A Synergistic Consortium Involved in rac-Dichlorprop Degradation as Revealed by DNA Stable Isotope Probing and Metagenomic Analysis.

rac-Dichlorprop, a commonly used phenoxyalkanoic acid herbicide, is frequently detected in environments and poses threats to environmental safety and human health. Microbial consortia are thought to play key roles in rac-dichlorprop degradation. However, the compositions of the microbial consortia involved in rac-dichlorprop degradation remain largely unknown. In this study, DNA stable isotope probing (SIP) and metagenomic analysis were integrated to reveal the key microbial consortium responsible for rac-dichlorprop degradation in a rac-dichlorprop-degrading enrichment. OTU340 (Sphingobium sp.) and OTU348 (Sphingopyxis sp.) were significantly enriched in the rac-[13C]dichlorprop-labeled heavy DNA fractions. A rac-dichlorprop degrader, Sphingobium sp. strain L3, was isolated from the enrichment by a traditional enrichment method but with additional supplementation of the antibiotic ciprofloxacin, which was instructed by metagenomic analysis of the associations between rac-dichlorprop degraders and antibiotic resistance genes. As revealed by functional profiling of the metagenomes of the heavy DNA, the genes rdpA and sdpA, involved in the initial degradation of the (R)- and (S)-enantiomers of dichlorprop, respectively, were mostly taxonomically assigned to Sphingobium species, indicating that Sphingopyxis species might harbor novel dichlorprop-degrading genes. In addition, taxonomically diverse bacterial genera such as Dyella, Sphingomonas, Pseudomonas, and Achromobacter were presumed to synergistically cooperate with the key degraders Sphingobium/Sphingopyxis for enhanced degradation of rac-dichlorprop. IMPORTANCE Understanding of the key microbial consortium involved in the degradation of the phenoxyalkanoic acid herbicide rac-dichlorprop is pivotal for design of synergistic consortia used for enhanced bioremediation of herbicide-contaminated sites. However, the composition of the microbial consortium and the interactions between community members during the biodegradation of rac-dichlorprop are unclear. In this study, DNA-SIP and metagenomic analysis were integrated to reveal that the metabolite 2,4-dichlorophenol degraders Dyella, Sphingomonas, Pseudomonas, and Achromobacter synergistically cooperated with the key degraders Sphingobium/Sphingopyxis for enhanced degradation of rac-dichlorprop. Our study provides new insights into the synergistic degradation of rac-dichlorprop at the community level and implies the existence of novel degrading genes for rac-dichlorprop in nature.

Different leaf-mediated deposition, absorbed and metabolism behaviors of 2,4-D isooctyl ester between Triticum aestivum and Aegilops tauschii Coss.

Tausch's goatgrass (Aegilops tauschii Coss.), is a major weed species, infesting wheat (Triticum aestivum) fields in China. 2,4-D isooctyl ester is widely used for broadleaf weed control and selected as a tool to study the differences between, A. tauschii and T. aestivum. In this study, we measured the growth responses of these species to 2,4-D isooctyl ester and found that T. aestivum was more sensitive to the herbicide than A. tauschii. To clarify the reasons for this difference, we measured the leaf-mediated deposition, absorption and metabolism of 2,4-D isooctyl ester and the expression of auxin receptor transport inhibitor response (TIR1) gene in T. aestivum and A. tauschii. The results indicated that the deposition of 2,4-D isooctyl ester droplets may be lower on A. tauschii than on T. aestivum, because of the increased contact angle and greater density of trichomes on the leaves of the former. A distinct increase in 2,4-D isooctyl ester uptake was detected in T. aestivum during the entire experimental period, and the rate was 2.2-fold greater than that in A. tauschii at 6 h after treatment. Compared with A. tauschii, T. aestivum exhibited a greater accumulation of primary metabolite 2,4-D in plants, which may be responsible for the different responses of the two species. Additionally, the absolute expression level of TIR1 was clearly greater in T. aestivum than that in A. tauschii. These data will be helpful to further understand the differences between T. aestivum and A. tauschii, which may provide a unique perspective for the development and identification of new target compounds that are effective against this weed species.

