Microorganism Ecology And Actinomycetes Resources In Saline-Alkali Extreme Environments

The melioration of saline and alkali soils is a difficult topic faced in the world. For long time, in the study filed of saline and alkali soil ecology, the research has been focused on plant physiology and bio-ecology. However, the research on microorganism in saline and alkali extreme environments is little because there are some old ideas that saline and alkali soils were life forbidden zone and shortage of the suitable methods for microorganism isolation and culture. Therefore, the exploitation and application of microorganism resources in saline and alkali extreme environments are very scarce. In order to investigate the secret and mechanisms of microorganism-ecological disciplines and exploitation and application of microorganism resources in plant, fishing and animal feeding for disease preventing and killing, saline and alkali soils in Ningxia, Shaanxi was used in this research for systematically studying the microorganism ecology and resource, and detecting the isolation and culture methods which are suitable for microorganism in saline-alkali extreme environment. At the same time, polyphasec techniques were used for classification and identification of microorganism in extreme environments and applied the techniques to practice. These will push the study on microorganism resource in saline-alkali extreme eco-environment advanced and forward the exploitation of actinomycetes resource. All that will be the solid foundation for the research of microorganism resource in saline-alkali extreme environment.This dissertation started with the studies on characteristics of saline and alkali soils in different typical ecology environments, analyzed the composition and population of microorganism and actinomycetes, and the relationships between them to enzyme activity, the primary properties of saline and alkali soils, salt ions constituent and other factors. DGGE as a method was employed in microorganism-ecological studies. The isolation methods of actinomycetes in saline-alkali extreme environments were built so as to obtaining a lot of actinomycetes. Meanwhile, application research of antagonistic were done by adopting the most common and dangerous botanical and animal pathogens in agricultural production as target pathogens in order to get some antagonistic actinomycetes with application values; At the same time, polyphasec techniques were used for classification of actinomycetes with special morphology or physiological characteristics and strains with high antagonistic function. The classification status of the actinomycetes was determined and several potential new species were obtained. The main results are as follows:1. The distribution principle of microbial ecology in saline and alkali extreme environments was obtainedBased on this research, the results showed that the distribution of microbial ecology in soil samples was greatly affected by soil types, salt ions constituent and soil physical and chemical properties. The bacterial and fungi amounts have significant positive correlation with nutrient content and [CO₃^2-+HCO^3-]/[Cl^-+SO₄^2-], and it was significant negative correlation with pH value and water-soluble salt content. However, actinomycetes was significant positive correlation with nutrient content, but was not correlated with the other factors. These indicate that actinomycetes have strong toleration to salt content and pH value. There was large population proportion of actinomycetes in different saline-alkali soils. This further revealed the importance of obtaining actinomycetes resources from extreme environments.The population distribution of shalotolerant actinomycete was different from various salinization degrees of soils. As a whole, population of shalotolerant actinomycete distributed in heavy salinization soil was the most, and followed by in moderate salinization soil and in light salinization soil. Based on the development of actinomycetes depended on salt content, actinomycetes can be divided into halophilic actinomycete developing well only in saline-alkali soil and halotolerant actinomycete with different susceptivity to salt content. The population distribution of actinomycete in extreme environment and the characteristic of halophilic and halotolerant actinomycete will provide the theoretical support and science foundation for the studies on actinomycete resources.2. Detection of the application of DGGE method in microbial ecology researchSoil microbial diversity in five typical saline-alkali soils was studied with the DGGE free-culture method. The total DNA indirectly extracted from the enrichment culture and the total DNA extracted directly from soil samples using DGGE method were compared. And it was also compared with the traditional plate count method. The results showed that the enrichment culture of microbial diversity analysis was better than directly extraction from soil samples and plate count. DNA direct extraction from environment access to the population and types of micro-organisms had obvious advantages, but the methods and effects of DNA extraction should be noted. This approach effectively reveals the microbial species diversity in saline environment, and also provides a theoretical basis for further explore the actinomycetes resources in extreme environments.3. Establishment of the isolation method system of actinomycetes in salinized extreme environmentsThis study investigated the effects of pretreatment of samples, activators, enrichment culture, medium design, salt types and concentrations design, different pH and others on the isolation effects of actinomycetes. The pretreatment methods to different saline samples and feasible isolation mediums were obtained. The isolation method systems of actinomycetes in saline environment were preliminarily established. These are very important for obtaining more actinomyceres in saline environment as well as for providing great technical support for the actinomyceres research.4. Analysis the components and population of actinomycetes flora in saline soil and selection of some strains with special physiological characteristicsThe relationshiip between actinomycetes and other ecological factors in saline soil was analyzed systematically and the distribution of actinomycetes in different environments was studied. The relationship between actinomycetes population and soil pH, [CO₃^2-], polyphenol oxidase activity are very significant positive correlated. Aactinomycetes composition in waste land is more complex than in arable land. In saline soil or in alkali soil, actinomycetes composition is more complex than other soil type. The ecological distribution of actinomycetes provides good theoretical basis for further studies on actinomycetes resources in saline-alkali soil. Simultaneously, 33 rare actinomycetes with specific form and 22 actinomycetes with salt-tolerant more than 18 percent salt content were identified, which providing a strain for the further study by the multi-phase classification technologies in order to obtain new taxonomy system.5. Determination of the characteristics of antibacterial spectrum of habitat salinity actinomycetes to nine kind animal and plant pathogens and Selection of some strains which had good antagonism.It was obtained that the characteristics of antibacterial spectrum of habitat salinity actinomycetes to nine kind animal and plant pathogens. Some strains with good antagonistic effects on animal pathogens were screened, which could be better strains for the biological prevention of major disease in animal husbandry. Three strains of actinomycetes 02D01, 43A04 and 13H06 with good antagonism spectrum were selected according to screening and re-screening.A strain 02D01 was found that its fermentation solution had good Inhibition effect on Point Aeromonas causing Fish bacterial enteritis. Dosage of 2 mg /kg·d could reach good treatment, and the fermentation solution had no acute toxicity but good temperature and acid stability.Through fermentation technology optimization, the best culture contained 2% soluble starch, 2% fish powder, 2% NaCl, 0.05% K₂HPO₄, 0.05% MgSO₄·7H₂O, 0.1% CaCO₃, in which the strain could grow and produce antibacterial substances, and the activity were best at the seed age of 48 h, inoculum of 5%, temperature of 25℃, initial pH of 9, fermentation time for 120 h, fermentation solution titer could reach 34197.94μg/ml, improved 27 times, which could be basis for deep production and application.6. The multiple taxonomy technology of actinomycetes habitated in salinity was perfected and classification status of some special actinomycetes was determined.In the study, morphology, physiological and biochemical characteristics, cytochemistry and 16S rDNA sequence and other multiple taxonomy technologies were used for the classification of actinomycetes isolated and screened from saline-alkali soils. The strains 02d01,13h06,14j18,43a04 and 56h02 producing antibacterial substances were identified as Streptomyces, and strain09j02 with special morphology as Amycolatopsis, salinity-resistance strain 59h05 as Nocardia, other strains all as Nocardiopsis.Strain 09j03, 47d01 and N.synnemataformans, 04d01 and N.synnemataformans, 68d14 and N.dassonvillei subsp. Albirubida, 20f03, 22f02 and N.exhalans may be the same species. Strain 07j03 and 21f15 may be a new species, but need be confirmed through DNA-DNA hybrid.Based on revising many methods and ecological characteristics of salinity environment a multiple taxonomy technology system was founded, which was adapted to the classification and identification of actinomycetes in salinity environment. This technology system set up the foundation for the classification of actinomycetes in extreme environments.