Isolation and characterization of novel bacterial taxa from extreme alkali-saline soil

In northeast China there are large areas of nearly bare soda saline-alkali soil, in which plant survival is exteremely difficult because the land has a pH greater than 10.5. In order to obtain resistant microbial resources able to grow in such conditions, we analyzed environmental microbial samples from this extreme saline-alkali soil region. Through selective culture conditions, 8 bacterial strains were isolated from medium with no less than 1.5 M NaCl, 12 were isolated in the medium with a pH value of no less than 11.0, and 8 were isolated in the medium with of no less than 200 mM Na2CO3. Based on 16S rRNA gene sequence analysis 20 novel strains of bacteria were identified and classified into four groups: 8 group I strains belong to the genus Bacillus; three group II belong to genus Nesterenkonia (2 strains) and genus Zhihengliuella (1 strain); eight group III strains belong to the genera Halomonas (6), Stenotrophomonas (1) and Alkalimonas (1); one group IV strain belongs to genus Litoribacter. These four groups belong to the phylum Firmicutes, phylum Actinobacteria, phylum Gamma-Proteobacteria and phylum Bacteroidetes, respectively. The sequence homology of 16S rRNA in strains ANESC-S, H.sp.C4 and H.sp.C6 with that of known strains was 93.2, 96.5 and 96.5%, respectively. Based on the 97.0% identity cutoff commonly used to discriminate bacterial species, our data suggest that H. sp.C4 and H.sp.C6 may be new species, and ANESC-S may be belong to a new genus of classified into order Cytophagales. The morphological characteristics by scanning electron microscopy (SEM) showed that, in addition to the coccal N.sp.N3, the majority (19) of the isolates are bacilli of various lengths. In culture the colonies appeared red, orange, yellow, light yellow, and milk-white, with milk-white being predominant. Based on the resistance to NaCl and pH, the 20 novel strains were classified into obligate alkaliphilic and halophilic bacteria, obligate alkaliphilic halotolerant bacteria, and facultative alkalophilic salt-tolerant bacteria. This is the first study reporting the isolation and characterization of bacterial resources from extreme saline-alkali soils from northeast China.