Preparation of anti-2,4-dichlorophenol and 2,4-dichlorophenoxyacetic acid monoclonal antibodies

The ratios of hapten and bovine serum albumin (BSA) in an antigen conjugate were determined by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Hybridomas secreting monoclonal antibodies against 2,4-dichlorophenoxyacetic acid (2,4-D) were produced by fusing 2,4-D-BSA conjugate-immunized splenocytes with a HAT-sensitive mouse myeloma cell line, P3-X63-Ag8-653. A substantial cross-reaction was observed for 2,4-dichlorophenol (2,4-DP) when compared with that observed for 2,4-D. The full measurement range for this assay is 0.2–3 μg ml⁻¹ for 2,4-DP. On the other hand, the range for 2,4-D is between 1 and 20 μg ml⁻¹. This revised version was published online in July 2006 with corrections to the Cover Date.     

Electrokinetic movement and biodegradation of 2,4-dichlorophenoxyacetic acid in silt soil

The coupling of electrokinetic movement of an organic contaminant, 2,4-dichlorophenoxyacetic acid (2,4-D), through soil and its biodegradation in situ has been demonstrated. In a first experiment, the direction and rate of movement of 2,4-D were determined using homogeneously contaminated soil (864 mg 2,4-D/kg dry weight soil) compacted into six individual compartments, 6 cm long, 3 cm wide, and 4 cm deep. Each compartment was bordered by a carbon felt anode and a stainless steel cathode. The application of a current density of 3.72 A/m(2) led to migration of 2,4-D towards the anode at a rate of approximately 4 cm/day. In a second experiment, electrokinetic movement and biodegradation were combined in situ. Sterilized silt soil contaminated with ring-labeled 14C-2,4-D (811 mg 2,4-D/kg dry weight soil) was compacted into a single soil compartment, 22 cm long, 7 cm wide, and 4 cm deep, in a 4.5 cm region adjacent to the cathode. The remainder of the compartment was filled with sterilized soil (to a total weight of 1,015 g). Burkholderia spp. RASC c2 (1.88 x 10(11) cells), a tetracycline-resistant bacterium with chromosomally encoded degradative genes for 2,4-D, was inoculated into the soil at a position 14-16 cm from the cathode. The reactor was placed within a sealed perspex box, with a constant air flow connected to sodium hydroxide traps. Under an applied current density of 0.89 A/m(2), the pollutant moved towards the bacteria. As it reached the inoculated region, its concentration decreased in the soil and 14CO2 was recovered in the traps. At the end of the experiment, 87.1% of radiolabel had been removed from the soil, 5.8% of which was recovered as 14CO2. A third, control, experiment showed a significant contrast in the absence of an electric current, where a slow rate of diffusion controlled the movement of both 2,4-D and bacteria in the soil and biodegradation occurred at the interface between the diffusing fronts.     

Metabolism of 2,4-dichlorophenoxyacetic acid, 4-chloro-2-methylphenoxyacetic acid and 2-methylphenoxyacetic acid by Alcaligenes eutrophus JMP 134

Of eleven substituted phenoxyacetic acids tested, only three (2,4-dichloro-, 4-chloro-2-methyl- and 2-methylphenoxyacetic acid) served as growth substrates for Alcaligenes eutrophus JMP 134. Whereas only one enzyme seems to be responsible for the initial cleavage of the ether bond, there was evidence for the presence of three different phenol hydroxylases in this strain. 3,5-Dichlorocatechol and 5-chloro-3-methylcatechol, metabolites of the degradation of 2,4-dichlorophenoxyacetic acid and 4-chloro-2-methylphenoxyacetic acid, respectively, were exclusively metabolized via the ortho-cleavage pathway. 2-Methylphenoxyacetic acid-grown cells showed simultaneous induction of meta- and ortho-cleavage enzymes. Two catechol 1,2-dioxygenases responsible for ortho-cleavage of the intermediate catechols were partially purified and characterized. One of these enzymes converted 3,5-dichlorocatechol considerably faster than catechol or 3-chlorocatechol. A new enzyme for the cycloisomerisation of muconates was found, which exhibited high activity against the ring-cleavage products of 3,5-dichlorocatechol and 4-chlorocatechol, but low activities against 2-chloromuconate and muconate.Non-standard abbreviations MCPA 4-chloro-2-methylphenoxyacetic acid - 2MPA 2-methylphenoxyacetic acid - PA phenoxyacetic acid     

