Isolation, screening, and degradation characteristics of 17β-estradiol obligate degradation bacteria in the soil of marsh irrigation

Abstract: Biogas slurry has been usually reused on-site in intensive livestock and poultry farms. It can also inevitably bring the accumulation and pollution of Steroid Estrogens (SEs) while recycling the organic matter during production. This study aims to reduce the pollution risk of SEs to the soil and water environment after biogas irrigation. The enrichment and purification were also adopted to isolate and screen the estrogen-degrading bacteria in the soil of a dairy farm in the southwest China of the biogas irrigation areas. A strain of estrogen-degrading bacteria was obtained, where the 17β-estradiol (17β-E2) was used as the only carbon source for growth and reproduction. The strain was homologated by the 16S rDNA gene sequences, further to identify the species for the subsequent 17β-E2 degradation tests. The strains were then characterized at different temperatures (10, 20, 25, 30, 35, 37, and 40 ℃), pH values (5, 6, 7, 8, and 9), substrate concentrations (1, 3, 5, 7, and 10 mg/L) degradation characteristics. A three-factor and three-level orthogonal test was continued to optimize the degradation conditions of the strain. The results show that a dominant strain isolated from the sample was identified as Hyphomicrobium sp., named Hyphomicrobium sp. SS-1. There were varying degrees of degrading the 17β-E2 under the conditions of 10-40 ℃, pH value of 5 ~ 9, and substrate concentration of 1-10 mg/L. Specifically, the degradation rate of 17β-E2 by strains showed a trend of increasing first and then decreasing, with the increase of temperature, pH value, and substrate concentration. The optimal growth temperature of the strain was achieved at 30 ℃ in the environmental temperature test, indicating that the high temperature was not conducive to the growth and metabolism of the strain. The degradation rate of 17β-E2 increased first and then decreased, with the increase in temperature, where the maximum degradation was 46.4% at 30 ℃. Furthermore, the presence of E1 and E3 estrogenic products was accompanied by the degradation of 17β-E2. In addition, the strain was more adapted to the alkaline condition rather than the acidic in the pH value test. The optimal sequence of pH values for the growth of the strain was ranked in the descending order of 7, 8, 9, 6, 5. The degradation rate was higher in the alkaline environment than that in the acidicones. Particularly, the degradation rate of 17β-E2 and the removal rate of total estrogen were 45.1% and 37.5%, respectively, in the pH value of the 7 test group. In the substrate concentration, the growth state of the strain at 5 mg/L substrate concentration was much better than the others, while the degradation rate of 17β-E2 reached 71% after 7 days of incubation. There was also less toxic than E2 degradation products E1 and E3 generation, where the total estrogen removal rate was 56.8%. The metabolism was inhibited significantly, with the increase in the substrate concentration strain growth. The orthogonal test showed that the influencing factors on the degradation ability of the strain were ranked in the order ofsubstrate concentration > temperature > (pH value, indicating all significant effects. The optimal degradation conditions were achieved for the strain, including the temperature 35℃, pH 7, and substrate concentration of 5 mg/L. Consequently, the degradation rate of 17β-E2 reached 97.09% by the strain after being cultured for 7 days under optimum conditions. This finding can provide high-quality strain resources for the microbial degradation of SEs in complex substrate environments. Anyway, the effective way can be widely expected for the estrogen pollution remediation in methane irrigated soil.