Zr-MOF modified cotton fiber for pipette tip solid-phase extraction of four phenoxy herbicides in complex samples.

Phenoxy herbicides are widely applied in agricultural weeding. The determination of herbicides is important in environmental protection, agricultural production, food safety, and public health. In this study, a facile and efficient analytical method was proposed for the trace detection of phenoxy herbicides in soil, cucumber, and tap water samples by coupling pipette tip solid phase extraction (PT-SPE) with high performance liquid chromatography. UiO-66-funtionalized cotton (Cotton@UiO-66) was packed into pipette-tip as sorbent to fabricate extraction device. The modification of UiO-66 on cotton fiber was confirmed using scanning electron microscope, Fourier transform infrared spectroscopy, and X-ray diffraction. The main factors affecting the adsorption of Cotton@UiO-66 for four phenoxy herbicides were evaluated by response surface methodology in detail. Under optimized conditions, Cotton@UiO-66 displayed excellent properties in the extraction of phenoxy herbicides with good peak shape. Linear ranges of 4-chlorophenoxyacetic acid, dicamba, 2,4-dichlorophenoxyacetic acid, and 2-(2,4-dichlorophenoxy) propionic acid were 1.4-72 µg/L, 5.6-280 µg/L, 2.8-140 µg/L and 3.2-160 µg/L (RSDs < 6.3%), respectively. The recoveries were between 83.3 and 106.8% with RSDs <6.7%, with detection limits ranging from 0.1 µg/L to 0.3 µg/L. The results show that Cotton@UiO-66 in PT-SPE is an effective method for monitoring phenoxy herbicides in complex samples.

Retention Modelling of Phenoxy Acid Herbicides in Reversed-Phase HPLC under Gradient Elution.

Phenoxy acid herbicides are used worldwide and are potential contaminants of drinking water. Reversed phase high-performance liquid chromatography (RP-HPLC) is commonly used to monitor phenoxy acid herbicides in water samples. RP-HPLC retention of phenoxy acids is affected by both mobile phase composition and pH, but the synergic effect of these two factors, which is also dependent on the structure and pKa of solutes, cannot be easily predicted. In this paper, to support the setup of RP-HPLC analysis of phenoxy acids under application of linear mobile phase gradients we modelled the simultaneous effect of the molecular structure and the elution conditions (pH, initial acetonitrile content in the eluent and gradient slope) on the retention of the solutes. In particular, the chromatographic conditions and the molecular descriptors collected on the analyzed compounds were used to estimate the retention factor k by Partial Least Squares (PLS) regression. Eventually, a variable selection approach, Genetic Algorithms, was used to reduce the model complexity and allow an easier interpretation. The PLS model calibrated on the retention data of 15 solutes and successively tested on three external analytes provided satisfying and reliable results.

Impact of dichlorprop on soil microbial community structure and diversity during its enantioselective biodegradation in agricultural soils.

Enantioselective biodegradation of racemic dichlorprop in two soils was investigated in the laboratory. Chiral separation of racemic dichlorprop was achieved by using HPLC with Phenomenex Lux Amylose-2. The first-order kinetic model fitted well the dissipation data of racemic dichlorprop and its pure R- and S-enantiomers. S-dichlorprop was preferentially degraded in both soils and enantioselectivity was affected by soil pH. The half-lives (DT50) of S-dichlorprop were 8.22 days in soil A and 8.06 days in soil D, while R-dichlorprop was more persistent with DT50 of 12.93 days in soil A and 12.38 days in soil D, respectively. Dichlorprop dissipated faster in soil D with lower organic matter content. In sterilized soils, neglected dissipation was observed and enantiomer fraction values remained constant, indicating that the enantioselective degradation was mainly controlled by soil microorganisms. Soil microbial community structure and diversity was assessed by Illumina MiSeq sequencing of 16S rRNA genes from dichlorprop and no dichlorprop contaminated microcosms. Compared with controls, dichlorprop application had no significant effect on microbial community structures at phylum level, but increased bacterial diversity and dichlorprop degradation related taxa in both soils. S-dichlorprop preferential degradation might be attributed to the S-enantiomer preferred degraders in the family of Sphingomonadaceae.

