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Bacterial contamination of raw diffusion juice poses unique challenges during the sugar extraction process. This study profiled bacterial communities by using full-length 16S rRNA amplicon sequencing and quantified the carbohydrate concentrations in raw diffusion juice samples received from sugar factory regions across the USA and Canada. Juice samples were collected at four time points during the 2021 and 2022 processing campaigns. Firmicutes was the dominant phylum from the raw diffusion juice samples collected during both campaigns and comprised 85.5% of total bacterial abundance. Lactic acid bacteria such as Leuconostoc and Lactobacillus were among the core genera which also dominated the bacterial community in raw diffusion juice. Positive correlations in the abundance of functionally and taxonomically related bacterial communities were identified. During the 2021 campaign, 44 bacterial genera were differentially abundant in raw diffusion juice extracted from sugarbeet roots in Periods 1 to 4. This number declined sixfold during the 2022 campaign to three genera. The concentration of raffinose in raw diffusion juice positively correlated to the relative abundance of Leuconostoc . Furthermore, an in vitro assay was performed to assess the growth dynamics of Leuconostoc mesenteroides in sucrose or raffinose-rich medium and observed the rapid consumption of both carbohydrates by this bacterium. This finding is important for deciphering microbial growth dynamics in raw diffusion juice that can be useful in minimizing sugar loss during the factory processing.IMPORTANCEFindings additionally provide baseline information that can be used to develop mitigation strategies that reduce losses due to microbial contamination of sucrose processing streams., Competing Interests: The authors declare no conflict of interest.