Isolation And Identification Of A Siderophore-Producing And Cadmium-resistant Bacteria And Its Bioremediation Of Cd-contaminated Soil With Ryegrass

With the aggravation of global industrialization,the heavy metal contaminated soil has become an urgent environmental problem to be solved worldwide.As a result of long-term mining activities and produced into tailings in mining areas,which have serious heavy metal pollution around the county,including Fe,Pb,Mn,Cd,etc.The cadmium pollution of soil is becoming increasingly severe,which lead to unpredictable threats for ecological environment and human’s life and health,therefore,the remediation to cadmium contaminated soil is pressingly needed.In recent years,the studies found that there are strong chelation between siderophore and heavy metal ions,also the SPB has a high tolerance to heavy metals,which have piqued more researches’interests on the application of SPB in the remediation of heavy metal contaminated soil.The remediation of heavy metal contaminated soil by SPB not only improves the absorption and enrichment of heavy metal irons in plants,but also has a significant effect on remediation that combined application of SPB with Ryegrass,which provides a new idea and technological support for the remediation study of heavy metal contaminated soil.In this study,five strains of producing siderophore were isolated and purified from a mining area soil by using the initial screening technology that isolated and purified of microorganisms and CAS plate method at Hongxin Mining Area,Linze county,Gansu province.And then testing Cd²⁺tolerance of the strains as the rescreening method.According to quantitative detection of siderophore using the CAS blue liquid detection method,to obtain a bacteria named W-STS-8,which has a high Cd²⁺tolerance and optimal siderophore production.The growth characteristics of the strain and the structure type of chelating groups of siderophore were measured.And then separated and purified the siderophore of the strain and the characteristic absorption peak of siderophore was measured.And the optimum conditions for siderophore production of siderophore-producing bacteria were studied.The effects of siderophore and its producing bacteria on the germination of ryegrass under cadmium stress were investigated,and finally the remediation efficiency of Cd-contaminated soil was studied with the combined the W-STS-8 strain and ryegrass by the laboratory soil midsize ecosystem experiment.The following research results are obtained:Five SPB strains were isolated from a mining area soil by the CAS blue plate method,the W-STS-8 possess the highest biomass,siderophore production and Cd²⁺tolerance in the Cd²⁺concentration of 0-150mg/L,and the yield of siderophore was positively correlated with the biomass of the strain.The siderophore yield of strain reached the maximum value about 68.38% at the 60mg/L concentration of Cd²⁺,compared the blank control(without Cd²⁺),it’s siderophore yield was increased by 17.65%.The strain was identified as Pseudomonas sp.W-STS-8 by using 16S rRNA gene sequence.The growth of the strain reached the stable stage at24h after inoculation,and entered the decline stage at 108h.The siderophore yield began to increase significantly at the exponential stage of growth and reached the maximum at 72h.The siderophore,which has two types chelating groups structures,hydroxamates type and catecholates type,respectively through FeCl₃ and Arnow’s experiment methods,so the strain produces a mixed siderophore.The strain W-STS-8 could secrete a yellow-green siderophore—pyoverdine.It shows that the purity of siderophore is good according to the fluorescence intensity under UV light and the strong yellow phenomenon with CAS detection solution of siderophore after separated and purified technology.And the characteristic absorption peak of siderophore was at 400nm by ultraviolet spectrum scanning.Study on the optimum conditions for siderophore production of siderophore-producing bacteria.The results showed that:when the medium carbon source for sucrose,nitrogen source for urea,added proline,3% inoculated quantity,pH of 8,28℃ of the temperature,the W-STS-8 strains reached the best growth of OD₆₀₀ at 2.24,and the siderophore yield also had the optimal value about 75.32%.Through studying the germination of ryegrass seed under cadmium stress,the results showed that the germination rate of ryegrass seed increased significantly when treated with the bacterial suspension(OD₆₀₀=1.4)and siderophore(OD₄₀₀=1.3),which increased by 73.14% and 150.92%,respectively,compared with the blank control(Cd²⁺concentration of 90mg/L,the seed germination rate of 36%).Through studying the combined remediation efficiency of Cd-contaminated soil by Pseudomonas sp.W-STS-8 and ryegrass by setting up a laboratory soil midsize ecosystem experiment.The results showed that:（1）The growth state of ryegrass:compared with the blank control（P）,after inoculation the fresh weight of ryegrass in P+B treatment group increased by(Cd_low was 7.34%,Cd_highwas 11.20%),the dry weight increased by(Cd_low was 18.84%,Cd_high was 23.16%),chlorophyll content increased by(Cd_low was 45.12%,Cd_high was 29.08%),the content of malondialdehyde in ryegrass decreased by(Cd_low was 44.02%,Cd_high was 42.68%),the content of proline content decreased by(Cd_low was 32.07%,Cd_high was 41.92%).（2）The activity of Cd-contaminated soil:compared with Cd-contaminated soil blank control,soil urease activity increased by(Cd_low was 92.07%,Cd_high was 217.01%),sucrase activity increased by(Cd_low was 86.50%,Cd_high was 343.64%),Alkaline phosphatase activity increased by(Cd_low was 69.95%,Cd_high was 216.54%)in B+P treatment group.Compared with Cd-contaminated soil blank control,the B,P and B+P treatment groups can also increase the number of soil microorganisms,but in which B+P treatment group increased the most significantly,the number of soil bacteria increased by(Cd_low was 133.91%,Cd_high was360.81%),the number of fungi increased by(Cd_low was 57.18%,Cd_high was 150.04%),the number of actinomycetes increased by(Cd_low was 53.87%,Cd_high was 70.03%).（3）Remediation efficiency:compared with control group（P）,after inoculation the bioaccumulation factor of ryegrass in P+B treatment group increased by(Cd_low was 1.32 times,Cd_high was 1.47 times);The phytoextraction ratio of plant was increased by(Cd_low was 1.57times,Cd_high was 1.81 times).The total content of Cd extraction from ryegrass increased by(Cd_low was 57.00%,Cd_high was 82.79%).