Modification of auxinic phenoxyalkanoic acid herbicides by the acyl acid amido synthetase GH3.15 from Arabidopsis.

Herbicide-resistance traits are the most widely used agriculture biotechnology products. Yet, to maintain their effectiveness and to mitigate selection of herbicide-resistant weeds, the discovery of new resistance traits that use different chemical modes of action is essential. In plants, the Gretchen Hagen 3 (GH3) acyl acid amido synthetases catalyze the conjugation of amino acids to jasmonate and auxin phytohormones. This reaction chemistry has not been explored as a possible approach for herbicide modification and inactivation. Here, we examined a set of Arabidopsis GH3 proteins that use the auxins indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) as substrates along with the corresponding auxinic phenoxyalkanoic acid herbicides 2,4-dichlorophenoxylacetic acid (2,4-D) and 4-(2,4-dichlorophenoxy)butyric acid (2,4-DB). The IBA-specific AtGH3.15 protein displayed high catalytic activity with 2,4-DB, which was comparable to its activity with IBA. Screening of phenoxyalkanoic and phenylalkyl acids indicated that side-chain length of alkanoic and alkyl acids is a key feature of AtGH3.15's substrate preference. The X-ray crystal structure of the AtGH3.15·2,4-DB complex revealed how the herbicide binds in the active site. In root elongation assays, Arabidopsis AtGH3.15-knockout and -overexpression lines grown in the presence of 2,4-DB exhibited hypersensitivity and tolerance, respectively, indicating that the AtGH3.15-catalyzed modification inactivates 2,4-DB. These findings suggest a potential use for AtGH3.15, and perhaps other GH3 proteins, as herbicide-modifying enzymes that employ a mode of action different from those of currently available herbicide-resistance traits.

In-situ photopolymerized polyhedral oligomeric silsesquioxane-derived monolithic capillary columns with quinidine functionality for enantioseparation by nano-liquid chromatography.

The successful fabrication of monolithic capillary columns for enantiomer separations was achieved within vinylized fused silica capillaries via fast "one-pot" photo-initiated free radical polymerization reaction. A mixture consisting of polyhedral oligomeric silsesquioxane, O-[2-(methacryloyloxy)ethylcarbamoyl]-10,11-dihydroquinidine was copolymerized in the presence of n-butanol, ethylene glycol and photo-initiator 2,2-dimethoxy-2-phenylacetophenone. The morphology of the resultant polymeric hybrid inorganic-organic material and its permeability as well as porosity can be controlled by adjusting the composition of the monomers and binary porogenic solvent. The chromatographic characteristics of the columns have been investigated. Separation factors of N-acetyl-phenylalanine (Ac-Phe) and dichlorprop dropped with decrease of chiral functional monomer. Permeability was better when the macroporogen ethyleneglycol was present at higher concentrations during the polymerization. In general, the chiral compounds were well separated (dichlorprop: α = 1.53, Rs up to 4.14; Ac-Phe: α = 1.36, Rs up to 2.69) by nano-HPLC with an optimized enantioselective monolithic capillary column which can be prepared within a few minutes.

Enantioselective Phytotoxic Disturbances of Fatty Acids in Arabidopsis thaliana by Dichlorprop.

