Herbicide-resistant HPPD variants identified via directed evolution.

Herbicides play a crucial role in boosting crop yields, yet the emergence of herbicide-resistant weeds and the susceptibility of crops to herbicides have posed significant challenges to their efficacy. ß-triketone herbicides specifically target the enzyme 4-Hydroxyphenylpyruvate dioxygenase (HPPD) essential for plant growth. Remarkably, few resistant weeds have been identified against these herbicides. In this study, we aimed to identify mutations within the cotton HPPD gene that confer resistance to mesotrione, a widely used triketone herbicide. Through the establishment of a high-throughput mutant screening system in E. coli, we identified four single nucleotide changes leading to amino acid substitutions in HPPD, resulting in mesotrione resistance while preserving native enzymatic activity. Various combinations of these mutations displayed synergistic effects on herbicide resistance. Additionally, the HPPD variants were able to complement the Arabidopsis athppd mutant, indicating their retention of sufficient native activity crucial for plant growth and development. Expression of these cotton HPPD variants in Arabidopsis resulted in heightened herbicide resistance. These findings offer critical insights into the target amino acids of HPPD for gene editing, paving the way for the development of herbicide-resistant cotton in the future.

Data derived extrapolation factors (DDEFs) for rat to human interspecies extrapolation for the HPPD inhibitor mesotrione.

In the risk assessment of agrochemicals, there has been a historical paucity of using data to refine the default adjustment factors, even though large datasets are available to support this. The current state of the science for addressing uncertainty regarding animal to human extrapolation (AFA) is to develop a "data-derived" adjustment factor (DDEF) to quantify such differences, if data are available. Toxicokinetic (TK) and toxicodynamic (TD) differences between species can be utilized for the DDEF, with human datasets being ideal yet rare. We identified a case for a currently registered herbicide, mesotrione, in which human TK and TD are available. This case study outlines an approach for the development of DDEFs using comparative human and animal data and based on an adverse outcome pathway (AOP) for inhibition of 4-hydroxyphenol pyruvate dioxygenase (HHPD). The calculated DDEF for rat to human extrapolation (AFA) for kinetics (AFAK = 2.5) was multiplied by the AFA for dynamics (AFAD = 0.3) resulting in a composite DDEF of ∼1 (AFA = 0.75). This reflects the AOP and available scientific evidence that humans are less sensitive than rats to the effects of HPPD inhibitors. Further analyses were conducted utilizing in vitro datasets from hepatocytes and liver cytosols and extrapolated to whole animal using in vitro to in vivo extrapolation (IVIVE) to support toxicodynamic extrapolation. The in vitro datasets resulted in the same AFAD as derived for in vivo data (AFAD = 0.3). These analyses demonstrate that a majority of the species differences are related to toxicodynamics. Future work with additional in vitro/in vivo datasets for other HPPD inhibitors and cell types will further support this result. This work demonstrates utilization of all available toxicokinetic and toxicodynamic data to replace default uncertainty factors for agrochemical human health risk assessment.

Paenibacillus lacisoli sp. nov., a mesotrione-degrading strain isolated from lakeside soil.

A Gram-stain-positive, facultatively anaerobic, rod-shaped bacterium, designated JX-17T, was isolated from a soil sample collected in Jiangxi Province, PR China. Growth was observed at 15-48 °C (optimum 37 °C), at pH 5.0-9.0 (optimum pH 7.0) and with 0-6.0% (w/v) NaCl (optimum 1.0%). Strain JX-17T could degrade approximately 50% of 50 mg/L mesotrione within 2 days of incubation, but could not use mesotrione as sole carbon source for growth. Strain JX-17T showed less than 95.3% 16S rRNA gene sequence similarity with type strains of the genus Paenibacillus. In the phylogenetic tree based on 16S rRNA gene and genome sequences, strain JX-17T formed a distinct lineage within the genus Paenibacillus. The ANI values between JX-17T and the most closely related type strains P. lentus CMG1240T and P. farraposensis UY79T were 70.1% and 71.4%, respectively, and the dDDH values between them were 19.0% and 23.3%, respectively. The major cellular fatty acids were anteiso-C15:0, iso-C16:0, anteiso-C17:0 and C16:0, the predominant respiratory quinone was MK-7, the major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, an unidentified glycolipid, an aminophospholipid and a phosphatidylinositol. The diagnostic diamino acid of the peptidoglycan was meso-diaminopimelic acid, and the DNA G + C content was 50.1 mol%. Based on the phylogenetic, phenotypic and chemotaxonomic characteristics, strain JX-17T represents a novel species within the genus Paenibacillus, for which the name Paenibacillus lacisoli sp. nov is proposed, with strain JX-17T (= GDMCC 1.3962T = KCTC 43568T) as the type strain.

