Effect of 2,4-dichlorophenoxyacetic acid on invertases in chicory root

The invertase present in roots of chicory (Cichoriun intybus) has a pH optimum of 7.5 and a MW of ca 260 000. It requires relatively high ionic strength to remove it from DEAE cellulose. Treatment of chicory root tissue with 2,4-dichlorophenoxyacetic acid gives rise to a highly active invertase with pH optimum of 5.6 and MW of ca 61 000. It is more easily removed from DEAE cellulose.     

Effect of 2,4-dichlorophenoxyacetic acid on the structure and function of developing chloroplasts

Dark-grown radish seedlings (Raphanus sativus L.) were sprayed with 10^-3 mol·l^-1 2,4-dichlorophenoxyacetic acid and then were exposed to a 14:10 light: dark cycle. Cotyledon samples from these seedlings and unsprayed controls were taken for electron microscopy, chlorophyll determinations, and photosynthetic rate measurements at regular intervals for 72 h. A normal development of etioplasts to chloroplasts with formation of typical grana-fret work system was observed in the control cotyledons. The chloroplasts in the 2,4-D-treated cotyledons showed changes in the organization of the grana thylakoids; these thylakoids being more appressed to each other than in the controls. The chlorophyll content of treated plants was less than that of controls but the rate of chlorophyll biosynthesis was unaffected. The photosynthetic rate/mg chlorophyll was considerably higher for treated plants suggesting that 2,4-D treatment resulted in decreased size of the photosynthetic unit.     

A bioluminescent whole-cell reporter for detection of 2, 4-dichlorophenoxyacetic acid and 2,4-dichlorophenol in soil

A bioreporter was made containing a tfdRP(DII)-luxCDABE fusion in a modified mini-Tn5 construct. When it was introduced into the chromosome of Ralstonia eutropha JMP134, the resulting strain, JMP134-32, produced a sensitive bioluminescent response to 2, 4-dichlorophenoxyacetic acid (2,4-D) at concentrations of 2.0 microM to 5.0 mM. This response was linear (R(2) = 0.9825) in the range of 2.0 microM to 1.1 x 10(2) microM. Saturation occurred at higher concentrations, with maximal bioluminescence occurring in the presence of approximately 1.2 mM 2,4-D. A sensitive response was also recorded in the presence of 2,4-dichlorophenol at concentrations below 1.1 x 10(2) microM; however, only a limited bioluminescent response was recorded in the presence of 3-chlorobenzoic acid at concentrations below 1.0 mM. A significant bioluminescent response was also recorded when strain JMP134-32 was incubated with soils containing aged 2,4-D residues.     

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 a gene probe with classical methods for detecting 2,4-dichlorophenoxyacetic acid (2,4-D)-biodegrading bacteria in natural waters

Colony hybridizations with a gene probe for enumeration of 2,4-dichlorophenoxy-acetic acid (2,4-D)-degrading bacteria were compared with classical enrichment and radiolabel most-probable-number (MPN) assay methods. Two natural water samples (rivers) and raw sewage were tested by each method. UV scans of enrichment cultures revealed 2,4-D degradation with raw sewage occurred in 4–11 days, 4–>22 days with Mary's River water, and 5–>22 days with Willamette River water. [¹⁴C]-2,4-D MPN analysis, measuring release of¹⁴CO₂, yielded estimates of bacteria per milliliter able to degrade 2,4-D. Raw sewage estimates were 1.4 × 10⁵ 2,4-D degraders/ml, Mary's River >1.6 × 10⁵/ml, and Willamette River water 1.6 × 10⁴/ml. Activities noted by UV scan enrichment data supported these results.Autoradiograms of colony blots were also used to estimate numbers of 2,4-D-degrading bacteria. These estimates were also supported by the UV scan data from enrichment cultures. Raw sewage gave counts between 5 × 10⁴ and 2.9 × 10⁵ 2,4-D-degrading bacteria/ml, which correlates well with the estimates obtained by¹⁴C-MPN analyses. River waters, both much lower in total bacterial counts and organic carbon than raw sewage, yielded fewer 2,4-D-degrading bacteria than estimated by¹⁴C-MPN. Media composition and cometabolism may account for discrepancies in estimates for 2,4-D-degrading bacteria observed when colony blot and¹⁴C-MPN analyses were compared.Replica plating made it possible to test for 2,4-D biodegradation from colonies reactive in autoradiograms. Five of 12 (42%) colonies reacting in the colony hybridization exhibited biodegradation activities. Nonreactive colonies failed to degrade 2,4-D.     

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.     

Influence of temperature on growth, 2,4-dichlorophenoxyacetic acid degradation and plasmid transfer in Azotobacter chroococcum

Summary Degradation of 2,4-D by Azotobacter chroococcum is profoundly influenced by incubation temperature. At 30 C the generation time was 1.4 h. Residual 2,4-D was detected when grown at 20 C. At 35 C the plasmid transfer was maximum.     

Effects of dissolved oxygen concentration on biodegradation of 2,4-dichlorophenoxyacetic acid

Batch experiments were conducted to examine the effects of dissolved oxygen concentration on the degradation of 2,4-dichlorophenoxyacetic acid (2,4-D) by an enrichment culture of 2,4-D-utilizing bacteria. A modified Monod equation was found to describe the relationship between the specific growth rate and the concentrations of both the organic substrate and dissolved oxygen. Values for the maximum specific growth rate, yield, and Monod coefficient for growth on 2,4-D were 0.09 h-1, 0.14 g/g, and 0.6 mg/liter, respectively. The half-saturation constant for dissolved oxygen was estimated to be 1.2 mg/liter. These results suggest that dissolved oxygen concentrations below 1 mg/liter may be rate limiting for the biodegradation of chlorinated aromatic compounds such as 2,4-D, which have a requirement for molecular oxygen as a cosubstrate for metabolism.     