Plant fatty acids have indispensable physiological functions and nutritional value. However, the overuse of herbicides may cause phytotoxic disturbances of fatty acids in nontarget plants while spraying for weeds. Evidence has shown that the herbicide dichlorprop can inhibit the activity of acetyl-CoA carboxylase (ACCase), a key enzyme involved in fatty acid synthesis. However, the enantioselective phytotoxic effects of dichlorprop enantiomers ((R)-dichlorprop and (S)-dichlorprop) on fatty acids and their related mechanisms remain unclear. To solve this issue, the enantioselective phytotoxicity of dichlorprop in the model plant species Arabidopsis thaliana (A. thaliana) with a focus on fatty acids was evaluated for the first time. The results indicated a significant difference in enantioselectivity and that exposure to (R)-dichlorprop can cause marked fatty acid disturbances in nontarget plant species. Specifically, (R)-dichlorprop decreased the content of three fatty acids by more than 50% by inhibiting the activity of ACCase. In addition, increased malondialdehyde (MDA) and lipid hydroperoxides (LOOHs) contents and membrane permeability reflected herbicide-induced lipid peroxidation, which decreased the unsaturation of fatty acids in membranes and further influenced membrane composition and function. Moreover, an increased level of glutathione peroxidase (GPX) and cytochrome P450 (CYP450) reflected a plant stress-induced response. To summarize, fatty acids represent a new perspective for evaluating the toxicity of chiral pesticides, contributing to a better understanding of the enantioselective phytotoxicity and mechanisms of dichlorprop, and providing evidence for herbicide security and risk assessments.

Methanotrophic contribution to biodegradation of phenoxy acids in cultures enriched from a groundwater-fed rapid sand filter.

Drinking water supply is in many parts of the world based on groundwater. Groundwater often contains methane, which can be oxidized by methanotrophs upon aeration. Sand from rapid sand filters fed with methane-rich groundwater can remove some pesticides (Hedegaard and Albrechtsen in Water Res 48:71-81, 2014). We enriched methanotrophs from filter sand and investigated whether they could drive the degradation of various pesticides. To enrich for methanotrophs, we designed and operated four laboratory-scale, continuously methane-fed column reactors, inoculated with filter sand and one control column fed with tap water. When enrichments were obtained, methane was continuously supplied to three reactors, while the fourth was starved for methane for 1 week, and the reactors were spiked with ten pesticides at groundwater-relevant concentrations (2.1-6.6 µg/L). Removal for most pesticides was not detected at the investigated contact time (1.37 min). However, the degradation of phenoxy acids was observed in the methanotrophic column reactor starved for methane, while it was not detected in the control column indicating the importance of methanotrophs. Phenoxy acid removal, using dichlorprop as a model compound, was further investigated in batch experiments with methanotrophic biomass collected from the enrichment reactors. Phenoxy acid removal (expressed per gram of matrix sand) was substantially improved in the methanotrophic enrichment compared to parent filter sand. The presence of methane did not clearly impact dichlorprop removal but did impact mineralization. We suggest that other heterotrophs are responsible for the first step in dichlorprop degradation, while the subsequent steps including ring-hydroxylation are driven by methanotrophs.

Monodisperse cobalt(II) based metal-organic coordination polymer beads as a sorbent for solid-phase extraction of chlorophenoxy acid herbicides prior to their quantitation by HPLC.

Metal-organic coordination polymer beads (MOCBs) are described for use as a sorbent for solid-phase extraction of chlorophenoxy herbides. By applying regulation of Co(II) ions, micro-sized monodisperse MOCBs were obtained through the microwave heating. The MOCBs-based method displays excellent extraction efficiency towards chlorophenoxy herbicides, specifically of 2-chlorophenoxyacetic acid, 4-chlorophenoxyacetic acid, 4-chloromethylphenoxyacetic acid, 2,4-dichlorophenoxyacetic acid, 2,4,5-trichlorophenoxyacetic acid and 2-(2,4-dichlorophenoxy)propionic acid. Following extraction, the herbicides were eluted with 8% formic acid in methanol and quantified by HPLC. The method, when applied to analyze spiked cereals, exhibits a wide linear range (from 0.6 to 1000 ng g-1) and low limits of quantification (ranging from 0.10 to 0.25 ng g-1). For a single column, the inter-day and intra-day precisions, expressed as the relative standard deviation are in the range of 2.5-6.8%. The batch-to-batch reproducibility (for n = 3) is <4.6%. For spiked cereal samples, relative recoveries are very good (90.3-102.3%, for n = 4). The extraction efficiency of MOCBs remains unchanged after reusing for 40 times. Graphical abstractSchematic presentation of Co(II)-doped metal-organic coordination polymer beads (Co(II)@MOCB) using for solid-phase extraction (SPE).

