Screening And Resistance-analysis Of Microorganisms In Extreme Saline Soil Environments

Songnen Plain is one of the world's three concentrated regions of saline-alkali soil, and its main salt compositions are sodium carbonate and sodium bicarbonate. In the heavily saline-alkali region of Songnen Plain, all are the contiguous saline-alkali spots most of which are bare spots except for sporadic growth of some seepweed, Artemisia, Kochia scoparius and so on. The environment which common creature cannot survive in is termed extreme environment, and saline-alkali spot soil environment is extremely alkaline. Extreme environment is often the main habitat of extremophiles. Due to the long-term pressure of natural selection, microorganisms in extreme saline-alkali soil environments enjoy the special microbial species and their associated functions which just lack of in the general environmental, and its saline-alkali tolerant characteristics suggest a wealth of saline-alkali tolerant genes. These resources will be useful for selective breeding saline-alkali resistance new varieties of plants. Therefore, the related research of soil microorganisms and their resistance characteristics in this extreme saline-alkali environment is a prerequisite to obtain good gene resistant to saline-alkali.Compared with NaCl stress, the impact of Na2CO3and NaHCO3stress on microorganism is more complex. In this paper, extreme saline soil and non-saline soil were choosed as experimental material and control respectively, and we compared and analyzed the different tolerance level of bacteria between these two soils. Based on three types of selection pressure(high salt and low pH, high salt and high pH, high pH), we screened a variety of bacteria with high saline-alkali tolerance from extreme saline soil. And then, through microscopes and scanning electron microscopy, the colony morphology and individual morphology of bacteria obtained were analyzed, and by bacterial16S rDNA sequence analysis, the molecular identification of the bacteria isolated was completed. Morver, we studied the resistance characteristics of these strains on NaCl, pH and Na2CO3in details. This study reveals the extreme saline soil microbial resources in the overall characteristics of bacterial community composition and resistance characteristics. The result has practically instructional significance on understanding of physiological and ecological mechanisms of salt-tolerance of bacteria. More importantly, it is the first time to complete a systematic study in the resistance characteristics of bacteria which survived in extreme saline soil without plant cover of Songnen Plain soda saline-alkali soil on saline-alkali stress, and this study laid a theoretical foundation for microbial resistance resource development of Songnen plain.Main results of the above research are as follows:1. In conclusion, there were more high salinity bacteria in extreme soda saline-alkali soil than in non-saline soil. There were significant differences between these two soil types, when take Na2CO3and pH as stress factors. The bacteria were disappeared in repeated cultivated non-saline soil, but many bacterial colonies were survived in extreme soda saline-alkali soil when the concentration of Na2CO3was higher than30mmol/L and pH value was higher than10.0. When take NaC1as a stress factor, most of bacteria was moderately salt-tolerant bacteria in extreme soda saline-alkali soil. Compared with bacteria in extreme soda saline-alkali soil, the higher concentrate of NaHCO3has inhibited bacteria in non-saline soil, but the inhibitory potency was lighter than the other inhibiting factors.2. Twenty-eight of bacteria which had high saline-alkali resistance were selected through various filter selection pressure. Physical microscope image showed that there were some certain differences in these bacteria's size, color and edges. Scanning electron microscope showed that the individual morphological characteristics of the bacteria from alkali spot soil were mainly rod-shaped, only one spherical bacterium was separated.3. Twenty-eight of the isolated strains were analyzed by the method which based on phylogenetic development of16S rRNA gene sequence. The results showed that the isolates are members of twenty one species of five families(Micrococcaceae, Cytophagaceae, Halomonadaceae, Xanthomonadaceae) and an unclassified family in the Gamma-Proteobacteria group of seven genera(Bacillus, Nesterenkonia, Zhihengliuella, Halomonas, Stenotrophomonas, Alkalimonas, Litoribacter) in four major phylogenetic groups(Bacteroidetes, Firmicutes, Actinobacteria, Gamma-Proteobacteria). Most of the strains belonged to Firmicutes and Gamma-Proteobacteria category, followed by Bacteroidetes and Actinobacteria category.4.Strains H1was characterized using a polyphasic approach. Strain H1was Gram- negative, strictly aerobic and non-motile and formed orange red colonies. Range growth conditions were10～37℃, pH7.5-9.2and0.5～4.0%NaCl. Phylogenetic analysis based on16S rRNA gene sequence comparisons showed that the isolate formed a distinct line within a clade containing the genus Litoribacter in the phylum Bacteroidetes and was related to the species Litoribacter ruber YIM CH208T(sequence similarity93.2%). The DNA G+C content was39.1mol%. The predominant cellular fatty acids were C15:0iso(27.50%)C17:0iso3OH(13.95), summed feature9(C17:1iso w9c or C16:010-methyl10.63%) and C17:1iso w9c10.63%. On the basis of the phenotypic, chemotaxonomic and phylogenetic data, strain H1represents a novel species of a new genus in the Bacteroidetes.5.Based on the resistance to sodium carbonate and sodium chloride, the bacteria under alkaline conditions of Songnen Plain could be divided into six types:halophilic bacteria of strong resistance to sodium carbonate, halophilic bacteria of moderate resistance to sodium carbonate, halophilic bacteria of slight resistance to sodium carbonate, salt-tolerant bacteria of strong resistance to sodium carbonate, salt-tolerant bacteria moderate resistance to sodium carbonate, salt-tolerant bacteria of slight resistance to sodium carbonate.