Biodegradability testing using standardized microbial communities as inoculum

Abstract

Reference materials are important tools for maintaining high-quality assurance standards, including for biological materials. A significant number of environmental international standards, including biodegradability and toxicity, involve utilization of activated sludge (AS) inocula. The absence of inoculum standardization in these tests is a potential source of error influencing the results. In this study three defined microbial consortia, two commercial inocula and a designed bacterial inoculum, were evaluated as an AS alternative seed for the Zahn-Wellens test, using diethylene glycol as the reference chemical. The results showed that to achieve diethylene glycol biodegradation with these inocula, a number of 10(5) colony-forming units per milliliter of effective degrader microorganisms had to be present. Moreover, the addition of AS supernatant to the test mixtures improved inocula performance (the biodegradability curves), bringing them closer to those obtained with AS inocula. Among the three defined consortia, the designed inoculum replicates bested the AS behavior in the range of concentrations tested, with degradation completed in 12-14 days. Comparisons of the community substrate utilization profiles corroborated these results, showing that the designed inoculum profile was the most similar to the AS profile. The biodegradability and metabolism results provided support for the assertion that the designed inoculum can be used in the Zahn-Wellens test and as a base to develop reference inocula for other biodegradability and toxicity tests.

Standardization of activated sludge for biodegradation tests

“…Several workers have pointed out the need for a certified biological material with the same metabolic potential as an activated sludge that can be used for quality control and to provide traceability [38][39][40][41]. So, the use of surrogate cultures has been proposed to fulfil the need of an activated sludge with constant characteristics.…”

“…The potential of Bi-Chem and Biolen to be used as alternative inocula to activated sludge was evaluated by assessing the biodegradability of diethylene glycol in the Zahn-Wellens assay [40]. Whereas complete biodegradation was achieved in 10-12 days with activated sludge, none of the commercial seeds reached the test validity criterion (biodegradation >70% on the 14th day, [25]).…”

“…Because four of these mixtures (composed of four to six strains) had repeatable functional diversity similar to that of an activated sludge, they were suggested as potential biological reference material in activated sludge-based bioassays. In a study already mentioned [40], a defined consortium composed of equal parts of six bacterial strains (i.e., Acinetobacter sp., Enterobacter sp., S. capitis, P. putida, B. subtilis and B. cereus) was compared with activated sludge samples using diethylene glycol as reference chemical in the Zahn-Wellens assay. Among the surrogates, the defined consortium behaviour was the most similar to that of activated sludge, although not as fast.…”

Assessing Microbial Communities for a Metabolic Profile Similar to Activated Sludge

“…The use of synthetic medium to grow each of these three consortia was the best methodology, because it enhanced the microbial metabolic responses for substrate use—higher net areas and higher number of substrates used (i.e., increased the metabolic potential). These three model communities, with CLPPs similar to activated sludge CLPPs, have already been tested as a biological reference material in toxicity and biodegradability tests, in which their potential to be used as alternative inocula to activated sludge was demonstrated ( Paixão et al, 2000 , 2006 ; Paixão, Santos, Baeta‐Hall, Tenreiro, and Anselmo, 2003 ; Paixão, Santos, Tenreiro, and Anselmo, 2003 ).…”

New Concepts in the Evaluation of Biodegradation/Persistence of Chemical Substances Using a Microbial Inoculum

“…The first approach is “standardization.” To this end several authors have suggested an increase in the cell density to control the F/M ratio or to acclimate the inocula, but unfortunately without creating sufficient interest amongst regulators (Thouand et al, 1995, 1996, 1999; Ingerslev et al, 2000; Vasquez-Rodriguez et al, 2007). Other ways have been attempted, especially the use of commercial seeds, but the undefined inocula were only of interest in relation to readily biodegradable substances (Tabka et al, 1993; Thouand et al, 1999; Sharma et al, 2000; Paixão et al, 2006). …”