Click Chemistry-Assisted Synthesis of a ß-d-Galactose-Targeted SiO2@RC Shell-Core Structure as a Nanoplatform for Metal-Based Complex Delivery.

A facile reversed-phase microemulsion method was used to synthesize shell-core nanospheres of SiO2@RCs (SiO2-encapsuled rare-earth metal complexes). ß-d-Galactose was then grafted onto the surfaces of the nanospheres through the copper(I)-catalyzed azide-alkyne cycloaddition click reaction for targeted delivery. The chemical characteristics and surface profiles of the nanocarriers were investigated by Fourier transform infrared spectroscopy, dynamic light scattering, transmission electron microscopy, and scanning electron microscopy. A high-efficiency microwave synthesis method was applied to prepare five complex cores by the reaction of different rare-earth metal salts with two isomeric ligands, o-CPA (2-chlorophenoxyacetic acid) and m-CPA (3-chlorophenoxyacetic acid). The crystal structures of the five synthesized RC cores were confirmed through X-ray diffraction, which revealed the formulas of five RCs, [Dy( o-CPA)3(H2O)]·H2O RC1, [Ho( o-CPA)3(H2O)]·H2O RC2, 2[Er( m-CPA)3(H2O)]·3H2O RC3, 2[Gd( m-CPA)3(H2O)]·3H2O RC4, and [Ce2( m-CPA)6(H2O)3]·2H2O RC5. An in vitro cell study revealed that all RCs exhibited certain anticancer activities. RC2, in particular, showed the strongest cytotoxicity against HepG2 cells. The enhanced cell permeability and drug retention considerably improved the cytotoxicity of all SiO2@RC2-gal relative to that of RC2. The selective uptake of the ß-d-galactose-conjugated nanospheres by HepG2 cells through mechanisms mediated by cell surface receptors resulted in fewer side effects on extrahepatic tissues. Our contribution provides a novel design concept of a target SiO2@RCs-gal nanocarrier for delivering affordable antitumor complexes in cancer therapy.

Rapid monitoring of plant growth regulators in bean sprouts via automated on-line polymeric monolith solid-phase extraction coupled with liquid chromatography tandem mass spectrometry.

An automated on-line solid-phase extraction (SPE) following liquid chromatography tandem mass spectrometry was established for the fast determination of plant growth regulator residues in soybean sprout and mung bean sprout. The crude extracted specimens were directly purified on a poly (2-(dimethylamino) ethyl methacrylate-co-ethylene dimethacrylate) monolithic column which was well-defined as the on-line SPE adsorbent. Under the optimized conditions, the developed method gave the linear range of 0.3-50 ng/mL for gibberellin and 2,4-dichlorophenoxyacetic acid, 0.2-50 ng/mL for 4-chlorophenoxyacetic acid, and 0.5-50 ng/mL for 1-naphthaleneacetic acid (r ≥ 0.998). The detection limits (S/N = 3) ranged from 1.0 to 2.5 µg/kg and the recoveries for spiked soybean sprout samples were in the range of 75.0-93.3%. Besides, the total time for one analysis was 16 min. The reusability of the monolith was up to 600 extractions. The proposed process facilitated fully automated SPE and accurate determination in one step with rapidity, simplicity, and reliability. Graphical abstract ᅟ.

CATALASE2 functions for seedling postgerminative growth by scavenging H2 O2 and stimulating ACX2/3 activity in Arabidopsis.

Increased fatty acid ß-oxidation is essential for early postgerminative growth in seedlings, but high levels of H2 O2 produced by ß-oxidation can induce oxidative stress. Whether and how catalase (CAT) functions in fine-tuning H2 O2 homeostasis during seedling growth remain unclear. Here, we report that CAT2 functions in early seedling growth. Compared to the wild type, the cat2-1 mutant, with elevated H2 O2 levels, exhibited reduced root elongation on sucrose (Suc)-free medium, mimicking soils without exogenous sugar supply. Treatment with the H2 O2 scavenger potassium iodide rescued the mutant phenotype of cat2-1. In contrast to the wild type, the cat2-1 mutant was insensitive to the CAT inhibitor 3-amino-1,2,4-triazole in terms of root elongation when grown on Suc-free medium, suggesting that CAT2 modulates early seedling growth by altering H2 O2 accumulation. Furthermore, like cat2-1, the acyl-CoA oxidase (ACX) double mutant acx2-1 acx3-6 showed repressed root elongation, suggesting that CAT2 functions in early seedling growth by regulating ACX activity, as this activity was inhibited in cat2-1. Indeed, decreased ACX activity and short root of cat2-1 seedlings grown on Suc-free medium were rescued by overexpressing ACX3. Together, these findings suggest that CAT2 functions in early seedling growth by scavenging H2 O2 and stimulating ACX2/3 activity.

