Studies On The Identification And Application Of A Copper-hyperaccumulation Bacillus Sp.HNU1

Copper is an essential microelement for the growth and development of plants and animals.Both deficiency and excess cause corresponding damage to the organism.The high concentration of copper in agricultural soil can be transmitted and accumulated through the food chain,which threat the safeties of ecological environment and human health.Microbial remediation is one of the remediation methods for heavy metal pollution in soil,which has the characteristics of low cost,simple operation and no secondary pollution.However,the poor stability of microbial strains and the unclear remediation mechanism restrict its large-scale application.In order to screen a novel engineering microorganism and clarify the remediation mechanism of biofertilizer to copper contaminated soil.In this study,a copper-hyperaccumulation bacteria HNU1strain was identified by molecular biology,physiological and biochemical characteristics and morphology techniques.The optimal culture conditions and medium component of HNU1 were screened out by using single-factor and orthogonal design test methods.The adsorption capacity of HNU1 strain to copper ions was determined by single factor test.Biofertilizer was prepared by solid fermentation method,and the effect and mechanism of biofertilizer on the growth of Brassica campestris ssp.chinensis L under copper stress was analyzed by the pot experiment.The results are as follows:1,The bacteria,named HNU1,was isolated and purified from the soil of Danzhou,Hainan Province by the Key Biology Laboratory of Hainan University.The 16S r RNA gene and gyr B gene were amplified by using universal primers 27f and 1492r and primers UP-1 and UP-2r,respectively.The alignment results of the two target gene sequences on NCBI showed that the highest homology between HNU1 strain and Bacillus.amyloliquefacien reached 99.59%and 99.2%,respectively.The phylogenetic trees constructed from the 16S r RNA and gyr B gene sequences showed that the strain HNU1 was clustered with Bacillus.amyloliquefacien,so the HNU1 strain was named Bacillus.amyloliquefacien HNU12,The optimum culture conditions of HNU1 strain were as follows:culture temperature 37℃,shaking speed 180rpm,medium p H8.0,inoculation of 0.1%and liquid volume of 40%.The optimum medium composition and ratio of HNU1 strain were sucrose 2.0g/L,yeast powder 4.0g/L,peptone 6.0g/L and Mg SO₄ 5.0g/L.3,The adsorption capability of Cu²⁺by strain HNU1 was mainly affected by p H,initial copper concentration and biomass dose.The adsorption capacity of strain HUN1to copper increased with the increase of p H value and initial copper concentration,but decreased with the increase of biomass dose.The maximum adsorption capacity of copper by strain HNU1 was 54.82 mg/g.4,Five biofertilizers（WF1,WF2,WF3,WF4 and WF5）with different formulas were prepared by solid-state method,and the effects of them on the growth of B.chinensis L were determined.When the copper concentration was 0 mg/kg,there were no significant differences in the growth indexes of B.chinensis L treated with WF1,WF2,WF3 and WF4,respectively,but they were significantly higher than WF5.When the copper concentration was 200 mg/kg,the growth index of B.chinensis L treated with WF4 was significantly higher than that of the other four biofertilizers.5,Comparing with the control,the increasing rates of plant height,fresh weight and dry weight of B.chinensis L treated with biofertilizer were 10.37%,65.3%and67.78%,respectively,indicating that biofertilizer could significantly promote the growth of B.chinensis L.In the copper concentration range of 50 to 200 mg/kg,the biomass of B.chinensis L decreased significantly with the increase of copper concentration,but the biomass of B.chinensis L treated with biofertilizer was significantly higher than that of the control.6,The application of biofertilizer significantly promoted the expression level of antioxidant enzyme genes in B.chinensis L and increased the soil p H.Based on the above results,the following conclusions can be drawn.1,Identification of the core strains Bacillus amyloliquefaciens HNU1 of biofertilizer by molecular biological methods,physiological and biochemical characteristics,and morphological methods;2,Bacillus amyloliquefaciens HNU1 has a wide adaptability to culture conditions,which is beneficial to large-scale application and reduces production costs;3,Bacillus amyloliquefaciens HNU1 strain has a high adsorption capacity of copper;4,Biofertilizer（WF4）prepared by solid fermentation method has a good fertilizer effect;5,The application of biofertilizer can significantly promote the growth of B.chinensis L.The mechanism of biofertilizers alleviating the copper stress on B.chinensis L is to improve the resistance of B.chinensis L and improve the soil properties.