Sequencing and Identification of Multispecies Biofilm Microbiota from Rhizosphere Samples

To begin, establish a multi-species rhizosphere biofilm, and obtain the microbiota cell suspension. Mix triptych soy broth media with minimal salts glycerol glutamate media in a one-to-one ratio. Incubate the cell suspension in the medium at 30 degrees Celsius overnight to activate the bacteria.

Place 100 micrometer nylon cell filters on a 24 well plate. Inoculate the bacterial suspension in three replicates and cultivate the pellicle biofilm for 36 hours at 30 degrees Celsius. Remove the filter containing the pellicle biofilm and add two milliliters of PBSS buffer to a new 24 well cell plate.

Place the filter into the plate to wash the biofilm and remove the free cells. After identification and cultivation of the main five species in the biofilm, incubate the 31 combinations of synthetic communities in the medium. Following the mentioned procedure, significant gradients in biofilm-forming capacity from cucumber rhizosphere soil microbiota were observed.

Biofilm amplicon sequencing confirmed the presence of various species in the pellicle.