The Influence of Mesotrione on Human Colorectal Adenocarcinoma Cells and Possibility of Its Toxicity Mitigation by Cichoric Acid.

Mesotrione, as a widely used herbicide, is present in the environment in detectable amounts, causing serious damage. Here, we aimed to investigate the effect of mesotrione on Caco-2 cells and the possibility of its toxicity mitigation by cichoric acid. Therefore, we analyzed the cytotoxicity of both these compounds and the selected oxidative stress parameters, apoptosis and interaction of both the tested compounds with the cell membrane and their accumulation within the cells. In cytotoxicity studies, the stimulating activity of mesotrione was observed, and simultaneously, the inhibitory effect of cichoric acid was noticed. This effect was related to the results of oxidative stress analysis and apoptosis measurements. The activity level of key enzymes (glutathione peroxidase, catalase and superoxide dismutase) in Caco-2 cells exposed to cichoric acid was higher as compared to that of the control. The treatment with mesotrione did not induce apoptosis in the Caco-2 cells. The penetration of the studied compounds into the Caco-2 cells was measured by using an HPLC methodology, and the results indicate mesotrione's high penetration capacity. The distribution of charge on the surface of the cell membranes changed under the influence of both compounds. Considering the mutual interactions of beneficial and potentially toxic food ingredients, it should be noted that, despite the observed favorable trend, cichoric acid is not able to overcome the toxic and cancer-stimulating effects of this pesticide.

Selectivity and control of Euphorbia heterophylla in sugarcane by herbicide in post-emergence.

To obtain good control of wild poinsettia (Euphorbia heterophylla) in post-emergence in sugarcane crop, we evaluate the herbicides association on post-emergence of E. heterophylla and the ratoon cane selectivity. The experimental scheme was in randomized blocks with 6 treatments and 4 replications. The treatments were: control; ametryn + mesotrione + sulfentrazone (1,500 + 144 + 800 g i.a ha-1); ametryn + mesotrione + diclosulan (1,500 + 144 + 200 g i.a ha-1); ametryn + mesotrione (2,500 + 144 g i.a ha-1: Highest dose); ametryn + mesotrione (2,000 + 144 g i.a ha-1: Lowest dose) and ametryn + mesotrione + diuron (1,000 + 144 + 1,250 g i.a ha-1). The percentage of control, dry mass, height and percentage of germination of E. heterophylla and injury level, yield and technological quality of sugarcane were evaluated. The best control of E. heterophylla was: ametryn + mesotrione +sulfentrazone; ametryn + mesotrione + diclosulan and ametryn + mesotrione (Lowest dose). As for the ratoon cane selectivity the best yield was achieved with the association ametryn + mesotrione +diclosulan. An appropriate association of herbicide molecules provides successful control of E. heterophylla, especially the association of sulfentrazone or diclosulan together with ametryn and mesotrione.

Non-destructive estimation of weed response to bleaching herbicides by Raman spectroscopy.

