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May 9, 2016 - that severely affects human health.1,2 With the extensive use of. TCF, residues ... such as leukemia, non-Hodgkin,s lymphoma, lung cance...
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Biodegradation of the Organophosphate Trichlorfon and Its Major Degradation Products by a Novel Aspergillus sydowii PA F‑2 Jiang Tian,† Qiaofeng Dong,§ Chenlei Yu,† Ruixue Zhao,† Jing Wang,† and Lanzhou Chen*,† †

School of Resource and Environmental Sciences, Hubei Key Laboratory of Biomass-Resources Chemistry and Environmental Biotechnology, Wuhan University, Wuhan, Hubei 430079, Peoples’ Republic of China § Institute of Wuhan Modern Urban Agriculture Planning and Design, Wuhan, Hubei 430072, People’s Republic of China S Supporting Information *

ABSTRACT: Trichlorfon (TCF) is an important organophosphate pesticide in agriculture. However, limited information is known about the biodegradation behaviors and kinetics of this pesticide. In this study, a newly isolated fungus (PA F-2) from pesticide-polluted soils was identified as Aspergillus sydowii on the basis of the sequencing of internal transcribed spacer rDNA. This fungus degraded TCF as sole carbon, sole phosphorus, and sole carbon−phosphorus sources in a mineral salt medium (MSM). Optimal TCF degradation conditions were determined through response surface methodology, and results also revealed that 75.31% of 100 mg/L TCF was metabolized within 7 days. The degradation of TCF was accelerated, and the mycelial dry weight of PA F-2 was remarkably increased in MSM supplemented with exogenous sucrose and yeast extract. Five TCF metabolic products were identified through gas chromatography−mass spectrometry. TCF could be initially hydrolyzed to dichlorvos and then be degraded through the cleavage of the P−C bond to produce dimethyl hydrogen phosphate and chloral hydrate. These two compounds were subsequently deoxidized to produce dimethyl phosphite and trichloroethanal. These results demonstrate the biodegradation pathways of TCF and promote the potential use of PA F-2 to bioremediate TCF-contaminated environments. KEYWORDS: trichlorfon, biodegradation, Aspergillus sydowii, mycelial dry weight, biodegradation pathway

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INTRODUCTION Trichlorfon (TCF), O,O-dimethyl-(2,2,2-trichloro-1-hydroxyethyl) phosphonate, is a commonly used broad-spectrum organophosphate pesticide (OP) produced increasingly at a large scale because of its agricultural benefits. However, this compound is also a potential source of environmental pollution that severely affects human health.1,2 With the extensive use of TCF, residues may be produced and may persist in contaminated soil, air, and groundwater. TCF may also pose serious public health problems because of its mutagenic and carcinogenic properties.3 This compound induces toxicity mainly by inhibiting acetylcholinesterase. Furthermore, exposure to TCF has been associated with severe human diseases, such as leukemia, non-Hodgkin’s lymphoma, lung cancer, and aplastic anemia in children.4 TCF is a well-known OP exhibiting a cholinesterase inhibitor activity in numerous fields and fruit crop protection strategies. It is an effective drug for Alzheimer’s disease and anticholinesterase in the medical industry.5,6 However, TCF in aqueous media (pH >6) is nonenzymatically dehydrochlorinated to a more toxic OP insecticide, namely, dichlorvos (DDCV), which is about 8 times more toxic than TCF and a more potent inhibitor of cholinesterase and inducer of organophosphorus-induced delayed neuropathy.7,8 TCF and DDCV can infiltrate surface and underground water through the soil in agricultural practices because of their stability and solubility under aqueous conditions. As a consequence, these substances contaminate the aqueous environment.9 Therefore, effective methods should be developed to reduce TCF and DDCV residue levels in the environment. © XXXX American Chemical Society

Hydrolysis, photolysis, and biodegradation are the three main degradation pathways of TCF in the environment. TCF and DDCV are easily O-demethylated to desmethyl compounds, dimethylphosphate, and monomethylphosphate through hydrolysis, pyrolysis, and other chemical processes.5,10−12 However, these conventional techniques are limited because of the undesired removal efficiency and toxic organic intermediate products.13 TCF in soil is initially degraded by the microbial community to produce environmentally friendly and weakly toxic products. Microorganisms can also utilize TCF as an energy source. As a result, the degradation rate of TCF in the environment is increased.14 Thus far, few TCF degradation microbes have been isolated,14 and limited information has described the biodegradation pathways of TCF. This study aimed to isolate and culture novel TCFdegrading microorganisms from polluted soils, to calculate their TCF degradation kinetic curves and degradation efficiencies with exogenous carbon and nitrogen sources, and to investigate possible TCF biodegradation pathways. As a model system, a novel TCF degradation fungus named PA F-2 was screened and used.

