Study On Degradation Characteristics Of P-nitrophenol By Rhodobacter Sphaeroides H Strain

With the rapid progress of world industrialization,p-nitrophenol?PNP?,as a typical nitroaromatic compound,has been widely used in the production of explosives,dyes and other industries.PNP,as a priority pollutant,has strong chemical stability and strong toxicity to organisms.By comparing different treatment methods including physical,chemical and microbiological methods,it is more economical to degrade PNP by microbiological method,which will not cause harm to the environment and has higher treatment efficiency.The strain selected in this paper is?Rhodobacter sphaeroides?H strain of photosynthetic bacteria,which is used to study the degradation characteristics of substrate PNP,and to analyze the utilization ways of the strain.The main research results are as follows:?1?Firstly,in different reaction systems,it is verified that the living body of strain H is the main part in the degradation process.The growth kinetics of strain H was further studied by fitting the growth inhibition kinetics equation of Haldane.The effect of H strain on the degradation of PNP was studied under different oxygen supply and illumination conditions respectively.The experimental results showed that the highest degradation rate of PNP was89.0%under anaerobic illumination conditions.On this basis,experiments of different physical and chemical factors were set up,and the optimum degradation conditions were obtained.The concentration of PNP,a degradation substrate,was 80 mg.L^-11 in the culture medium,15%of H strain was inoculated,the culture was required at 30?,and the optimum pH value was 7.0.?2?In the above different single-factor experiments,three factors were selected according to the degree of influence on PNP degradation rate,and the optimal conditions for H strain to degrade PNP were obtained through response surface optimization of degradation conditions.According to the experimental data obtained from the above physical and chemical factors experiments,pH value,PNP concentration and temperature are selected.These factors are subjected to response surface optimization analysis experiments.The experimental data are fitted and analyzed through regression equations,and the optimal conditions and degradation rate after optimization are predicted.Response surface optimization prediction results:the concentration of substrate PNP added to the medium is81.01 mg.L^-1,pH value is 8.09 and temperature is 30.49?.The maximum degradation rate of PNP is predicted to reach 92.3%.In order to verify the reliability of response surface prediction data,verification experiments need to be carried out under the best conditions.The experimental results show that the degradation rate of PNP degraded by strain H can reach 91.1%,and the difference between the predicted and measured degradation rates is 1.2%?the error is less than 2%?,within the allowable range of error,so the response surface prediction value is reliable.?3?Under the optimized conditions,the concentration of degrading substrate PNP is 81mg.L^-1,and 15%H strain is inoculated in the culture medium.The experimental temperature is 30?and pH value is 8.0.The relationship between the growth of H strain and the degradation rate of PNP is analyzed,and the kinetic characteristics of degradation of PNP by H strain are simulated.The results showed that the concentration of substrate PNP degraded by H strain decreased from 81 mg.L^-11 to 20 mg.L^-11 during the growth adaptation period.At this time,the degradation rate of substrate PNP was 74.9%,and finally the degradation rate of PNP was 91.1%after H strain entered the stabilization period.At the same time,the first-order kinetic equation was used to fit the relationship between PNP concentration and reaction time.After fitting the data with the equation,the kinetic parameters of half-life and reaction rate constant were obtained:t_1/2/2 was 43.3 h and K was 0.0144 h^-1,respectively.After the optimal degradation conditions were obtained through optimization analysis,different kinds of nitrogen sources and phenol mixtures were added to the culture medium to study the effect on degradation substrate PNP.According to the experiment,adding?NH₄?₂SO₄ and yeast extract as nitrogen sources simultaneously in the culture medium can effectively improve the degradation rate of PNP by 91.1%.Four phenolic compounds?catechol,phenol,hydroquinone and cresol?were selected and added to the culture medium.The experimental results showed that these phenolic compounds could inhibit the degradation of PNP substrate.Through orthogonal experiment analysis,the probability P value?sig.?of catechol was 0.025 at the minimum,and the inhibition of PNP degradation was the most significant.?4?High performance liquid chromatography-mass spectrometry?HPLC-MS?is used to detect the intermediate products.The detected substances include hydroquinone?HQ?,4-hydroxymucofuroic acid semialdehyde?4-HS?and maleamic acid?MA?.According to the detected substance types,it is presumed that PNP may first generate HQ under the catalysis of enzyme,and HQ is converted into 4-HS under the catalysis of hydroquinone 1,2-dioxygenase.The activity of hydroquinone 1,2-dioxygenase in H strain cells is detected by using an ultraviolet spectrophotometer.To understand the effect of hydroquinone 1,2-dioxygenase in the metabolic process of strain H,the results showed that the absorption peak of HQ at 289 nm gradually shifted to the absorption peak of 4-HS at 320 nm,indicating that HQ produced 4-HS under the effect of hydroquinone 1,2-dioxygenase.Combined with the detected intermediate products and hydroquinone 1,2-dioxygenase,it is analyzed that hydroquinone pathway may be used when strain H degrades PNP.The effects of different carbon sources,NaCl concentration and metal ions on PNP degradation and enzyme production of H strain were further studied.The results showed that malic acid was used as carbon source to promote PNP degradation,the degradation rate was91.0%,and the growth of H strain OD₅₉₀90 was 2.151.When 20 mg.L^-11 NaCl was added,it produced inhibitory effect,PNP degradation rate was 60.0%,and H strain growth OD₅₉₀90 was0.704.The addition of metal ions Ca²⁺promoted the degradation and Cu²⁺inhibited the degradation.The PNP degradation rates were 91.0%and 15.9%respectively,and the growth OD₅₉₀90 was 2.201 and 0.410 respectively.Through the whole cell protein electrophoresis experiment of strain H,the effects of different factors on enzyme protein yield were analyzed.The results showed that the gray value of 90KD protein band was 6620 and 2952 respectively in the lanes added with 20 mg.L^-11 NaCl and sucrose,and the protein content was significantly lower,which indicated that NaCl and sucrose had obvious inhibitory effect on the secretion and enzyme production of H strain.