AimsTo utilize a three-tiered approach to provide insight into the microbial community structure, the spatial distribution and the metabolic capabilities of organisms of a biofilm in the two towers of a high-rate biological contact reactor treating winery wastewater. Methods and ResultsNext-generation sequencing indicated that bacteria primarily responsible for the removal of carbohydrates, sugars and alcohol were more abundant in tower 1 than tower 2 while nitrifying and denitrifying bacteria were more abundant in tower 2. Yeast populations differed in each tower. Fluorescent insitu hybridization coupled with confocal microscopy showed distribution of organisms confirming an oxygen gradient across the biofilm depth. The Biolog system (ECO plates) specified the different carbon-metabolizing profiles of the two biofilms. ConclusionsThe three-tiered approach confirmed that the addition of a second subunit to the bioreactor, expanded the treatment capacity by augmenting the microbial and metabolic diversity of the system, improving the treatment scope of the system. Significance and Impact of the StudyA three-tiered biofilm analysis provided data required to optimize the design of a bioreactor to provide favourable conditions for the development of a microbial consortium, which has optimal waste removal properties for the treatment requirements at hand.