Salt accumulation in soil caused by using excess amounts of nitrogen fertilizers deteriorates the growth and development of crops and vegetables, known as 'salt stress'. To maintain high quality and productivity, excess nitrate should be removed from glass and greenhouse soils. In this study, an eff...
Salt accumulation in soil caused by using excess amounts of nitrogen fertilizers deteriorates the growth and development of crops and vegetables, known as 'salt stress'. To maintain high quality and productivity, excess nitrate should be removed from glass and greenhouse soils. In this study, an effective way to remove soil nitrate was developed using soil microorganism. The microorganisms with high capability of nitrate uptake were isolated from upland soils and identified by characterizing 16S rRNA gene sequences. The optimal incubation condition for the maximal nitrate removal activity was developed and applied to lettuce cultivation. For the commercial purpose, mass production of the microorganism and quality inspection were also developed. Identification of the isolated strain was performed by biochemical methods and a comparison of the 16S rRNA genomic sequences. Biochemical characteristics obtained by conventional and API tests revealed that it was a Gram-negative aerobic bacterium, and the API 20E tests identified the strain GG0461 as a member of the family Enterobacteriaceae. The sequence of 1,434 bp of 16S rRNA gene was determined by PCR analysis and registered as GenBank accession No. EF426859. In the sequence analysis of 16S rRNA gene, the strain GG0461 was identified as Enterobacter amnigenus by 98% sequence similarity with the phylogenetic relationship derived from a neighbor-joining analysis of the pairwise comparisons among the 16S rRNA gene sequences of 11 validly described species of four genera. Although three microorganisms, Enterobacter amnigenus, Bacillus arbutinivorans and unidentified Bacillus species, were isolated and identified as excellent in nitrate uptake activity. The various characteristics of E. amnigenus GG0461 were investigated, since nitrate uptake activity of E. amnigenus has not been reported so far. The nitrate uptake capability of GG0461 was investigated at various conditions. The addition of nitrogen sources was helpful for the growth, but not required, and the microbial growth was stimulated by increasing the concentration of nitrate in the media. The maximal nitrate uptake was obtained at 50 mM and the rate of uptake was decreased to 60% at 10,000 ppm. The growth and nitrate uptake activity of GG0461 were maximal at 37℃. Kinetics of nitrate uptake were analyzed at various soil temperatures. Nitrate uptake activity was increased under alkaline conditions of culture media and was accompanied with the acidification of media. These results imply that the major factor mediating bacterial nitrate uptake is a nitrate/proton anti-porter. Effect of the strain GG0461 on nitrate removal was measured during cultivations of lettuce and Arabidopsis. When 5x108 cfu·ml-1 bacteria were treated on the soil, cell density was decreased rapidly during the first 5 days and then remained constant at 1x106 cfu·g-1 soil. Nevertheless, at the salt stress conditions, the growth of lettuce was improved by the treatment of the strain GG0461. When the soil was treated with nitrate solutions at the concentrations from 100 to 200 mM once a week, the growth of lettuce was improved. When the nitrate concentration was increased to 800 mM, the growth became deteriorated and, however, positive effect on lettuce growth by the GG0461 treatment was observed at this condition. Similar bacterial effect on Arabidopsis growth was also observed in the soil treated with nitrate solution up to 1 M or higher. A method for the large-scale bacterial culture was developed for the field application. PAF media was good enough for GG0461 in a small-scale cultivation. Therefore, each nutrient of PAF meida was replaced with a commercial product. When the commercial products were used, GG0461 took 35 h for the uptake of 5,000 ppm nitrate although the growth rate of GG0461 was increased at the same level as that in PAF media. Therefore, fish-fermented amino acids were replaced with tryptone, yeast extract, and soybean meal. The optimal conditions for the growth and nitrate uptake by GG0461 were obtained with 2% tryptone and 1% glycerol. In summary, the soil bacterium, Enterobacter amnigenus GG0461, showed high capability of nitrate uptake in the greenhouse soil, and it was able to survive as a major strain maintaining significant population density in soil. For the industrial application, large-scale production was also possible by using commercial media. These data indicate that the Enterobacter amnigenus GG0461 can be a good candidate for the microbial remediation of the removal of excess soil nitrate.
