Marcha and thiat are traditionally prepared amylolytic starters use for production of various ethnic alcoholic beverages in Sikkim and Meghalaya states in India. In the present study we have tried to investigate the bacterial and fungal community composition of marcha and thiat by using high throughput sequencing. Characterization of bacterial community depicts phylum Proteobacteria is the most dominant in both marcha (91.4%) and thiat (53.8%), followed by Firmicutes, and Actinobacteria. Estimates of fungal community composition showed Ascomycota as the dominant phylum. Presence of Zygomycota in marcha distinguishes it from the thiat. The results of NGS analysis revealed dominance of yeasts in marcha whereas molds out numbers in case of thiat. This is the first report on microbial communities of traditionally prepared amylolytic starters of India using high throughput sequencing.

Preparation of dry starters for alcohol production is an age-old traditional technology in the Eastern Himalayan regions of east Nepal, the Darjeeling hills, Sikkim, and Arunachal Pradesh in India, and Bhutan. We studied the bacterial diversity in 35 samples of traditionally prepared dry starters, represented by marcha of Nepal, Sikkim, the Darjeeling hills, and Bhutan, phab of Bhutan, and paa, pee, and phut of Arunachal Pradesh, respectively. Populations of bacteria in these starters were 105 to 108 cfu/g. A total of 201 bacterial strains were isolated from starter samples, phenotypically characterized, and their identities confirmed by the 16S rRNA sanger sequencing method. The dominant phylum was Firmicutes (85%), followed by Proteobacteria (9%), and Actinobacteria (6%). Lactic acid bacteria (LAB) (59%) formed the most abundant group, followed by non-LAB (32%) and Gram-negative bacteria (9%). Based on the 16S rRNA gene sequencing result, we identified LAB: Enterococcus durans, E. faecium, E. fecalis, E. hirae, E. lactis, Pediococcus acidilactici, P. pentosaceus, Lactobacillus plantarum subsp. plantarum, Lb. pentosus, Leuconostoc mesenteroides, and Weissella cibaria; non-LAB: Bacillus subtilis subsp. inaquosorum, B. circulans, B. albus, B. cereus, B. nakamurai, B. nitratireducens, B. pseudomycoides, B. zhangzhouensis, Kocuria rosea, Staphylococcus hominis subsp. hominis, S. warneri, S. gallinarum, S. sciuri, Lysinibacillus boronitolerans, Brevibacterium frigoritolerans, and Micrococcus yunnanensis; Gram-negative bacteria: Pseudomonas putida, Klebsiella pneumoniae, Enterobacter hormaechei subsp. xiangfangensis, E. hormaechei subsp. steigerwaltii, and Stenotrophomonas maltophilia. We characterized diversity indexes of the bacterial community present in traditionally prepared dry starters. This is the first report on the bacterial diversity of traditionally dry starters of the Eastern Himalayas by sanger sequencing.

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