Phenotypic and Genotypic Identification of Bacteria Isolated From Traditionally Prepared Dry Starters of the Eastern Himalayas

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 10(5) to 10(8) 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.