Mineralization of 2,4-dichlorophenoxyacetic acid in soil simultaneously enriched with saccharides

Detoxication of 2,4-dichlorophenoxyacetic acid (2,4-D) in samples of chernozem soil was determined by a biological test and the time course of production of¹⁴CO₂ a product of microbial degradation of 2-¹⁴C-2,4-D, was measured during 38-d incubation at 28°C in the dark. Enrichment of the soil with glucose (1000 ppm), two exocellular bacterial glucan and glucomannan polysaccharides (750 ppm), or a mixture of glucose with (NH₄)₂SO₄ (C:N=5∶1) brought about acceleration of both detoxication and mineralization of 2,4-D (50 ppm) added simultaneously with the saccharides. Mineralization of the saccharides always preceded the degradation of the herbicide. The lag phase of 2,4-D mineralization, did not exceed 3 d. In samples with saccharides the doubling time of the mineralization activity in the exponential phase of the process was substantially shortened and the mineralization of 2,4-D was accelerated even when the soil was inoculated with a suspension of soil in which microbial 2,4-D decomposers had accumulated. The extent, of mineralization was not affected by the presence of saccharides (about 1/3 of the introduced radioactive carbon was transformed into¹⁴CO₂). All saccharides had a similar effect which reflected an increase in the overall bacterial count and in the relative abundance of bacterial 2,4-D decomposers. The role of other mechanisms such as co-metabolism in the stimulation of the degradation process is discussed.     

Polymerase chain reaction and gene probe detection of the 2,4-dichlorophenoxyacetic acid degradation plasmid, pJP4.

Specific and sensitive detection of indigenous and introduced degradative organisms is an essential prerequisite to their use in remediation of toxic waste and soil systems. Procedures were employed for the use of polymerase chain reaction and gene probes for sensitive detection of the 2,4-dichlorophenoxyacetic-acid-degrading bacterium, Alcaligenes eutrophus JMP134(pJP4). Two 20-mer oligonucleotide primers were identified for amplification of a 205-bp region of the tfdB gene of pJP4, and optimum conditions for amplification were determined. Both the polymerase chain reaction amplification process and hybridization with the 5'-end-labelled probe were found to be specific to organisms containing plasmid pJP4 or its derivative pRO103. Detection limits were determined for the template supplied either as bacterial cells or purified plasmid DNA. The detection was sensitive up to an initial inoculum of 3,000 CFU or 156 pg of total plasmid DNA. However, when the amplified product was transferred to a nylon membrane and hybridized with the 5'-end-labelled probe, the detection sensitivity increased to 300 CFU or 15.6 pg of plasmid DNA. This sensitive detection method is more specific than use of traditional indicator media (M. A. Loos, Can. J. Microbiol. 21:104-107, 1975). An oligonucleotide (20 bases) complementary to a sequence internal to the 205-bp region was synthesized and utilized as a probe to confirm the specificity of the detection.     

Polymerase chain reaction and gene probe detection of the 2,4-dichlorophenoxyacetic acid degradation plasmid, pJP4.

