Laccase-induced Rhizospheric Humification Reduces 17β-estradiol Contamination In Maize

Environmental estrogens are a kind of endocrine disrupting chemicals perturbing living organism endocrine systems,and the exposure to ecosystems not only causes negative effects of plant growth and metabolism but also increases the risk of the human diet.Laccase,as a green catalyst,can induce the oxidative polymerization and humification of various substrates,such as estrogens,phenols,and amino acids by using molecular oxygen as the only electron acceptor.This reaction has the advantages of simple and controllable operation,high catalytic efficiency,and eco-friendliness.However,there are few international reports about whether laccase-driven rhizospheric humification（L-DRH）can be used to reduce the risk of estrogen contamination in plant?In this study,17β-estradiol（E2）and maize were respectively selected as a representative environmental estrogen and tested plant.The effect of L-DRH on the attenuation kinetics of E2 in maize rhizosphere was studied by greenhouse hydroponic pot experiments,and the physiological and biochemical mechanisms of reducing E2 pollution in maize by L-DRH were revealed.The main results are summarized as follows:（1）L-DRH removed E2 in the rhizosphere of maize,and the attenuation of E2 followed first-order kinetics during the water environment.Compared with laccase-free,removal rates were increased from 54.72%to 97.48%;the kinetic constant（k）values of E2 removal were increased from 0.04 h^-1 to 0.33 h^-1 within 12 h of incubation in the 10μM E2+maize+laccase treated group.However,in the 50μM E2+maize+laccase treated group,the removal rates of E2 were increased from 40.10%to 80.00%;the k values were increased from 0.02 h^-1 to 0.04h^-1.The contents of humic precursors（HPs,such as total phenols,total amino acids,and total sugars）were increased with the extension of incubation;the contents of HPs were higher in laccase-free than in the treated group with laccase.For example,after 120 h of incubation,the contents of total phenols,total amino acids,and total sugars in the 10μM E2+maize treatment group increased to 214.00,14.34,and 276.26μg·m L^-1,respectively,while those in the 10μM E2+maize+laccase treatment group were 205.11,13.16,and 223.22μg·m L^-1.The result of high-resolution mass spectrometry（HRMS）identification had revealed that L-DRH could not only convert E2 into the oxidation products such as estrone（E1）and estriol（E3）,but also polymerized E2 into dimers,trimers,tetramers,oligomers,and polymers.The contents of E2 oxidation products and autopolymerization products were increased in the early stage of the humification reaction and then tended to decrease.Besides,in the E2+maize+laccase treated group,the contents of E2 oxidation and autopolymerization products were decreased significantly.Therefore,L-DRH could promote the oxidative polymerization of E2 and HPs through single-electron oxidation,which reduced E2 pollution in maize rhizosphere effectively.（2）L-DRH promoted the copolymerization of E2 and HPs in maize rhizosphere to produce macromolecular humic precipitates.The produced copolymerization precipitates in L-DRH were collected by large-scale experimental methods,and their micromorphology,elemental proportion,functional group composition,chemical structure,and organic carbon component distribution were analyzed.Scanning electron microscopy combined with energy spectrum observed that the surface of the precipitates was rough and showed an agglomeration phenomenon,and the E2+corn+laccase treatment group exhibited more obvious agglomeration,higher C/N ratio,and stronger hydrophilic degree.Fourier transform infrared spectroscopy and ¹³C-nuclear magnetic resonance spectroscopy demonstrated that these precipitates had aromatic C=C and conjugated carbonyl C=O stretching,C-O-C aromatic ether bond stretching,and aromatic carbon,phenolic hydroxyl,carboxyl carbon functional groups,suggesting obvious aromatics and acidity.Corrected scanning transmission electron microscopy revealed that the carbon signals of aromatic carbon,phenolic carbon,aliphatic carbon,and carboxyl carbon formed in the humified precipitates by E2+corn+laccase treatment group were stronger than those of the laccase-free treatment group.It follows that L-DRH contributed to the copolymerization between E2 and HPs in the rhizosphere,which formed large molecules of C-C,C-O-C,or C-N-C covalent humification products.（3）L-DRH effectively reduced the biotoxicity and environmental risk of E2 in the maize seedlings caused by the high concentration of E2.Compared with 50μM E2+maize treatment group,the plant height,root length,fresh weight,and photosynthetic pigment contents of maize seedlings were increased significantly in the 50μM E2+maize+laccase treated group（p<0.05）.In addition,L-DRH also attenuated the effect of E2 uptake and accumulation by maize seedlings.For example,within 0-120 h,in 10μM E2+maize and 10μM E2+maize+laccase treated group,the concentrations of E2 reached maximum values of17.53,10.84μmol·kg^-1（FW）respectively at 12 h and 8 h in maize root and reached maximum values of 3.11,2.89μmol·kg^-1（FW）at 24 h and 12 h in shoot.Meanwhile,E2uptake and accumulation in different parts of the maize showed that the roots were larger than the stems and leaves.For example,E2 accumulation in root and shoot reached maximum values of 2.19 and 0.76 nmol·plant^-1 at 13 and 24 h in the 10μM E2+maize treated group,respectively;however,in the 10μM E2+maize+laccase treated group,the accumulation of E2 in maize root and shoot reached maximum values of 1.27 and 0.59nmol·plant^-1 at 8 h and 12 h,respectively.The identification of E2 in maize root showed that oxidation products（E1 and E3）and polymerization products of E2 were detected by HRMS and their production gradually increased in the early stages of incubation,followed by a decreased trend.The yield of oxidation and autopolymerization products of E2 in maize root decreased more significantly in L-DRH compared to the E2+maize treatment group.Therefore,L-DRH had double effects which could detoxify E2 phytotoxicity and mitigated the risk of E2 contamination in plants.Based on all the results,L-DRH was effective in reducing E2 concentrations in maize rhizosphere.The results of study provide theoretical insights and scientific guidance for avoiding the risk of estrogen contamination in crop,increasing the content of environmental carbon,and promoting agricultural yields with enzymatic humification.