Enantioselective Phytotoxicity of Dichlorprop to Arabidopsis thaliana: The Effect of Cytochrome P450 Enzymes and the Role of Fe.

The ecotoxicology effects of chiral herbicides have long been recognized and have drawn increasing attention. The toxic mechanisms of herbicides in plants are involved in production of reactive oxygen species (ROS) and cause damage to target enzymes, but the relationship between these two factors in the enantioselectivity of chiral herbicides has rarely been investigated. Furthermore, even though cytochromes P450 enzymes (CYP450s) have been related to the phytotoxicity of herbicides, their roles in the enantioselectivity of chiral herbicides have yet to be explored. To solve this puzzle, the CYP450s suicide inhibitor 1-aminobenzotriazole (ABT) was added to an exposure system made from dichlorprop (DCPP) enantiomers in the model plant Arabidopsis thaliana. The results indicated that different phytotoxicities of DCPP enantiomers by causing oxidative stress and acetyl-CoA carboxylase (ACCase) damage were observed in the presence and the absence of ABT. The addition of ABT decreased the toxicity of (R)-DCPP but was not significantly affected that of (S)-DCPP, resulting in smaller differences between enantiomers. Furthermore, profound differences were also observed in Fe uptake and distribution, exhibiting different distribution patterns in A. thaliana leaves exposed to DCPP and ABT, which helped bridge the relationship between ROS production and target enzyme ACCase damage through the function of CYP450s. These results offer an opportunity for a more-comprehensive understanding of chiral herbicide action mechanism and provide basic evidence for risk assessments of chiral herbicides in the environment.

[Determination of 4 kinds of plant growth regulator in bean sprout by solid phase extraction column coupled with ultra-high performance liquid chromatography].

OBJECTIVE: A method for the determination of 6-benzylaminopurine( 6-BAP), isopentennyladenine( z-IP), 4-fluorophenoxyacetic acid( 4-FPA), 4-chlorophenoxyacetic acid( 4-CPA) in bean sprout was developed using solid phase extraction column with ultra-high performance liquid chromatography. METHODS: The sample was extracted by acetonitrile,dehydrated by salt,then centrifugation,and purified by PXC/PWA solid phase extraction column. The chromatographic analysis was carried out on C18 chromatographic column( 100 mm ×2. 1 mm,1. 8 µm),acetonitrile and sodium dihydrogen phosphate for gradient elution,diode array detector for detection,and quantified with external standard method. RESULTS: The calibration curves showed good linearity in the range of 0. 25-25 µg/mL( 6-BAP and z-IP) and 0. 50-50 µg/mL( 4-FPA and 4-CPA) with correlation coefficients greater than 0. 999. Three levels spiked recoveries were carried out using blank bean sprout extraction as substrate,the recoveries ranged from70. 0% to 96. 4%,and the relative standard deviations( RSDs) ranged from 2. 84% to12. 10%( n = 6). The qualitative limits of detections were 0. 0082-0. 075 mg/kg and the quantitative limits were 0. 027-0. 25 mg/kg for the 4 PGRs. CONCLUSION: The method is simple and easy to operate using solid phase extraction column coupled,simultaneous determination of 4 PGRs by ultra-high performance liquid chromatography,can ensure the corresponding accuracy,sensitivity and precision.

Establishment of Bacterial Herbicide Degraders in a Rapid Sand Filter for Bioremediation of Phenoxypropionate-Polluted Groundwater.

