-sensing measurements in non-stationary conditions, as well as for the determination of the mechanical durability of the BOD biosensor in time. The non-steady response of the experimental output of the BOD biosensor was fitted according to the developed model that enables to determine the stability of the biosensor output and dependency on biodegradable organic substrate concentration. The calibration range of the studied BOD biosensor in OECD synthetic wastewater was 15-110 mg O(2) L(-1). Repeatability tests showed relative standard deviation (RSD) values of 2.8% and 5.8% for the parameter τ(d), characterizing the transient output of the amperometric oxygen sensor in time, and τ(s), describing the dependency of the transient response of the BOD biosensor on organic substrate concentration, respectively. BOD biosensor experiments for the evaluation of the biochemical oxygen demand of easily degradable and refractory municipal wastewater showed good concurrence with traditional BOD(7) analysis.

Immobilized multi-species based biosensor for rapid biochemical oxygen demand measurement

To improve the practicability of rapid biochemical oxygen demand (BOD) method, we proposed a stable BOD sensor based on immobilizing multi-species BODseed for wastewater monitoring in the flow system. The activation time of the biofilm was greatly shortened for the biofilm prepared by BODseed in the organic–inorganic hybrid material. Some influence factors such as temperature, pH, and concentration of phosphate buffer solution (PBS) were investigated in detail in which high tolerance to environment was validated for the BOD sensor permitted a wide pH and PBS concentration ranges. The minimum detectable BOD was around 0.5 mg/l BOD under the optimized 1.0 mg/ml BODseed immobilized concentration. The as-prepared BOD sensor exhibited excellent stability and reproducibility for different samples. Furthermore, the as-prepared BOD biosensor displayed a notable advantage in indiscriminate biodegradation to different organic compounds and their mixture, similar to the character of conventional BOD 5 results. The results of the BOD sensor method are well agreed with those obtained from conventional BOD 5 method for wastewater samples. The proposed rapid BOD sensor method should be promising in practical application of wastewater monitoring.

Development of mediated BOD biosensor system of flow injection mode for shochu distillery wastewater

Although microbial biochemical oxygen demand (BOD) sensors utilizing redox mediators have attracted much attention as a rapid BOD measurement method, little attempts have been made to apply the mediated BOD biosensors to the flow injection analysis system. In this work, a mediated BOD sensor system of flow injection mode, constructed by combining an immobilized microbial reactor with an electrochemical flow cell of three electrodes configuration, has been developed to estimate BOD of shochu distillery wastewater (SDW). It was demonstrated consequently that the mediated sensing was realized by employing phosphate buffer containing potassium hexacyanoferrate as the carrier. The output current was found to yield a peak with a sample injection, and to result from reoxidation of reduced mediator at the electrode. By employing the peak area as the sensor response, the effects of flow rate and pH of the carrier on the sensitivity were investigated. The sensor system using a microorganism of high SDW-assimilation capacity showed good performance and proved to be available for estimation of BOD of SDW.

An innovative reactor-type biosensor for BOD rapid measurement

Biochemical oxygen demand (BOD) is one of the most important and widely used parameters for characterizing the organic pollution of water and wastewater. In this paper, a novel reactor-type biosensor for rapid measurement of BOD was developed, based on using immobilized microbial cell (IMC) beads as recognition bio-element in a completely mixed reactor which was used as determining chamber, replacing the traditionally used membrane as recognition bio-element. The IMC beads were freely suspended in the aqueous solution, so the mass transfer resistance for dissolved oxygen and organic compounds significantly reduced, and the quantity of the microbial cells used as recognition element can be easily adjusted, in comparison with the traditional membrane-type BOD biosensor, in which exists a unadjustable contradiction between the quantity of biomass and the thickness of the bio-membrane, thus limiting the stability and the detection limit. This novel kind of BOD biosensor significantly increased the sensitivity of the response, the detecting precision and prolonged the life time of the recognition element. The experimental data showed that the most appropriate temperature for biochemical reaction in the reactor was 30°C, and the IMC beads could keep the bioactivity for about 70d at the detecting frequency of 8 times every day. The standard deviation of repeatability and the reproducibility of responses were within ±6.4% and ±5.0%, respectively, which are within acceptable bias limits, and meet the requirement of BOD rapid measurement.

A novel microbial BOD biosensor developed by the immobilization of P. Syringae in micro‐cellular polymers

AbstractBACKGROUND: A biochemical oxygen demand (BOD) sensor, based on an immobilized Pseudomonas syringae in highly porous micro‐cellular polymer (MCP) in combination with a dissolved oxygen electrode, has been developed for the analysis of biodegradable organic compounds in aqueous samples. Microorganisms were immobilized in a molded MCP disk and a wastewater sample was injected into the biocomposite disk by a flow injection system. Dissolved oxygen (DO) changes as a measure of soluble BOD was read with a DO probe placed into a flow cell carrying biocatalytically activated disk.RESULTS: Optimal response of the MCP BOD sensor was obtained at pH 6.8 and 25 °C with a typical response time of 3–5 min for a 2 mm thick molded polymeric disk. The sensor showed detection linearity over the range 5–100 mg L−1 BOD5 (r2 > 0.99) at a flow rate of 0.6 mL min−1. The repeatability and reproducibility of the sensor response were found to be 3.08% and 7.77%, respectively. BOD values produced with this biosensor for various municipal and industrial wastewaters correlated well with those determined by the conventional 5‐day BOD test.CONCLUSION: This new biosensor was different from present amperometric BOD biosensor configurations in which the biocatalyst (microbial/enzymatic) is placed between cellulose and Teflon membranes installed on a DO probe. The use of a molded MCP disk coniainng microbial activity offers better stability and lifetime for commercial use in environmental monitoring. Copyright © 2008 Society of Chemical Industry