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MATERIALS AND METHODS

Soil and Chemicals. The soil samples used in this research were collected from a pesticide-contaminated factory (30°02′ N, 115°37′ E) Received: February 25, 2016 Revised: May 5, 2016 Accepted: May 9, 2016

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DOI: 10.1021/acs.jafc.6b00909 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry in Hubei province, China. The soil samples were air-dried, sieved through a 20-mesh screen, and stored at −20 °C. Standard TCF (≥98% purity) was purchased from Shanghai Civi Chemical Technology Co., Ltd. (Shanghai, China). Chromatography grade methanol was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). All other solvents and chemicals used in this study were of analytical grade. Screening, Culturing, and Identification of TCF-Degrading Microorganisms. The initial enrichment culture tryptone yeast cystine (TYC) and enrichment procedures were prepared, as described by Zhang et al.15 with minor modifications. The TYC medium containing 5 g/L tryptone, 5 g/L yeast extract, 1 g/L K2HPO4, and 1 g/L glucose was used to isolate TCF-degrading microorganisms. In brief, 10 g of previously treated soil samples was added to 250 mL flasks with 100 mL of TYC. After the samples were incubated for 5 days in a rotary shaker (150 rpm) in a darkened thermostatic chamber maintained at 28 °C, 1 mL of soil suspension was transferred to 250 mL flasks containing a fresh TYC medium supplemented with sterile TCF (50 mg/L) and further incubated for 5 days under the same conditions. After five subsequent transfers into the same TYC medium supplemented with different TCF concentrations (50, 100, 200, 400, and 500 mg/L), 200 μL of serial dilutions (10−4, 10−5, and 10−6) of the soil suspensions was plated onto mineral salt medium (MSM) agar plates containing 1 g/L NH4Cl, 1 g/L NaCl, 1 g/L KH2PO4, 1 g/L K2HPO4, and 1 g/L MgSO4·7H2O supplemented with TCF (500 mg/ L) to isolate individual colonies. The colonies with distinct morphological characteristics were purified by streaking on the same agar plates. One TCF-degrading fungus designated PA F-2 was isolated and used for further research. Fungal identification was based on the internal transcribed spacer (ITS) sequence in 18S rDNA. DNA extraction and ITS rDNA amplification methods were performed, as described by Hebart16 and Gardes,17 respectively. Amplification results were sequenced by the BGI Gene Co. (Wuhan, China). A BLAST search was conducted at the National Center for Biotechnology Information (http://www.ncbi. nlm.nih.gov/). Sequencing data were phylogenetically analyzed using Mega 5. Inoculum Preparation. A pure culture of the isolate was inoculated in a potato dextrose agar (PDA) slant and incubated at 28 °C. After 72 h of cultivation, 10 mL of sterile distilled water was added, and the spore suspension was harvested. The spore suspension was then diluted and quantified via a hemocytometer counting method to set the density to 109/mL. Afterward, 3% (v/v) of the spore suspension was used as an inoculum for further TCF degradation analyses. Effect of Spore Concentration on the TCF Degradation by PA F-2. MSM with a final TCF concentration of 100 mg/L in aqueous solution was used to investigate the effect of spore density on TCF degradation by PA F-2. Spore suspensions were prepared at three concentrations, namely, 109, 108, and 107/mL. The treatments were inoculated in 50 mL Erlenmeyer flasks containing 20 mL of sterile broth medium. As a control, a medium without inoculated fungus was also cultured under the same conditions. The Erlenmeyer flasks were incubated at 30 °C and 150 rpm in a rotary shaker for 7 days, and the TCF concentration was determined at one-day intervals. Microbial biomass was harvested through centrifugation at 12000 rpm, washed with distilled water, and dried at 70 °C for 0.5 h. Optimization of TCF Degradation Conditions. Medium pH, temperature, and initial pesticide concentrations are significant variables in pesticide degradation.18 Response surface methodology (RSM) based on a Box−Behnken design was utilized to optimize the three important factors that significantly affect TCF degradation by PA F-2. The factors and optimized ranges chosen for independent variables were temperature (24, 28, and 32 °C), initial TCF concentration (50, 100, and 200 mg/L), and initial medium pH (6.0, 7.0, and 8.0). Seventeen experimental runs with three replicates at the center point and range values (Table S1) were generated and designed in Expert Design 8.0. All of the treatments were incubated at 150 rpm in a rotary shaker for 7 days. The TCF concentration and mycelial dry weight were determined. The dependent variable was the