Specific and sensitive detection of indigenous and introduced degradative organisms is an essential prerequisite to their use in remediation of toxic waste and soil systems. Procedures were employed for the use of polymerase chain reaction and gene probes for sensitive detection of the 2,4-dichlorophenoxyacetic-acid-degrading bacterium, Alcaligenes eutrophus JMP134(pJP4). Two 20-mer oligonucleotide primers were identified for amplification of a 205-bp region of the tfdB gene of pJP4, and optimum conditions for amplification were determined. Both the polymerase chain reaction amplification process and hybridization with the 5'-end-labelled probe were found to be specific to organisms containing plasmid pJP4 or its derivative pRO103. Detection limits were determined for the template supplied either as bacterial cells or purified plasmid DNA. The detection was sensitive up to an initial inoculum of 3,000 CFU or 156 pg of total plasmid DNA. However, when the amplified product was transferred to a nylon membrane and hybridized with the 5'-end-labelled probe, the detection sensitivity increased to 300 CFU or 15.6 pg of plasmid DNA. This sensitive detection method is more specific than use of traditional indicator media (M. A. Loos, Can. J. Microbiol. 21:104-107, 1975). An oligonucleotide (20 bases) complementary to a sequence internal to the 205-bp region was synthesized and utilized as a probe to confirm the specificity of the detection.     

Mineralization of 2,4-dichlorophenoxyacetic acid in soil previously enriched with organic substrates

Samples of chernozem soil were enriched with vanillic acid, protocatechuic acid glucose, a mixture of glucose and (NH₄)₂SO₄ (C∶N = 5∶1), ethanol and 2,4-dichlorophenoxyacetic acid (2,4-D). After a 6-d (with 2,4-D 35-d) incubation during which primary oxidation of the introduced substrates occurred, the soil was supplied with a solution of 2-¹⁴C-2,4-D (50ppm; 6.7kBq) and production of¹⁴CO₂ (product of microbial degradation of 2,4-D) was measured. Previously enriched samples exhibited a higher degradation rate; both the lag phase and doubling time of mineralization activity in the exponential phase of the process were markedly higher. This reflected an overall proliferation of bacteria and the increased relative proportion of bacterial strains capable of mineralizing 2,4-D in enriched samples. The stimulation of 2,4-D degradation may involve specific adaptation and selection mechanisms (as in the case with samples previously enriched with 2,4-D or its structural analogues—aromatic monomers, ethanol) as well as nonspecific mechanisms. The extent of mineralization of 2,4-D was not affected by soil pretreatment, about 1/3 of introduced radioactive carbon being invariably transformed to¹⁴CO₂.     

Enhancement of 2,4-dichlorophenoxyacetic acid (2,4-D) degradation in soil by dissemination of catabolic plasmids

Few studies have been done to evaluate the transfer of catabolic plasmids from an introduced donor strain to indigenous microbial populations as a means to remediate contaminated soils. In this work we determined the effect of the conjugative transfer of two 2,4-D degradative plasmids to indigenous soil bacterial populations on the rate of 2,4-D degradation in soil. We also assessed the influence of the presence of 2,4-D on the number of transconjugants formed. The two plasmids used, pEMT1k and pEMT3k, encode 2,4-D degradative genes (tfd) that differ in DNA sequence as well as gene organisation, and confer different growth rates to Ralstonia eutropha JMP228 when grown with 2,4-D as a sole carbon source. In an agricultural soil (Ardoyen) treated with 2,4-D (100 ppm) there were ca. 10⁷CFU of transconjugants per gram bearing pEMT1k as well as a high number of pEMT3k bearing transconjugants (ca. 10⁶ CFU/g). In this soil the formation of a high number of 2,4-D degrading transconjugants resulted in faster degradation of 2,4-D as compared to the uninoculated control soil. In contrast, only transconjugants with pEMT1k were detected (at a level of ca. 10³ CFU/g soil) in the untreated Ardoyen soil. High numbers of transconjugants that carried pEMT1k were also found in a second experiment done using forest soil (Lembeke) treated with 100 ppm 2,4-D. However, unlike in the Ardoyen soil, no transconjugants with pEMT3k were detected and the transfer of plasmid pEMT1k to indigenous bacteria did not result in a higher rate of decrease of 2,4-D. This may be because 2,4-D was readily metabolised by indigenous bacteria in this soil. The results indicate that bioaugmentation with catabolic plasmids may be a viable means to enhance the bioremediation of soils which lack an adequate intrinsic ability to degrade a given xenobiotic.     

