An Approach to Constructing Multispecies Biofilm Communities from Rhizosphere Soil

In our research, we focused on rhizosphere bacteria and biofertilizers, and we aim to develop synthetic biobacterial communities that will be used as second-generation biofertilizers. The focus of our research on rhizosphere bacteria has shift from single-strain to community-level. However, there is no standard methods for building synthetic bacterial communities.

Currently, there is still uncertainty regarding the compatibility and colonization ability of synthetic bacterial communities predicted by microbiome analysis. Thus, it is also a major bottleneck in the development of second-generation biofertilizers. Compared to large sequencing data of conventional methods, our culture first, identify later approach greatly reduces the amount of work involved in 60 SRR gene sequencing.

It is cost-effective to create synthetic bacterial communities that colonize use quickly and efficiently. Our future research will focus on biological function of bacterial communities based on multi-species biofilms, and we will pay particular attention to promoting plant growth, improve plant stress resistance, and protecting plant health. To begin, obtain cucumber seeds, and disinfect them with 75%ethanol, followed by 2%sodium hypochlorite solution.

After rinsing the seeds in sterile water, germinate them under sterile conditions. Transplant the consistently sprouted, two-leaf stage seedlings into pots containing two kilograms of black soil. When the seedlings reach the four-leaf stage, invert the pot and isolate the roots, along with two-millimeter thick rhizosphere soil, using sterilized gloves.

Place the roots in 30 milliliters of PBSS buffer in a 50-milliliter centrifuge tube. Shake the tube for 20 minutes, and filter the suspension through a 100-micrometer nylon cell filter to remove roots or large sediments. Transfer the filtrate to another 50 milliliter centrifuge tube, and centrifuge it at 3, 200 g for 15 minutes at room temperature.

Temporarily store the tube at four degrees Celsius before extraction of rhizosphere soil bacterial cells. To begin, establish a multi-species rhizosphere biofilm, and obtain the microbiota cell suspension. Mixed Triptyc 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.