degradation of 100 mg/L TCF in MSM by PA F-2 after 7 days of culture. The experiment was conducted in a randomized block design to minimize the effect of unpredictable variability in response, and data were analyzed through response surface regression in Expert Design 8.0 to fit the quadratic polynomial equation (eq 1)17 Yi = b0 +

∑ biXi + ∑ bijXiXj + ∑ biiXi2

(1)

where Yi is the predicted response, b0 is a constant, Xi and Xj are the variables, bi is a coefficient, bij is the interaction coefficient, and bii is the quadratic coefficient. Effect of Exogenous Sources on TCF Degradation. The sterilized MSM with TCF (100 mg/L) was supplemented with exogenous carbon source sucrose (1%, m/v) and exogenous nitrogen source yeast extract (1%, m/v). Triplicate cultures were inoculated with PA F-2 spore suspension (approximately 109/mL) and incubated at 28 °C and 150 rpm for 7 days. Mycelial growth and TCF degradation were examined at an interval of 1 day. Chemical Analysis. The TCF concentrations in MSM were measured through high-performance liquid chromatography (HPLC). In brief, 2 mL of cell-free MSM samples was centrifuged (12000 rpm), suspended, and passed through a 0.22 μm PTFE membrane filter. The samples were then analyzed using an HPLC system (Agilent 1100, USA) equipped with a variable-wavelength UV detector and a C18 reverse-phase column (Hypersil ODS2 5 μm × 4.6 mm × 250 mm). The mobile phase used for chemical detection was methanol/distilled water (70:30, v/v) injected at a flow rate of 1.0 mL/min and detected at 210 nm. The sample injection volume was 10 μL. The concentrations of TCF residues in MSM were calculated on the basis of a peak area from the calibration curve, which was found to be linear within the range of 50−1000 mg/L TCF with R2 = 0.9998. Metabolites were identified in a Shimadzu QP2010 plus gas chromatography−mass spectrometry (GC-MS) system (Shimadzu, Japan) equipped with an on-column, split/splitless capillary injection system, and an RTX-5MS capillary column (30 m × 250 μm × 0.25 μm). Cell-free MSM (20 mL) was extracted using 20 mL of dichloromethane in a rotary shaker for 1 h. After partitioning was observed, the supernatants were transferred to new centrifuge tubes, dehydrated with anhydrous sodium sulfate, and evaporated to dryness in a nitrogen stream. The dry residues were dissolved in methanol, filtered through a 0.22 μm PTFE membrane, and analyzed through GC-MS. The GC-MS operating conditions were as follows. The injector and transfer line temperatures were 250 and 280 °C, respectively. Helium (99.999% purity) was used as a carrier gas at a constant flow rate of 1.0 mL/min. The column was held at 50 °C for 2 min, ramped at 15 °C/min to 260 °C, and held at 260 °C for 5 min. The ion source temperature was 230 °C, and the ionization energy was 70 eV. The solvent cut time was set at 3 min, and the injection volume was 1.0 μL with splitless sampling.12 Kinetic and Statistical Analysis. The trials were all performed in triplicates. The control groups supplemented with at least three gradient TCF concentrations were set in each experiment to the calculated calibration curve. The concentration of TCF residues was calculated on the basis of a peak area from the calibration curve, and the TCF biodegradations were calculated from the equation (eq 2)

Dt = (Cck − Cexptl)/C0 × 100%

(2)

where Dt is the TCF biodegradation rate at time t, C0 is the initial PNP concentration, and Cck and Cexptl are the TCF concentrations in control and experimental treatments measured at time t by the calibration curve, respectively. The biodegradation of TCF in liquid MSM was fitted to a firstorder kinetic model (eq 3)

Ct = C0 × e−kt

(3)

where Ct represents the TCF concentration at time t (days), C0 represents the concentration of TCF at time zero, and k and t are the rate constant (per day) and degradation period in days, respectively. The time at which the TCF concentration in MSM was reduced by B

DOI: 10.1021/acs.jafc.6b00909 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Figure 1. Phylogenetic dendrogram obtained by the distance matrix analysis of ITS rDNA sequences showing the position of PA F-2 among phylogenetic neighbors. Bootstrap values (%) are indicated at the nodes. The scale bar indicates 0.01 substitution per nucleotide position. Accession numbers are shown in parentheses.