In this study, we investigated the establishment of natural bacterial degraders in a sand filter treating groundwater contaminated with the phenoxypropionate herbicides (RS)-2-(4-chloro-2-methylphenoxy)propanoic acid (MCPP) and (RS)-2-(2,4-dichlorophenoxy)propanoic acid (DCPP) and the associated impurity/catabolite 4-chlorophenoxypropanoic acid (4-CPP). A pilot facility was set up in a contaminated landfill site. Anaerobic groundwater was pumped up and passed through an aeration basin and subsequently through a rapid sand filter, which is characterized by a short residence time of the water in the filter. For 3 months, the degradation of DCPP, MCPP, and 4-CPP in the sand filter increased to 15 to 30% of the inlet concentration. A significant selection for natural bacterial herbicide degraders also occurred in the sand filter. Using a most-probable-number (MPN) method, we found a steady increase in the number of culturable phenoxypropionate degraders, reaching approximately 5 × 10(5) degraders per g sand by the end of the study. Using a quantitative PCR targeting the two phenoxypropionate degradation genes, rdpA and sdpA, encoding stereospecific dioxygenases, a parallel increase was observed, but with the gene copy numbers being about 2 to 3 log units higher than the MPN. In general, the sdpA gene was more abundant than the rdpA gene, and the establishment of a significant population of bacteria harboring sdpA occurred faster than the establishment of an rdpA gene-carrying population. The identities of the specific herbicide degraders in the sand filter were assessed by Illumina MiSeq sequencing of 16S rRNA genes from sand filter samples and from selected MPN plate wells. We propose a list of potential degrader bacteria involved in herbicide degradation, including representatives belonging to the Comamonadaceae and Sphingomonadales.

Microbial degradation of 2,4-dichlorophenoxyacetic acid: Insight into the enzymes and catabolic genes involved, their regulation and biotechnological implications.

A considerable progress has been made to understand the mechanisms of biodegradation of 2,4-dichlorophenoxyacetic acid (2,4-D). 2,4-D biodegradation pathway has been elucidated in many microorganisms including Cupriavidus necator JMP134 (previously known as Wautersia eutropha, Ralstonia eutropha and Alcaligenes eutrophus) and Pseudomonas strains. It generally involves the side chain removal of 2,4-D by α-ketoglutarate-dependent 2,4-D dioxygenase (tfdA) to form 2,4-dichlorophenol (2,4-DCP); hydroxylation of 2,4-DCP by 2,4-DCP hydroxylase (tfdB) to form dichlorocatechol; ortho or meta cleavage of dichlorocatechol by chlorocatechol 1,2-dioxygenase (tfdC) to form 2,4-dichloro-cis,cis-muconate; conversion of 2,4-dichloro-cis,cis-muconate to 2-chlorodienelactone by chloromuconate cycloisomerase (tfdD); conversion of 2-chlorodienelactone to 2-chloromaleylacetate by chlorodienelactone hydrolase (tfdE) and, finally, conversion of 2-chloromaleylacetate to 3-oxoadepate via maleylacetate by chloromaleylacetate reductase and maleylacetate reductase (tfdF), respectively, which is funnelled to the tricarboxylic acid cycle. The latest review on microbial breakdown of 2,4-D, other halogenated aromatic pesticides, and related compounds was compiled by Haggblom, however, a considerable progress has been made in this area of research since then. Thus, this review focuses on the recent advancement on 2,4-D biodegradation, the enzymes, and genes involved and their biotechlogical implications.

Predicting the Sorption of Aromatic Acids to Noncarbonized and Carbonized Sorbents.

