Analytica Chimica Acta, 135 (1982) 61-67 Elsevier Scientific Publishing Company, Amsterdam -
Printed in The Netherlands
and SHUECHI SUZUKI
Research Laboratory of Resources Utilization. Tokyo Institute of Technology, Nagatsuta-cho, Midori-ku, Yokohama 227 (Japan) (Received 14th April 1981)
SUMMARY A bacterial sensor system based on Methylomonas flagellata AJ 3670 is described for methane determinations. The system consists of a bacterial, reactor, a reference reactor and two oxygen sensors. The current decreases with time until a steady state is reached within 30 s at 30°C; the maximum current difference is obtained at 30°C and pH 7.2. The response time for the determination of methane is less than 1 min. A linear relationship is obtained between the current difference and the methane concentration below 6.6 mM; the lower limit of determination is 5 PM, and the current decrease is reproducible within 5%. The current output of the sensor is almost stable for more than 10 days and 250 assays.
Methane is an attractive energy source and a main component of natural gases. It is used as gaseous fuel in many fields, and it is well known that
methane forms explosive mixtures with air (5-14%). Therefore, rapid methods for the determination of methane in air are required in various fields such as coal mining and gasification processes_ Methane is also produced by methanogenie bacteria. The biological formation of methane is the result of a specific type of bacteria energy-yielding metabolism. World-wide interest has arisen in the production of methane by fermentation of biomass, renewable resources. Control of the fermentation process is needed for effective production of these useful materials. The determination of raw materials and products in the media is required for process control, spectrophotometric methods normally being applied. However, as molasses and other natural materials are used as the raw materials for fermentation, the media are not optically clear. Electrochemicalmonitoring of these compounds may have definite advantages_ For some time, the utilization of micro-organisms has not been restricted to the fermentation industries [l] and many valuable applications have been found, including new electrochemical uses. As previously reported, many microbial sensors have been developed for the determination of BOD [Z--4], assimilable sugars (glucose, fructose and sucrose) [ 51, and volatile or gaseous compounds such as alcohols 161, acetic acid f73 and ammonia [ES]_ These electrodes consist of micro-organisms and oxygen electrodes, Assimilation of organic compounds by micro-organisms can be determined from the respiratory activity of the micro-organisms, which can be directly measured by an oxygen electrode. 0003-2670/82/0000-0000/$02.75
0 1982 Elsevier Scientific Publishing Company
Whittenbury et al. [9, lo] isoIated more than 100 gram-negative, strictly aerobic, methane-utilizing bacteria_ More recently, an easily-grown methaneoxidizing bacterium was isolated in a pure culture from a natural source through studying the production of single cell protein 1111. This bacterium was identified as a new species Methylomonas flagellata, capable of growing by oxidizing methane as the main carbon and energy source. In general, methane-oxidizing bacteria utilize methane, and oxygen is consumed by the respiration as follows [12,13] CH4 + 02
In this study, the characteristics of a methane sensor system are described,
and the sensor system is applied to the determination of methane in air. EXPERIMENTAL Materials and micro-organisms
Reagents were commercially available analytical-grade chemicals. Deionized water was used for all procedures. The micro-organism used was M. flagellata, which was kindly provided by the Fermentation Research Institute, Ministry for Industrial Trade and Industry, Tsukuba_ The M_ frageliata was maintained on a gel (pH 7.2) containing 0.5 g (NH4)$04, 0.3 g KH2P04, 1.8 g Na,HPO, - 12Hz0, 0.2 g MgSO,-7&O, 10 mg F’eS04.7H10, 1.0 mg CuS04-5HrO and 30 g of agar per liter of distilled water. Cultivation. The M. flagellata was cultured under aerobic conditions for 48 h in the same medium (pH 7.2) as the above gel except for agar. Methane was provided as a growth substrate by incubating the culture under an atmosphere of 80% air-20% methane at 30°C in 500-mI suction flasks sealed with rubber stoppers and placed in a rotary shaker incubator_ The cells were centrifuged at 5°C and SOOOg,and washed twice with physiological saline solution.
