Motohiko Hikuma

Amperometric estimation of BOD by using living immobilized yeasts

SummaryA microbial electrode consisting of immobilized living whole cells of yeasts, porous membrane and an oxygen electrode was prepared for continuous estimation of biochemical oxygen demand (BOD). Immobilized Trichosporon cutaneum was employed for the microbial electrode sensor for BOD. When a sample solution containing the equivalent amount of glucose and glutamic acid was injected into the sensor system, the current of the electrode decreased markedly with time until steady state was reached. The response time was within 18 min. A linear relationship was observed between the current decrease and the concentration below 41 mg l− of glucose and 41 mg l− glutamic acid (5-day BOD 60 mg l−). The current decrease was reproducible within ± 6% of the relative error when a sample solution containing 27 mg l− of glucose and 27 mg l− of glutamic acid (5-day BOD 40 mg l−) was employed. The microbial electrode sensor was applied to untreated waste waters from a fermentation factory. Good comparative results were obtained between BOD estimated by the microbial electrode and that determined by the conventional 5-day method (regression coefficient was 1.2). Furthermore, the effect of various compounds on BOD estimation was also examined. The current output of the microbial electrode sensor was almost constant for 17 d and 400 tests.

Amperometric determination of acetic acid with immobilized Trichosporon brassicae

A microbial sensor consisting of immobilized Trichosporon brassicae, a gas-permeable Teflon membrane and an oxygen electrode is suitable for the continuous determination of acetic acid in fermentation broths. When an acetic acid solution is pumped through the flow system, the current decreases to a steady state with a response time of 8 min; shorter pumping times give peaks which can also be measured. The relationship between the current decrease and the acetic acid concentration is linear up to 54 mg l-1, with a relative standard deviation of about 6% at the higher concentrations. Selectivity is satisfactory. Results obtained with this sensor and by gas chromatography for a glutamic acid fermentation broth were in good agreement (regression coefficient 1.04). The sensor was stable for more than 3 weeks and 1500 assays.

A potentiometric microbial sensor based on immobilized escherichia coli for glutamic acid

The sensor consists of immobilized E. coli (which contains glutamate decarboxylase) and a carbon dioxide gas-sensor. Continuous introduction of sample solution into a flow system incorporating the sensor gives a potential which increases until a steady state is reached after 5 min. Measurements can also be made with only a 1- or 3-min introduction period with little loss of sensitivity. Calibration plots of mV measurements vs. logarithmic glutamic acid concentration are linear in the range 100–800 mg l-1. The sensor is highly selective, stable and reproducible. It has been applied to the determination of glutamic acid in fermentation broths.

Amperometric determination of total assimilable sugars in fermentation broths with use of immobilized whole cells

A microbial sensor consisting of immobilized living whole cells of Brevibacterium lactofermentum and an oxygen electrode was prepared for continuous determination of total assimilable sugars (glucose, fructose and sucrose) in a fermentation broth for glutamic acid production. Total assimilable sugars were evaluated from oxygen consumption by the immobilized microorganisms. When a sample solution containing glucose was applied to the sensor system, increased consumption of oxygen by the microorganisms caused a decrease in the dissolved oxygen around the Teflon membrane of the oxygen electrode and the current of the electrode decreased markedly with time until steady state was reached. The response time was ≈ 10 min by the steady state method and 1 min by the pulse method. A linear relationship was found between the decrease in current and the concentration of glucose (<1 mM), fructose (<1 mM) and sucrose (<0.8 mM). The ratio of the sensitivity of the microbial sensor to glucose, fructose and sucrose was 1.00:0.80:0.92. The decrease in current was reproducible to within 2% of the relative standard deviation when a sample solution containing glucose (0.8 mM) was employed for experiments. The selectivity of the microbial sensor for assimilable sugars was satisfactory for use in the fermentation process. The additivity of the response of the microbial sensor for glucose, fructose and sucrose was examined. The difference between the observed and calculated values was within 8%. The microbial sensor was applied to a fermentation broth for glutamic acid production. Total assimilable sugars can be determined by the microbial sensor which can be used for more than 10 days and 960 assays.