Figure 2. Effects of initial inoculum spore concentrations on (A) TCF residues ((■) 109/mL, (●) 108/mL, (▲) 107/mL) and (B) mycelial dry weight of PA F-2. Data presented are means of three replicates ± standard deviation. Different letters indicate significant differences (∗∗ at P < 0.001 and ∗ at P < 0.05). 50% (DT50) was measured from the linear equation obtained from the regression between ln(Ct/C0) of the chemical data and time (t).19 Data were analyzed through one-way ANOVA and Duncan’s multiple-range test to compare the means. Multiple mean comparisons were performed with a significant difference test at P < 0.05 or P < 0.001. The values shown in tables and figures are the means of three replicates ± standard deviation.

Aspergillus sydowii (GenBank accession no. KJ173528) with homology of >99.6%. A phylogenetic tree was constructed via a neighbor-joining method based on the ITS rDNA sequences of PA F-2 and related species. The phylogenetic analysis also indicated that the isolate was closely similar to the Aspergillus group (Figure 1). Aspergillus species are ubiquitous molds in the environment. Aspergillus sp. is a well-known broad-spectrum enzyme producer within the microbial kingdom. Several A. sydowii strains were isolated and utilized in producing industrial enzymes, for example, β-glucosidase,20 α-galactosidase,21 cellulase,22 and xylanase.23 Nevertheless, the OP biodegradation ability of this species has been rarely investigated.24,25 To our knowledge, our study is the first to describe the TCF biodegradation by a novel A. sydowii isolate. Effects of Spore Concentration on Growth and TCF Degradation. The initial amount of inocula is considered an

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RESULTS AND DISCUSSION Isolation and Characterization of TCF-Degrading PA F-2 Fungus. After performing the soil enrichment procedure, we successfully isolated one fungal strain, designated PA F-2, which can utilize TCF as sole carbon source, sole phosphorus source, and sole carbon−phosphorus source on MSM (Figure S1). PA F-2 was an obligate aerobe on MSM agar plates. In the first 24 h of incubation, the colonies initially appeared white and then turned green entirely after 72 h of culture. On the basis of the ITS sequence result, we found that PA F-2 is C

DOI: 10.1021/acs.jafc.6b00909 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Figure 3. Response surface plot showing the effect of temperature and medium pH on TCF degradation by PA F-2 at an initial TCF concentration of 125 mg/L.

important factor of pesticide degradation.18 In this study, 81.8% of 100 mg/L TCF was degraded in MSM with a 109/mL fungal spore concentration within 7 days of culture. This finding was significantly different (P < 0.05 and P < 0.001) from the two other treatments (Figure 2A). The mycelial biomass (P < 0.05) at the initial inoculum amount of 109/mL was higher than that at the other initial amounts of inocula. The maximum mycelial dry weight (3.67 mg/mL) was also obtained (Figure 2B). The TCF degradations in these three experiments were well fitted to the first-order kinetics (kinetic equations and kinetic parameters not shown). On the basis of the similar variation trend of growth between TCF degradation and mycelial biomass, we can hypothesize that biomass growth, which is remarkably affected by initial inoculum amount and TCF toxicity,26 possibly influences the TCF degradation under aqueous conditions. A high initial spore concentration may also compensate for the population decline affected by the TCF solution, and survivors in the medium can multiply and degrade OPs.27 Optimization of TCF Degradation Conditions. The interactive effects of different variables and the optimum values of temperature (X1), TCF concentration (X2), and medium pH (X3) on the maximum TCF biodegradation were determined by analyzing the Box−Behnken design. The designed matrix of variables and the corresponding response values of TCF degradation rate (Y1) and PA F-2 dry weight (Y2) are listed in Table S1. The data were analyzed in Expert Design, and the obtained results were fitted with two second-degree polynomial equations (eqs 4 and 5) to explain the TCF biodegradation and mycelial dry weight of PA F-2, respectively.