Apparatus Figure 1 shows a schematic diagram of the system. The system consisted of two oxygen electrodes (model U-l, Ishikawa Seisakujo, Tokyo), two reactors, an electrometer (Hokuto Denko model 0.552, Iwaki) and arecorder (Riken Denki model SPJGC). The reactors (55~ml capacity) contained 41 ml of the culture medium, one with and one without bacterial cells. The oxygen eIectrodes consisted of a teflon membrane (50~rzrnthick), a platinum cathode, a lead anode and sodium hydroxide electrolyte (30% w/v). These electrodes were fixed to custom-made teflon flow-through cells. The system was connected up with glass and teflon tubing (0.3 cm diameter)_ The system contained two vacuum pumps, one to evacuate the gas sample tube (glass, diameter O-4 cm, length 80 cm, volume 40 ml) and the other to
6 Fig. 1. Scheme of microbial sensor system for methane. 1, vacuum pump; 2, sample gas bag; 3, gas sample line; 4, cotton filter; 5, control reactor; 6, methane-oxidizing bacteria reactor; 7, oxygen electrode; 8, amplifier; 9, recorder; 10,vacuum pump; 11-17, glassstopcocks.
transport the sample gas through the system. The flow rate of the sample gas through the reactors was controlled with the glass valves at 80 ml min-‘. The cotton filter served to remove other micro-organisms in the sample gas and to prevent contamination of the two reactors and gas lines. The sample gas exiting from the system was passed into the laboratory extraction hoods via glass tubes. All the lines were made gas-tight with teflon tubes and glass tubes, and careful checks were made for leakage. The lines were designed so as to maintain symmetry between the measuring and reference flow lines. The flow rates in each line were balanced by adjusting valves 15 and 16. The reactors were maintained at 30°C + O.l”C by a thermostatted bath. Procedure for measurement In a typical procedure, pump 10 was switched on and valves 14-17 were opened with valve 13 closed, so that air flowed through the system. A steady current -was obtained within a few minutes, corresponding to the concentration of oxygen at the outer surface of the electrodes_ Euring passage through the reactor containing micro-organisms, some oxygen was consumed but the oxygen concentration at the outlet of the reactor reached a constant level within a few minutes. Valves 12 and 13 were then closed, valve 11 was opened
and the gas sample line was evacuated by pump 1. Closing valve 11 and opening valve 12 allowed the sample gas containing methane to flow into the gas sample line. Vacuum pump 1 was then removed and valve 12 closed. Valves 11 and 13 were then opened and valve 14 was closed simultaneously, so that the sample gas was transferred to the measuring lines by pump 10. During the passage of the sample gas containing methane, the difference in current recorded for the two electrodes depended on the concentration of methane_ Each sample was measured in duplicate by repeating the procedure.
Preparation of methane standards The methane used was obtained commercially (99.99% purity). A known volume (V) of this methane was injected into a 2-1 gas bag (vinyl fluoride film), which was then filled with air from a gas-tight syringe. The molarity of methane (at 25”C, 1 atm.) in the gas bag was then calculated from x (mole) = 273 V/(22.4 X 298 X 2). Methane was determined by gas chromatography (Shimazu Seisakujo, model GC3BT) with 5A molecular sieve (60-80 mesh) in a 3 m X 0.3 cm i-d_ column at 40°C with argon as the carrier gas and thermal conductivity detection in the usual manner. RESULTS
Response and calibration of the electrode Figure 2 shows typical response curves of the sensor system. When the sample gas containing methane entered the reactor, methane was assimilated by the microorganisms with consumption of oxygen_ The current decreased to a minimum, which indicated that the consumption of oxygen by the micro-organisms, and the supply of oxygen from the air was in equilibrium_ As the system contained two oxygen electrodes, the maximum difference of current depended on the concentration of methane in the sample gas. When pure air was again passed through the reactors, the current of the sensor returned to its initial level within 1 min. The response time required for the determination of methane was within 1 mm, and the total time required for an assay of methane was 2 min. The calibration graphs for the system were strictly linear for concentrations of methane in the range O--6-6 mM, the current difference ranging from 0 to 0.35 PA. The minimum concentration for determination was 5 PM. The current difference measured for the same sample (0.66 mM) was reproducible within 5%; the standard deviation was 9.40 nA in 25 experiments.
