| Environmental estrogen belongs to a biological exogenous substance for a living body,which can break the steady state of the living body itself and make each stable running system lose its stable state.Studies have shown that excessive intake of estrogen can lead to a significant increase in the incidence rate of tumor diseases such as breast cancer and cervical cancer in women,and can also lead to a decrease in semen volume and decrease in sperm concentration.As early as 2003,the World Health Organization recommended that the mass concentration of estrogen in drinking water should not be higher than 1.0 ng·L-1.The production of estrogen in the environment is very extensive,and its main source is the intensive production in the chemical,pharmaceutical,agricultural,forestry and animal husbandry industries.At present,the scale of animal husbandry industry is huge,and the large amount of livestock and poultry manure produced by the intensive production model is difficult to be standardized,which makes it become one of the pollution sources to be solved.Therefore,how to achieve the harmless treatment and resource utilization of livestock and poultry manure has become one of the hot spots at home and abroad.Functional microorganisms have the advantages of efficiently degrading estrogen in sewage and livestock and poultry manure,and can effectively reduce the risk of environmental estrogen pollution,it has been widely concerned.The purpose of this article is to detect and degrade estrogen pollutants in chicken feed and chicken manure.High-performance liquid chromatography?HPLC?was used to detect the content of estrogen pollutants in laying hen feed and chicken manure.Natural strains and genetically engineered engineering strains were used to study the biodegradation of estrogen pollutants.We chose the dominant strains that degrade steroids:Commanonas testosteroni CT1and KF-1,Vibrio sp.H5,Buttiauxella sp.S19-1,Butmonaux putida LN12 and CT1 engineering strains Ca3,Cb7,M6,M2,Mact1 and A3.By comparing the ability of single strains and mixed strains to degrade estrogen,we obtained the strains or flora with the optimal degradation effect,and optimized the degradation conditions.This research mainly includes the following contents:1. A HPLC method for the determination of estrogen was established.The estrogens were determined by HPLC,and the standard curves were established using estradiol?E2?standards with different concentrations of 10,20,30,40,and 50?mol·L-1.The results showed that estradiol?E2?was eluted at 4.1 min and an absorption peak appeared.The limit of detection?LOD?was 8.2 ng·m L-1,the limit of quantification?QOD?was 27.2 ng·m L-1,and the precision RSD was?5%.2. The detection of estrogen in laying hen manure and laying hen feed was carried out.The extract of chicken manure and feed was analyzed by HPLC.The absorption peak of E2?4.1 min?was detected in laying hen manure.According to the standard curve,the content of E2was as high as 230 ng·g-1,but there was no E2 absorption peak in the feed.3. Methylsilanization treatment and test of estrogen.It was found by HPLC that the E2 absorption peak of E2?4.1 min?in the chicken manure extract was weakened after methylsilylation.Interestingly,the absorption peaks at 2.8,3.0 and3.2 min also almost disappeared simultaneously.It can be inferred that the absorption peaks of2.8,3.0 and 3.2 min may be the absorption peaks of estrogen analogues or the intermediate metabolites of estrogen and analogues.4. Degradation of natural bacteria in chicken manureThe results of the liquid chromatogram of chicken manure cultured for 10 days without inoculation were compared with those extracted immediately after collection:when the chicken manure was cultured for 10 days,the absorption peak of E2?4.1 min?in the sample disappeared.The absorption peaks at 2.8 and 3.2 min increased,while the absorption peak decreased at 3.0min.We infer that some natural strains in chicken manure may also play a positive role in the degradation of E2 and estrogen analogues.Combined with the results of methylsilanization treatment,we further proved that the absorption peaks at 3.0 min were estrogen analogues,and the absorption peaks at 2.8 and 3.2 min were intermediate metabolites of estrogen.5. Comparative study on the degradation efficiency of different strainsBy comparing the differences in the degradation efficiency of 11 single strains and mixed flora on estrogen under the same conditions,the results showed that 11 single strains and mixed flora all had degradation effects on estrogen,but the degradation efficiency was different,and the degradation efficiency of mixed flora was higher than that of single strains.6. Optimization of estrogen biodegradation conditions.The results of the study on the degradation factors of E2 through bacterial flora optimization showed that when the concentration of the bacterial solution was OD600nm=1.0,the p H was 8.0,the degradation temperature was 27?,and the degradation time was 10 days,the degradation efficiency of E2 by the mixed flora was 78.6%,reaching the highest value.To sum up:the presence of E2 was detected in laying hen manure by HPLC,and its content was 230 ng·g-1;the E2 absorption peak was not detected in the laying hen feed,but the absorption occurred at 3.0 min.Through methylsilanization experiments,it was proved that the absorption peak was an estrogen analogue.Similarly,estrogen analogues were also detected in laying hen manure.It could be concluded that the laying hen feed can increase the egg production of laying hens by adding estrogen analogues.In this paper,the degradation of natural bacteria in chicken manure was compared before and after 10 days without inoculation.By comparing the degradation efficiency of 11 single strains and mixed flora on estrogen,it was proved that the flora had the highest degradation efficiency on estrogen.Through the study of the factors affecting the degradation of E2 by the flora,the results showed that the degradation efficiency of the flora to E2 reached a maximum of 78.6%when the p H was 8.0,the degradation temperature was 27?,and the degradation was 10 days. |
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