Chromobacterium violaceum R1-ferricyanide mediated biosensor for determination of biochemical oxygen demand

Abstract

Biochemical oxygen demand (BOD) is an important organic pollution parameter in water system because it indicates the amount of organic matters in water sample. Since the standard method of BOD measurement (BOD5) takes 5 days to complete, it is not practical for rapid environmental pollution monitoring. In the present study, an effective ferricyanide-mediated microbial biochemical oxygen demand (BOD) biosensor was constructed and used for rapid BOD determination in a water system. Prior to biosensor fabrication, microorganisms were first isolated from various water systems and then screened for their ability to use a redox mediator for organic degradation. The selected microorganism was immobilized onto the surface of the electrode tip for BOD measurement. The performance of the developed biosensor was then optimized, characterized and validated using synthetic organic solutions and real water samples. 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. Chromobacterium violaceum R1 (isolated from river water) was found to be a potential microorganism to be used as a biological sensing element in the biosensor. The technology of ultramicroelectrode (UME) was incorporated into the biosensor during transducer fabrication. The electrode tip (transducer) was fabricated in two-electrode configuration (10-µm Pt working electrode and 1-mm Pt counter electrode). Living C. violaceum R1 cells were immobilized onto the surface of the UME working electrode by using calcium alginate gel and further enclosed by a layer of polyamide membrane. Glucose-glutamic acid (GGA) solution and OECD synthetic wastewater were used as the standard solutions. The amperometric measurement was optimized at +450 mV operating potential and 30 mM ferricyanide in a 0.1 M phosphate buffer (pH 7.0) at 26°C. The developed biosensor exhibited a linear response ranging from 20–225 mg O2 L-1 BOD5 for standard GGA solution and 25–230 mg O2 L-1 BOD5 for OECD synthetic wastewater, with a response time of 30 min. Repeatability and reproducibility of the biosensor were within the limits set by the APHA—less than 15.4%. The biosensor is applicable for rapid BOD measurement of samples with a high content of fast and easily assimilated compounds. When used to estimate the BOD of various wastewaters, the developed biosensor gave values comparable to those obtained using the conventional BOD5 method. Hence, a biosensor has been successfully developed for rapid determination of BOD in water samples.