Microbial BOD sensor for monitoring treatment of wastewater from a rubber latex industry

A cell-based biosensor system was designed for monitoring an anaerobic process for treatment of high biochemical oxygen demand (BOD) levels in wastewater samples from a factory processing concentrated rubber latex. The BOD biosensor used immobilized mixed culture of microorganisms as the biological sensing element and an oxygen electrode as the transducer. The assay principle is based on the determination of the oxygen consumption rate caused by microbial respiration. Synthetic wastewater according to the OECD specifications was used as standard solution for calibration of the BOD biosensor. Response time of the sensor was 10–15 min. The BOD of the influent and the effluent from an anaerobic reactor was measured using both the cell-based biosensor system and a standard method (BOD 5). Good agreement was achieved between the results from the two assay methods with a percentage difference of less than 10%. However, when exposing the mixed culture to wastewaters from other industrial plants the agreement between the results of the two assays was poor. The anaerobic treatment of the wastewater from the concentrated latex process resulted in a COD removal efficiency of 97% at a hydraulic retention time (HRT) of 50 days. The BOD biosensor was successfully applied to off-line and on-line monitoring of the anaerobic reactor treatment process.

High-Throughput Determination of Biochemical Oxygen Demand (BOD) by a Microplate-Based Biosensor

The use of the conventional 5-day biochemical oxygen demand (BOD5) method in BOD determination is greatly hampered by its time-consuming sampling procedure and its technical difficulty in the handling of a large pool of wastewater samples. Thus, it is highly desirable to develop a fast and high-throughput biosensor for BOD measurements. This paper describes the construction of a microplate-based biosensor consisting of an organically modified silica (ORMOSIL) oxygen sensing film for high-throughput determination of BOD in wastewater. The ORMOSIL oxygen sensing film was prepared by reacting tetramethoxysilane with dimethyldimethoxysilane in the presence of the oxygen-sensitive dye tris(4,7-diphenyl-1,10-phenanthroline)ruthenium-(II) chloride. The silica composite formed a homogeneous, crack-free oxygen sensing film on polystyrene microtiter plates with high stability, and the embedded ruthenium dye interacted with the dissolved oxygen in wastewater according to the Stern-Volmer relation. The bacterium Stenotrophomonas maltophilia was loaded into the ORMOSIL/ PVA composite (deposited on the top of the oxygen sensing film) and used to metabolize the organic compounds in wastewater. This BOD biosensor was found to be able to determine the BOD values of wastewater samples within 20 min by monitoring the dissolved oxygen concentrations. Moreover, the BOD values determined by the BOD biosensor were in good agreement with those obtained by the conventional BOD5 method.

A new BOD estimation method employing a double-mediator system by ferricyanide and menadione using the eukaryote Saccharomyces cerevisiae

A new biochemical oxygen demand (BOD) sensing method employing a double-mediator (DM) system coupled with ferricyanide and a lipophilic mediator, menadione and the eukaryote Saccharomyces cerevisiae has been developed. In this study, a stirred micro-batch-type microbial sensor with a 560 μL volume and a two-electrode system was used. The chronamperometric response of this sensor had a linear response between 1 μM and 10 mM hexacyanoferrate(II) ( r 2 = 0.9995, 14 points, n = 3, average of relative standard deviation and R.S.D. av = 1.3%). Next, the optimum conditions for BOD estimation by the DM system (BOD DM) were investigated and the findings revealed that the concentration of ethanol, used to dissolve menadione, influenced the sensor response and a relationship between the sensor output and glucose glutamic acid concentration was obtained over a range of 6.6–220 mg O 2 L −1 (five points, n = 3, R.S.D. av 6.6%) when using a reaction mixture incubated for 15 min. Subsequently, the characterization of this sensor was studied. The sensor responses to 14 pure organic substances were compared with the conventional BOD 5 method and other biosensor methods. Similar results with the BOD biosensor system using Trichosporon cutaneum were obtained. In addition, the influence of chloride ion, artificial seawater and heavy metal ions on the sensor response was investigated. A slight influence of 20.0 g L −1 chloride ion and artificial seawater (18.4 g L −1 Cl −) was observed. Thus, the possibility of BOD determination for seawater was suggested in this study. In addition, no influence of the heavy metal ions (1.0 mg L −1 Fe 3+, Cu 2+, Mn 2+, Cr 3+ and Zn 2+) was observed. Real sample measurements using both river water and seawater were performed and compared with those obtained from the BOD 5 method. Finally, stable responses were obtained for 14 days when the yeast suspension was stored at 4 °C (response reduction, 93%; R.S.D. for 6 testing days, 9.1%).