2,4-二氯苯酚在土壤与河流底泥中降解动力学

以南京化学工业园内四柳河沿岸土壤与河流底泥为研究对象,通过土壤灭菌、温度与污染物初始浓度调控,研究了2,4-二氯苯酚在土壤与河流底泥中降解动力学及其影响因子。结果表明:微生物对2,4-二氯苯酚降解起主导作用,在45d内,非灭菌土壤和河流底泥的降解率分别是灭菌条件下的1.5～3倍、1.4～2.8倍,土壤和河流底泥中的2,4-二氯苯酚微生物降解量分别为0.128～0.599和0.113～0.718mg·kg-1,非灭菌处理半衰期时间短于灭菌处理;(10±1)℃～(30±1)℃范围内,随着温度的增高,2,4-二氯苯酚降解加快,在(30±1)℃土壤与河流底泥中残留量最小,分别为0.305和0.203mg·kg-1,半衰期也最短;土壤与河流底泥中的2,4-二氯苯酚均在其浓度为0.5mg·kg-1时降解最快,随着初始浓度的增加,2,4-二氯苯酚降解速度呈现递减趋势,半衰期增长。     

Introduction of 2,4-dichlorophenoxyacetic acid into soil with solvents and resulting implications for bioavailability to microorganisms

Slow equilibration of introduced chemicals through tortuous pore space limits uniform substrate distribution in soil biodegradation studies. The necessity of introducing poorly soluble xenobiotics via organic solvents, the volume of which is minimized to limit toxicity, likely also affects xenobiotic distribution. Our objective was to investigate relative effects of carrier solvent choice and volume on xenobiotic distribution, apparent solvent toxicity, and soil degradation of 2,4-dichlorophenoxy acetic acid. Incubations using four carrier solvents ranging in properties showed that the fraction of 2,4-D mineralized was a hyperbolic function of solvent volume used (0.02–10 μl g⁻¹), attributed to compensating effects of herbicide bioavailability and solvent toxicity. Substrate concentration influenced mineralization of herbicide introduced with organic carriers, but not water. Fraction of material readily desorbed increased when water was the carrier. Results suggest that solvent toxicity should be balanced with uniformity of substrate distribution when using organic carriers in soils. Substrate bioavailability is a ubiquitous issue in terrestrial microbiology research, thus limitations observed herein broadly apply to microbiology questions about introduced substances in soil. We advocate the development of tools to characterize variable conditions among soil compartments, estimates of substrate bioavailability, and linkage of this information to microbial data.     

Effect of glucose on the amount of bacteria mineralizing 2,4-dichlorophenoxyacetic acid in soil

Plate numbers of bacteria and relative incidence of strains capable of mineralization of 2,4-dichlorophenoxyacetic acid (2,4-D) in chernozem samples incubated for 14 d with the herbicide (50 ppm) in the presence or absence of glucose (1000 ppm) were compared. Whereas the total number of bacteria increased 1.2-fold in the variant with 2,4-D and 2.4-fold in the variant with glucose and the herbicide, the number of 2,4-D-mineralizing bacteria increased 12.1-fold and 34.2-fold, respectively. In a collection of 96 isolates of soil bacteria substantially more strains capable of degradation of 2,4-D in the presence of glucose were detected as compared with the variant without it, indicating that processes of cometabolic type are involved during the degradation of this herbicide in the soil.     

Comparison of 2,4-dichlorophenoxyacetic acid degradation and plasmid transfer in soil resulting from bioaugmentation with two different pJP4 donors

