MICREDOX® - Reporting biochemical oxygen demand of synthetic and real wastewaters

Since 1912, the five-day biochemical oxygen demand test (BOD5) has been used to measure the amount of biodegradable organic compounds present in wastewaters (Tchobanoglous and Schroeder, 1987). The major limitation of this test is the five-day duration due to the low aqueous solubility of oxygen. Lincoln Ventures Ltd has developed an alternative assay, called MICREDOX®, to measure the biochemical oxygen demand (BOD) of water samples. MICREDOX® is a rapid, sensitive, and inexpensive assay which replaces oxygen with a highly soluble artificial electron acceptor (potassium ferricyanide) that is reduced by the microorganisms during the oxidation of organic substrates. The high solubility of the electron acceptor allows for much higher microbial concentrations to be used without the rapid depletion of the electron acceptor and consequently the time required to degrade significant amounts of organic matter is greatly reduced. Limiting-current micro electrode amperometry was used to measure the quantity of reduced electron acceptor produced during the MICREDOX® incubation. The resulting limiting-currents were converted into MICREDOX® BOD values which were equivalent to those obtained from the BOD5 test. Additionally the percentage conversion of the glucose/glutamic acid (GGA) standard was calculated. While previous MICREDOX® assays have reported good conversions for easily oxidised substrates such as the GGA standard, low correlations between the MICREDOX® and BOD5 values for synthetic and real wastewater samples have been reported. This study aimed to improve the correlation of the MICREDOX® assay and the BOD5 test for synthetic and real wastewaters by culturing the microbial biocomponent of the MICREDOX® assay under a range of conditions and then observing which conditions produced microorganisms capable of determining MICREDOX® BOD values closest to the BOD5 test values for the GGA standard, the Organisation for Economic Cooperation and Development synthetic sewage (OECD, 1993) standard and real wastewater samples. This study was performed using four microorganisms, Pseudomonas fluorescens, Bacillus subtilis, Klebsiella oxytoca and Arthrobacter globiformis. Initial screening experiments, i.e. API ZYM and BIOLOG Microplate™ tests, were conducted to obtain an overview of the types of enzymes secreted and substrates used by each microorganism. These tests divided the microorganisms into two groups. Group one included P. fluorescens, B. subtilis and K. oxytoca, which expressed and metabolised a similar and wide range of enzymes and substrates. Group two consisted of A. globiformis, which expressed a smaller range of enzymes and metabolised fewer substrates. Growth studies also revealed discrimination between group one and group two microorganisms; P. fluorescens, B. subtilis and K. oxytoca preferentially grew in growth media containing glucose and glutamic acid as carbon sources while A. globiformis preferentially grew in media containing peptones as the carbon source. A factorial experimental design was developed to systematically evaluate the factor or combination of factors that produced microorganisms capable of accurately determining MICREDOX® BOD values which were the same as those values reported by the conventional BOD5 test. Additionally the percentage conversion of the GGA standard was calculated to evaluate which factor(s) gave microorganisms capable of degrading the highest proportion of the GGA standard. The factors tested in the factorial design were microorganisms (P. fluorescens, B. subtilis, K. oxytoca and A. globiformis), growth media composition (glucose, glutamic acid and OECD media), media strength (1 and 10 mM), and microbial growth phase (exponential and stationary). The factorial experiments showed that microbial strain was the dominant factor to influence the MICREDOX® BOD values for the BOD standards. For example A. globiformis determined MICREDOX® BOD values for the OECD standard of up to 154 (c.f. BOD5 values of 171 mg-1 obtained from BOD5 test), while P. fluorescens, B. subtilis and K. oxytoca did not achieve MICREDOX® BOD values greater than 48 for this standard. Media composition and harvest stage also had a significant effect on the MICREDOX® BOD values. Generally cells cultured in glucose media and harvested at late stationary phase gave the highest BOD values for the OECD standard. The factorial experimental results also showed that microbial strain was the dominant factor influencing the percentage conversion of the GGA standard. In this case, however, P. fluorescens, B. subtilis, and K. oxytoca exhibited greater percentage conversion of GGA than A. globiformis. Microorganisms cultured in glucose, glutamic acid, or OECD media reported similar percentage conversions of the GGA standard. Microorganisms harvested at exponential growth phase gave higher percentage conversion of the GGA standard than cells harvested at stationary growth phase. MICREDOX® assays using mixtures of microorganisms as the biocomponent failed to report MICREDOX® BOD values for the OECD standard as high as those determined by single cultures of A. globiformis. Additionally single cultures of K. oxytoca gave the highest percentage conversion of the GGA standard (90 %). Freeze dried cells determined similar MICREDOX® BOD values for the GGA and OECD standards as the free cells and these values aligned well with the BOD5 values. The MICREDOX® assay, however, tended to under report primary wastewater and over report secondary wastewater BOD values when both free A. globiformis cells or freeze dried A. globiformis cells were used as the biocomponent. Further studies are required to investigate these limitations of the MICREDOX® assay