The aim of our study was to evaluate the use of Raman spectroscopy for pre-diagnostic estimation of weed response to bleaching herbicides. Model plants were Chenopodium album and Abutilon theophrasti treated with mesotrione (120 g a.i. ha-1). Raman single-point measurements were taken 1, 2, 3, and 7 days after herbicide application from different points on the leaves. Principal component analysis (PCA) was carried out on data normalized by the highest intensity band at 1522 cm-1 and using spectral region from 950 to 1650 cm-1 comprising mainly contributions of carotenoids. The carotenoids by intensive band at ∼1522 cm-1 and bands with lower intensity at ∼1155 and 1007 cm-1 in treated plants were confirmed. According to PC1 (the first principal component) and PC2 (the second principal component), the highest intensity bands responsible for treatment differentiation in C. album could be assigned to chlorophyll, lignin, and carotenes. According to PC1 in A. theophrasti leaves the treatment differences could be observed 7 days after mesotrione treatment and PC2 gave a clear separation between all control and treated leaf samples. Raman spectroscopy may be a good complement to invasive analytical methods, in assessing the plant abiotic stress induced by bleaching herbicides.

Cytochrome P450 CYP81A10v7 in Lolium rigidum confers metabolic resistance to herbicides across at least five modes of action.

Rapid and widespread evolution of multiple herbicide resistance in global weed species endowed by increased capacity to metabolize (degrade) herbicides (metabolic resistance) is a great threat to herbicide sustainability and global food production. Metabolic resistance in the economically damaging crop weed species Lolium rigidum is well known but a molecular understanding has been lacking. We purified a metabolic resistant (R) subset from a field evolved R L. rigidum population. The R, the herbicide susceptible (S) and derived F2 populations were used for candidate herbicide resistance gene discovery by RNA sequencing. A P450 gene CYP81A10v7 was identified with higher expression in R vs. S plants. Transgenic rice overexpressing this Lolium CYP81A10v7 gene became highly resistant to acetyl-coenzyme A carboxylase- and acetolactate synthase-inhibiting herbicides (diclofop-methyl, tralkoxydim, chlorsulfuron) and moderately resistant to hydroxyphenylpyruvate dioxygenase-inhibiting herbicide (mesotrione), photosystem II-inhibiting herbicides (atrazine and chlorotoluron) and the tubulin-inhibiting herbicide trifluralin. This wide cross-resistance profile to many dissimilar herbicides in CYP81A10v7 transgenic rice generally reflects what is evident in the R L. rigidum. This report clearly showed that a single P450 gene in a cross-pollinated weed species L. rigidum confers resistance to herbicides of at least five modes of action across seven herbicide chemistries.

Identification, characterization and expression of rice (Oryza sativa) acetyltransferase genes exposed to realistic environmental contamination of mesotrione and fomesafen.

The plant acetyltransferases (ACEs) belong to a super family of proteins that contribute to secondary metabolisms and involve various abiotic and biotic stress responses. However, how rice ACEs respond to toxic agrochemicals is largely unknown. This study demonstrates that 86 and 83 genes coding ACEs in the transcriptome profiling were expressed under mesotrione (MTR) and fomesafen (FSA) exposure, respectively. Of these, 18 and 8 ACE differentially expressed genes (DEGs) were identified in MTR- and FSA-exposed rice transcriptome datasets. Some of the ACE genes were validated by quantitative RT-PCR analysis. Analysis of biochemical properties of ACEs revealed that many genes have various cis-elements and structural domain which may cope with a variety of biotic and abiotic stress responses and detoxification of xenobiotics. Moreover, the ACE activities in rice were induced under MTR and FSA exposure and reached out to the highest value at the 0.1 mg L-1. The ACE activities in the MTR and FSA treated roots were 2.6 and 3.5 fold over the control and those in shoots with MTR and FSA were 4.0 and 26.1 fold over the control, respectively. These results indicate that the ACE-coding genes can respond to the MTR and FSA stress by increasing their transcriptional level, along with the enhanced specific ACE protein activities in rice tissues.

Comparison of quintrione and quinclorac on mechanism of action.

