Microbial diversity in different stages of the ethanol production process using culture-independent techniques

Although the ethanol production process is well established, microbial contamination can be a major obstacle to high productivity. The goal of this work was to characterize the microbial diversity of contaminants in different stages of the industrial ethanol production process using metagenomics. Samples from six stages of the ethanol production process were collected: sugarcane juice, mixed juice, clarified juice, evaporated juice, must and wine. DNA extraction of samples was performed, and used for pyrosequencing of Bacteria and Archaea 16S rRNA genes, and Fungi ITS (Internal Transcribed Spacer) region. Pyrosequecing revealed, in all the samples together, 355 groups at genus level for the domain Bacteria, 22 for the domain Archaea and 203 for the kingdom Fungi. Analysis of bacterial sequences showed that community changes are related to temperature rising in certain stages of the production process. After fermentation, Lactobacillus and unclassified Lactobacillales account for nearly 100% of the sequences. For Fungi as well as Archaea, sequence data was obtained only for sugarcane juice and mixed juice. The predominant Fungi groups were Unclassified Fungi and Candida. For Archaea, the predominant group was an unclassified Soil Crenarchaeotic group. Good’s coverage estimator showed that the number of sequences obtained was not enough to cover microbial diversity in all the stages. This is the first work to characterize the diversity of Bacteria, and Archaea and Fungi associated with the process of ethanol production from sugarcane using culture-independent techniques.