MICREDOX ®—development of a ferricyanide-mediated rapid biochemical oxygen demand method using an immobilised Proteus vulgaris biocomponent

Abstract

Biochemical oxygen demand (BOD) is an international regulatory environmental index for monitoring organic pollutants in wastewater and the current legislated standard test for BOD monitoring requires 5 days to complete (BOD 5 test). We are developing a rapid microbial technique, MICREDOX ®, for measuring BOD by eliminating oxygen and, instead, quantifying an equivalent biochemical co-substrate demand, the co-substrate being a redox mediator. Elevated concentrations of Proteus vulgaris, either as free cells or immobilised in Lentikat ® disks, were incubated with an excess of redox mediator (potassium hexacyanoferrate(III)) and organic substrate for 1 h at 37 °C without oxygen. The addition of substrate increased the catabolic activity of the microorganisms and the accumulation of reduced mediator, which was subsequently re-oxidised at a working electrode generating a current quantifiable by a coulometric transducer. The recorded currents were converted to their BOD 5 equivalent with the only assumption being a fixed conversion of substrate and known stoichiometry. Measurements are reported both for the BOD 5 calibration standard solution (150 mg l −1 glucose, 150 mg l −1 glutamic acid) and for filtered effluent sampled from a wastewater treatment plant. The inclusion of a highly soluble mediator in place of oxygen facilitated a high ferricyanide concentration in the incubation, which in turn permitted increased concentrations of microorganisms to be used. This substantially reduced the incubation time, from 5 days to 1 h, for the biological oxidation of substrates equivalent to those observed using the standard BOD 5 test. Stoichiometric conversion efficiencies for the oxidation of the standard substrate by P. vulgaris were typically 60% for free cells and 35–50% for immobilised cells.