BIOSENSORS FOR SENSING ORGANIC SOLUTES AND BIOCHEMICAL OXYGEN DEMAND USING THERMALLY-KILLED MICROBIAL CELLS

Abstract

Bacillus subtilis and Bacillus licheniformis 7B were killed by exposing the living cells to a constant high temperature in the range of 160 °C-280 °C. The exposure time required to completely kill an entire cell population decreased from 30 mins to 2.5 mins with increasing temperature. The enzyme system in the dead cells was shown to remain viable and active by the oxidation of lactic, formic and glutamic acids in the presence of a free suspension of the dead cells. The oxidation kinetics of the acids followed the Michaelis-Menten rate equation including the effect of pH described by one of the Michaelis pH functions based on the protonation and deprotonation of the active sites of the enzymes. The activity of the enzyme system in these dead cells was observed to be comparable with that in the living cells. Better enzymic activity was obtained when the cells were killed with shorter time exposure at higher temperature. Higher oxidation rate was observed under conditions facilitating the diffusion of the substrates through the cell membranes. These results are significant to bioprocessing and biosensor fabrication since dead cells with their enzyme systems remaining viable and active are relatively free from maintenance and control of the growth environment which are often necessary when living cells are used. They also have the advantage of the protection of the enzyme by the cell membrane compared with the completely exposed purified enzymes. " Bacillus subtilis, thermally killed by exposing its vacuum-dried cells to 280 °C for 2.5 minutes, was used for sensing biochemical oxygen demand (BOD) of organic solutes, waters and wastewaters. The BOD sensor was fabricated by covering a dissolved oxygen probe with a biofilm, containing the dead cells. The dead cell biosensor showed good sensing characteristics for the BOD5 of aqueous organic solutions and wastewaters in regards to its response and recovery times, sensitivity, reproducibility and stability. It has a significantly long life span of several months, requiring only simple storage at room temperature in phosphate buffer in between measurements and even over extended idling period. In contrast, living cells sensors would require food and nutrients additions for their sustenance, particularly over extended idling period. The sensitivities of the dead (Bacillus subtilis) cell biosensor towards 24 organic solutes were generally comparable with those of the living cell biosensor and those based on the APHA (American Public Health Association, 1992) method of measurement using activated sludge. The low response observed for soluble starch of both the living and dead cell sensors was confirmed experimentally to be the cause of high diffusion resistance of the membrane resulting in a significantly longer response time than those for other solutes. The BOD5 values of some real wastewater and synthetic water samples were determined and compared with those obtained by the APHA method. The deviation of the biosensor value from the APHA value would depend on the difference in the sensitivity of the microbial system and the device towards the compounds in the test sample. Nevertheless the deviation was found to be of the same order as that generally observed among the APHA values reported for a given substrate by different analysts and laboratories. These results also confirmed that Bacillus subtilis killed by short-time exposure to high temperature still retained sufficient enzymic viability and activity for the bio-oxidation of the organic compounds and for BOD sensing. The response of the dead Bacillus subtilis cell biosensor in a multicomponent organic solution was described mathematically taking into consideration the mass transfer and bio-oxidation rate processes of the organic solutes and the dissolved oxygen in the biosensor. The model described the biochemical oxygen demand (BOD5) of an organic solution based on the BOD value of 220 mg L-1 assigned to a BOD check solution containing 150 mg L-1 of glucose and the same concentration of glutamic acid. The model was extended to describe the effects of cell population immobilized in the biofilm and substrate temperature on the BOD-sensing characteristics of the sensor. Experimental data were obtained to determine the model parameters for the sensing of a single organic solute. The validity of the model in predicting the sensor response to multicomponent organic mixtures and their biochemical oxygen demand based on the single solute parameters was substantiated by the experimental results. These results illustrated the usefulness of the mathematical model for the characterization, design and fabrication of a bio-oxidation related biosensor for sensing multicomponent organic solutions and in particular the 5-day biochemical oxygen demand of these solutions.