Description of Microbacterium luticellae sp. nov., Microbacterium alcoholitolerans sp. nov. and Microbacterium daqui sp. nov. isolated from baijiu pit mud and daqu

Four Gram-stain-positive bacterial strains, designated as YY-01 T , YY-02 T , YY-03 T and YY-04, were isolated from pit mud (YY-01 T , YY-02 T and YY-04) and daqu (YY-03 T ) samples collected at a winery in Shandong Province, China. Phylogenetic and genomic analyses, along with bioassays and antibiotic susceptibility testing, revealed that the four strains, YY-01 T , YY-02 T , YY-03 T and YY-04, represent three novel species within the genus Microbacterium . Phylogenetic reconstruction based on 16S rRNA gene sequences revealed the closest relatives as follows: strain YY-01 T shares closest affinity with Microbacterium pseudoresistens CC-005209 T (97.1% similarity), strains YY-02 T and YY-04 cluster with Microbacterium esteraromaticum DSM 8609 T (98.3–98.4% similarity) and strain YY-03 T aligns with Microbacterium mitrae M4-8 T (97.6% similarity). Genome phylogeny further resolved strain YY-01 T as a sister lineage to Microbacterium laevaniformans DSM 20140 T , while strains YY-02 T , YY-03 T and YY-04 exhibited phylogenetic relationships congruent with the 16S rRNA topology. Genomic relatedness analysis demonstrated that strains YY-02 T and YY-04 share an OrthoANIu value of 99.9% and a digital DNA–DNA hybridization (dDDH) value of 100%, confirming their classification as conspecific. In contrast, OrthoANIu and dDDH values between strains YY-01 T , YY-02 T and YY-03 T and other Microbacterium species were ≤82.1 and 28.3%, respectively, supporting their distinct species status. Chemotaxonomic analysis revealed distinct profiles for each strain. Strain YY-01 T was characterized by MK-13 menaquinones; major fatty acids anteiso-C 15 : 0 , anteiso-C 17 : 0 and iso-C 16 : 0 ; and the polar lipids diphosphatidylglycerol (DPG) and phosphatidylglycerol (PG). Strain YY-02 T contained predominant menaquinones MK-11 and MK-13 and major fatty acids anteiso-C 17 : 0 , anteiso-C 15 : 0 and iso-C 16 : 0 , along with DPG and PG. Strain YY-03 T possessed major menaquinones MK-11 and MK-13; major fatty acids anteiso-C 15 : 0 , iso-C 15 : 0 and iso-C 16 : 0 ; and polar lipids DPG and PG. Integrative genomic, phenotypic and phylogenetic evidence supports the proposal of three novel species: Microbacterium luticellae sp. nov. (type strain YY-01 T =GDMCC 1.5102 T =KCTC 59478 T ), Microbacterium alcoholitolerans sp. nov. (type strain YY-02 T =GDMCC 1.5103 T =KCTC 59479 T ) and Microbacterium daqui sp. nov. (type strain YY-03 T =GDMCC 1.5104 T =KCTC 59480 T ).