Approaches based on the octanol-water partition coefficient are commonly used to describe sorption of neutral organic compounds in environmental systems, but they are not suitable for organic acids, which can dissociate to form anions. We here investigate the applicability of an alternative approach based on the pH-dependent distribution ratio (DOW) to describe sorption of aromatic acids to sorbents representing different degrees of carbonization. Sorption isotherms for four structurally similar acids ((2,4-dichlorophenoxy)acetic acid (2,4-D), 4-chloro-2-15 methylphenoxy)acetic acid (MCPA), 4-(2,4-dichlorophenoxy)butanoic16 acid (2,4-DB), and 5-chloro-2-(2,4-dichlorophenoxy)phenol (triclosan)) were measured for 15 sorbents: fresh and carbonized wood shavings, pig manure, sewage sludge, carbon nanotubes, and activated carbon. Dissociation greatly affected the sorption of all acids. Sorption coefficients measured in the high pH range indicated that sorption of the anions ranged over several orders of magnitude and should not be neglected. Sorption trends for all sorbates and carbonized sorbents could be very well described by a single regression equation that included DOW of the sorbate and the specific surface area of the sorbent (R(2) > 0.89).

[Determination of 6 kinds of plant growth regulator in bean sprout by ultra high performance liquid chromatography-tandem mass spectrometry].

OBJECTIVE: A method for the simultaneous determination of 6 plant growth regulator (PGR) residues in bean sprout was developed by ultra high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). 6-Benzylaminopurine, isopentennyladenine, 4-chlorophenoxyacetic acid, 4-fluorophenoxyacetic acid, indole-3- acetic acid and indole-3-butyric acid were concerned. METHODS: Bean sprout samples were extracted by acetonitrile and QuEChERS extraction kit, purified by C18 powers. After centrifugation, the sample liquids was diluted 10 times by ultrapure water. The chromatographic analysis was carried out on an waters acquity UPLC BEH C18 column( 100 mm x 2.1 mm, 1.7 microm). The analyzer confirmed and quantified by mass spectrum of triple quadrupole in the multiple reaction monitoring (MRM) mode and quantified by matrix-matched external standard method. RESULTS: The calibration curves showed good linearity in each range with correlation coefficients greater than 0.998. 3 levels spiked recoveries were carried out using blank bean sprout extraction as substrate, the recoveries ranged from 84.2% to 107.5%, the relative standard deviations (RSDs) ranged from 3.08% to 12.71%. The qualitative limits of detections (S/N = 3) were 0.03-3.0 microg/kg and the quantitative limits(S/N = 10) were 0.1-10.0 microg/kg for the 6 PGRs. CONSLUSION: The method is simple and easy to operate, with less organic reagent, high sensitivity and good stability. It is suitable for the detection of 6 kinds of plant growth regulators in bean sprouts.

Systematic phenotypic screen of Arabidopsis peroxisomal mutants identifies proteins involved in ß-oxidation.

Peroxisomes are highly dynamic and multifunctional organelles essential to development. Plant peroxisomes accommodate a multitude of metabolic reactions, many of which are related to the ß-oxidation of fatty acids or fatty acid-related metabolites. Recently, several dozens of novel peroxisomal proteins have been identified from Arabidopsis (Arabidopsis thaliana) through in silico and experimental proteomic analyses followed by in vivo protein targeting validations. To determine the functions of these proteins, we interrogated their transfer DNA insertion mutants with a series of physiological, cytological, and biochemical assays to reveal peroxisomal deficiencies. Sugar dependence and 2,4-dichlorophenoxybutyric acid and 12-oxo-phytodienoic acid response assays uncovered statistically significant phenotypes in ß-oxidation-related processes in mutants for 20 of 27 genes tested. Additional investigations uncovered a subset of these mutants with abnormal seed germination, accumulation of oil bodies, and delayed degradation of long-chain fatty acids during early seedling development. Mutants for seven genes exhibited deficiencies in multiple assays, strongly suggesting the involvement of their gene products in peroxisomal ß-oxidation and initial seedling growth. Proteins identified included isoforms of enzymes related to ß-oxidation, such as acyl-CoA thioesterase2, acyl-activating enzyme isoform1, and acyl-activating enzyme isoform5, and proteins with functions previously unknown to be associated with ß-oxidation, such as Indigoidine synthase A, Senescence-associated protein/B12D-related protein1, Betaine aldehyde dehydrogenase, and Unknown protein5. This multipronged phenotypic screen allowed us to reveal ß-oxidation proteins that have not been discovered by single assay-based mutant screens and enabled the functional dissection of different isoforms of multigene families involved in ß-oxidation.