Quintrione is a new post-emergence herbicide developed for use in rice; however, the mechanism of action remains unclear. We determined the phytotoxicity of quintrione, and the contributions of hormone levels and lipid peroxidation to phytotoxicity, by comparing them to those induced by quinclorac. We also investigated 4-hydroxyphenylpyruvate dioxygenase (HPPD) activity and carotenoid content following treatment with quintrione by comparing them to those induced by quinclorac and mesotrione. We found that quintrione and quinclorac both inhibited the growth of Echinochloa crusgalli var. zelayensis, but that quinclorac was a little more effective. At 24 h, quintrione and quinclorac significantly increased ethylene production and the contents of abscisic acid (ABA) and indole acetic acid (IAA) compared with the control. No significant differences were observed between quintrione and quinclorac on the three plant hormones. Quintrione and quinclorac also induced the formation of malondialdehyde (MDA), which is associated with lipid peroxidation, with no significant difference between them. Carotenoid content was reduced in E. crusgalli var. zelayensis following treatments with quintrione, quinclorac, and mesotrione. At 120 h, carotenoid contents were significantly higher following the quintrione and quinclorac treatments, in comparison with mesotrione treatment. There were no significant differences between quintrione and quinclorac in the inhibition of HPPD activity, and the effects of both were significantly less than the effect of mesotrione. In summary, E. crusgalli var. zelayensis was susceptible to both quintrione and quinclorac. The mechanism of action of quintrione, like that of quinclorac, was related to levels of plant hormones and lipid peroxidation; however, quintrione was a poor inhibitor of HPPD activity compared to mesotrione.

Terbuthylazine herbicide: an alternative to atrazine for weed control in glyphosate-tolerant maize.

It is proposed that the herbicide terbuthylazine is more effective than atrazine in controlling weeds in maize. This study aimed to evaluate the efficacy of terbuthylazine and atrazine in a mixture with glyphosate in glyphosate-tolerant maize for post-emergence application. The experiment was conducted over three trials using randomized blocks with 4 repetitions and 10 treatments, composed by terbuthylazine rates + glyphosate, atrazine rates + glyphosate, [atrazine + mesotrione] + glyphosate, atrazine + tembotrione, isolated glyphosate, and nontreated control. Trial 1 were infested with Bidens subalternans DC. and Commelina benghalensis L; trial 2 with Urochloa plantaginea (Link) R. D. Webster, Ipomoea spp., volunteer soybean, B. subalternans, and grasses; and trial 3 infestation with C. benghalensis, U. plantaginea, Ipomoea spp., volunteer soybean, B. subalternans, Amaranthus hybridus L., and grasses. Weed control, crop injury, and yield were evaluated. Terbuthylazine + glyphosate showed an efficacy equivalent to that of atrazine or [atrazine + mesotrione] + glyphosate in the control of broadleaves and C. benghalensis. In contrast, the efficacy of terbuthylazine was similar or greater than that observed for atrazine in controlling grasses, depending on the location. Terbuthylazine is an important partner of glyphosate in controlling weeds in maize and is an alternative to atrazine.

Charge-transfer complex-based spectrophotometric method for the determination of mesotrione in environmental samples.

A simple, accurate, sensitive, and selective spectrophotometric method has been developed for the determination of mesotrione. This method is based on the reaction of mesotrione with Fe(III) to form a charge transfer metal complex having λmax at 348 nm. Beer's law was obeyed in the concentration range of 0.2-10.0 µg mL-1 with limit of detection (LOD) and limit of quantification (LOQ) equal to 0.053 and 0.162 µg mL-1, respectively. The percent recovery of mesotrione from different environmental and agricultural samples was found to be 95.00-106.50% at various levels. Notably, the developed method was successfully employed for the determination of mesotrione in environmental (pond water, canal water, tap water, and soil) and agricultural (maize grains) samples.

Synthesis of molecularly imprinted fluorescent probe based on biomass-derived carbon quantum dots for detection of mesotrione.

A novel fluorescent probe based on molecularly imprinted polymers (MIPs) coupled with carbon quantum dots (CQDs) was fabricated and successfully used for selective recognition of mesotrione. In this probe, the biomass-derived CQDs were prepared through a hydrothermal method using mango peels as carbon source, and the whole synthesis procedure was green without chemical reagents. The CQDs were encapsulated into MIPs by using sol-gel technology. After removal of the template molecule mesotrione, specific binding sites are formed and there is electrostatic attraction between the probe and the template molecule. The synthetic CQDs@MIPs were able to selectively capture the target mesotrione with fluorescence quenching via the specific interaction between mesotrione and the recognition cavities. The probe was used for determination of mesotrione in corn to verify the practicality of the proposed method. The detection limit of mesotrione was 4.7 nmol L-1, and the linear range was 15 nmol L-1 to 3000 nmol L-1. Meanwhile, the recoveries of this method for mesotrione were 91.4-96.2%, and the relative standard deviations (RSDs) were 3.2-6.1%. This work provides a novel research method to synthesize CQDs@MIPs with high selectivity (imprinting factor = 5.6), and which can be used for convenient, rapid recognition and sensitive detection of trace compounds from complex matrices.