A pilot field study was conducted to assess the impact of bioaugmentation with two plasmid pJP4-bearing microorganisms: the natural host, Ralstonia eutropha JMP134, and a laboratory-generated strain amenable to donor counterselection, Escherichia coli D11. The R. eutropha strain contained chromosomal genes necessary for mineralization of 2,4-dichlorophenoxyacetic acid (2,4-D), while the E. coli strain did not. The soil system was contaminated with 2,4-D alone or was cocontaminated with 2,4-D and Cd. Plasmid transfer to indigenous populations, plasmid persistence in soil, and degradation of 2,4-D were monitored over a 63-day period in the bioreactors. To assess the impact of contaminant reexposure, aliquots of bioreactor soil were reamended with additional 2,4-D. Both introduced donors remained culturable and transferred plasmid pJP4 to indigenous recipients, although to different extents. Isolated transconjugants were members of the Burkholderia and Ralstonia genera, suggesting multiple, if not successive, plasmid transfers. Upon a second exposure to 2,4-D, enhanced degradation was observed for all treatments, suggesting microbial adaptation to 2,4-D. Upon reexposure, degradation was most rapid for the E. coli D11-inoculated treatments. Cd did not significantly impact 2,4-D degradation or transconjugant formation. This study demonstrated that the choice of donor microorganism might be a key factor to consider for bioaugmentation efforts. In addition, the establishment of an array of stable indigenous plasmid hosts at sites with potential for reexposure or long-term contamination may be particularly useful.     

Investigation of highly sensitive piezoelectric immunosensors for 2,4-dichlorophenoxyacetic acid

The improved highly sensitive piezoelectric immunosensor has been developed and evaluated using a model interaction of antibody with the model hapten-herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). For immobilization of 2,4-D, the self-assembled layers of cystamine, 4-aminothiophenol or 3,3'-dithio-bis(propionic acid N-hydroxysuccinimide ester) were formed on smooth and rough crystals coated with gold or silver electrodes. The immunochemical interactions performed well in all cases, the aminothiophenol on gold was chosen as the optimum with regard to regeneration of immunosensing surfaces. The kinetics of interaction of surface-bound 2,4-D with free antibody provided significantly higher kinetic parameters (kinetic association rate constant) when using optically smooth crystals compared to common rough crystal. Therefore, the smooth crystal should be preferred for future kinetic studies. The competitive assay of the herbicide 2,4-D achieved a limit of detection of 10 ng/l using the monoclonal anti-2,4-D antibody F6C10. Finally, a direct assay format has been evaluated using a thicker layer of glutaraldehyde-crosslinked antibody on the sensing surface. The direct binding of a small herbicide molecule was followed in real time. The detected concentration of 2,4-D (5 microg/l) was low enough for future direct monitoring of this herbicide in water.     

Rhizodeposition and the enhanced mineralization of 2,4-dichlorophenoxyacetic acid in soil from the Trifolium pratense rhizosphere

Enhanced biodegradation of organic xenobiotic compounds in the rhizosphere is frequently recorded although the specific mechanisms are poorly understood. We have shown that the mineralization of 2,4-dichlorophenoxyacetic acid (2,4-D) is enhanced in soil collected from the rhizosphere of Trifolium pratense[e.g. maximum mineralization rate=7.9 days-1 and time at maximum rate (t1)=16.7 days for 12-day-old T. pratense soil in comparison with 4.7 days-1 and 25.4 days, respectively, for non-planted controls). The purpose of this study was to gain a better understanding of the plant-microbe interactions involved in rhizosphere-enhanced biodegradation by narrowing down the identity of the T. pratense rhizodeposit responsible for stimulating the microbial mineralization of 2,4-D. Specifically, we investigated the distribution of the stimulatory component(s) among rhizodeposit fractions (exudates or root debris) and the influence of soil properties and plant species on its production. Production of the stimulatory rhizodeposit was dependent on soil pH (e.g. t1 for roots grown at pH 6.5 was significantly lower than for those grown at pH 4.4) but independent of soil inorganic N concentration. Most strikingly, the stimulatory rhizodeposit was only produced by T. pratense grown in non-sterile soil and was present in both exudates and root debris. Comparison of the effect of root debris from plant species (three each) from the classes monocotyledon, dicotyledon (non-legume) and dicotyledon (legume) revealed that legumes had by far the greatest positive impact on 2,4-D mineralization kinetics. We discuss the significance of these findings with respect to legume-rhizobia interactions in the rhizosphere.