Hanstruepera marina sp. nov. and Hanstruepera flava sp. nov., two novel species in the family Flavobacteriaceae isolated by a modified in situ cultivation technique from marine sediment

A modified in situ cultivation technique was developed and applied to resource mining of uncultured microbes from marine sediments of Meishan Island in the East China Sea. Two novel strains NBU2968(T) and NBU2984(T) were isolated by this method but not standard Petri dish, which indicated the modified technique was more effective compared to conventional approaches for isolating uncultured microbes and could be popularized and applied to other aquatic environments. The two novel strains were identified by the polyphasic taxonomic approach. Cells of both strains were observed to be Gram-staining-negative, rod-shaped, nonmotile, aerobic, and yellow-pigmented. Catalase and oxidase activities and hydrolysis of Tweens 40, 60, and 80 of two novel strains were positive. Methyl red reaction, H(2)S production, and hydrolysis of Tween 20 were negative. According to 16S rRNA gene sequence analysis, two novel strains shared the highest similarities (96.4–97.7%) to the species with a validated name in the genus Hanstruepera, while shared lower sequence similarities (<95.6%) to all other genera. Phylogenetic analysis revealed that strains NBU2968(T) and NBU2984(T) were affiliated with the genus Hanstruepera. ANI and dDDH values between the two novel strains and Hanstruepera species were 77.4–78.3% and 20.4–20.9%, respectively, which were below the thresholds for species delineation. The 16S rRNA gene sequence similarity, ANI, and dDDH values between the two novel strains were 99.3, 88.9, and 36.3%, respectively, indicating that the two strains represent different species. The genomes of NBU2968(T) and NBU2984(T) were 3.28 Mbp with a G+C content of 34.2% and 3.09 Mbp with a G+C content of 34.4%, respectively. The only respiratory quinone was menaquinone-6 (MK-6). The major cellular fatty acids were iso-C(15:0), iso-C(15:1)G, and iso-C(17:0) 3-OH. The major polar lipids of the two strains were phosphatidylethanolamine, unidentified amino lipids, and unidentified lipids. Based on the above polyphasic characteristics, strains NBU2968(T) and NBU2984(T) represent two novel species within the genus Hanstruepera, for which the names Hanstruepera marina sp. nov. and Hanstruepera flava sp. nov. are proposed. The type strains are NBU2968(T) (= MCCC 1K06392(T)= KCTC 82913(T)) and NBU2984(T) (= MCCC 1K07472(T)= KCTC 92511(T)), respectively.