Method for the detachment of culturable bacteria from wetland gravel

The study of bacterial communities in microbially-mediated water treatment systems is becoming increasingly popular. Aquatic bacterial communities are often found in fixed-film environments, residing within a matrix of extracellular polymeric substances commonly referred to as a biofilm. A method for detaching the biofilm is required to either enumerate or characterize these bacterial communities. There are a variety of detachment methods including scraping, swabbing, shaking, sonication, blending, and digestion. The objective of this work was to develop an agitation-based protocol for detachment of culturable bacterial communities from the biofilm surrounding pea gravel from constructed wetland mesocosms. Three different protocol factors were systematically investigated using a triplicated 23 factorial design to determine the most effective detachment protocol. Factors studied included: the use of either tap water or phosphate buffer as the shaking/detachment solution; the use of either manual-shaking at room temperature or mechanical shaking at 30 °C; and the presence or absence of an enzyme cocktail consisting of lipase, β-galactosidase and α-glucosidase. The resulting suspensions were evaluated for organics, inorganics, culturable bacteria, community level physiological profile (CLPP) and several BIOLOG™ ECO plate substrate related diversity indices. Using these metrics, the most effective shaking/detachment protocol was identified as mechanical shaking for 3 h at 30 °C using a phosphate buffer with an enzyme cocktail.