Development of a standard bacterial consortium for laboratory efficacy testing of commercial freshwater oil spill bioremediation agents

Six crude oil-degrading bacterial strains isolated from different soil and water environments were combined to create a defined consortium for use in standardized efficacy testing of commercial oil spill bioremediation agents (OSBA), The isolates were cryopreserved in individual aliquots at pre-determined cell densities, stored at -70 degrees C, and thawed for use as standardized inocula as needed, Aliquots were prepared with precision (typically within 10% of the mean) ensuring reproducible inoculation, Five of the six strains displayed no appreciable loss of viability during cryopreservation exceeding 2.5 years, and five isolates demonstrated stable hydrocarbon-degrading phenotypes during inoculum preparation and storage. When resuscitated, the defined consortium reproducibly biodegraded Alberta Sweet Mixed Blend crude oil (typically +/- 7% of the mean of triplicate cultures), as determined by quantitative gas chromatography-mass spectrometry of various analyte classes, Reproducible biodegradation was observed within a batch of inoculum in trials spanning 2.5 years, and among three batches of inoculum prepared more than 2 years apart, Biodegradation was comparable after incubation for 28 days at 10 degrees C or 14 days at 22 degrees C, illustrating the temperature tolerance of the bacterial consortium. The results support the use of the synthetic consortium as a reproducible, predictable inoculum to achieve standardized efficacy tests for evaluating commercial OSBA.