ELUCIDATING THE DIVERSITY OF MICROBIAL RETTING COMMUNITIES ON HIBISCUS CANNABINUS USING NEXT-GENERATION SEQUENCING

Purpose (a): Global environmental concerns have led to a growing interest in renewable resources such as plant-based fibers. Beyond textiles and cordage, plant fibers have the potential for incorporation into renewable, bio-based composite materials for the building and manufacturing sectors. Successful commercialization of fiber production requires optimization of fiber extraction. Retting is the traditional method of fiber extraction, whereby endogenous microorganisms break down heteropolysaccharides to release fiber bundles. Previous studies have analyzed the retting solution for its bacterial constituents, but none have followed changes in the microbial community through the retting process. This research aims to track the bacterial components of the retting community through time, and determine the effects of bacterial augmentation with isolated pectinolytic bacteria using next generation sequencing of 16S rRNA gene amplicons. Methods (b): Batch C1 included plant material and an inoculum of pond water represented a “traditional” retting environment, while C2 contained autoclaved pond water to represent the endogenous microorganisms associated with the plant material. Experimental batch E1 included the addition of three pectinolytic bacterial isolates: Bacillus DP1, Paenibacillus DP2, and Bacillus K1. Total DNA was extracted from the surface-adhering biofilm bacteria and used for PCR with full-length 16S primers 27F and 1492R. Amplification products were used as templates for a nested PCR with primers 786F and 939R targeting variable region 5 of the 16S molecule. The nested 16S rRNA amplicons were then sequenced on the next-generation Ion Torrent PGM platform. Results (c): The E1 environment showed a marked increase in phylum Firmicutes (55 to 94%), while phylum Proteobacteria showed a progressive decrease from Day 1 to 4 (36% to 5%). This was correlated with easy separation of fibers by mechanical movement. At a finer taxonomic level, E1 showed a rapid loss of inoculated Bacillus species DP1 and K1, and a more gradual loss of the family Paenibacillaceae 1 (P. DP2) as the time course progressed. In contrast, C1 was co-dominated by phyla Firmicutes and Proteobacteria, while C2 was composed in large part by the phylum Bacteroidetes. Additionally, comparison of the microbial communities under the different conditions revealed differences in diversity and composition at day 4 time points between the three conditions. Conclusions (d): Introduction of pectinolytic bacteria into the batch reactions increased production rate and increased fiber quality. Introduction of Paenibacillus DP2 is likely the driving force behind the community shifts detected in E1, which warrants further study to determine the mechanism of action. The findings confirmed previous studies that suggest a gradual replacement of aerobic organisms to an environment that is dominated by strict anaerobes. Moreover, the efforts shed light on conditions and mechanisms for the manipulation of microbiomes. These approaches may have relevance to the treatment of dysbiosis in the gut flora in humans and the treatment of related diseases.