Salt accumulation in soil caused by using excess amounts of nitrogen fertilizers deteriorates the growth and development of crops and vegetables, known as 'salt stress'. To maintain high quality and productivity, excess nitrate should be removed from glass and greenhouse soils. In this study, an effective way to remove soil nitrate was developed using soil microorganism. The microorganisms with high capability of nitrate uptake were isolated from upland soils and identified by characterizing 16S rRNA gene sequences. The optimal incubation condition for the maximal nitrate removal activity was developed and applied to lettuce cultivation. For the commercial purpose, mass production of the microorganism and quality inspection were also developed. Identification of the isolated strain was performed by biochemical methods and a comparison of the 16S rRNA genomic sequences. Biochemical characteristics obtained by conventional and API tests revealed that it was a Gram-negative aerobic bacterium, and the API 20E tests identified the strain GG0461 as a member of the family Enterobacteriaceae. The sequence of 1,434 bp of 16S rRNA gene was determined by PCR analysis and registered as GenBank accession No. EF426859. In the sequence analysis of 16S rRNA gene, the strain GG0461 was identified as Enterobacter amnigenus by 98% sequence similarity with the phylogenetic relationship derived from a neighbor-joining analysis of the pairwise comparisons among the 16S rRNA gene sequences of 11 validly described species of four genera. Although three microorganisms, Enterobacter amnigenus, Bacillus arbutinivorans and unidentified Bacillus species, were isolated and identified as excellent in nitrate uptake activity. The various characteristics of E. amnigenus GG0461 were investigated, since nitrate uptake activity of E. amnigenus has not been reported so far. The nitrate uptake capability of GG0461 was investigated at various conditions. The addition of nitrogen sources was helpful for the growth, but not required, and the microbial growth was stimulated by increasing the concentration of nitrate in the media. The maximal nitrate uptake was obtained at 50 mM and the rate of uptake was decreased to 60% at 10,000 ppm. The growth and nitrate uptake activity of GG0461 were maximal at 37℃. Kinetics of nitrate uptake were analyzed at various soil temperatures. Nitrate uptake activity was increased under alkaline conditions of culture media and was accompanied with the acidification of media. These results imply that the major factor mediating bacterial nitrate uptake is a nitrate/proton anti-porter. Effect of the strain GG0461 on nitrate removal was measured during cultivations of lettuce and Arabidopsis. When 5x108 cfu·ml-1 bacteria were treated on the soil, cell density was decreased rapidly during the first 5 days and then remained constant at 1x106 cfu·g-1 soil. Nevertheless, at the salt stress conditions, the growth of lettuce was improved by the treatment of the strain GG0461. When the soil was treated with nitrate solutions at the concentrations from 100 to 200 mM once a week, the growth of lettuce was improved. When the nitrate concentration was increased to 800 mM, the growth became deteriorated and, however, positive effect on lettuce growth by the GG0461 treatment was observed at this condition. Similar bacterial effect on Arabidopsis growth was also observed in the soil treated with nitrate solution up to 1 M or higher. A method for the large-scale bacterial culture was developed for the field application. PAF media was good enough for GG0461 in a small-scale cultivation. Therefore, each nutrient of PAF meida was replaced with a commercial product. When the commercial products were used, GG0461 took 35 h for the uptake of 5,000 ppm nitrate although the growth rate of GG0461 was increased at the same level as that in PAF media. Therefore, fish-fermented amino acids were replaced with tryptone, yeast extract, and soybean meal. The optimal conditions for the growth and nitrate uptake by GG0461 were obtained with 2% tryptone and 1% glycerol. In summary, the soil bacterium, Enterobacter amnigenus GG0461, showed high capability of nitrate uptake in the greenhouse soil, and it was able to survive as a major strain maintaining significant population density in soil. For the industrial application, large-scale production was also possible by using commercial media. These data indicate that the Enterobacter amnigenus GG0461 can be a good candidate for the microbial remediation of the removal of excess soil nitrate.
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