Microbial degradation of 2,4-dichlorophenoxyacetic acid on the Greenland ice sheet

The Greenland ice sheet (GrIS) receives organic carbon (OC) of anthropogenic origin, including pesticides, from the atmosphere and/or local sources, and the fate of these compounds in the ice is currently unknown. The ability of supraglacial heterotrophic microbes to mineralize different types of OC is likely a significant factor determining the fate of anthropogenic OC on the ice sheet. Here we determine the potential of the microbial community from the surface of the GrIS to mineralize the widely used herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). Surface ice cores were collected and incubated for up to 529 days in microcosms simulating in situ conditions. Mineralization of side chain- and ring-labeled [(14)C]2,4-D was measured in the samples, and quantitative PCR targeting the tfdA genes in total DNA extracted from the ice after the experiment was performed. We show that the supraglacial microbial community on the GrIS contains microbes that are capable of degrading 2,4-D and that they are likely present in very low numbers. They can mineralize 2,4-D at a rate of up to 1 nmol per m(2) per day, equivalent to ～26 ng C m(-2) day(-1). Thus, the GrIS should not be considered a mere reservoir of all atmospheric contaminants, as it is likely that some deposited compounds will be removed from the system via biodegradation processes before their potential release due to the accelerated melting of the ice sheet.     

Enhanced degradation of phenoxyacetic acid in soil by horizontal transfer of the tfdA gene encoding a 2,4-dichlorophenoxyacetic acid dioxygenase

Few studies have investigated the possible impact of in situ gene transfer on the degradation of xenobiotic compounds in natural environments. In this work we showed that horizontal transfer of the tfdA gene, carried on plasmid pRO103, to phenol degrading recipient strains significantly increased the degradation rate of phenoxyacetic acid in sterile and non-sterile soil microcosms. The tfdA gene encodes a 2,4-dichlorophenoxyacetic acid/2-oxoglutarate dioxygenase and by complementation with the phenol degradation pathway an expanded catabolic substrate range, now including phenoxyacetic acid, is evolved. Presence of selective pressure had a positive effect on the emergence of transconjugants. However, even in the absence of phenoxyacetic acid transconjugant populations were detected and were kept at a constant level throughout the experimental period. The residuesphere (interface between decaying plant material and soil matrix) of dry leaves of barley was shown to be a hot-spot for gene transfer and presence of barley straw increased the conjugation frequencies in soil microcosms to the same extent as presence of organic nutrients. The results of this study indicate that dissemination of catabolic plasmids is a possible mechanism of genetic adaptation to degradation of xenobiotic compounds in natural environments, and that complementation of catabolic pathways possibly plays an important role in the evolution of new degradative capabilities. The application of horizontal gene transfer as a possible tool in bioremediation of contaminated sites is discussed.     

Plasmid as a measure of microbial degradation capacity for 2,4-dichlorophenoxyacetic acid

The purpose of this research was to pursuit the quantification of microbial degradation capacity for 2,4-dichlorophenoxyacetic acid (2,4-D) by detecting and quantifying a prominent 2,4-D degradation encoding plasmid. Batch reactor acclimation, de-acclimation, and re-acclimation tests were conducted during which periods the courses of 2,4-D dissipation and plasmid evolution were quantitatively measured. Pure cultures of bacterial strains were detected to give rise to a plasmid approximately the size of 90 kb after acclimation. The 90 kb plasmid content of Arthrobacter sp. increased when degradation occurred after acclimation, with a rate that corresponded closely to the degradation rate. During de-acclimation, plasmid content declined exponentially at a half-life of approximately 3.5 days. Re-acclimation saw a renewed induction of plasmid, but substrate consumption limited the rise of plasmid to a level much lower than after the first acclimation. This research recommends a method for measuring the microbial degradation capability for a xenobiotic.     

Duplication of a 2,4-dichlorophenoxyacetic acid monooxygenase gene in Alcaligenes eutrophus JMP134(pJP4).

The Alcaligenes eutrophus JMP134 plasmid pJP4 contains genes necessary for the complete degradation of 2,4-dichlorophenoxyacetic acid (2,4-D) and 3-chlorobenzoic acid. tfdA encodes 2,4-D monooxygenase, the initial enzyme in the 2,4-D catabolic pathway. The tfdA locus has recently been localized to a region on pJP4 13 kilobases away from a cluster of five genes, tfdB to tfdF, which encode the enzymes responsible for the further degradation of 2,4-D to chloromaleylacetic acid (W.R. Streber, K. N. Timmis, and M. H. Zenk, J. Bacteriol. 169:2950-2955, 1987). A second, dissimilar locus on pJP4, tfdAII, has been observed which encodes 2,4-D monooxygenase activity. Gas chromatographic analysis of the 2,4-D metabolites of A. eutrophus harboring pJP4 or subclones thereof localized tfdAII to within a 9-kilobase SstI fragment of pJP4 which also carries the genes tfdBCDEF. This fragment was further characterized in Escherichia coli by deletion and subcloning analysis. A region of 2.5 kilobases, adjacent to tfdC, enabled E. coli extracts to degrade 2,4-D to 2,4-dichlorophenol. Hybridization under low-stringency conditions was observed between tfdA and tfdAII, signifying that the 2,4-D monooxygenase gene was present as two related copies on pJP4.     

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.