Characterization of 4-hydroxyphenylpyruvate dioxygenases, inhibition by herbicides and engineering for herbicide tolerance in crops.

4-Hydroxyphenylpyruvate dioxgenase (HPPD) enzymes from rat and from several plants contained only about a single inhibitor-binding active site per dimer which matched the content of iron in the purified Arabidopsis thaliana and Avena sativa enzymes. The dimeric HPPDs were about 10 fold more catalytically active than the tetrameric P. fluorescens enzyme with kcat/KmHPP values ranging from 0.8 to 2.5 s-1 µM-1. Most were also highly sensitive to herbicides with, for example, Ki values for mesotrione ranging from 25 to 100 pM. Curiously HPPDs from cool climate grasses were much less herbicide-sensitive. When likewise expressed in Nicotinia tabacum, Avena sativa HPPD, Ki value of 11 nM for mesotrione, conferred far greater tolerance to mesotrione (CallistoTM) than did any of the more sensitive HPPDs. Targeted mutagenesis of the Avena HPPD led to the discovery of 4 mutations imparting improved inherent tolerance, defined as the ratio of Ki to KmHPP, by about 16 fold without any loss of catalytic activity. The Nicotinia line with the highest expression of this quadruple mutant exhibited substantial resistance even up to a 3 kg/ha post-emergence application of mesotrione. The maximum observed expression level of heterologous plant HPPDs in tobacco was ca. 0.35% of the total soluble protein whereas the endogenous tobacco HPPD constituted only ca. 0.00075%. At such high expression even HPPDs with impaired catalytic activity could be effective. A quintuple mutant Avena sativa HPPD conferred substantial tolerance across a broad range of HPPD herbicide chemistries despite being only ca. 5 % as catalytically active as the wild type enzyme. Testing various wild type and mutant HPPDs in tobacco revealed that tolerance to field rates of herbicide generally requires about two order of magnitude increases in both inherent herbicide tolerance and expression relative to endogenous levels. This double hurdle may explain why target-site based resistance to HPPD-inhibiting herbicides has been slow to evolve in weeds.

Enhanced disappearance of mesotrione and fomesafen by water hyacinth (Eichhornia crassipes) in water.

This study aimed to investigate the potential use of water hyacinth (Eichhornia crassipes) in removing two herbicides (mesotrione and fomesafen) with long degradation cycles in water. The relative growth rate (RGR) of water hyacinth in the presence of 100-mg/L mesotrione and fomesafen was significantly lower than that in their absence, particularly with fomesafen. Moreover, the RGRFW and RGRDW with treatment with fomesafen were 1.47- and 1.58-fold lower than those with treatment with mesotrione, respectively. The disappearance rate constants of mesotrione and fomesafen in natural water were, respectively, 0.1148 and 0.0276 d-1 with plants and 0.0038 and 0.0005 d-1 without plants. The disappearance rate constants with and without plants were significantly different, indicating that uptake by plants combined with degradation by plant-associated bacteria account for 96.7% and 98.2% of the removal of mesotrione and fomesafen, respectively. The bioconcentration factor for mesotrione and fomesafen in living water hyacinth plants ranged 0.38-16.97 and 1.05-3.50 L/kg, respectively, whereas the residues of mesotrione and fomesafen in water decreased by 70-92 and 22-34%, respectively, after the plants were grown for 14 d in culture solution with 100-mg/L mesotrione and fomesafen. These results show that uptake by plants combined with degradation by plant-associated bacteria may be the dominant process in the removal of mesotrione and fomesafen from water by plants. Water hyacinth may be applied as an efficient, economical, and ecological alternative to accelerate the removal and degradation of agro-industrial waste water polluted with mesotrione and fomesafen.

