Study On The Restoration Of Petroleum Contaminated Sediments By Immobilized Laccase

With the rapid development of shipping economy,the demand for petroleum has increased dramatically.There are many marine petroleum pollution incidents occurring frequently,which seriously affect the marine sediment environment.Recently,laccase has drawn much attention in the field of petroleum pollution control due to its outstanding petroleum degradability and simple requirements for environmental conditions.However,the shortcomings of poor stability and easy inactivation of free laccase have greatly restricted the application of laccase.In this paper,immobilized laccase was prepared by adsorption technology with biochar as the support material,and tried to apply to the treatment of petroleum-contaminated sediments.Firstly,the preparation conditions of immobilized laccase were optimized.The results indicated that the key factors affecting the activity of immobilized laccase were sizes of biochar,immobilization time,mass ratio of biochar to laccase and immobilization temperature.Through orthogonal experiments,it was found that the mass ratio of biochar to laccase had the greatest effect on laccase activity,followed by immobilization time,sizes of biochar,and immobilization temperature.As a result,the immobilized laccase activity reached the maximum of 40.0 U/g when the biochar was 80 meshes,the immobilization time was 9 hours,the mass ratio of biochar to laccase was 1:1 and the immobilization temperature was 4?C.Comparing the enzymatic properties of immobilized laccase and free laccase,it was known that immobilized laccase prepared with biochar with large specific surface area and porous structure as the carrier greatly improved the acid-base stability,thermal stability and storage stability.Then,the immobilized laccase prepared at the optimal condition was applied to the restoration of petroleum-contaminated sediments.The results showed that,the degradation reaction of immobilized laccase to the petroleum in sediments reached equilibrium at almost54 hours,and the degradation rate of petroleum could reach 60.3%.The kinetic analysis results showed that during a certain concentration,the degradation reaction of petroleum in sediments conformed to pseudo-first-order kinetics,with a reaction rate constant of 0.019h^-1.The analysis result of degradation products showed that the immobilized laccase had a wide range for substrates,which could degrade chain hydrocarbons and even refractory polycyclic aromatic hydrocarbons efficiently,and was suitable for the restoration of petroleum-contaminated sediment.Lastly,the influencing factors affecting the degradation of petroleum in sediments were investigated.It could be seen that,the temperature one of environmental conditions had the greatest influence on the degradation of petroleum in sediments.In the range of 10-30?C,the degradation rate of petroleum in sediment increased continuously from 30.9%to 74.8%with the increase of temperature.Hence,the restoration process should be conducted during the season with high temperature in practical applications.The degradation rate of petroleum gradually decreased with the increase of pH and salinity,and the minimum removal rate of petroleum could also reach 58.1%when the pH was in the range of 7.0-8.5and the salinity was in the range of 0-40‰.Mn²⁺,Al³⁺,Mg²⁺,Cu²⁺,and Zn²⁺showed promoting effects on the degradation of petroleum,while Fe²⁺showed an inhibitory effect.In addition,the amount of enzyme and the initial concentration of petroleum also had influence on the degradation of petroleum in sediments.For sediments with a petroleum concentration of 2500 mg/kg,the degradation rate of petroleum in sediment could reach more than 50%when the enzyme dosage was 50 g/kg,and the optimal enzyme dosage was150 g/kg.Immobilized laccase could play a good role in petroleum degradation when the initial concentration of petroleum was in the range of 1000-3000 mg/kg,and the degradation rate of petroleum was between 60%and 84%.In summary,the immobilized laccase has a significant promotion effect on the restoration of sediments polluted by petroleum,and exhibits excellent degradation ability in most sedimental environments.Moreover,the immobilized laccase is a combination of biochar and laccase,both of which are cheap and pollution-free.It has strong application potential in the restoration of petroleum-contaminated sediments.