Amperometric determination of sodium nitrite by a microbial sensor

SummaryA microbial sensor was prepared to determine sodium nitrite. This microbial sensor consisted of immobilized Nitrobacter sp. and an oxygen electrode. When a sample solution containing sodium nitrite was tested, nitrite was changed to NO2 gas in the buffer (pH 2.0) and the current of the electrode decreased with time until a steady state was reached. The steady state current was attained within 10 min and the maximum decrease in current was obtained at 30°C and pH 2.0. A linear relationship was observed between the current decrease and the sodium nitrite concentration below 0.59 mM, the minimum sodium nitrite concentration that could be determined was 0.01 mM. The current decrease was reproducible (5% relative error). The current output of the sensor was almost constant for more than 21 days and 400 assays.

Microbial sensors for volatile compounds

A microbial sensor consisting of immobilized microorganisms, a gas permeable membrane, and an oxygen electrode was prepared for the continuous determination of methyl alcohol. Immobilized methyl alcohol utilizing bacteria was employed for the sensor. The response time of the sensor was within 10 min by the steady-state method. A linear relationship was observed between the current decrease and the concentration of methyl alcohol below 22.5 mg l-1. The selectivity of the microbial sensor for methyl alcohol was satisfactory. Microbial sensors using Trichosporon brassicae for ethyl alcohol and acetic acid are also described. A microbial sensor consisting of immobilized nitrifying bacteria (isolated from activated sludges), a gas-permeable Teflon membrane and an oxygen electrode was prepared for the amperometric determination of ammonia. When the sensor was inserted in a solution containing ammonia, the current decreased to a steady-state with a response time of 4 min. The relationship between the current decrease and the ammonia concentration was linear up to 42 mg l-1. The minimum concentration for the determination was 3.5 mg l-1. The current decrease was reproducible within 4 per cent of relative error. The current output of the sensor was almost constant for over 10 days and 200 assays.

A rapid electrochemical method for assimilation test of microorganisms

SummaryA microbial electrode consisting of the immobilized microorganisms to be tested and an oxygen electrode was used to study the assimilation characteristics of microorganisms. When a sample solution containing a substrate was injected into the microbial sensor system, the current of the sensor markedly decreased with time if the microorganisms assimilated the substrate. On the other hand, no current decrease was observed if the microorganisms could not assimilate the substrate. Assimilation characteristics of various microorganisms such as molds, yeasts, bacteria, actinomycetes and activated sludges were tested with various substrates. The time required for a test was 30 min per substrate by the pulse method (sample injection period, 5 min). Good correlations were obtained between this electrochemical method and the conventional growth test. The fundamental differences between the two methods and the application of the electrochemical method are discussed.

Photoresponse of a reconstituted membrane containing bacteriorhodopsin observed by using an ion-selective field effect transistor

Abstract A method of observing the functions of a reconstituted membrane containing bacteriorhodopsin(bR) by using an ion-selective field effect transistor (ISFET) is proposed. bR was located on the surface of the ISFET with an acetylcellulose (AC) membrane containing bacteriorhodopsin (PC) and cholesterol. The gate potential of this device rose rapidly and increased gradually during illumination. It showed the photoresponse, especially a gradual increase, for which the proton pump of bR was responsible. The effects of light intensity, temperature, buffer action and the inhibitor La3+ were examined.

Sugar (Glucose, Fructose, Sucrose) Sensor for Fermentation Control

Cane and beet molasseses, which are used as the raw materials of fermentation processes, mainly contain three kinds of sugars, glucose, fructose and sucrose. Accordingly, on-line measurements of those sugars would be required for advanced fermentation control.’ A glucose consisting of immobilized glucose oxidase and an oxygen electrode can be used for the determination of glucose. However, the determination of fructose is quite tedious. On the other hand, three sugars can be specifically determined by a glucose sensor system consisting of chemical isomerization and chemical hydrolyzation reactors. The schematic diagram of a sugar sensor system is shown in FIGURE 1. The principles of each sugar measurement are as follows: (1) Glucose measurement4lucose + 0, Glucono-lactone + glumrc oxidass

Application of Microbial Electrode to Analysis of Waste Water

The biochemical oxygen demand(BOD) test and determination of nitrogenous compounds are required to control the pollution of water. Microbial electrodes consisting of immobilized microorganisms and electrochemical devices have been developed for rapid estimation of BOD (1) and determination of ammonia (2), nitrite (3), and nitrate with response times of 4–20 min. They were prepared according to procedures previously reported (1–3).