Potential application of Pistia stratiotes for the phytoremediation of mesotrione and its degradation products from water.

The aim of the present work is to estimate remediation potential of Pistia stratiotes, its ability to uptake mesotrione (MES) - one of the most frequently used herbicides, and its main degradation products: 2-amino-4-methylsulfonyl benzoic acid (AMBA) and 4-methylsulfonyl-2-nitrobenzoic acid (MNBA). This research focuses on model experiments performed under laboratory conditions. The results show that Pistia stratiotes can uptake up to 75% of degradation products from 1 L of surface water samples polluted with 0.4 µg/L of each analyte during 7 days without significant phytotoxic effect. Under the same experimental conditions, the effectiveness of mesotrione sorption is in the range of 42-58%. The phytotoxicity of this compound is higher in comparison to its degradation products (decrease of chlorophyll concentration in plant tissues exposed to MES 27-32% vs 4-13% in case of exposition to AMBA and MNBA). The adequate nutrition of the plants is crucial to their well-being and thus the sorption of pollutants.

Effects of the herbicide mesotrione on soil enzyme activity and microbial communities.

Mesotrione (2-[4-(methylsulfonyl)-2-nithobenzoyl]-1, 3-cyclohexanedione) is a selective triketone herbicide that has been widely used in corn production for the past 15 years. However, its potential for risk to soil ecosystems is poorly documented. The present study investigated the soil enzyme activity and soil microbial community responses to a 20 days' mesotrione exposure at doses of 0.1, 1.0 and 5.0 mg/kg. On days 2, 5, 10 and 20, activities of soil ß-glucosidase, urease and acid phosphatase, soil microbe abundances, soil microbial community structure and abundance of the AOA-amoA and AOB-amoA genes were measured. Results showed that activities of urease and acid phosphatase were relatively stable, with no difference found between the mesotrione-treated group and control at the end of exposure. But ß-glucosidase activity was reduced in the 5.0 mg/kg mesotrione treatment. In the 1.0 and 5.0 mg/kg mesotrione-treated soil, abundance of bacteria, fungi and actinomycetes all were reduced. In the 0.1 mg/kg mesotrione-treated soil, only fungi abundance was reduced by the end of the exposure. The analysis of terminal restriction fragment length polymorphism (T-RFLP) revealed soil microbial community structure could be affected by mesotrione at all experimental doses, and microbial diversity declined slightly after mesotrione exposure. Abundance of AOA-amoA and AOB-amoA genes were reduced markedly in 1.0 and 5.0 mg/kg mesotrione-treated soil. The present study suggests that mesotrione at higher doses might induce negative impacts on soil microbes, a finding which merits additional research and which should be accounted for when application of this herbicide is considered.

Functional and structural characterization of two Bacillus megaterium nitroreductases biotransforming the herbicide mesotrione.

Mesotrione is a selective herbicide belonging to the triketone family, commonly used on maize cultures since 2003. A mesotrione-transforming Bacillus megaterium Mes11 strain isolated from an agricultural soil was used as a model to identify the key enzymes initiating the biotransformation of this herbicide. Two enzymes (called NfrA1 and NfrA2/YcnD) were identified, and functionally and structurally characterized. Both belong to the NfsA FRP family of the nitro-FMN reductase superfamily (type I oxygen-insensitive nitroreductase) and show optimal pH and temperature of 6-6.5 and 23-25°C, respectively. Both undergo a Ping Pong Bi Bi mechanism, with NADPH and NADPH/NADH as cofactors for NfrA1 and NfrA2/YcnD, respectively. It is interesting that both can also reduce various nitro compounds including pesticides, antibiotics, one prodrug and 4-methylsulfonyl-2-nitrobenzoic acid, one of the mesotrione metabolites retrieved from the environment. The present study constitutes the first identification of mesotrione-transforming enzymes. These enzymes (or their corresponding genes) could be used as biomarkers to predict the capacity of ecosystems to transform mesotrione and assess their contamination by both the parent molecule and/or the metabolites.