The statistical significance of eqs 3 and 4 was also evaluated by performing ANOVA and F test in Expert Design (Table S2). The accuracy of the developed model of TCF degradation was supported by the response coefficient (R2 = 0.9614). This finding indicated that 96.14% of the response variability could be covered by this model. The nonsignificant lack of the fit value confirmed that the developed model could sufficiently predict the TCF biodegradation rate (P > 0.01). The preciseness and reliability of this model were supported by the low coefficient of variation (CV = 7.45%). On the basis of the regression parameter estimates, we noted that the linear and square terms of medium pH (X3) significantly affected (P < 0.0001) the TCF biodegradation. By contrast, the initial TCF concentration (X2) and the interaction terms did not significantly influence the TCF biodegradation (P > 0.05). Temperature and medium pH are also two relevant parameters in the biodegradation of other OPs.19,27 The three-dimensional response surface was plotted as the effects of temperature and medium pH at a fixed initial TCF concentration of 125 mg/L to demonstrate the TCF degradation by PA F-2 (Figure 3). The plot of TCF degradation revealed the theoretical maximum and minimum values of 75.31 and 30.11%, respectively. At the theoretical maximum point, the optimum levels of the two variables were obtained as 0.27 and −0.496 in terms of the coded units, that is, at 28.9 °C and pH 6.5, respectively. Therefore, the optimal conditions for the PA F-2-facilitated TCF degradation were determined as 28.9 °C, pH 6.5, and 125 mg/L initial TCF concentration. These parameters indicated the neutral and acidic conditions of TCF concentration. The adjusted R2 of 0.7237 demonstrated that the predicted values of the developed model of mycelial dry weight were not consistent with the experimental values (data not shown). This result was also supported by the high coefficient of variation (CV = 20.29%). Likewise, regression analysis revealed that the linear and square terms of temperature (X1), TCF concentration (X2), and medium pH (X3) elicited nonsignificant effects (P > 0.05) on the mycelial dry weight. Table S1 shows that the rapid increase in the TCF degradation rate was not related to the mycelial dry weight of PA F-2. This result is different from previous findings, which revealed the rapid increase in pesticide degradation rate and bacterial biomass.27

Y1 = 67.49 + 3.57X1 + 0.20X 2 − 13.05X3 + 1.75X1X 2 − 1.78X1X3 + 5.48X 2X3 − 10.29X12 + 0.49X 22 − 13.55X32

(4)

Y2 = 1.73‐0.013X1 + 0.13X 2 + 0.21X3 + 0.17X1X 2 − 0.083X1X3 + 0.26X 2X3 − 0.17X12 − 0.23X 22 + 0.58X32

(5)

Y1 and Y2 are the predicted TCF degradation (%) and mycelial dry weight (mg/mL) of PA F-2, respectively. X1, X2, and X3 are the coded values of temperature (°C), TCF concentration (mg/L), and medium pH, respectively. D

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Journal of Agricultural and Food Chemistry Effect of Exogenous Sources on TCF Degradation. The kinetic curves of the TCF degradation by PA F-2 supplemented with exogenous sucrose and yeast extract are shown in Figure 4A. In the cultures without exogenous compounds, TCF

Alternate exogenous chemicals, such as carbon and nitrogen sources, are reported as crucial factors that determine the efficiencies of pesticide biodegradation.25,27,28 Sucrose and yeast extract are two of the most common nutrients used to culture microbes. Ahmed et al.29 and Chen et al.19 observed similar enhanced OP degradation and growth characteristics in the presence of these two exogenous compounds. The TCF degradation and mycelial dry weight growth described in this paper could also indicate that additional carbon and nitrogen sources could significantly increase mycelial growth, but the increment in TCF degradation is variable. However, this finding is different from previous results observed in the biodegradation of other OPs.30,31 This result suggests that co-metabolism with exogenous chemicals can be an effective mechanism to improve microbial growth. By contrast, TCF degradation, which is conducted by specific proteins and enzymes, is positively influenced by microbial growth and enzymatic activity simultaneously. The additional yeast extract containing nitrogen sources, multiple polypeptides, and vitamins improves PA F-2 growth and specific enzyme synthesis.29 Such effects led to complete TCF degradation. Degradation Products and Degradation Pathway of TCF by PA F-2. The retention times (RT), characteristic ions of the mass spectra (m/z), and chemical structural formula of each degradation product are summarized in Table 2. The

