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 2(3) 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 degrees C; and the presence or absence of an enzyme cocktail consisting of lipase, beta-galactosidase and alpha-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 3h at 30 degrees C using a phosphate buffer with an enzyme cocktail.