Improved quantification of aggregated bacteria by combined enzymatic and mechanical treatment of flocs and biofilm from a rotating drum bioreactor

For estimates by plate counts of cell numbers of aggregated bacteria growing in a rotating drum bioreactor (RBR) used to examine biological nitrate elimination (denitrification) it was necessary to develop or to improve treatment methods for the disaggregation of bacterial flocs and biofilm into individual cells. In order to propagate and identify single strains (1) conventional mechanical methods and (2) a combination of enzymatic and mechanical methods were tested. Ultrasonic treatment of bacterial flocs at 50 W for 120 sec yielded the highest number of colony-forming units (CFU) as determined by plate counts. Cellulase pre-treatment of bacterial aggregates or biofilms consisting mainly of pseudomonads was used to loosen the extracellular polymeric substances (EPS) which are responsible for the stability of these bacterial aggregates. Subsequent mechanical treatment led to substantially improved disaggregation without significant impairment or disrupture of cells. For the detachment of bacterial biofilm growth on the packing material, ultrasonication was used for post-treatment, whereas an ultra-turrax homogenizer was sufficient for disaggregation of the bacterial flocs.