Effects of temperature, air flow and cell content on the current difference Figure 3 shows the effect of temperature on the current difference of the microbial system. The current difference was determined after 30-min incubation of the reactors at various temperatures. The concentration of dissolved oxygen decreased with increasing temperature, and the results were
Fig. 2. Response curves of the microbial sensor for methane. The sample gas was transferred into the gas sampler at time 0: (0) 0.66 mM; (0) 0.39 mM methane. Conditions as in Experimental; 300 mg of wet cells per reactor. Fig_ 3. Effect of temperature tions as for Fig. 2.
of the microbial
corrected on the basis of the concentration of dissolved oxygen determined at various temperatures_ As shown, the currents decreased above 35” C because the bacteria were inactivated by heat, but because the respiration activity of the bacteria decreased below 25°C the current increased with decreasing temperature. The maximum current difference was observed at 30°C. The effect of the air flow rate on the current difference of the sensor is shown in Fig. 4. The current difference was almost constant below flow rates of 80 ml min-I, but above this flow rate the current difference decreased rapidly. Insofar as the respiration activity of M. fZageZiata is measured in the sys+&m, the cell content in the reactor will obviously affect the current output_ The wet cell content of each reactor was changed from 100 mg to 400 mg and the effects on the current difference were examined. Figure 5 shows that the current difference increased as the wet cell content increased up to 300 mg per reactor, and a wet cell content of 300 mg per reactor is recommended_ The long-term stability of the microbial sensor system was examined with sample gases containing 0.66 or 0.39 mM of methane_ The current output was almost constant for more than 10 days and 250 assays, but showed better reproducibility at the higher concentration over that time. Comparison with gas chromatography The microbial sensor system was applied to the determination of methane in air samples that were also analyzed by conventional gas chromatography. Over the range 0.2-3.5 mM methane in air the correlation coefficient between the results of the two methods was 0.97.
Fig. 4. Effect of sample gas flow rate on current difference of the microbial sensor for methane concentrations of 0.66 mM ( o ) and 0.39 mM (a)_ Other conditions as for Fig. 2. Fig. 5_ Effect of cell content on current difference of the microbial sensor. Other conditions as in Fig. 2.
Conclusions A rapid and continuous method for the determination of methane is desirable in many fields, and on-line measurement of methane is required for its effective production by fermentation. Although gas chromatography can be used, it seems basically unsuitable for such on-line measurements. In the proposed method, the respiration of micro-organisms is converted immediately to a current signal. The sensitivity of this amfierometic signal is almost the same as that of the gas chromatographic signal; the minimum measurable concentration is 3 FM by gas chromatography with a flame ionization detector, and 5 ,uM by the proposed microbial sensor. Accordingly, the microbial sensor system with M_ fZugeZZafa seems promising for the rapid on-line determinations of methane. REFERENCES 1 J. Levy, J. Campbell Jr. and T. H. Blackbum, Introductory Microbiology, J. Wiley, New York, 1973. 2 I_ Ka.rube,T. Matsunaga, S. Mitsuda and S. Suzuki, Biotechnol. Bioeng., 19 (1977) 1535. 3 I. Karuhe, S. Mitsuda, T. Matsunaga and S. Suzuki, J. Ferment. Technol., 55 (1977) 243. 4 M. Hiiuma, H. Suzuki, T. Yasuda, I. Karube and S. Suzuki, Eur_ J. AppL Microhiol. Biotechnol., 8 (1979) 289. 5 M. Hikuma, H. Obana, T. Yasuda, I. Karube and S. Suzuki, Enzyme Microb. Technol., 2 (1930) 234_ 6 M. Hikuma, T. Kubo, T. Yasuda, I. Karube and S. Suzuki, Biotechnol. Bioeng., 21 (1979) 1845. 7 M. Hikuma, H. Suzuki, T. Yasuda, I. Karube and S. Suzuki, Anal. Chim. Acta, 109 (1979) 33.
67 8 9 10 11 12 13
I. Karube, T. Okada and S. Suzuki, Anal. Chem., 53 (1981) 1852. R. Whittenbury, K. C. Phillips and J. F. Wilkinson, J. Gen. Microbial., 61 (1970) 205. R. Whittenbury, S. L. Davies and J. F. Davy, J. Gen. Microbial., 61 (1970) 219. Y. Morinaga, S. Yamanaka, S. Otsuka and Y. Hirose, Agric. Biol. Chem., 40 (1976) 8. D. W. Ribbons, J. Bacterial., 122 (1975) 1351. I. J. Higgins and J. R. Quayle, Biochem. J., 188 (1970) 201.