Degradation of Mesotrione Affected by Environmental Conditions.

With the widespread use of mesotrione, its residues have become increasingly serious and caused a series of environmental problems in northern China. To reduce the harm of these residues, we investigated the degradation effect of mesotrione in typical soils in northern China at different temperatures, soil moisture, pH values and initial concentrations. We also examined the influence of soil type, microorganisms and the use of organic matter and biogas slurry as soil amendments. Mesotrione degradation rates increased as the temperature, soil moisture, soil pH and the content of biogas slurry increased; and decreased as the organic content and the initial concentration of mesotrione increased. The degradation rates were different in the three soils. Microorganisms played an important role in the degradation process. These result may offer a theoretical basis for decreasing mesotrione residue when using this product in northern China.

Sugarcane bagasse as support for immobilization of Bacillus pumilus HZ-2 and its use in bioremediation of mesotrione-contaminated soils.

The degrading microorganisms isolated from environment usually fail to degrade pollutants when used for bioremediation of contaminated soils; thus, additional treatments are needed to enhance biodegradation. In the present study, the potential of sugarcane bagasse as bacteria-immobilizing support was investigated in mesotrione biodegradation. A novel isolate Bacillus pumilus HZ-2 was applied in bacterial immobilization, which was capable of degrading over 95 % of mesotrione at initial concentrations ranging from 25 to 200 mg L(-1) within 4 days in flask-shaking tests. Scanning electron microscope (SEM) images showed that the bacterial cells were strongly absorbed and fully dispersed on bagasse surface after immobilization. Specially, 86.5 and 82.9 % of mesotrione was eliminated by bacteria immobilized on bagasse of 100 and 60 mesh, respectively, which indicated that this immobilization was able to maintain a high degrading activity of the bacteria. Analysis of the degradation products determined 2-amino-4-methylsulfonylbenzoic acid (AMBA) and 4-methylsulfonyl-2-nitrobenzoic acid (MNBA) as the main metabolites in the biodegradation pathway of mesotrione. In the sterile soil, approximately 90 % of mesotrione was degraded after supplementing 5.0 % of molasses in bacteria-bagasse composite, which greatly enhanced microbial adaptability and growth in the soil environment. In the field tests, over 75 % of mesotrione in soil was degraded within 14 days. The immobilized preparation demonstrated that mesotrione could be degraded at a wide range of pH values (5.0-8.0) and temperatures (25-35 °C), especially at low concentrations of mesotrione (5 to 20 mg kg(-1)). These results showed that sugarcane bagasse might be a good candidate as bacteria-immobilizing support to enhance mesotrione degradation by Bacillus p. HZ-2 in contaminated soils.

Construction of Immobilized Laccase System Based on ZnO and Degradation of Mesotrione.

Mesotrione (MES) is a new environmental pollutant. Some reports have indicated that microbial enzymes could be utilized for MES degradation. Laccase is a green biocatalyst whose potential use in environmental pollutant detoxification has been considered limited due to its poor stability and reusability. However, these issues may be addressed using enzyme immobilization. In the present study, we sought to optimize conditions for laccase immobilization, to analyze and characterize the characteristics of the immobilized laccase, and to compare its enzymatic properties to those of free laccase. In addition, we studied the ability of laccase to degrade MES, and analyzed the metabolic pathway of MES degradation by immobilized laccase. The results demonstrated that granular zinc oxide material (G-ZnO) was successfully used as the carrier for immobilization. G-ZnO@Lac demonstrated the highest recovery of enzyme activity and exhibited significantly improved stability compared with free laccase. Storage stability was also significantly improved, with the relative enzyme activity of G-ZnO@Lac remaining at about 54% after 28 days of storage (compared with only 12% for free laccase). The optimal conditions for the degradation of MES by G-ZnO@Lac were found to be 10 mg, 6 h, 30 °C, and pH 4; under these conditions, a degradation rate of 73.25% was attained. The findings of this study provide a theoretical reference for the laccase treatment of 4-hy-droxyphenylpyruvate dioxygenase (HPPD)-inhibiting herbicide contamination.