Figure 4. (A) TCF degradation by PA F-2 in MSM supplemented with (■) TCF, (●) TCF + sucrose, and (▲) TCF + yeast extract. (B) Mycelial dry weight of PA F-2 in MSM supplemented with exogenous compounds within 7 days. Different letters indicate significant differences (∗∗ at P < 0.001 and ∗ at P < 0.05).

degradation increased rapidly, and an apparent lag phase was observed in the first 24 h. Furthermore, approximately 68.6% of TCF was degraded after 7 days of incubation. The TCF degradation proceeded much more rapidly in the sucrosecontaining medium than in the sucrose-free medium within 2 days. A stable level of approximately 60% was reached within 4 days of incubation without a lag phase. The maximum TCF degradation of 79.3% was obtained within 7 days. However, the most efficient TCF degradation condition was obtained in the medium supplemented with yeast extract as an exogenous nitrogen source. Furthermore, 100 mg/L TCF was completely degraded within 4 days. The degradation of TCF in the liquid medium supplemented with or without sucrose and yeast extract was fitted to the firstorder kinetic model (Table 1). The kinetic data for all runs calculated from eq 2 revealed that the experimental data were well correlated with the model (the value of R2 = 0.9106− 0.9899). A larger degradation rate constant (k = 0.769/day, P < 0.05) and degradation rate (v = 30.318 mg/day, P < 0.05) were observed in the yeast extract-containing medium. The degradation time (DT50) in the yeast-containing medium was 0.9 day, which was significantly shorter (P < 0.05) than those in the sucrose-containing medium (3.1 days) and nonexogenous chemical-containing medium (4.2 days). The mycelial dry weights in the three experiments were compared. The results revealed that additional sucrose and yeast extract remarkably improved the growth of PA F-2 during the 7 days of incubation (Figure 4B). In the presence of sucrose, the mycelial dry weight rapidly increased and reached the maximum value of 147 mg/mL within 7 days. The maximum mycelial dry weight was obtained at 40 mg/mL within 5 days in the yeast extract-containing medium. The dry weight declined to 16 mg/mL after 7 days of incubation.

Table 2. Chromatographic Properties of Metabolites of TCF during Degradation by PA F-2

degradation products of TCF were identified on the basis of the mass spectra and were matched with authentic standard compounds from the National Institute of Standards and

Table 1. Kinetic Parameters of Degradation of TCF by PA F-2 in MSM Supplemented with Exogenous Compoundsa treatment

kb (days)

vc (mg/day)

regression eq (first-order)

R2

DT50d (days)

PA F-2 + TCF PA F-2 + TCF + sucrose PA F-2 + TCF + yeast extract

0.165a 0.225a 0.769b

8.921a 19.547b 30.318c

ln(Ct/C0) = −0.5081t + 0.2196 ln(Ct/C0) = −0.4259t − 0.4806 ln(Ct/C0) = −1.3118t + 0.6525

0.9726 0.9106 0.9899

4.2b 3.1c 0.9d

a

The values are means of three replicates. Different letters indicate significant difference between treatments (p < 0.05). bk, degradation rate constant. cv, TCF degradation rate. dDT50, time in which the TCF concentration in MSM was reduced by 50%. E

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Figure 5. Proposed biodegradation pathways of TCF in PA F-2. Solid arrows denote the proposed pathways derived from the metabolites detected in this study. Dotted arrows signify the proposed pathways derived from the metabolites detected in previous references. (∗) Dimethyl phosphite and dimethyl hydrogen phosphite are tautomeric forms, whereas phosphoryl tautomer dimethyl phosphite predominates and dimethyl hydrogen phosphite is less stable in equilibrium solution.

intermediate of TCF and DDCV decomposed by microorganisms.5 In this study, this compound was deoxidized to produce two phosphite compound, namely, dimethyl phosphite and dimethyl hydrogen phosphite. These two compounds have been found as tautomeric forms and important intermediates in phosphorus reactions. However, it is well established that the phosphoryl tautomer dimethyl phosphite predominates as these two compounds are in equilibrium, presumably for less stable dimethyl hydrogen phosphite and the very strong energy of PO bond in dimethyl phosphite.32 The phosphoryl tautomer dimethyl phosphite is then degraded to water, carbon dioxide, and phosphate radical, which is supplied as a phosphorus compound to microorganisms in media. This proposed pathway may demonstrate the growth of PA F-2 in the solid plate supplemented with TCF as sole phosphorus and sole carbon−phosphorus sources (Figure S1). Chloral hydrate is spontaneously converted to trichloroethanal, and these two chlorides are then utilized in glucuronide synthesis by organisms.10,33 The synthesized glucuronide could be used as a carbon source by PA F-2. However, trimethyl orthophosphate and dichloromethane in the DDCV biodegradation pathways were proposed as primary metabolites. The methoxy group (−CH2O) in trimethyl orthophosphate is easily removed through free radical attacks, followed by the production of dimethyl hydrogen phosphate.13 Trimethyl orthophosphate was degraded to phosphate radical, carbon dioxide, and water. Dichloromethane was a common organic solvent that cannot be converted to glucuronide. The production of dichloromethane directly decreased the percentage of biologically utilized carbon in DDCV. As a result, microbial growth was restricted. Therefore, the mycelial dry weight of PA F-2 increased slightly in all of the experiments in which TCF was supplemented as solo carbon source as TCF was spontaneously converted to DDCV under aqueous conditions in this study.

Technology (NIST, USA). The mass spectra of the metabolic products of TCF degradation are illustrated in Figure S2. The GC-MS analysis revealed that the peak at a RT of 10.87 min corresponded to the TCF standard, and the peak at a RT of 9.49 min corresponded to DDCV, an important intermediate product. Metabolite HCl, the main decomposition product from TCF to DDCV, was eluted from the capillary column before methanol12 because no signal of HCl was detected in the mass spectrum system. TCF and DDCV decreased over time as the five compounds were formed. Compound C, which was similar to the characteristic parental ions of trimethyl orthophosphate in the NIST library database (>80% matching), showed a prominent protonated molecular ion at m/z 110 and RT of 6.142 min. Compounds D and E were identified as dimethyl hydrogen phosphite and dimethyl phosphite with approximately equal RTs of 4.175 and 4.117 min and were characterized by ions at m/z 79 and 80, respectively. Compounds F and G with RTs of 3.558 and 3.083 min showed the same molecular ions at m/z 82, which correspond to chloral hydrate and trichloroethanal, respectively. Among these degradation products, trichloroethanal, dimethyl phosphite, and dimethyl hydrogen phosphite have also been reported during the pyrolytic decomposition of TCF and oxygen plasma treatment of DDCV.12,13 In the control group without PA F-2 inoculates, these metabolites were detected in very low concentrations. This finding suggested that TCF decomposed under aqueous conditions, but the reaction was accelerated by the incubation of PA F-2. On the basis of the chemical structures and the metabolic products of TCF in Table 2, we proposed the TCF biodegradation pathways (Figure 5). Under neutral and alkaline conditions, TCF was initially hydrolyzed to produce DDCV and HCl (eluted before detection). With the inoculation of PA F-2, TCF was hydrolyzed through the cleavage of the P−C bond to produce dimethyl hydrogen phosphite and chloral hydrate. Dimethyl hydrogen phosphate is a dominant F

DOI: 10.1021/acs.jafc.6b00909 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.6b00909. Table S1, Box−Behnken experimental design and response of dependent variable for TCF degradation and dry weight; Table S2, analysis of variance for the response surface quadratic model for TCF degradation and figures; Figure S1, growth of fungus PA F-2 on MSM agar plates; Figure S2, mass spectra of the metabolic products from TCF degradation (PDF)

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AUTHOR INFORMATION

Corresponding Author

*(L.C.) E-mail: [email protected]. Phone: +86-27-87152713. Fax: +86-27-68778893. Funding

This work was supported by the National Natural Science Foundation of China (31370421), the Wuhan basic research plan (2014060101010060), and the National Talent Plan of Science Foundation (Wuhan University, J1103409). Notes

The authors declare no competing financial interest.

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DOI: 10.1021/acs.jafc.6b00909 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.jafc.6b00909 J. Agric. Food Chem. XXXX, XXX, XXX−XXX